**Part 2**

## **Application**

58 Genetic Engineering – Basics, New Applications and Responsibilities

Singh T., Hayashi M., Mano S., Arai Y., Goto S., & Nishimura M. (2009). Molecular

Tanaka Y., Nakamura S., Kawamukai M., Koizumi N., & Nakagawa T. (2011). Development

Waadt R., Schmidt L.K., Lohse M., Hashimoto K., Bock R., & Kudla J. (2008). Multicolor

Walter M., Chaban C., Schutze K., Batistic O., Weckermann K., Nake C., Blazevic D., Grefen

Xu X., Soutto M., Xie Q., Servick S., Subramanian C., von Arnim A.G., & Johnson C.H.

Ye X., Al-Babili S., Kloti A., Zhang J., Lucca P., Beyer P., & Potrykus I. (2000). Engineering

Zamyatnin A.A., Jr., Solovyev A.G., Bozhkov P.V., Valkonen J.P., Morozov S.Y., & Savenkov

Zelazny E., Borst J.W., Muylaert M., Batoko H., Hemminga M.A., & Chaumont F. (2007).

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signals in mixed DNA cloning by the Multisite Gateway system. *Journal of* 

components required for the targeting of PEX7 to peroxisomes in Arabidopsis thaliana. *The Plant Journal*, Vol.60, No.3, (Nov 2009), pp. 488-498, ISSN 1365-313X

of a series of gateway binary vectors possessing a tunicamycin resistance gene as a marker for the transformation of Arabidopsis thaliana. *Bioscience, Biotechnology, and Biochemistry*, Vol.75, No.4, (May 2011), pp. 804-807, ISSN 1347-6947 (Electronic),

bimolecular fluorescence complementation reveals simultaneous formation of alternative CBL/CIPK complexes in planta. *The Plant Journal*, Vol.56, No.3, (Nov

Vidal M. (2000). GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes. *Methods in Enzymology*,

C., Schumacher K., Oecking C., Harter K., & Kudla J. (2004). Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. *The Plant Journal*, Vol.40, No.3, (Nov 2004), pp. 428-438, ISSN

(2007). Imaging protein interactions with bioluminescence resonance energy transfer (BRET) in plant and mammalian cells and tissues. *P Natl Acad Sci USA*, Vol.104, No.24, (Jun 12 2007), pp. 10264-10269, ISSN 0027-8424 (Print), 0027-8424

the provitamin A (beta-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. *Science*, Vol.287, No.5451, (Jan 14 2000), pp. 303-305, ISSN 0036-8075

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The cauliflower mosaic virus (CaMV) 35S promoter sequence alters the level and patterns of activity of adjacent tissue- and organ-specific gene promoters. *Plant Cell* 

### **Thermostabilization of Firefly Luciferases Using Genetic Engineering**

Natalia Ugarova and Mikhail Koksharov

*Lomonosov Moscow State University, Faculty of Chemistry Moscow, Russia* 

#### **1. Introduction**

Bioluminescence is light emission from living organisms that is based on chemiluminescent reaction catalyzed by a specific kind of enzymes, luciferases. Bioluminescence accompanies the oxidation of an organic substrate called luciferin in the presence of a luciferase enzyme (Wilson & Hastings, 1998). Luciferin and luciferase are generic rather than structuralfunctional terms that describe substrates and enzymes that interact with each other with the emission of light. Emission of light energy in chemical and biological processes is observed rather often, especially in the reactions with participation of free radicals. However, in most cases the quantum yield (the number of light quanta emitted on the oxidation of one molecule of a substrate) is not higher than tenth or thousandth percent. The distinctive feature and great advantage of bioluminescent systems is high quantum yield (from 1 to 60%), which is achieved by the participation of luciferase in the reaction. Luciferases, on the one hand, act as a matrix, which is covalently or non-covalently bound to emitting chromophore. On the other hand, they play the role of biocatalyst that makes possible the formation of electronically excited product. In most cases luciferases are exceptionally specific to their substrates. The protein structures of bioluminescent enzymes impose strict requirements to the structure of luciferins. This is probably necessary for the maximum concentration of energy of the enzymatic reaction on the chromophore and for the formation of the electronically excited reaction product, which further returns to the ground state with the emission of quantum of visible light.

Luciferases have been isolated from a large number of organisms (insects, fish, bacteria, jellyfish, protozoa, etc) and characterized (Wilson & Hastings, 1998). All of them are oxidoreductases. Molecular air oxygen or hydrogen peroxide acts as an oxidizing agent. The structures of oxidizing substrates, luciferins, are rather different. In most cases, luciferins are heterocyclic compounds. The products of their oxidation have rather low excitation energy, and this is indeed the reason that the energy of enzymatic reaction appears to be enough for the formation of the product in electronically excited state. Therefore, one may conclude that luciferin-luciferase systems are unique, high-performance converters of the energy of biochemical reactions into light, and the interest in the study of structures and functions of luciferases and elucidation of their biological role in nature does not diminishes (Fraga, 2008; Seliger & McElroy, 1960).

The interest for bioluminescent systems is also determined by their great practical importance. For number of luciferases, such important metabolites as flavin mononucleotide (FMN) or adenosine triphosphate (ATP) are necessary participants (co-substrates) of the enzymatic reaction. High quantum yield of bioluminescence in the luciferase-catalyzed reactions simplifies the procedure of light registration. Due to the practically absolute specificity of luciferases to the substrates, luciferases are widely used as highly efficient analytical reagents. The intensity of light emitted in the course of the reaction (bioluminescence intensity) is proportional to the rate of the formation of electronically excited reaction product, and hence is proportional to the concentration of a substrate provided that the substrate concentration is lower than *Km*. Since every molecule of the substrate entering the reaction gives from 0.3 to 1 quantum of light, the sensitivity of bioluminescent methods of substrate detection is 10–13–10–18 moles of substrates in test solution that is by many orders of magnitude higher than the sensitivity of other detection methods. In addition, the construction of instruments for light measuring (luminometers) is much easier than that of spectrophotometers or spectrofluorimeters, which also simplifies practical application of bioluminescent systems (Ugarova, 2005).

#### **2. Overview of bioluminescent system of fireflies**

#### **2.1 Scheme of luciferase-catalyzed reaction**

Beetle luciferase [luciferin 4-monooxygenase (ATP hydrolysis); luciferin: oxygen 4 oxidoreductase (decarboxylation, ATP hydrolysis), EC 1.13.12.7] catalyzes firefly luciferin oxidation by air oxygen in the presence of MgATP (Fraga, 2008; Ugarova, 1989). The reaction is accompanied by the emission of visible light with the quantum yield of 40-60% (Niwa et al., 2010*)*. As shown in Fig. 1, at the first step the enzyme binds its substrates luciferin (1) and ATP. After the formation of the triple complex luciferin reacts with ATP to

Fig. 1. Scheme of the reaction catalyzed by firefly luciferase (Ugarova et al., 1993)

The interest for bioluminescent systems is also determined by their great practical importance. For number of luciferases, such important metabolites as flavin mononucleotide (FMN) or adenosine triphosphate (ATP) are necessary participants (co-substrates) of the enzymatic reaction. High quantum yield of bioluminescence in the luciferase-catalyzed reactions simplifies the procedure of light registration. Due to the practically absolute specificity of luciferases to the substrates, luciferases are widely used as highly efficient analytical reagents. The intensity of light emitted in the course of the reaction (bioluminescence intensity) is proportional to the rate of the formation of electronically excited reaction product, and hence is proportional to the concentration of a substrate provided that the substrate concentration is lower than *Km*. Since every molecule of the substrate entering the reaction gives from 0.3 to 1 quantum of light, the sensitivity of bioluminescent methods of substrate detection is 10–13–10–18 moles of substrates in test solution that is by many orders of magnitude higher than the sensitivity of other detection methods. In addition, the construction of instruments for light measuring (luminometers) is much easier than that of spectrophotometers or spectrofluorimeters, which also simplifies

Beetle luciferase [luciferin 4-monooxygenase (ATP hydrolysis); luciferin: oxygen 4 oxidoreductase (decarboxylation, ATP hydrolysis), EC 1.13.12.7] catalyzes firefly luciferin oxidation by air oxygen in the presence of MgATP (Fraga, 2008; Ugarova, 1989). The reaction is accompanied by the emission of visible light with the quantum yield of 40-60% (Niwa et al., 2010*)*. As shown in Fig. 1, at the first step the enzyme binds its substrates luciferin (1) and ATP. After the formation of the triple complex luciferin reacts with ATP to

> - O

O

O N


λ max λ max


<sup>O</sup> o

*h*ν

>600 nm ≅ 560 nm

N N

Fig. 1. Scheme of the reaction catalyzed by firefly luciferase (Ugarova et al., 1993)

 (1) (2) PPi

N

N

S S

2' 2

S S

S S

<sup>O</sup> **.**

N

N

N

S S

N


OH \*

O C O

<sup>C</sup> OAMP O

+O2

CO2

practical application of bioluminescent systems (Ugarova, 2005).

**2. Overview of bioluminescent system of fireflies** 

**2.1 Scheme of luciferase-catalyzed reaction** 

N


> - O


> - O

S S

2' 2 1

N

(3)

*h*ν N

3

N

S S

SS

S S

N

N

≈

N

O C O

(4б) (4а)

COOH

4 5 +ATP

O

form mixed anhydride of carboxylic and phosphoric acids, luciferyl adenylate (2) and pyrophosphate (РPi). The luciferyl adenylate is oxidized with air oxygen to form a cyclic peroxide, dioxytanone (3), through a series of intermediate steps. The dioxytanone molecule has a remarkable feature: one portion of the molecule is a readily oxidizable heterocyclic structure (luciferyl) with low ionizing potential and another portion (peroxide) has a high affinity to electron. Due to the intramolecular electron transfer from the phenolate group to the peroxide, a resonance charge-transfer structure is formed. The break of the O-O bond causes decarboxylation of dioxytanone and formation of biradical (ketone anion-radical and phenolate cation-radical). As a result of intramolecular recombination of the radicals, the reaction product, oxyluciferin (4), is formed in singlet electronically excited state. Depending on the properties of microenvironment, oxyluciferin may exist in the form of ketone (4a), enol (4b), or enolate-anion. The electronically excited oxyluciferin reactivates with the emission of quantum of light with λmax from 536 to 623 nm depending on the firefly species and pH (Viviani, 2002).

#### **2.2 Primary structure of firefly luciferases**

Up to the middle of 1980s, the studies were carried out with native firefly luciferases, which were isolated from desiccated firefly lanterns. As a rule, composition of enzyme preparations was not homogeneous due to the presence of different modified forms of enzyme, which, probably, appeared during the functioning of the enzyme. For this reason, even primary amino acid sequence of luciferases remained unknown for a long time. A new phase began in 1985 when cDNA of luciferase for the North American *Photinus pyralis* fireflies was isolated (De Wet et al., 1985*)*. In 1987, the amino acid sequence of this luciferase was determined (De Wet et al., 1987*)*, and in 1989 four luciferases from Jamaican click beetles were cloned (Wood et al., 1989). At the beginning of 1990th, the luciferase from the East-European (north-Caucasian) *Luciola mingrelica* fireflies was cloned and the homogeneous preparation of the recombinant *L. mingrelica* luciferase was obtained (*Devine* et al., 1993). At the present time, primary structures of more than 40 luciferase genes are known that were isolated from different species of fireflies, click-beetles, and railroad worms inhabiting in the USA, Russia, Japan, South-American countries, *etc* (Arnoldi et al., 2010; Oba et al., 2010a; Viviani, 2002). The scheme of the chemical reaction catalyzed and the structure of the emitter are identical for all firefly luciferases. Luciferase molecules consist of one polypeptide chain (542-552 residues), do not contain cofactors and have similar amino acid composition. More than half of the amino acids are non-polar or ambivalent. The number of charged residues is also practically identical for all firefly luciferases, and the main difference between them is in the number of Trp and Cys residues. The amino acid sequence identity for different luciferases usually correlates with phylogenetic relationships of the corresponding fireflies (Li et al, 2006). For luciferases from Luciolini tribe of fireflies (*Luciola* and *Hotaria* genus's) the sequence identity is near 80%, and it is more than 90%for the part of the protein after a 200th residue. The structure of *L. mingrelica* luciferase is very close to the structure of Japanese *Hotaria* (*Luciola*) *parvula* luciferase (98% identity) despite the huge geographic separation of the original firefly species. The *Luciola* luciferases show less identity with luciferases from other firefly subfamilies (67% identity), for example, *P. pyralis* or *L. noctiluca*, and with luciferases from click beetles (43% identity) (Ugarova & Brovko, 2002). At the same time, the identity between the luciferases isolated from Jamaican and Brazil beetles reaches 80% (Oba et al, 2010b; Viviani et al., 1999).

Beetle luciferases belong to the superfamily of enzymes catalyzing formation of acyladenylates from ATP and compounds with a carboxyl group. This superfamily includes the families of non-ribosomal peptide synthetases, acyl-CoA-ligases, *etc* (Gulick, 2009). In 1990s the search of conserved motifs in the amino acid sequences of the proteins within this superfamily has been carried out in our laboratory using computer analysis. In addition to the previously known motif 1 (residues 197-210 in the *L. mingrelica* luciferase), another conserved motif 2 (residues 410-460) has been found. It was proposed that the conserved amino acid residues in motifs 1 and 2 perform key functions in the catalysis and that both motifs belong to a single conserved structural element. It was also proposed that the 197-220 region forms the ATP binding center and the 410-460 region is important for the interaction of the luciferase with its activator coenzyme A (Morozov & Ugarova, 1996*)*. Later structural findings have confirmed these conclusions.

#### **2.3 Spatial structure of firefly luciferase and its complexes with substrates**

In 1996 the spatial structure of the luciferase from the American *P. pyralis* fireflies without substrates was obtained using X-Ray analysis (Conti et al., 1996). This opened a new stage in the studies of firefly luciferases. The X-Ray data confirmed the supposition given above about the important role of motifs 1 and 2. Both motifs are loops with undefined electron density indicating high mobility of these fragments, and the motif 2 is partly involved in a polypeptide connecting the two domains of the protein globule. Mutual arrangement of the domains plays an important role in the catalysis as it will be shown below. Unfortunately, one may only assume the possible binding centers for luciferin and ATP. In 1997 the data about the crystal structure of another enzyme belonging to the superfamily of adenylating proteins (adenylase) were published (Conti et al., 1997). In this case the crystals of the complex of the enzyme with its substrates AMP and phenylalanine (analog of the complex of the enzyme with the adenylation product) were analysed. As one may expect, spatial structures of adenylase and luciferase, which both have the same ability for adenylation of substrate carboxyl group with ATP, appeared to be rather similar although the amino acid sequence homology of these enzymes is low. Both enzymes are composed of two domains, large N-domain, which, in turn, may be divided into three sub-domains, and small C-domain. The two domains are connected with very flexible and disordered loop. Each domain and even sub-domain of the adenylase has similar topology with the corresponding domain or sub-domain of the luciferase. The only significant difference is mutual rearrangement of the N- and C-domains. The domains of adenylase are drawn together and rotated by 90o with respect to their orientation in the firefly luciferase (Conti et al., 1997). It was proposed that the observed rotation of domains relative to each other is a consequence of change in the globule conformation upon binding of substrates. In this case the threedimensional structure of the luciferase-luciferin-ATP complex should be similar to that of the adenylase-phenyl alanine-AMP complex. The computer modeling on the basis of this hypothesis allowed to construct a model of the enzyme-substrate complex for the *P. pyralis* luciferase (Sandalova & Ugarova, 1999*)*. The structures of the luciferase before and after binding of substrates are shown in Fig. 2.

Neither ATP, nor luciferin can bind with the enzyme without the rotation of the domains. The catalytically important residues of the C-domain (Lys529 and Thr527) approach the substrate molecules only after the rotation of the domains. Similar model structure was obtained by Branchini and coworkers (Branchini et al. 1998). All the residues that are in

Beetle luciferases belong to the superfamily of enzymes catalyzing formation of acyladenylates from ATP and compounds with a carboxyl group. This superfamily includes the families of non-ribosomal peptide synthetases, acyl-CoA-ligases, *etc* (Gulick, 2009). In 1990s the search of conserved motifs in the amino acid sequences of the proteins within this superfamily has been carried out in our laboratory using computer analysis. In addition to the previously known motif 1 (residues 197-210 in the *L. mingrelica* luciferase), another conserved motif 2 (residues 410-460) has been found. It was proposed that the conserved amino acid residues in motifs 1 and 2 perform key functions in the catalysis and that both motifs belong to a single conserved structural element. It was also proposed that the 197-220 region forms the ATP binding center and the 410-460 region is important for the interaction of the luciferase with its activator coenzyme A (Morozov & Ugarova, 1996*)*. Later structural

**2.3 Spatial structure of firefly luciferase and its complexes with substrates** 

In 1996 the spatial structure of the luciferase from the American *P. pyralis* fireflies without substrates was obtained using X-Ray analysis (Conti et al., 1996). This opened a new stage in the studies of firefly luciferases. The X-Ray data confirmed the supposition given above about the important role of motifs 1 and 2. Both motifs are loops with undefined electron density indicating high mobility of these fragments, and the motif 2 is partly involved in a polypeptide connecting the two domains of the protein globule. Mutual arrangement of the domains plays an important role in the catalysis as it will be shown below. Unfortunately, one may only assume the possible binding centers for luciferin and ATP. In 1997 the data about the crystal structure of another enzyme belonging to the superfamily of adenylating proteins (adenylase) were published (Conti et al., 1997). In this case the crystals of the complex of the enzyme with its substrates AMP and phenylalanine (analog of the complex of the enzyme with the adenylation product) were analysed. As one may expect, spatial structures of adenylase and luciferase, which both have the same ability for adenylation of substrate carboxyl group with ATP, appeared to be rather similar although the amino acid sequence homology of these enzymes is low. Both enzymes are composed of two domains, large N-domain, which, in turn, may be divided into three sub-domains, and small C-domain. The two domains are connected with very flexible and disordered loop. Each domain and even sub-domain of the adenylase has similar topology with the corresponding domain or sub-domain of the luciferase. The only significant difference is mutual rearrangement of the N- and C-domains. The domains of adenylase are drawn together and rotated by 90o with respect to their orientation in the firefly luciferase (Conti et al., 1997). It was proposed that the observed rotation of domains relative to each other is a consequence of change in the globule conformation upon binding of substrates. In this case the threedimensional structure of the luciferase-luciferin-ATP complex should be similar to that of the adenylase-phenyl alanine-AMP complex. The computer modeling on the basis of this hypothesis allowed to construct a model of the enzyme-substrate complex for the *P. pyralis* luciferase (Sandalova & Ugarova, 1999*)*. The structures of the luciferase before and after

Neither ATP, nor luciferin can bind with the enzyme without the rotation of the domains. The catalytically important residues of the C-domain (Lys529 and Thr527) approach the substrate molecules only after the rotation of the domains. Similar model structure was obtained by Branchini and coworkers (Branchini et al. 1998). All the residues that are in

findings have confirmed these conclusions.

binding of substrates are shown in Fig. 2.

direct contact with the substrates are absolutely conservative. These models were further confirmed by the independent studies: the residual activity of the luciferase mutant, in which Lys529 was changed to Ala, was less than 0.1% (Branchini et al., 2000). The role of others residues of the luciferase active site was supported by mutagenesis methods (Branchini et al., 1999, 2000, 2001, 2003).

Fig. 2. Superimposition of the spatial structures of firefly luciferase without substrates (grey tubes) and the luciferase-luciferin-ATP complex (magenta tubes). The substrates are shown as CPK models (Sandalova & Ugarova, 1999). Molecular graphics were created with YASARA (www.yasara.org) and PovRay (www.povray.org) software

In 2006 three structures of *Luciola cruciata* luciferase were solved (Nakatsu et al., 2006): in complex with Mg-ATP, in complex with the analog of intermediate product of the adenylation step – DLSA, and in complex with the reaction products – LO and AMP. This allowed to correct the data obtained previously by homology modeling. Firefly luciferase consists of two domains, large N-terminal domain (1-436 amino acid residues) and small Cterminal domain (443-544 amino acid residues), which are connected by the flexible loop (337-442 amino acid residues). By-turn N-terminal domain is composed of two distinct subdomains (Wang et al., 2001): A (1-190) and B (191-436) forming a strong hydrophobic interface (Fig. 3).

When luciferase is in complex with DLSA, the C-domain is rotated by 90° relative to the Ndomain compared to the free enzyme. This leads to the transformation from open to closed conformation of luciferase so that both domains are in close contact. The structures obtained allowed to determine the structure of the firefly luciferase active site and the environment of the substrates at the adenylation step of the reaction. Later findings revealed that yet another rotation of the C-domain by 140° is required for the oxidation step (Gulick, 2009). In this conformation the residue K445 assumes the role of the group K531 (Branchini et al., 2005; Branchini et al., 2011).

Fig. 3. 3D-structure of *L. cruciata* firefly luciferase in complex with DLSA (Nakatsu et al., 2006). Subdomains A, B and C are depicted in blue, magenta and orange, respectively

#### **3. Stability of firefly luciferases in solution**

Firefly luciferases are currently used for microbial contamination testing, as very sensitive reporter genes and in pyrosequencing. They are also promising labels in immunoassays and biosensors for the study of protein-protein interactions (Binkowski et al., 2009; Lundin, 2000; Viviani & Ohmiya, 2006). The stability and activity of enzymes are basic parameters defining the efficiency of their applications. Nevertheless, applications of wild-type firefly luciferases are often limited by the insufficient stability of the enzyme. Firefly luciferases demonstrate low stability in water solutions (Dementieva et al., 1989; White et al., 1996) and can aggregate as well as can be easily adsorbed on the surfaces (Hall & Leach, 1988; Herbst et al., 1998; Suelter & DeLuca, 1983).

The *P. pyralis* luciferase loses up to 99% of activity within 24 hours at 4-8°C as a result of protein adsorption on the container surface when low concentrations of luciferases are used. The addition of bovine serum albumin (0.1 mg/ml) reduces activity loss associated with adsorbtion to 10% even at very low concentrations of luciferase (10 ng/ml). An increase of ionic strength increased the stability of luciferase and an addition of Triton X-100 (0.2 mM)

the substrates at the adenylation step of the reaction. Later findings revealed that yet another rotation of the C-domain by 140° is required for the oxidation step (Gulick, 2009). In this conformation the residue K445 assumes the role of the group K531 (Branchini et al.,

Fig. 3. 3D-structure of *L. cruciata* firefly luciferase in complex with DLSA (Nakatsu et al., 2006). Subdomains A, B and C are depicted in blue, magenta and orange, respectively

Firefly luciferases are currently used for microbial contamination testing, as very sensitive reporter genes and in pyrosequencing. They are also promising labels in immunoassays and biosensors for the study of protein-protein interactions (Binkowski et al., 2009; Lundin, 2000; Viviani & Ohmiya, 2006). The stability and activity of enzymes are basic parameters defining the efficiency of their applications. Nevertheless, applications of wild-type firefly luciferases are often limited by the insufficient stability of the enzyme. Firefly luciferases demonstrate low stability in water solutions (Dementieva et al., 1989; White et al., 1996) and can aggregate as well as can be easily adsorbed on the surfaces (Hall & Leach, 1988; Herbst et al.,

The *P. pyralis* luciferase loses up to 99% of activity within 24 hours at 4-8°C as a result of protein adsorption on the container surface when low concentrations of luciferases are used. The addition of bovine serum albumin (0.1 mg/ml) reduces activity loss associated with adsorbtion to 10% even at very low concentrations of luciferase (10 ng/ml). An increase of ionic strength increased the stability of luciferase and an addition of Triton X-100 (0.2 mM)

**3. Stability of firefly luciferases in solution** 

1998; Suelter & DeLuca, 1983).

2005; Branchini et al., 2011).

and glycerol (10-50%) prevented the adsorption of luciferase on the surface (Suelter & DeLuca, 1983). The stabilizing effect of salt addition was also observed for *L. mingrelica* firefly luciferase. In presence of 0.4 M MgSO4 and 0.5 M Na2SO4 the thermal stability of *L. mingrelica* luciferase was 2 and 10 times higher respectively compared with a buffer solution without the salt added (Brovko et al., 1982). However, even in the presence of stabilizing salts at 10°C *L. mingrelica* luciferase loses about 90% of activity after 10-15 days storage at the concentration of 0.1 mg/ml. But after the addition of 10% ethylene glycol and the antimicrobial agent (0,02% NaN3) the luciferase can be stored more than 100 days without loss of activity (Dementieva et al., 1989). The presence of osmolytes in the solution such as glycine betaine and polyols can lead to a significant stabilization of luciferase at the elevated temperatures (Eriksson et al, 2003; Mehrabi et al., 2008; Moroz et al., 2008). Luciferases from the *Luciola* genus readily inactivate at the low ionic strength though *P. pyralis* luciferase is crystallized at the same conditions (Dementieva et al., 1989; Simomura et al., 1977). Native firefly luciferases as well as recombinant enzyme during eukaryotic expression are localized in peroxisomes (De Wet et al., 1987; Gould et al., 1987; Keller et al., 1987) due to the presence of C-terminal signal peptide (AKM, SKL) that is recognized by the cellular peroxisomal transport system (Rainer, 1992).

Firefly luciferase is sensitive to proteolysis, which leads to a short *in vivo* half-life of the enzyme. This effect is especially pronounced at high temperatures when partial enzyme unfolding leads to the higher accessibility of proteolytic sites (Frydman, 1999). Two protease sensitive regions (206-220 and 329-341 residues) were identified in *P. pyralis* luciferase amino acid sequence (Thompson et al., 1997). Half-life of luciferase activity *in vivo* in mammalian cells is only 3-4 hours at 37°C (Leclerc et al., 2000; Thompson et al., 1991). Substrate and competitive inhibitors change the conformation of luciferase, when introduced to prokaryotic and eukaryotic cells, leading to a several-fold decrease of enzyme degradation (Thompson et al., 1991). *P. pyralis* luciferase quickly inactivates in eukaryotic cells at 40-45°C with a half-life of 4-20 min (Forreitor et al., 1997; Souren et al., 1999b). Nevertheless, the stability can be relatively high at moderate temperatures. For example, *L. mingrelica* luciferase produced in frog oocytes had a half-life of about 2 days (Kutuzova et al., 1989).

At the same time even in the solutions with high ionic strength and in the presence of glycerol (10%) and bovine serum albumin (10 mg/ml) *P. pyralis* luciferase loses >90% of activity *in vitro* within 6-20 min at 37-42°C (Nguyen et al., 1989; Thulasiraman & Matts, 1996; Tisi et al., 2002; White et al., 1996). Similar results were obtained for *L. mingrelica* luciferase (Lundovskikh et al., 1998; Ugarova et al., 2000). It was demonstrated that inactivated luciferase is virtually unable to restore activity after cooling and usually aggregates (Minami & Minami, 1999; Schumacher et al., 1996; Souren et al., 1999a, 1999b). Appreciable spontaneous reactivation was only observed for diluted solutions of luciferase that was fully inactivated by guanidine chloride. But reactivation rate was very low and equilibrium was reached after 72 hours. In case of partial denaturation by guanidine chloride the degree of reactivation was low apparently due to aggregation of the enzyme (Herbst et al., 1998). Denatured luciferase can still be effectively reactivated in the presence of different chaperone systems. For instance, in rabit reticulocyte lysate a refolding of luciferase occurs with a half-period of 8 minutes (Nimmesgern et al., 1993). A detailed mechanism of luciferase inactivation in solution is still unknown. In several works (Frydman et al., 1999; Herbst et al., 1998; Wang et al., 2001) different unfolding intermediates were studied that are formed during chemical denaturation and refolding of *P. pyralis* luciferase. Thermoinactivation kinetics was studied in detail in case of *L. mingrelica* luciferase (Brovko et al., 1982; Ugarova et al., 2000). It was shown that at elevated temperature *L. mingrelica* luciferase undergoes two-step inactivation: at the first stage a reversible dissociation of luciferase homodimers probably occurs, which is followed by an irreversible inactivation of monomers (Lundovskikh et al., 1998). Luciferases of *Luciola cruciata* and *Luciola lateralis* were isolated and purified in 1992. *L. cruciata* luciferase was found to be similar to *P. pyralis* luciferase in stability, whereas *L. lateralis luciferase* showed significantly higher thermal and pH stability compared with the other two. *L. cruaciata* and *P. pyralis* luciferases were completely inactivated after incubation at 50°C for 30 min. Under the same conditions (10 mМ Naphosphate, 0.2 mМ EDTA, 100 mМ NaCl, 10% glycerol) the remaining activity of *L. lateralis* luciferase was about 20% (Kajiyama et al., 1992).

Thus, an optimization of the enzyme microenvironment in the solution may increase the stability of luciferases. The more promising approach is thermostabilization of luciferases by genetic engineering methods, which opens wide opportunities of new enzyme forms with enhanced resistance to such environment factors as temperature, organic solvents, and other chemical agents (Ugarova, 2010). It is important to emphasize that comparison of thermostability of mutant and wild-type luciferases must be performed under identical conditions, because the buffer composition and different stabilizing agents (BSA, ammonium sulfate, phosphates, glycerol, etc.) can greatly affect the rate of thermal inactivation.

#### **4. Thermostabilization of firefly luciferases by random and site-directed mutagenesis**

In the absence of structural information on a luciferase, random mutagenesis was the most common and efficient approach to obtain mutant proteins with improved stability to the action of temperature, pH, etc. Firefly luciferase is particularly suitable for activity screens owing to the ease with which its bioluminescence activity can be detected (Wood & DeLuca, 1987). Kajiyama and Nakano were the first to use random mutagenesis in an attempt to isolate thermostable mutants of the luciferase from *L. cruciata* fireflies (Kajiyama & Nakano, 1993). The mutagen-treated plasmid was transformed into *E.coli* JM101. After 12 h at 37°C, colonies on LB/ampicillin plates were transferred on nitrocellulose filters, which were put on new agar plates and incubated at 60°C for 30 min. Remaining luciferase activity was determined on filters by photographic method using X-ray films. Three isolated mutants carried the same an amino acid substitution Thr217Ile and were superior to wild-type in thermal and pH stability. Furthermore, its specific activity was increased to 130% of that of the wild-type. In order to examine the effect of amino acid residue substitution at position 217 on the thermostability of *L. cruciata* luciferase, the authors have replaced the residue Thr217 with all possible amino acid residues by site-directed mutagenesis. The thermostability of these mutants correlated well with hydrophobicity of the substituted amino acid residue. Especially, Thr217Leu and Thr217Val luciferases still retained over 75% of activity after 10 min incubation at 50°C. The fact that hydrophilic and large hydrophobic (Trp and Tyr) substitution resulted in an expression yield >1000-fold less than wild-type is also consistent with residue 217 being in a buried, hydrophobic environment (Kajiyama & Nakano, 1993). The amino acid sequences of *L. cruciata* and *L. lateralis* luciferases are 94% identical. To examine the effect of hydrophobic amino acid at position 217 on the thermostability of *L. lateralis luciferase*, three mutants, Ala217Ile, Leu, Val were constructed,

kinetics was studied in detail in case of *L. mingrelica* luciferase (Brovko et al., 1982; Ugarova et al., 2000). It was shown that at elevated temperature *L. mingrelica* luciferase undergoes two-step inactivation: at the first stage a reversible dissociation of luciferase homodimers probably occurs, which is followed by an irreversible inactivation of monomers (Lundovskikh et al., 1998). Luciferases of *Luciola cruciata* and *Luciola lateralis* were isolated and purified in 1992. *L. cruciata* luciferase was found to be similar to *P. pyralis* luciferase in stability, whereas *L. lateralis luciferase* showed significantly higher thermal and pH stability compared with the other two. *L. cruaciata* and *P. pyralis* luciferases were completely inactivated after incubation at 50°C for 30 min. Under the same conditions (10 mМ Naphosphate, 0.2 mМ EDTA, 100 mМ NaCl, 10% glycerol) the remaining activity of *L. lateralis*

Thus, an optimization of the enzyme microenvironment in the solution may increase the stability of luciferases. The more promising approach is thermostabilization of luciferases by genetic engineering methods, which opens wide opportunities of new enzyme forms with enhanced resistance to such environment factors as temperature, organic solvents, and other chemical agents (Ugarova, 2010). It is important to emphasize that comparison of thermostability of mutant and wild-type luciferases must be performed under identical conditions, because the buffer composition and different stabilizing agents (BSA, ammonium

sulfate, phosphates, glycerol, etc.) can greatly affect the rate of thermal inactivation.

**4. Thermostabilization of firefly luciferases by random and site-directed** 

In the absence of structural information on a luciferase, random mutagenesis was the most common and efficient approach to obtain mutant proteins with improved stability to the action of temperature, pH, etc. Firefly luciferase is particularly suitable for activity screens owing to the ease with which its bioluminescence activity can be detected (Wood & DeLuca, 1987). Kajiyama and Nakano were the first to use random mutagenesis in an attempt to isolate thermostable mutants of the luciferase from *L. cruciata* fireflies (Kajiyama & Nakano, 1993). The mutagen-treated plasmid was transformed into *E.coli* JM101. After 12 h at 37°C, colonies on LB/ampicillin plates were transferred on nitrocellulose filters, which were put on new agar plates and incubated at 60°C for 30 min. Remaining luciferase activity was determined on filters by photographic method using X-ray films. Three isolated mutants carried the same an amino acid substitution Thr217Ile and were superior to wild-type in thermal and pH stability. Furthermore, its specific activity was increased to 130% of that of the wild-type. In order to examine the effect of amino acid residue substitution at position 217 on the thermostability of *L. cruciata* luciferase, the authors have replaced the residue Thr217 with all possible amino acid residues by site-directed mutagenesis. The thermostability of these mutants correlated well with hydrophobicity of the substituted amino acid residue. Especially, Thr217Leu and Thr217Val luciferases still retained over 75% of activity after 10 min incubation at 50°C. The fact that hydrophilic and large hydrophobic (Trp and Tyr) substitution resulted in an expression yield >1000-fold less than wild-type is also consistent with residue 217 being in a buried, hydrophobic environment (Kajiyama & Nakano, 1993). The amino acid sequences of *L. cruciata* and *L. lateralis* luciferases are 94% identical. To examine the effect of hydrophobic amino acid at position 217 on the thermostability of *L. lateralis luciferase*, three mutants, Ala217Ile, Leu, Val were constructed,

luciferase was about 20% (Kajiyama et al., 1992).

**mutagenesis** 

and all of them demonstrated enhanced thermostability (Kajiyama & Nakano, 1994). The *L. lateralis* luciferase mutant Ala217Leu retained over 70% of the initial activity after 60 min incubation at 50°C. Its half-life was about 20 times longer than that of the wild type *L. lateralis* luciferase. Its thermostability was superior to that of the *L. cruciata* luciferase mutant Thr217Leu.

Random mutagenesis was also used to obtain thermostable mutant of *P.pyralis* luciferase. The substitution Glu354Lys increased thermostability of the enzyme 5-fold (White et al., 1996). The substitution of Glu354 with all possible amino acid residues by site-directed mutagenesis showed that the most stable mutants contained Lys or Arg residues. Thus, the substitution of negatively charged residue to positive one in this part of enzyme molecule increased the thermostability of *P.pyralis* luciferase. Thermostable *P.pyralis* luciferase was also obtained by a combination of random and site-directed mutagenesis. The double mutant was constructed that contained the substitutions Glu354Lys and Ala215Leu (similar to Ala217Leu in *L. lateralis* luciferase). In this case the effect of thermostabilization was not as high as for *. lateralis* luciferase. At 37°C the single mutants retained 10-15% of activity after 5 hours, whereas the wild type luciferase was completely inactivated. The double mutant combined the thermostability gains of the single mutants and retained greater than 50% activity for over 5 h. At 42°C the half life of the double mutant was reduced to 20 minutes. At 50°C it was only 4 min (Price et al., 1996). Other point mutations have been identified (largely by random mutagenesis) that significantly increase the thermostability of the *P.pyralis* luciferase: T214A, I232A and F295L. Combining these point mutations with the E354K mutation into the *P.pyralis* gene resulted in mutant luciferase (rLucx4ts) that had an increase in thermostability of about 7°C relative to the wild-type enzyme. Hence, in this case the multiple point mutations led to a cumulative increase in thermostability (Tisi et al., 2002).

After the spatial structure of luciferase was published, it became possible to rationally select specific positions for mutagenesis. For example, in molecule of *P.pyralis* luciferase five bulky hydrophobic solvent-exposed residues, which are all non-conserved and do not participate in secondary-structure formation, were substituted by hydrophilic ones, in particular by charged groups. These substitutions (F16R, L37Q, V183K, I234K and F465R) led to the enzyme with greatly improved pH-tolerance and stability up to 45°C. The mutant showed neither a decrease in specific activity relative to the wild-type luciferase (Law et al., 2006). Introduction of almost all known point mutations (12 residues) enhancing the thermostability of *P. pyralis* luciferase resulted in a highly stable mutant with half-time of inactivation of 15 min at 55°C, whereas wild-type luciferase inactivates within seconds at this conditions (Tisi et al., 2007).

#### **5. Rational protein design approach to produce the stable and active enzyme**

Mutations that are efficient in one particular luciferase do not always lead to successful results when applied to other homologous luciferases. For example, the mutation E354R increased the thermal stability of *P. pyralis* luciferase, whereas the corresponding E356R substitution did not affect *H. parvula* luciferase. The substitution A217L in *L. lateralis*, *L. cruciata* and in *P. pyralis* (A215L) firefly luciferases produced fully active and thermostable mutants, but in the case of *H. parvula luciferase* this mutation decreased activity to about 0.1% of the wild type in spite of some increase in thermal stability (Kitayama, et al. 2003). These results are of particular interest for the *L. mingrelica* luciferase because it shares 98% homology with *H. parvula*. Hence, both enzymes are considered to be almost identical, and the similar effect of this mutation could be expected for *L. mingrelica* luciferase. A rational protein design approach was used to increase thermal stability of *L. mingrelica* luciferase and prevent the detrimental effect of the of the A217L mutation on its activity by combining the mutation A217L with additional substitutions in its vicinity. The three-dimensional structure of the firefly luciferase and the multiple sequence alignment of beetle luciferases were analyzed to identify these additional substitutions (Koksharov & Ugarova, 2011a). Comparison of the A217 environment in *L. mingrelica* luciferase with that of *L. cruciata* and *L. lateralis* luciferases showed only 3 significant differences: G216N, I212L, S398M. Another difference was the change I212L, but it is unlikely to be important because the properties of Leu and Ile are very close. On the other hand, the neighboring residue G216 and the more remote S398 are characteristic for the small subgroup of luciferases very close in homology to *L. mingrelica* luciferase (including *H. parvula* luciferase). We surmised that the elimination of these differences between two groups of luciferases would lead to the A217 environment similar to that of *L. cruciata* and *L. lateralis* luciferases, which could possibly prevent the loss of activity accompanying the substitution A217L. First, we assumed that that changing the neighboring residue G216 would be sufficient to retain the enzyme activity/ Therefore, the double mutant G216N/A217L was constructed. Since this double mutant still showed low activity, we introduced the additional substitution S398M of the less close residue. This led to a stable and active mutant of *L. mingrelica* luciferase (Table 1).


Table 1. Thermal stability of luciferases with substitution of the residue 217 in a 0.05 M Naphosphate buffer, containing 0.4 M (NH4)2SO4, 2 mM EDTA, 0.2 mg/ml BSA, pH 7.8

The residues 216, 217, 398 are located near one of the walls of the luciferin-binding channel (Fig. 4). In the majority of beetle luciferases position 216 is normally occupied with a residue having a side group but in *L. mingrelica* and *H. parvula* luciferases it is occupied with Gly. Glycine is known to be a very destabilizing residue when in internal position of α-helices because of the absence of side group and excessive conformational freedom (Fersht & Serrano, 1993).

Since the G216 is located in the α-helix (Fig. 4) it can be suggested that it makes the surrounding structure less stable and more sensitive to the substitutions of the neighboring

homology with *H. parvula*. Hence, both enzymes are considered to be almost identical, and the similar effect of this mutation could be expected for *L. mingrelica* luciferase. A rational protein design approach was used to increase thermal stability of *L. mingrelica* luciferase and prevent the detrimental effect of the of the A217L mutation on its activity by combining the mutation A217L with additional substitutions in its vicinity. The three-dimensional structure of the firefly luciferase and the multiple sequence alignment of beetle luciferases were analyzed to identify these additional substitutions (Koksharov & Ugarova, 2011a). Comparison of the A217 environment in *L. mingrelica* luciferase with that of *L. cruciata* and *L. lateralis* luciferases showed only 3 significant differences: G216N, I212L, S398M. Another difference was the change I212L, but it is unlikely to be important because the properties of Leu and Ile are very close. On the other hand, the neighboring residue G216 and the more remote S398 are characteristic for the small subgroup of luciferases very close in homology to *L. mingrelica* luciferase (including *H. parvula* luciferase). We surmised that the elimination of these differences between two groups of luciferases would lead to the A217 environment similar to that of *L. cruciata* and *L. lateralis* luciferases, which could possibly prevent the loss of activity accompanying the substitution A217L. First, we assumed that that changing the neighboring residue G216 would be sufficient to retain the enzyme activity/ Therefore, the double mutant G216N/A217L was constructed. Since this double mutant still showed low activity, we introduced the additional substitution S398M of the less close residue. This led

to a stable and active mutant of *L. mingrelica* luciferase (Table 1).

Relative specific activity%

wild-type 100 13 ± 1 G216N/A217L 10 280 ± 28 S398M 106 16.1 ± 1.6

Table 1. Thermal stability of luciferases with substitution of the residue 217 in a 0.05 M Naphosphate buffer, containing 0.4 M (NH4)2SO4, 2 mM EDTA, 0.2 mg/ml BSA, pH 7.8

The residues 216, 217, 398 are located near one of the walls of the luciferin-binding channel (Fig. 4). In the majority of beetle luciferases position 216 is normally occupied with a residue having a side group but in *L. mingrelica* and *H. parvula* luciferases it is occupied with Gly. Glycine is known to be a very destabilizing residue when in internal position of α-helices because of the absence of side group and excessive conformational freedom (Fersht &

Since the G216 is located in the α-helix (Fig. 4) it can be suggested that it makes the surrounding structure less stable and more sensitive to the substitutions of the neighboring

*Luciola* wild-type 100 ~ 4

luciferase T217I 130 50 °C ~ 28

*Luciola* wild-type ~ 6

luciferase A217L 50 °C ~ 125

*Hotaria* wild-type 100 ~ 18

luciferase A217L 0.074 45°C ~ 60

G216N/A217L/S398M 60

Temperature of inactivation

45°C

Half-life, min Reference

276 ± 28

Kajiyama& Nakano, 1993

Kajiyama & Nakano, 1994

Kitayama et al., 2003

Koksharov & Ugarova, 2011a

Enzyme Mutant

*cruciata*

*lateralis*

*parvula*

*Luciola mingrelica* luciferase

Serrano, 1993).

residues. This can explain the unusual decrease in activity in case of the A217L mutation in *Hotaria parvula* luciferase (Kitayama, et al. 2003). The double mutation G216N/A217L resulted in the significant increase of the thermal stability of *L. mingrelica* luciferase, but this mutant retained only 10% of the wild-type activity. The comparison of the environment of residue 217 in the crystal structure of *L. cruciata luciferase* (Nakatsu, et al., 2006) with the homology model of *L. mingrelica* luciferase (Koksharov & Ugarova, 2008) (Fig. 4) shows that internal cavities probably exist in *L. mingrelica* luciferase near the 216 and 398 positions because of the smaller size side groups of the residues in this positions compared to *L. cruciata luciferase*. Additional cavity in the vicinity of S398 could potentially decrease the local conformational stability, make it more flexible and sensitive to the mutations and the changes in the environment. This hypothesis is supported by the higher resistance of the bioluminescence spectrum of the S398M mutant to pH and temperature, which indicates more rigid and stable microenvironment (Ugarova & Brovko, 2002).

Fig. 4. Structure of *L. mingrelica* luciferase in complex with oxyluciferin (LO) and AMP. The residues G216, A217, R220 and S398 are indicated by arrows. 7 Å microenvironment of A217 is indicated by ellipse (Koksharov & Ugarova, 2011a). The large N-terminal and the smaller C-terminal domains are depicted in grey and orange, respectively

The lowered local conformational stability in the vicinity of G216 and S398 residues can explain why the A217L mutation in *H. parvula* and *L. mingrelica* luciferaess leads to the decline in activity and red shift of λmax that were not observed in the cases of *L. cruciata*, *L. lateralis*, *P. pyralis* luciferases containing Asn or Thr at the position 216 and Met at the position 398. In the former case the enzymes are much more likely to loose the conformation optimal for the activity as a result of residue substitutions. As can be seen the G216, A217, S398 residues are located in one plane with the neighboring residue R220 (Fig. 5). The residue R220 (the residue R218 in *P.pyralis* luciferase) is highly conservative and necessary for the green emission of firefly luciferases. Its substitutions led to the red bioluminescence, 3-15-fold decrease in activity, extended luminescence decay times and dramatic increase in *Km* values (Branchini et al., 2001). The G216N/A217L double substitution in *L. mingrelica* luciferase caused the similar type of effects but of less extent. Thus, in *L. mingrelica* and *H. parvula* luciferases the proper alignment of the R220 residue can be affected by the substitution of A217L and lead to the observed detrimental effects. Placing Asn and Met at positions 216 and 398 respectively (as in the triple mutant G216N/A217L/S398M of *L. mingrelica* luciferase and in native *L. cruciata*, *L. lateralis* luciferases) makes local microenvironment of A217 sufficiently rigid to retain active conformation in the case of the A217L mutation.

Fig. 5. Residues 216, 217, 220 and 398 in the structures of *L. mingrelica* (A) and *L. cruciata* (B) luciferases (Koksharov & Ugarova, 2011a). Reproduced by permission of The Royal Society of Chemistry (RSC)

In conclusion it can be stated that rational protein design of the residue microenvironment can be an effective strategy when a single mutation in one firefly luciferase does not lead to the desirable effect reported for the mutation of the homologous residue in the another firefly luciferase. The constructed triple mutant G216N/A217L/S398M showed significantly improved thermal stability, high activity and bioluminescence spectrum close to that of the wild-type enzyme. The improved characteristics of this mutant make it a promising tool for *in vitro* and *in vivo* applications.

#### **6. Site-directed mutagenesis of cysteine residues of** *Luciola mingrelica* **firefly luciferase**

The number of Cys residues of luciferases is highly varied (from 4 to 13 residues) depending on the firefly species. Luciferases contain three absolutely conservative SH groups that do not belong to the active site. However their mutagenesis was shown to affect activity and stability of luciferases (Dement'eva et al., 1996; Kumita et al., 2000). For example, the mutant *Photinus pyralis* luciferase in which all the four Cys residues were substituted with Ser, retained only 6.5 % of activity, whereas mutants with single substitutions lost 20-60% of activity (Kumita et al., 2000; Ohmiya & Tsuji, 1997).

S398 residues are located in one plane with the neighboring residue R220 (Fig. 5). The residue R220 (the residue R218 in *P.pyralis* luciferase) is highly conservative and necessary for the green emission of firefly luciferases. Its substitutions led to the red bioluminescence, 3-15-fold decrease in activity, extended luminescence decay times and dramatic increase in *Km* values (Branchini et al., 2001). The G216N/A217L double substitution in *L. mingrelica* luciferase caused the similar type of effects but of less extent. Thus, in *L. mingrelica* and *H. parvula* luciferases the proper alignment of the R220 residue can be affected by the substitution of A217L and lead to the observed detrimental effects. Placing Asn and Met at positions 216 and 398 respectively (as in the triple mutant G216N/A217L/S398M of *L. mingrelica* luciferase and in native *L. cruciata*, *L. lateralis* luciferases) makes local microenvironment of A217 sufficiently rigid to retain active conformation in the case of the

Fig. 5. Residues 216, 217, 220 and 398 in the structures of *L. mingrelica* (A) and *L. cruciata* (B) luciferases (Koksharov & Ugarova, 2011a). Reproduced by permission of The Royal Society

In conclusion it can be stated that rational protein design of the residue microenvironment can be an effective strategy when a single mutation in one firefly luciferase does not lead to the desirable effect reported for the mutation of the homologous residue in the another firefly luciferase. The constructed triple mutant G216N/A217L/S398M showed significantly improved thermal stability, high activity and bioluminescence spectrum close to that of the wild-type enzyme. The improved characteristics of this mutant make it a promising tool for

**6. Site-directed mutagenesis of cysteine residues of** *Luciola mingrelica* **firefly** 

The number of Cys residues of luciferases is highly varied (from 4 to 13 residues) depending on the firefly species. Luciferases contain three absolutely conservative SH groups that do not belong to the active site. However their mutagenesis was shown to affect activity and stability of luciferases (Dement'eva et al., 1996; Kumita et al., 2000). For example, the mutant *Photinus pyralis* luciferase in which all the four Cys residues were substituted with Ser, retained only 6.5 % of activity, whereas mutants with single substitutions lost 20-60% of

A217L mutation.

of Chemistry (RSC)

**luciferase** 

*in vitro* and *in vivo* applications.

activity (Kumita et al., 2000; Ohmiya & Tsuji, 1997).

The *Luciola mingrelica* firefly luciferase contains eight cysteine residues, three of which correspond to the conservative cysteine residues of *P. pyralis* firefly luciferase - 82, 260, and 393. Mutant forms of *L. mingrelica* luciferase containing single substitutions of these cysteine residues to alanine were obtained previously (Dement'eva et al., 1996). These substitutions had no effect on bioluminescent and fluorescent spectra of the enzyme and on enzyme activity. The stability of the C393A mutant was 2-fold higher at 5-35C than that of the wildtype enzyme. The substitutions C82A, C260A did not affect the thermal stability of luciferase. The pLR plasmid, encoding firefly luciferase with the structure identical to that of the native enzyme, was previously used for the preparation of the mutant forms of the enzyme with single substitutions of the non-conserved cysteine residues C62S, C146S (Lomakina et al., 2008) and C164S (Modestova et al., 2010). These substitutions also had no significant effect on the catalytic and spectral properties of the luciferase, but they resulted in an increase of the enzyme thermal stability and in a decrease of the dependence of inactivation rate constant on the enzyme concentration (unlike the wild-type enzyme). Moreover, the DTT influence on luciferase stability was diminished. These effects were most pronounced for the enzyme with the substitution C146S.

The purification of recombinant luciferase obtained using the plasmid pLR is a complicated multistage process. Therefore, the recombinant *L. mingrelica* luciferase with C-terminal His6 tag was used for mutagenesis of cysteine residues (Modestova et al., 2011). The wild-type enzyme and its mutant forms were expressed in *E. coli BL21(DE3) cells* transformed with the pETL7 plasmid (Koksharov & Ugarova, 2011a). This approach led to the simpler scheme of the luciferase purification and to the increase of the enzyme yield due to the use of the highly efficient pET expression system. The influence of polyhistidine tag on luciferase properties was not previously analyzed in detail according to the literature. A number of publications indicate that while his-tags often don't affect enzyme function, in many cases the biological or physicochemical properties of the histidine tagged proteins are altered compared to their native counterparts (Amor-Mahjoub et al., 2006; Carson et al., 2007; Efremenko et al., 2008; Freydank et al., 2008; Klose et al., 2004; Kuo & Chase, 2011). The goal of this study was to elucidate the role of non-conserved cysteine residues in the *L. mingrelica* firefly luciferase, to study the mutual influence of these residues and the effect of His6-tag on the activity and thermal stability of luciferase (Modestova et al., 2011).

#### **6.1 Analysis of the fragments of luciferase amino acid sequences containing cysteine residues**

Among the firefly luciferases those amino acid sequences are known, firefly luciferases from *Luciola* and *Hotaria* genera, and the *Lampyroidea maculata* firefly luciferase form a separate group with more than 80% amino acid identity (Fig. 6). The second group includes luciferases from firelies of various genera: *Nyctophila*, *Lampyris*, *Photinus*, *Pyrocoelia,* etc. The sequence identity of luciferases from the first and the second group does not exceed 70%.

Amino acid sequences of the firefly luciferases belonging to these groups vary significantly. One of the most evident distinctions is the amount and location of cysteine residues. The residue С82 is absolutely conserved in all beetle luciferases, and the residue С260 is absolutely conserved in all firefly luciferases. The residue С393 is conserved in all beetle luciferases except the *Cratomorphus distinctus* (Genbank AAV32457) and one (Genbank U31240) of the *P. pennsylvanica* luciferases. The C62, 86, and 284 residues are also absolutely


Fig. 6. Fragments of amino acid sequence alignment of various firefly luciferases (the regions containing Cys residues). The numbering corresponds to that of *Luciola mingrelica*  luciferase

Fig. 7. Fragment of the 3D structure of *Luciola mingrelica* firefly luciferase containing the residues C62 and C164

conserved in all luciferases from the first group. The residue C146 is conserved in all luciferases of the first group, except for the *L. lateralis* and *L. cruciata* luciferases, in which alanine and tyrosine are located at the position 146. The residue C164 is conserved in luciferases of the first group except for the *L. lateralis* luciferase, which contains S146. The C86 residue is located in a highly conserved region of luciferases of the first group, near the C82 residue, which in its turn is located not far from the active site of the enzyme. Besides, the C86 residue is located near the surface of the protein, and the surface area of its side chain, that is accessible to the solvent, is about 11 Å2. The residue C146 is of particular interest because of its surface location. Its side chain is exposed to the solvent with the accessible surface area as high as 48 Å2. As a whole the *Luciola* luciferases possess high

Origin C62 C82, C86 C146 C164 C260 C284 C393 First group of luciferases *Luciola mingrelica* FDIT**C**RLAEAM IAL**C**SEN**C**EEFF VQKTVT**C**IKKIVI NFGGHD**C**METFI LGYFA**C**GYRVVML TLQDYK**C**TSVILV RRGEI**C**VKGPS *Luciola cruciata* LEKS**CC**LGKAL IAL**C**SEN**C**EEFF VQKTVTTIKTIVI DYRGYQ**C**LDTFI LGYLI**C**GFRVVML TLQDYK**C**TSVILV RRGEV**C**VKGPM *Hotaria parvula* FDIT**C**RLAEAM IAL**C**SEN**C**EEFF VQKTVT**C**IKTIVI NFGGHD**C**METFI LGYFA**C**GYRVVML TLQDYK**C**TSVILV RRGEI**C**VKGPS *Hotaria unmunsana* FDIT**C**RLAEAM IAL**C**SEN**C**EEFF VQKTVT**C**IKTIVI NFGGYD**C**METFI LGYFA**C**GYRVVML TMQDYK**C**TSVILV RRGEI**C**VKGPS *Hotaria tsushimana* FDIT**C**HLAEAM IAL**C**SEN**C**EEFF VQKTVT**C**IKTIVI NFGGYD**C**METFI LGYFA**C**GYRVVML TMQDYK**C**TSVILV RRGEI**C**VKGPS *Luciola italica* FDIT**C**RLAEAM IAL**C**SEN**C**EEFF VQKTVT**C**IKTIVI NFGGYD**C**VETFI LGYFA**C**GYRIVML TLQDYK**C**TSVILV RRGEI**C**VKGPS

*maculata* FDIS**C**RLAEAM IAL**C**SEN**C**EEFF VQKTVT**C**IKTIVI NFGGYD**C**VETFI LGYFA**C**GYRIVML TMQDYK**C**TSVILV RRGEI**C**VKGPS *Luciola lateralis* LEKS**CC**LGEAL IAL**C**SEN**C**EEFF VQKTVTAIKTIVI DYRGYQSMDNFI LGYLT**C**GFRIVML TLQDYK**C**SSVILV RRGEV**C**VKGPM *Luciola terminalis* LDVS**C**RLAQAM IAL**C**SEN**C**EEFF VQKTVT**C**IKTIVI DYQGYD**C**LETFI LGYLI**C**GFRIVML TLADYK**C**NSAILV RRGEI**C**VKGPM Second group of luciferases (illustrated by *Photinus pyralis* luciferase) *Photinus pyralis* FEMSVRLAEAM IVV**C**SENSLQFF VQKKLPIIQKIII DYQGFQSMYTFV LGYLI**C**GFRVVLM SLQDYKIQSALLV QRGEL**C**VRGPM

Fig. 6. Fragments of amino acid sequence alignment of various firefly luciferases (the regions containing Cys residues). The numbering corresponds to that of *Luciola mingrelica* 

Fig. 7. Fragment of the 3D structure of *Luciola mingrelica* firefly luciferase containing the

conserved in all luciferases from the first group. The residue C146 is conserved in all luciferases of the first group, except for the *L. lateralis* and *L. cruciata* luciferases, in which alanine and tyrosine are located at the position 146. The residue C164 is conserved in luciferases of the first group except for the *L. lateralis* luciferase, which contains S146. The C86 residue is located in a highly conserved region of luciferases of the first group, near the C82 residue, which in its turn is located not far from the active site of the enzyme. Besides, the C86 residue is located near the surface of the protein, and the surface area of its side chain, that is accessible to the solvent, is about 11 Å2. The residue C146 is of particular interest because of its surface location. Its side chain is exposed to the solvent with the accessible surface area as high as 48 Å2. As a whole the *Luciola* luciferases possess high

*Lampyroidea* 

luciferase

residues C62 and C164

amino acid sequence identity. However, there are several small areas in their amino acid sequences the composition of which varies significantly. It is in these areas that the residues C62 and C164 are located. These residues are positioned in two α-helixes and are in close proximity with each other (Fig. 7).

The cysteine residues 62, 86, 146, and 164 of *L. mingrelica* luciferase were chosen for the sitespecific mutagenesis. In terms of the molecule topology the most suitable substitutions of the Cys are Ser (hydrophilic amino acid) and Val (hydrophobic amino acid). The side chain sizes of these residues are similar to that of Cys. We considered Ser as the most suitable substitution for C86 and C146 residues because the side chains of these residues are in contact with aqueous solution. The residue C164 was also substituted by Ser because its microenvironment is weakly hydrophilic. Moreover, our previously results (Modestova et al., 2010) suggest that in certain conditions this residue becomes available to the solvent. In case of the residue Cys62 two mutants were obtained: C62S and C62V.

#### **6.2 Preparation and physicochemical properties of mutant luciferases**

The recombinant *L. mingrelica* firefly luciferase encoded by the plasmid pETL7 (GenBank No. HQ007050) (Koksharov & Ugarova, 2011a) served as the parent enzyme (wild-type). This form contains 4 additional amino acid residues (MASK) on N-terminus as compared to the native sequence of *L. mingrelica* firefly luciferase (GeneBank No. S61961). The sequence AKM at its C-terminus is replaced by the sequence SGPVEHHHHHH. A number of mutants were obtained by site-directed mutagenesis of the plasmid pETL7: the mutant luciferases with the single substitutions C62S, C62V, C86S, C146S, C164S, double substitutions C62/146S, C62/164S, C86/146S, and C146/164S; the triple substitution С62/146/164S. The wild-type luciferase and its mutant forms were purified using metal chelate chromatography. The expression level and the specific activity of wild-type and its mutants C62S, C62V, C164S, C62/146S, and C146S/C164S were the same within an experimental error. Specific activity of the mutant C146S was ~15% higher than that of the wild-type, while its expression level was unaltered. Meanwhile, the substitution C86S resulted in the decrease of the enzyme expression level (62% compared to wild-type) and its specific activity (30% compared to wild-type). The properties of the firefly luciferase with the double substitution C86S/146S were similar to those of the mutant C86S. Drastic decrease of the expression level and of the enzyme specific activity was observed at the introduction of the double mutation C62S/C164S and the triple mutation С62S/C146S/C164S. Bioluminescence and intrinsic fluorescence spectra of the wild-type luciferase and its mutant forms were identical. Single mutations had almost no effect on the *K*m values for both substrates (*K*mATP and *K*mLH2) with the exception of the mutant C86S, for which, as well as for the mutant C86S/C146S, 1.5-fold increase of both parameters was observed. The simultaneous substitution of the residues C62S and C164S in both double and triple mutants led to 30% increase of *K*mATP, but didn't affect *K*mLH2.

The irreversible inactivation of the wild-type luciferase and its mutant forms was measured in 0.05 М Тris-acetate buffer (2 мМ EDTA, 10 мМ MgSO4, pH 7.8) at 37° and 42°C at concentration range of 0.01-1.0 µM. The inactivation of the wild-type luciferase and its mutant forms followed the monoexponential first-order kinetics at all enzyme concentrations assayed. The *k*in values of the wild-type luciferase and its mutant forms did not depend on the initial luciferase concentration. The enzyme stabilization was only observed for the mutant C146S: the *k*in value decreased 2-fold at 37C and by 30% - at 42°C (Table 2). At 37°C the *k*in values of the mutants С62V, C164S and C146S/C164S were similar to the *k*in of the wild-type luciferase, but at 42°C the *k*in values of these mutants were higher than that of the wild-type enzyme. All other mutants were less stable than the wild-type enzyme. The substitution C86S caused a significant destabilizing effect on the enzyme: the *k*in value increased twofold both at 37° and 42°C. The double mutant C62S/C164S and the triple mutant С62S/C146S/C164S were the least stable among the mutants obtained.


Table 2. Rate constants of irreversible inactivation of wild-type luciferase and its mutant forms with single and multiple substitutions of the 62, 86, 146, 164 cysteine residues at 37 and 42°C

#### **6.3 The effect of polyhistidine tag on the properties of firefly luciferase**

 Comparison of the physicochemical properties of luciferases with single substitutions of the residues C62S, C146S and C164S that were obtained for *L. mingrelica* luciferase without His6 tag (Lomakina et al., 2008) with that of the mutant enzymes containing C-terminal His6-tag (Modestova et al., 2011) led to a conclusion that the His6-tag shows significant influence on the luciferase properties. Introduction of the His6-tag into the luciferase structure leads to the increase of the *K*mATP and *K*mLH2 values. The interaction of the enzyme with the substrates is known to involve the rotation of a big N-domain and a small C-domain of the luciferase against each other at almost 90° (Sandalova & Ugarova, 1999). This movement is necessary for the participation of the residue K531 from C-domain in the formation of enzyme-ATPluciferin active complex. The presence of the flexible His6-tag on the C-terminus of the protein molecule might somewhat impede the process of domains rotation, that may result in a slight increase of Km values for the both substrates.

Thermal inactivation of the firefly luciferase without His6-tag is a two-step process, which includes a fast and a slow inactivation stages. The *k*in values of both stages are dependent on the enzyme concentration, which is known to be a characteristic feature of oligomeric

observed for the mutant C146S: the *k*in value decreased 2-fold at 37C and by 30% - at 42°C (Table 2). At 37°C the *k*in values of the mutants С62V, C164S and C146S/C164S were similar to the *k*in of the wild-type luciferase, but at 42°C the *k*in values of these mutants were higher than that of the wild-type enzyme. All other mutants were less stable than the wild-type enzyme. The substitution C86S caused a significant destabilizing effect on the enzyme: the *k*in value increased twofold both at 37° and 42°C. The double mutant C62S/C164S and the triple mutant С62S/C146S/C164S were the least stable among the

*k*in, min-1 Enzyme 37° 42°

wild-type 0,022 ± 0,004 0,074 ± 0,006 C62V 0,024 ± 0,004 0,135 ± 0,004 C62S 0,036 ± 0,004 0,127 ± 0,004 C86S 0,040 ± 0,002 0,160± 0,006 C146S 0,011 ± 0,002 0,058 ± 0,003 C164S 0,018 ± 0,003 0,108 ± 0,005 C62S/C146S 0,042 ± 0,005 0,108 ± 0,005 C62S/C164S 0,052 ± 0,003 0,153 ± 0,005 C86S/C146S 0,047 ± 0,004 0,120 ± 0,006 C146S/C164S 0,023 ± 0,006 0,086 ± 0,005 C62S/C146S/C164S 0,055 ± 0,005 0,142 ± 0,006

Table 2. Rate constants of irreversible inactivation of wild-type luciferase and its mutant forms with single and multiple substitutions of the 62, 86, 146, 164 cysteine residues at 37

 Comparison of the physicochemical properties of luciferases with single substitutions of the residues C62S, C146S and C164S that were obtained for *L. mingrelica* luciferase without His6 tag (Lomakina et al., 2008) with that of the mutant enzymes containing C-terminal His6-tag (Modestova et al., 2011) led to a conclusion that the His6-tag shows significant influence on the luciferase properties. Introduction of the His6-tag into the luciferase structure leads to the increase of the *K*mATP and *K*mLH2 values. The interaction of the enzyme with the substrates is known to involve the rotation of a big N-domain and a small C-domain of the luciferase against each other at almost 90° (Sandalova & Ugarova, 1999). This movement is necessary for the participation of the residue K531 from C-domain in the formation of enzyme-ATPluciferin active complex. The presence of the flexible His6-tag on the C-terminus of the protein molecule might somewhat impede the process of domains rotation, that may result

Thermal inactivation of the firefly luciferase without His6-tag is a two-step process, which includes a fast and a slow inactivation stages. The *k*in values of both stages are dependent on the enzyme concentration, which is known to be a characteristic feature of oligomeric

**6.3 The effect of polyhistidine tag on the properties of firefly luciferase** 

in a slight increase of Km values for the both substrates.

mutants obtained.

and 42°C

enzymes. The single mutations С62S, С146S, С164S result in stabilization of the enzyme at the slow stage of inactivation and in a decrease of *k*in dependence on the enzyme concentration (Lomakina et al., 2008). The thermal inactivation of the His6-tag containing wild-type luciferase and its mutants is a one-step process. The *k*in values of these enzymes do not depend on luciferase concentration and coincide with the *k*in values of the respective mutants without His6-tag that were measured at the increased enzyme concentration (1 µM). This influence of the His6-tag on the inactivation kinetics of the wild-type luciferase and its mutants may be due to the fact that the presence of the His6-tag considerably alters the process of luciferase oligomerization.

#### **6.4 Effect of the cysteine substitutions on luciferase structure and thermal stability**

The substitution C146S results in a 2-fold stabilization of the enzyme at 37°C and in a 30% increase of the enzyme stability at 42°C. This effect is associated with the surface location of the side chain of this residue, its large solvent accessible area and the lack of interactions with other amino acid residues of the enzyme. The C164S substitution doesn't alter the enzyme stability at 37°C, but leads to some destabilization at 42°C, though this destabilization is less than that caused by the substitutions C62V, C62S and C86S. This effect is, on the one hand, due to the fact, that the C164 residue is located in an area, which is distant from the enzyme active site. On the other hand, the raise of temperature causes the increase of solvent accessibility and the replacement of cysteine residue by the hydrophilic serine improves interactions with the solvent.

Analysis of the luciferase 3D-model shows that it is hard to unambiguously estimate the properties of the C62 residue microenvironment. This residue contacts with both hydrophilic and hydrophobic amino acids. Therefore, two enzymes were obtained that carry a hydrophilic and a hydrophobic side chain in the position 62. The specific activity, the expression level and the kinetic parameters of the mutants C62S and C62V were similar to those of the wild-type enzyme. The *k*in values at 42°C were also similar, but the mutant C62V turned out to be 2-fold more stable than the mutant C62S at 37°C. Therefore, the hydrophobic valine residue is more advantageous at 37°C in terms of the enzyme stability. However, at temperature of 42°C the role of the amino acid residue microenvironment in the enzyme stabilization becomes less pronounced and both modifications – serine or valine – result in destabilization of the protein globule.

The substitution C86S shows the most significant influence on the luciferase properties. It results in a decrease of the luciferase expression level and the specific activity, a deterioration of the Km values for both substrates, and a decrease of the enzyme thermal stability. The C86 residue is located within an unstructured area of the amino acid chain of the enzyme (Fig. 8). The amino acid sequence forms a loop in this area due to the formation of a hydrogen bond between the SH-group of the residue C86 and the oxygen atom OE1 belonging to the residue E88. The SH-group of cysteine residue is known to have a tendency to form non-linear hydrogen bonds due to fact that the deformation of the valence angle has a relatively small energy cost (Raso et al., 2001). The OH-group of serine residues has no such tendency. Thereby it may be possible that the hydrogen bond between S86 and E88 residues can't be formed in the mutant C86S. This may lead to an increase in mobility of the chain fragment containing the abovementioned residues.

Fig. 8. Fragment of the 3D structure of *Luciola mingrelica* firefly luciferase containing C82 and C86 residues (Modestova et al., 2011)

It is important to underline that the C86 residue is located in an absolutely conserved area of luciferases *Luciola* genus, not far from the enzyme active site and at the distance of ~15 Å from T253, F249, F252 residues. These residues participate in the process of luciferase substrates binding, and it is known that their mutations lead to a drastic alteration of the enzyme catalytic properties and, in certain cases, to the disturbance of the enzyme expression process (Freydank et al., 2008). On the basis of the experimental data one can conclude that disturbance stripping-down of the protein structure (the "untwisting" of the helix) in the area of the localization of the residue C86 disrupts the native structure of the firefly luciferase active site area and leads to the deterioration of the luciferase activity and stability.

Analysis of the properties of the mutants with multiple amino acid substitutions indicates that in most of the cases the effect of such substitutions is additive. For instance, the C86S/C146S mutant possesses the properties of the luciferase with single C86S substitution, because it is the C86S substitution that affects the enzyme properties most significantly. The mutants C62S/C146S and C146S/C164S also possess the characteristic properties of the respective mutants with single replacements. However, the combination C62S/C164S leads to the drastic decrease of the enzyme expression level, to the lowering of its specific activity and stability and to the increase of the *K*mATP in comparison with the enzymes with the single substitutions C62S and C164S. These facts indicate that the effect of these substitutions is nonadditive. The analysis of luciferase 3D structure shows that C62 and C164 residues belong to two closely located α-helixes (Fig. 8). The single mutations of these residues have no significant effect on the enzyme properties, which is probably due to the enzyme ability to compensate the effects of these substitutions. Meanwhile, the double substitutions affect the mutual disposition of two α-helixes, in which these residues are located.

Thus, the role of each cysteine residue in luciferase molecule is different and is determined by its location relative to the active site, its microenvironment and even the oligomerization state of luciferase. For example, in some cases the introduction of Cys residues into internal protein core can increase the luciferase stability after replacement of hydrophilic residue by more hydrophobic Cys. Such examples will be shown below.

Fig. 8. Fragment of the 3D structure of *Luciola mingrelica* firefly luciferase containing C82 and

It is important to underline that the C86 residue is located in an absolutely conserved area of luciferases *Luciola* genus, not far from the enzyme active site and at the distance of ~15 Å from T253, F249, F252 residues. These residues participate in the process of luciferase substrates binding, and it is known that their mutations lead to a drastic alteration of the enzyme catalytic properties and, in certain cases, to the disturbance of the enzyme expression process (Freydank et al., 2008). On the basis of the experimental data one can conclude that disturbance stripping-down of the protein structure (the "untwisting" of the helix) in the area of the localization of the residue C86 disrupts the native structure of the firefly luciferase active site area and leads to the deterioration of the luciferase activity and

Analysis of the properties of the mutants with multiple amino acid substitutions indicates that in most of the cases the effect of such substitutions is additive. For instance, the C86S/C146S mutant possesses the properties of the luciferase with single C86S substitution, because it is the C86S substitution that affects the enzyme properties most significantly. The mutants C62S/C146S and C146S/C164S also possess the characteristic properties of the respective mutants with single replacements. However, the combination C62S/C164S leads to the drastic decrease of the enzyme expression level, to the lowering of its specific activity and stability and to the increase of the *K*mATP in comparison with the enzymes with the single substitutions C62S and C164S. These facts indicate that the effect of these substitutions is nonadditive. The analysis of luciferase 3D structure shows that C62 and C164 residues belong to two closely located α-helixes (Fig. 8). The single mutations of these residues have no significant effect on the enzyme properties, which is probably due to the enzyme ability to compensate the effects of these substitutions. Meanwhile, the double substitutions affect the mutual disposition of two α-helixes, in which these residues are

Thus, the role of each cysteine residue in luciferase molecule is different and is determined by its location relative to the active site, its microenvironment and even the oligomerization state of luciferase. For example, in some cases the introduction of Cys residues into internal protein core can increase the luciferase stability after replacement of hydrophilic residue by

more hydrophobic Cys. Such examples will be shown below.

C86 residues (Modestova et al., 2011)

stability.

located.

#### **7. Increase of** *P. pyralis* **luciferase thermostability by introduction of disulfide bridges**

It was mentioned above that luciferases are peroxisomal enzymes. They do not form structural disulfide bonds despite of containing SH-groups (Ohmiya & Tsuji, 1997). When expressed in *E. coli*, firefly luciferases cannot form any disulfide bonds due to the reducing environment of the cytoplasm. On the other hand, introduction of disulfide bridges was found to be one of the most efficient strategies for increasing protein stability (Eijsink et al., 2004). Recently, disulfide bridges were introduced into *P. pyralis* firefly luciferase (Imani et al., 2010) by site-directed mutagenesis. Two different mutant proteins were made with a single bridge. *P.pyralis* firefly luciferase contains four cysteine residues at the positions 81, 216, 258 and 391. To find the residues capable to form disulfide bridges after their mutation to cysteine, the crystal structure of *P. pyralis* luciferase was uploaded to the NCBS integrated Web Server. The results from server showed that there are 150 pairs that could potentially be selected for disulfide bridge formation. But only two pairs of residues were chosen due to their similar size to the Cys residues: A103 and S121, located distant from active site region of the enzyme, and A296 and A326, situated in the vicinity of the active site region. The ability of mutated sites to form disulfide bridges was analyzed in Swiss-PDB Viewer.

Two mutant luciferases, each containing one S-S bridge, were obtained: A103C/S121C and A296C/A326C. Relative specific activity showed a 7.25-fold increase for the mutant A296C/A326C whereas the mutant A103C/S121C showed only 80% of wild-type specific activity. Both mutants were more stable then the wild-type enzyme. For example, after incubation at 40°C for 5 min the mutants A296C/A326C and A103C/S121C retained ~88% and 22% of activity respectively, whereas the wild-type enzyme lost nearly all of its activity. Using circular dichroism spectropolarimetric and fluorescence spectroscopic analysis, the conformational changes of the enzyme structure were revealed, showing the more fixed structure of aromatic residues, more compactness of tertiary structure, and a remarkable increase in α-helix content.

It can be concluded that disulfide bridge formation in mutant A296C/A326C did not have a destabilizing effect on the enzyme and caused a remarkable change in both secondary and tertiary structure that is reflected in active site structure. These changes endow the enzyme with properties that show an increased resistance to pH and temperature without any stabilizer. On the other hand, the thermal stability of the mutant A103C/S121C arises from the change of tertiary structure. Finally, these results showed that the engineered disulfide bridge not only did not destabilize the enzyme but also in one mutant it improved the specific activity and led to pH-insensitivity of the enzyme (Imani et al., 2010).

#### **8. Thermostabilization of the** *Luciola mingrelica* **firefly luciferase by** *in vivo* **directed evolution**

Firefly luciferase can be simply screened for its *in vivo* bioluminescence activity (Wood & DeLuca, 1987). This makes a directed evolution approach the most promising for optimization of different luciferase properties including thermostability. This strategy was shown to successful improve of a wide range of properties for different enzymes, for example, thermal stability, enantioselectivity, substrate specicity, and activity in nonnatural environments (Jäckel et al., 2008; Turner, 2009). The critical part of a directed evolution experiment is the availability of a sensitive and efficient screening procedure. Otherwise identifying the desired mutants within large libraries can become very laborious and costly. However, there is only one example known when directed evolution was used for enhancing the thermostability of firefly luciferase. Wood & Hall obtained the exceptionally stable mutant of *Photuris pennsylvanica* luciferase by this approach. This mutant still remains the most stable firefly luciferase to date. In this case a sophisticated automatic robotic system was implemented to screen mutant libraries. It limits the possibility of wide application of this technique. However, that system was able to screen more than 10000 mutants per cycle with a precise measurement of *in vitro* properties of the mutants generated such as activity and Km. The developed ultra-stable mutant contained 28 substitutions and demonstrated a half-life of about 27 h at 65°C (Wood & Hall, 1999). The more simple, but efficient screening strategy was successfully used here to evolve a thermostable form of *L. mingrelica* luciferase (Koksharov & Ugarova, 2011b).

#### **8.1 Directed evolution of luciferase**

Wild-type *L. mingrelica* luciferase displays rather low thermostability with a half-life of 50 minutes at 37°C. So, the consecutive rounds of random mutagenesis and screening were used to considerably improve thermostability of *L. mingrelica* luciferase without compromising its activity. The fact that E*. coli* cells withstand temperatures up to about 55°C (Jiang *et al.*, 2003) and the availability of *in vivo* bioluminescence assay, allowed to identify thermostable mutants by a simple non-lethal *in vivo* screening of *E. coli* colonies that contained mutant luciferases. The incubation of *E. coli* colonies at elevated temperatures resulted in the inactivation of less stable luciferase mutants. Therefore, thermostable mutants displayed higher residual bioluminescence activity and could be efficiently detected by a simple photographic registration of *in vivo* bioluminescence of colonies. *E. coli* cells remained viable after the subjection to elevated temperatures and the subsequent detection of *in vivo* bioluminescence. Therefore, there was no need in using replica plates, which simplified the procedure. Each round of screening could be carried out in a simple and rapid manner (Koksharov & Ugarova, 2010, 2011b).

The plasmid pLR3 (GenBank No. HQ007051) (Koksharov & Ugarova, 2008), which contains *L. mingrelica* luciferase gene, was used in random mutagenesis performed by error-prone PCR. A mutation rate of about 1 amino acid change (2-3 base changes) per the region mutated is reported to be most desirable for an efficient selection of improved mutant (Cirino et al., 2003). It generally gives 30-40% of active clones in the library (Cirino et al., 2003), so this frequency was targeted in our work. Mutagenesis was applied to a 785 bp region of the luciferase gene, which corresponds to amino acid residues 130-390 out of 548 residues of *L. mingrelica* luciferase. This region was chosen because of the convenient restriction sites available (XhoI and BglII) and because most reported mutants, that increase the thermostability of firefly luciferases, are located in this region. The results indicate that the screening of 1000 colonies typically gives a couple of different thermostable mutants. Up to 2000-3000 mutant colonies could be conveniently screened on a single 90 mm Petri dish. The mutant S118C was used as a parent enzyme for directed evolution because it demonstrated slightly higher thermostability compared with the wild-type enzyme (Koksharov & Ugarova, 2008). The most thermostable mutant identified in each cycle of mutagenesis was used as a starting point in the following cycle (Table 3).

evolution experiment is the availability of a sensitive and efficient screening procedure. Otherwise identifying the desired mutants within large libraries can become very laborious and costly. However, there is only one example known when directed evolution was used for enhancing the thermostability of firefly luciferase. Wood & Hall obtained the exceptionally stable mutant of *Photuris pennsylvanica* luciferase by this approach. This mutant still remains the most stable firefly luciferase to date. In this case a sophisticated automatic robotic system was implemented to screen mutant libraries. It limits the possibility of wide application of this technique. However, that system was able to screen more than 10000 mutants per cycle with a precise measurement of *in vitro* properties of the mutants generated such as activity and Km. The developed ultra-stable mutant contained 28 substitutions and demonstrated a half-life of about 27 h at 65°C (Wood & Hall, 1999). The more simple, but efficient screening strategy was successfully used here to evolve a

Wild-type *L. mingrelica* luciferase displays rather low thermostability with a half-life of 50 minutes at 37°C. So, the consecutive rounds of random mutagenesis and screening were used to considerably improve thermostability of *L. mingrelica* luciferase without compromising its activity. The fact that E*. coli* cells withstand temperatures up to about 55°C (Jiang *et al.*, 2003) and the availability of *in vivo* bioluminescence assay, allowed to identify thermostable mutants by a simple non-lethal *in vivo* screening of *E. coli* colonies that contained mutant luciferases. The incubation of *E. coli* colonies at elevated temperatures resulted in the inactivation of less stable luciferase mutants. Therefore, thermostable mutants displayed higher residual bioluminescence activity and could be efficiently detected by a simple photographic registration of *in vivo* bioluminescence of colonies. *E. coli* cells remained viable after the subjection to elevated temperatures and the subsequent detection of *in vivo* bioluminescence. Therefore, there was no need in using replica plates, which simplified the procedure. Each round of screening could be carried out in a simple and rapid manner

The plasmid pLR3 (GenBank No. HQ007051) (Koksharov & Ugarova, 2008), which contains *L. mingrelica* luciferase gene, was used in random mutagenesis performed by error-prone PCR. A mutation rate of about 1 amino acid change (2-3 base changes) per the region mutated is reported to be most desirable for an efficient selection of improved mutant (Cirino et al., 2003). It generally gives 30-40% of active clones in the library (Cirino et al., 2003), so this frequency was targeted in our work. Mutagenesis was applied to a 785 bp region of the luciferase gene, which corresponds to amino acid residues 130-390 out of 548 residues of *L. mingrelica* luciferase. This region was chosen because of the convenient restriction sites available (XhoI and BglII) and because most reported mutants, that increase the thermostability of firefly luciferases, are located in this region. The results indicate that the screening of 1000 colonies typically gives a couple of different thermostable mutants. Up to 2000-3000 mutant colonies could be conveniently screened on a single 90 mm Petri dish. The mutant S118C was used as a parent enzyme for directed evolution because it demonstrated slightly higher thermostability compared with the wild-type enzyme (Koksharov & Ugarova, 2008). The most thermostable mutant identified in each cycle of

mutagenesis was used as a starting point in the following cycle (Table 3).

thermostable form of *L. mingrelica* luciferase (Koksharov & Ugarova, 2011b).

**8.1 Directed evolution of luciferase** 

(Koksharov & Ugarova, 2010, 2011b).


\*) For each cycle, the mutant showing the highest stability is shown in bold and underlined. It was used as a parent for the following cycle.

Table 3. Mutants of *Luciola mingrelica* firefly luciferase obtained during four cycles of directed evolution

At the first cycle of mutagenesis the screening of the mutant colonies was performed directly after their growth at 37°C. The wild-type *L. mingrelica* luciferase is insufficiently stable at these conditions, so the *in vivo* bioluminescence of its colonies is rather dim. Three clones were identified during screening that produced distinctly brighter colonies because of the increased thermostability (Table 3). During the second and third cycles of mutagenesis an additional incubation at 50°C for 40 min was required to detect mutants showing higher stability. Three mutants obtained at the third cycle displayed similar brightness after incubation at 50°C but increasing the incubation temperature to 55°C showed that the mutants 3T1, 3T2 are more stable than 3T3. After the fourth round of directed evolution the mutant 4TS was identified, which showed the highest *in vivo* thermostability among the mutants described in this study. It retained noticeable brightness of bioluminescence after incubation of its colonies at 55°C for 40 min while all the other mutants were completely inactivated. Moreover, the mutant 4TS displayed decreased but noticeable *in vivo* bioluminescence when its colonies were heated for 20 min at 60°C. *E. coli* cells completely lost their viability after 2 min at 60°C. Therefore, further selection of mutants with even higher stability will require the of replica plates.

#### **8.2 Expression and purification of mutant and wild-type luciferases**

The wild-type *L. mingrelica* luciferase and the mutant 4TS were expressed using the plasmid pETL7, which was described earlier. Average yields of the purified proteins (mg per 1 L of culture) were 160 mg for wild-type and 300 mg for te mutant 4TS. As a result of purification the enzymes were obtained in 20 mM Na-phosphate buffer containing 0.5 M NaCl, pH 7.5 containing 300 mM imidazole, 2 mM EDTA, 1 mM DTT. Generally the luciferases proteins remained fully active for at least 1 month in this buffer. For the long-term storage the proteins were transferred to 50 mM Tris-acetate buffer (pH 7.3) containing 100 mM Na2SO4, 2 mM EDTA and frozen at −80°C. This way they retained full activity for at least 2 years and tolerated several freeze-thaw cycles without inactivation. Despite the fact that the catalytic efficiency of the intermediate mutants was not monitored, the resultant mutant 4TS demonstrated the significant improvement of specific activity as well as *Km* for ATP.

#### **8.3 Thermostability**

Comparison of 4TS and wild-type *L. mingrelica* luciferase thermal stability at 42°C in Trisacetate buffer TsB1 (50 mM Tris-acetate buffer containing 20 mM MgSO4, 2 mM EDTA, 0.2 mg/ml BSA, pH 7.8) showed a 65-fold the increase in the half-life of *L. mingrelica* luciferase at 42°C (from 9.1 to 592 min). Thermal inactivation of the wild-type enzyme and 4TS was also studied in Na-phosphate buffer TsB2 (50 mM Na-phosphate buffer containing 410 mM (NH4)2SO4, 2 mM EDTA, 0.2 mg/ml BSA, pH 7.8) to compare these results with other literature data (Kajiyama & Nakano, 1994; Kitayama, et al., 2003; White, et al., 1996). At all the temperatures studied the mutant 4TS was significantly more stable than the wild-type. As can be seen from the Arrhenius plot, TsB2 buffer causes significant stabilization of both the wild-type enzyme and 4TS compared with TsB1 buffer (Fig. 9)

Fig. 9. Arrhenius plot showing the dependence of rates of inactivation on temperature for the wild-type luciferase (diamonds) and the mutant 4TS (circles) in buffer TsB1 (closed symbols) and TsB2 (open symbols) (Koksharov & Ugarova, 2011b). C(enzyme)=13 μg/ml

#### **8.4 Structural analysis**

The mutant 4TS contains 7 new substitutions compared with its parent form S118C: T213S, K156R, R211L, A217V, C146S, E356K, and S364C. All the substitutions are non-conservative among firefly luciferases. Judging from the order of appearance of these substitutions in the course of directed evolution (Table 3), literature data and their location in the 3D structure of the enzyme (Fig. 10), four of these substitutions were suggested to be the key mutations that cause the high stability of the mutant 4TS: R211L, A217V, E356K, and S364C. The mutations of the residues A217 (Kajiyama & Nakano, 1993) and E356 (White, et al., 1996) are known to significantly increase the thermostability of firefly luciferases according to the

proteins were transferred to 50 mM Tris-acetate buffer (pH 7.3) containing 100 mM Na2SO4, 2 mM EDTA and frozen at −80°C. This way they retained full activity for at least 2 years and tolerated several freeze-thaw cycles without inactivation. Despite the fact that the catalytic efficiency of the intermediate mutants was not monitored, the resultant mutant 4TS

Comparison of 4TS and wild-type *L. mingrelica* luciferase thermal stability at 42°C in Trisacetate buffer TsB1 (50 mM Tris-acetate buffer containing 20 mM MgSO4, 2 mM EDTA, 0.2 mg/ml BSA, pH 7.8) showed a 65-fold the increase in the half-life of *L. mingrelica* luciferase at 42°C (from 9.1 to 592 min). Thermal inactivation of the wild-type enzyme and 4TS was also studied in Na-phosphate buffer TsB2 (50 mM Na-phosphate buffer containing 410 mM (NH4)2SO4, 2 mM EDTA, 0.2 mg/ml BSA, pH 7.8) to compare these results with other literature data (Kajiyama & Nakano, 1994; Kitayama, et al., 2003; White, et al., 1996). At all the temperatures studied the mutant 4TS was significantly more stable than the wild-type. As can be seen from the Arrhenius plot, TsB2 buffer causes significant stabilization of both

Fig. 9. Arrhenius plot showing the dependence of rates of inactivation on temperature for the wild-type luciferase (diamonds) and the mutant 4TS (circles) in buffer TsB1 (closed symbols) and TsB2 (open symbols) (Koksharov & Ugarova, 2011b). C(enzyme)=13 μg/ml

The mutant 4TS contains 7 new substitutions compared with its parent form S118C: T213S, K156R, R211L, A217V, C146S, E356K, and S364C. All the substitutions are non-conservative among firefly luciferases. Judging from the order of appearance of these substitutions in the course of directed evolution (Table 3), literature data and their location in the 3D structure of the enzyme (Fig. 10), four of these substitutions were suggested to be the key mutations that cause the high stability of the mutant 4TS: R211L, A217V, E356K, and S364C. The mutations of the residues A217 (Kajiyama & Nakano, 1993) and E356 (White, et al., 1996) are known to significantly increase the thermostability of firefly luciferases according to the

demonstrated the significant improvement of specific activity as well as *Km* for ATP.

the wild-type enzyme and 4TS compared with TsB1 buffer (Fig. 9)

**8.3 Thermostability** 

**8.4 Structural analysis** 

previous studies. The effect of the residues R211 and S364 on thermostability is identified for the first time. The increase in stability by the substitutions R211L, A217V, S364C, and S364A, can be attributed to the improvement of the internal hydrophobic packing (Fersht & Serrano, 1993). In the case of R211L, S364C, and S364A, the increase of hydrophobicity of the protein core is achieved by the substitution of the non-conservative buried polar residues by the hydrophobic ones. As a result of the substitution A217V the larger side group of Val fills the internal cavity, which is otherwise occupied by a water molecule (Conti et al., 1996). The surface mutation C146S is known to increase the resistance to oxidative inactivation (Lomakina *et al.*, 2008). This mutation can explain the increased storage stability of 4TS in the absence of DTT compared with wild-type. The WT luciferase loses 70% of its activity within two weeks, whereas the mutant 4TS was remained fully active within one month at the same conditions (Koksharov & Ugarova, 2011b). The mutants T213S/S364C and S364A displayed similar *in vivo* properties. There, it the substitution T213S is unlikely to affect thermostability. The substitution of the surface residue 156 from positively charged Lys to similar in properties Arg is also unlikely cause a significant effect on luciferase. The starting mutant S118C showed only small 1.5-fold increase in stability at 42°C. The mutant 4TS and its variant without the mutation S118C showed indistinguishable *in vivo* thermostability at 60°C. Thus, the contribution of S118C seems insignificant. Interestingly, Ser118 is highly

Fig. 10. Homology model of *L. mingrelica* luciferase showing the location of substitutions in the mutant 4TS. Four key thermostabilizing mutations are underlined. LO и AMP – luciferyl and adenylate groups of DLSA (5'-*O*-[N-(dehydroluciferyl)-sulfamoyl] adenosine). Subdomains A, B and C are depicted in blue, magenta and orange, respectively

conservative in firefly luciferases. The only exceptions are the similar substitution S118C in the recently cloned juvenile luciferase from *L. cruciata* (Oba et al, 2010a) and the substitution S118T in the luciferase from *Lampyroidea maculata* (Emamzadeh et al., 2006). However, in luciferases from non-firefly beetles this position is usually occupied by His or Val.

All four key thermostabilizing substitutions (R211L, A217V, E356K, and S364C) are located in the second subdomain of firefly luciferase. According to the results of Frydman and coworkers (Frydman *et al.*, 1999), the fragments of firefly luciferase comprising residues 1- 190 and 422-544 possess high intrinsic stability. These fragments mainly correspond to the subdomains A and C of firefly luciferase (Fig. 10). That study demonstrated that the middle subdomain B (192-435) was significantly less stable and that it was the first to unfold under denaturating conditions. Hence, it likely that the stability of the second subdomain is the less stable "bottleneck" that determines the stability of the firefly luciferase protein. Therefore, most of the thermostabilizing mutations would tend to be located in the second subdomain or at the interface of this subdomain and the remaining parts of the protein. It is noteworthy that almost all thermostable mutants reported in the literature are located in this part of the luciferase structure, which is consistent with this hypothesis.

#### **8.5 Conclusion**

We have demonstrated that the *in vivo* directed evolution strategy is a simple and efficient method to increase thermal stability of firefly luciferase, which allows to obtain highly thermostable mutants without sacrificing catalytic efficiency. The final mutant obtained here even displayed superior catalytic properties such as higher specific activity, lower *Km* for ATP and increased temperature optimum. In typical applications, like ATP-related assays or reporter genes, beetle luciferases are used at room temperature or 37°C. The mutant 4TS retains 70% activity after two days of incubation at 37°C. Therefore, its stability is sufficient for most common *in vivo* and *in vitro* applications. The high specific activity, catalytic efficiency, and improved protein yield make the mutant 4TS an efficient tool for ATP determination (Ugarova *et al.*, 2010). The increased temperature optimum this mutant can be an advantage when used for *in vivo* imaging and in high temperature applications. The new positions identified in this study can be successfully used for the stabilization of other firefly luciferases, especially from the *Luciola* and *Hotaria* genus's. The non-lethal *in vivo* screening approach described here can be potentially implemented to other beetle or non-beetle luciferases when the development of thermostable forms of the enzyme is desirable.

#### **9. Acknowledgements**

This work was supported by the Russian Foundation for Basic Research (grants 08-04-00624 and 11-04-00698a).

#### **10. References**

Amor-Mahjoub, M.; Suppini, J.; Gomez-Vrielyunck, N. & Ladjimi, M. (2006). The effect of the hexahistidine-tag in the oligomerization of HSC70 constructs. *Journal of Chromatography. B. Analyt. Technol. Biomed. Life Sci*., Vol.844, No.2, (December 2006), pp. 328–334, ISSN 1570-0232

conservative in firefly luciferases. The only exceptions are the similar substitution S118C in the recently cloned juvenile luciferase from *L. cruciata* (Oba et al, 2010a) and the substitution S118T in the luciferase from *Lampyroidea maculata* (Emamzadeh et al., 2006). However, in

All four key thermostabilizing substitutions (R211L, A217V, E356K, and S364C) are located in the second subdomain of firefly luciferase. According to the results of Frydman and coworkers (Frydman *et al.*, 1999), the fragments of firefly luciferase comprising residues 1- 190 and 422-544 possess high intrinsic stability. These fragments mainly correspond to the subdomains A and C of firefly luciferase (Fig. 10). That study demonstrated that the middle subdomain B (192-435) was significantly less stable and that it was the first to unfold under denaturating conditions. Hence, it likely that the stability of the second subdomain is the less stable "bottleneck" that determines the stability of the firefly luciferase protein. Therefore, most of the thermostabilizing mutations would tend to be located in the second subdomain or at the interface of this subdomain and the remaining parts of the protein. It is noteworthy that almost all thermostable mutants reported in the literature are located in this

We have demonstrated that the *in vivo* directed evolution strategy is a simple and efficient method to increase thermal stability of firefly luciferase, which allows to obtain highly thermostable mutants without sacrificing catalytic efficiency. The final mutant obtained here even displayed superior catalytic properties such as higher specific activity, lower *Km* for ATP and increased temperature optimum. In typical applications, like ATP-related assays or reporter genes, beetle luciferases are used at room temperature or 37°C. The mutant 4TS retains 70% activity after two days of incubation at 37°C. Therefore, its stability is sufficient for most common *in vivo* and *in vitro* applications. The high specific activity, catalytic efficiency, and improved protein yield make the mutant 4TS an efficient tool for ATP determination (Ugarova *et al.*, 2010). The increased temperature optimum this mutant can be an advantage when used for *in vivo* imaging and in high temperature applications. The new positions identified in this study can be successfully used for the stabilization of other firefly luciferases, especially from the *Luciola* and *Hotaria* genus's. The non-lethal *in vivo* screening approach described here can be potentially implemented to other beetle or non-beetle

luciferases when the development of thermostable forms of the enzyme is desirable.

This work was supported by the Russian Foundation for Basic Research (grants 08-04-00624

Amor-Mahjoub, M.; Suppini, J.; Gomez-Vrielyunck, N. & Ladjimi, M. (2006). The effect of

the hexahistidine-tag in the oligomerization of HSC70 constructs. *Journal of Chromatography. B. Analyt. Technol. Biomed. Life Sci*., Vol.844, No.2, (December 2006),

luciferases from non-firefly beetles this position is usually occupied by His or Val.

part of the luciferase structure, which is consistent with this hypothesis.

**8.5 Conclusion** 

**9. Acknowledgements** 

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### **Genetic Engineering of Phenylpropanoid Pathway in** *Leucaena leucocephala*

Bashir M. Khan\* *National Chemical Laboratory (NCL) India* 

#### **1. Introduction**

92 Genetic Engineering – Basics, New Applications and Responsibilities

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*Appl. WO 1999/014336* 

containing the gene for firefly luciferase. *Anal. Biochem*., Vol.161, No.2, (March

beetle luciferases can elicit bioluminescence of different colors. *Science,* Vol.244,

Trees are reservoirs of many economically and biotechnologically significant products. Wood, one such gifts of nature, consists of lignin, hemicelluloses, and cellulose. Biochemistry of lignin, which being one of the most abundant biopolymers on earth, has been studied extensively, partly due to the significance, and interest of such knowledge from industrial point of view (Boerjan et al., 2003). Lignin has far reaching impacts on agriculture, industry and the environment, making phenylpropanoid metabolism, a major route for synthesis of lignin in plants, a globally important part of plant chemistry.

Besides its critical role in normal plant health and development, high levels of lignin are problematic in the agro-industrial exploitation of various plant species. It is considered an undesirable component in paper manufacture due to the cost, energy consumption, and pollutant generating processes required for its removal (Baucher et al*.,* 2003; Boerjan, 2005; Chiang, 2002). Thus, making it essential to provide designer plant species with altered lignin content, and hence, to diminish the pressure on the domestication of natural forest resources in the future. Considerable scientific interest has been focused on the development of trees with improved wood quality through modification of different genes involved in lignin biosynthesis, which could be important for the improved end use of wood material (Chiang, 2006; Higuchi, 2006). *Leucaena leucocephala*, one of the most versatile fast growing commercially important trees for paper and pulp industry in India, contributes nearly a quarter of the total raw material. The wider use of this tree species in the pulp industry is due to its high rate of biomass production and ability to adapt to a variety of soils and climatic conditions. Every step towards the development of this tree variety in terms of increased biomass and reduced lignin content would be of great help to pulp and paper industry as it will decrease cost and release of hazardous chemicals during the production of paper pulp.

This chapter will briefly cover, the chemistry of lignin deposition in plants, role of different monolignol biosynthesis pathway genes, followed by studies concentrated on genetic

<sup>\*</sup> Shuban K. Rawal2, Manish Arha1, Sushim K. Gupta1, Sameer Srivastava1, Noor M. Shaik1, Arun K. Yadav1, Pallavi S. Kulkarni1, O. U. Abhilash1, SantoshKumar1, Sumita Omer1, Rishi K. Vishwakarma1, Somesh Singh1, R. J. Santosh Kumar1, Prashant Sonawane.1, Parth Patel1, C. Kannan1, Shakeel Abbassi1 *1National Chemical Laboratory (NCL), 2Ajeet Seeds Pvt. Ltd.* 

engineering of phenylpropanoid pathway in *Leucaena leucocephala* as tool for altering its lignin composition, thereby its application in pulp and paper industries.

#### **2. Lignin: Occurrence, structure and function**

Lignin (from Latin *lignum* meaning wood), is one of the most abundant natural organic polymer next only to cellulose (Boerjan et al., 2003). It is a vital cell wall component of all vascular plants and represents on an average of 25 % of the terrestrial biomass. It plays crucial role in structural integrity of cell wall & stiffness and strength of stem (Chabannes et al., 2001; Jones et al., 2001). Lignin is primarily synthesized and deposited in the secondary cell wall of specialized cells such as xylem vessels, tracheids and fibers. It is also deposited in minor amounts in the periderm where association with suberin provides a protective role against pathogens. In addition, lignin waterproofs the cell wall; enabling transport of water and solutes through the vascular system (Sarkanen & Ludwig, 1971). Lignins are complex racemic aromatic heteropolymers synthesized from the dehydrogenative polymerization of monolignols, namely coumaryl, coniferyl and sinapyl alcohol monomers differing in their degree of methoxylation (Freudenberg & Neish, 1968). These monolignols produce respectively, p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) phenylpropanoid units when incorporated into the lignin polymer (Fig. 1). The amount and composition of lignins vary among taxa, cell types and individual cell wall layer, and are influenced by developmental and environmental cues (Campbell & Sederoff, 1996). Lignin content is higher in softwoods (27-33%) than in hardwood (18-25%) and grasses (17-24%). The highest amounts of lignin (35-40%) occur in compression wood on the lower part of branches and leaning stems (Sarkanen & Ludwig, 1971).

Fig. 1. Structure of a lignin oligomer. These structures consist of phenylpropanoid units H, G and S. A number of such oligomers cross-polymerizes to form a complex structure of lignin

#### **2.1 Lignin biosynthesis**

94 Genetic Engineering – Basics, New Applications and Responsibilities

engineering of phenylpropanoid pathway in *Leucaena leucocephala* as tool for altering its

Lignin (from Latin *lignum* meaning wood), is one of the most abundant natural organic polymer next only to cellulose (Boerjan et al., 2003). It is a vital cell wall component of all vascular plants and represents on an average of 25 % of the terrestrial biomass. It plays crucial role in structural integrity of cell wall & stiffness and strength of stem (Chabannes et al., 2001; Jones et al., 2001). Lignin is primarily synthesized and deposited in the secondary cell wall of specialized cells such as xylem vessels, tracheids and fibers. It is also deposited in minor amounts in the periderm where association with suberin provides a protective role against pathogens. In addition, lignin waterproofs the cell wall; enabling transport of water and solutes through the vascular system (Sarkanen & Ludwig, 1971). Lignins are complex racemic aromatic heteropolymers synthesized from the dehydrogenative polymerization of monolignols, namely coumaryl, coniferyl and sinapyl alcohol monomers differing in their degree of methoxylation (Freudenberg & Neish, 1968). These monolignols produce respectively, p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) phenylpropanoid units when incorporated into the lignin polymer (Fig. 1). The amount and composition of lignins vary among taxa, cell types and individual cell wall layer, and are influenced by developmental and environmental cues (Campbell & Sederoff, 1996). Lignin content is higher in softwoods (27-33%) than in hardwood (18-25%) and grasses (17-24%). The highest amounts of lignin (35-40%) occur in compression wood on the lower part of branches and

Fig. 1. Structure of a lignin oligomer. These structures consist of phenylpropanoid units H, G and S. A number of such oligomers cross-polymerizes to form a complex structure of

lignin composition, thereby its application in pulp and paper industries.

**2. Lignin: Occurrence, structure and function** 

leaning stems (Sarkanen & Ludwig, 1971).

lignin

Although lignin has been studied for over a century, many aspects of its biosynthesis still remain unresolved. The monolignol biosynthetic pathway has been redrawn many times, yet still remains a matter of debate. During the last two decades, significant headway has been made in isolating and characterizing a number of genes pertaining to monolignol biosynthesis from different plants. Several reviews on the advancements of monolignol biosynthesis pathways are also available (Boerjan et al., 2003; Humphreys & Chapple, 2002; Whetten & Sederoff, 1995; Whetten et al., 1998).

In plants, lignins are synthesized by the polymerization of monolignols, namely p-coumaryl, coniferyl and sinapyl alcohol monomers differing in their degree of methoxylation (Higuchi, 1985; Sederoff & Chang, 1991) via phenylpropanoid biosynthetic pathway (Gross, 1985). This pathway comprises a complex series of branching biochemical reactions responsible for synthesis of a variety of products like lignin, flavonoids and hydroxycinnamic acid conjugates. Many intermediates and end products of this pathway play important role in plant such as phytoalexins, antiherbivory compounds, antioxidants, ultra violet (UV) protectants, pigments and aroma compounds. Finally, the diverse functions of lignin and related products in resistance to biotic and abiotic stresses make this pathway vital to the health and survival of plants.

The synthesis of lignin represents one of the most energy demanding biosynthetic pathways in plants, requiring large quantities of carbon skeletons. Deposition of lignin in plants proceeds via the following steps:


#### **2.1.1 Biosynthesis of monolignols**

The biosynthesis of monolignols proceeds through the phenylpropanoid pathway starting with deamination of phenylalanine to produce cinnamic acid and involves successive hydroxylation reactions of the aromatic ring, followed by phenolic *o*-methylation and conversion of the side chain carboxyl to an alcohol group (Fig. 2). Immense amount of work has been done in characterizing the monolignol biosynthesis pathway in past two decades. It is a complex pathway comprising of enzymes with functions like methyltransferase, hydroxylase, reductase and dehydrogenase. Some of the important enzymes involved in monolignol biosynthesis have been discussed below.

#### **2.1.1.1 Phenylalanine ammonia-lyase (PAL)**

The enzyme phenylalanine ammonia-lyase (PAL; EC: 4.3.1.5) that catalyzes the conversion of phenylalanine to transcinnamic acid, is the initial step towards monolignol biosynthesis and other phenolic secondary plant metabolites. Genes encoding PAL have been studied in *Populus* species (Kao et al. 2002; Osakabe et al., 1995), loblolly pine and other plant species (Bate et al., 1994; Hatton et al., 1995; Jones, 1984; Kumar & Ellis, 2001; Leyva et al., 1992; Ohl et al., 1990). *PAL* exists as a multiple member gene family and the individual members can be involved in different metabolic pathways as suggested by their expression patterns in association with certain secondary compounds accumulated in specific tissue or

Fig. 2. An overview of monolignol biosynthesis pathway: PAL, Phenylalanine ammonialyase; C4H, Cinnamic acid 4-hydroxylase; C3H, p-Coumarate 3-hydroxylase; HCT, Hydroxycinnamoyltransferase; 4CL, 4-Coumarate-CoA ligase; CCoAOMT, Caffeoyl-CoA Omethyltransferase; CCR, Cinnamoyl CoA reductase; CAld5H, Coniferyl aldehyde 5 hydroxylase; AldOMT, 5- hydroxyconiferyl aldehyde O-methyltransferase; CAD, Cinnamyl alcohol dehydrogenase; SAD, Sinapyl alcohol dehydrogenase

developmental stage. The biochemical activity of all known PALs is verified as specific deamination of phenylalanine, but genetic and physiological function may vary among different *PAL* members. The expression of *PAL* genetic function is controlled by various genetic circuits and signaling pathways.

#### **2.1.1.2 Cinnamate 4-Hydroxylase (C4H)**

96 Genetic Engineering – Basics, New Applications and Responsibilities

Fig. 2. An overview of monolignol biosynthesis pathway: PAL, Phenylalanine ammonialyase; C4H, Cinnamic acid 4-hydroxylase; C3H, p-Coumarate 3-hydroxylase; HCT,

methyltransferase; CCR, Cinnamoyl CoA reductase; CAld5H, Coniferyl aldehyde 5-

alcohol dehydrogenase; SAD, Sinapyl alcohol dehydrogenase

Hydroxycinnamoyltransferase; 4CL, 4-Coumarate-CoA ligase; CCoAOMT, Caffeoyl-CoA O-

hydroxylase; AldOMT, 5- hydroxyconiferyl aldehyde O-methyltransferase; CAD, Cinnamyl

C4H (EC: 1.14.13.11) constitutes the CYP73 family of the large group of Cyt P450 monooxygenases. It catalyzes the 4-hydroxylation of *trans-*cinnamate, the central step in the generation of phenylalanine-derived substrates for the many branches of phenylpropanoid metabolism. The first and the last enzymes of this short sequence of closely related reactions, termed the general phenylpropanoid metabolism, are PAL and 4CL, respectively. A second metabolic link couples C4H to the membrane-localized Cyt P450 Reductase (CPR). The expression patterns of all three C4H-linked enzymes, PAL, 4CL, and CPR, and of the corresponding mRNAs have been analyzed in cell-suspension cultures and various intact tissues of parsley (Logemann et al., 1995) and *Arabidopsis* (Mizutani and Ohta, 1997). A reduction in PAL levels leads to an increase in the S/G ratio, whereas reduced C4H activity leads to a decrease in the S/G ratio. These observations support the existence of some sort of metabolic channeling between the enzymes of the central phenylpropanoid pathway and those of monolignol biosynthesis and also provide a basis for the development of new strategies for modified or reduced lignin content.

Similar to PAL, C4H is thought to be involved in a number of secondary metabolism pathways in addition to monolignol biosynthesis as *p*-coumarate is an intermediate for biosynthesis of many secondary compounds (Croteau et al., 2000). Multiple *C4H* gene members are identified in many plant species, however, only one *C4H* is known in the *Arabidopsis* genome (Raes et al., 2003). The expression study of two *C4H* members in quaking aspen indicated that one is strongly expressed in developing xylem tissues and the other is more active in leaf and young shoot tissues (Shanfa et al., 2006). In other species, *C4H* gene is expressed in a variety of tissues and the expression is induced by wounding, light, pathogen attacks and other biotic & abiotic stimuli (Bell-Lelong et al., 1997; Raes et al., 2003). The mechanisms that regulate the genetic function of *C4H* gene and its family members are yet unknown.

#### **2.1.1.3 Coumarate 3-hydroxylase (C3H)**

Early biochemical evidence suggested that conversion of coumarate to caffeate is catalyzed by a nonspecific phenolase, but that suggestion did not receive much support in other studies (Boniwell & Butt, 1986; Kojima & Takeuchi, 1989; Petersen et al., 1999; Stafford & Dresler, 1972). The gene encoding *p*-coumarate 3-hydroxylase (*C3H*) was cloned and an alternative pathway proposed based on the enzyme activity of *CYP98A3* gene from *Arabidopsis* (Franke et al., 2002 a; Nair et al., 2002; Schoch et al., 2001).

#### **2.1.1.4 p-hydroxycinnamoyl-CoA: quinate shikimate p-hydroxycinnamoyl- transferase (HCT)**

The enzyme *p*-coumarate 3-hydroxylase (C3H) converts *p*-coumaric acid into caffeic acid and has been shown to be a cytochrome p450-depenedent monooxygenase. It is interesting to note that enzymatic assays have demonstrated that the shikimate and quinate esters of *p*-coumaric acid are the preferred substrates for C3H over *p*-coumaric acid, *p*-coumaroyl-CoA, *p* coumaraldehyde, *p*-coumaryl alcohol, nor the 1–*O*-glucose ester and the 4–*O*-glucoside of *p*-coumaric acid are good substrates (Franke et al., 2002; Nair et al., 2002; Schoch et al., 2001). *p*-Coumarate is first converted to *p*-coumaroyl-CoA by 4CL, with subsequent conversion to *p*-coumaroyl-shikimate and *p*-coumaroyl-quinate, the substrates for C3H, by *p*-hydroxycinnamoyl-CoA:quinate-(CQT) or *p*-hydroxycinnamoyl-CoA:shikimate *p*-hydroxycinnamoyltransferase (CST) (Schoch et al., 2001). These enzymes, described as reversible enzymes, can convert caffeoyl-shikimate or caffeoyl-quinate (chlorogenic acid) into caffeoyl-CoA, the substrate for CCoAOMT. A reversible acyltransferase with both CQT and CST activity, designated HCT, has been purified and the corresponding gene cloned from tobacco (Hoffmann et al., 2002). Silencing of *HCT* through RNA interference (RNAi) lead to reduction in lignin, hyper accumulation of flavonoids and growth inhibition in *Arabidopsis* (Chapple, 2010; Hoffmann et al., 2004, 2007)

#### **2.1.1.5 Coumarate Coenzyme-A ligase (4CL)**

Genetic and biochemical functions of 4-Coumarate Coenzyme A ligase (4CL; EC: 6.2.1.12) genes have been clearly demonstrated in association with monolignol biosynthesis (Lewis and Yamamoto, 1990; Lee et al., 1997; Hu et al., 1998, 1999; Harding et al., 2002). *4CL* genes usually exist as a multi-gene family. Four *4CL* genes were detected in the *Arabidopsis* genome and the expression of each member was regulated differentially in tissues and development stages (Raes et al., 2003). In aspen trees, two *4CL* genes were cloned and their expression were clearly distinct, with one in epidermal & leaf tissue and the other specifically in developing xylem tissue (Harding et al., 2002; Hu et al., 1998). Furthermore, the enzymatic activities of *4CL* members from aspen, loblolly pine, tobacco, soybean, *Arabidopsis*, and many other species were found to have distinct substrate specificities (Hu et al., 1998; Lindermayr et al., 2003; Voo et al., 1995). Whether the substrate specificity of the *4CL* members relates to different metabolic pathways is unknown. As the 4CL catalytic kinetics vary among species, it is also likely that the mainstream pathway mediated by 4CL may not be exactly the same in all plant species or tissues. Nevertheless, monolignol biosynthesis is tightly controlled by *4CL*.

#### **2.1.1.6 O-methyltransferases (O-MT)**

S-adenosyl-L-methionine methyltransferases are key enzymes in the phenylpropanoid, flavanoid and many other metabolic pathways in plants. The enzymes Caffeate 3-O methyltransferase (COMT; EC: 2.1.1.68) and Caffeoyl CoA 3-O methyltransferase (CCoAOMT; EC: 2.1.1.104) control the degree of methoxylation in lignin precursors *i.e. p*- hydroxyphenyl (H), guaiacyl (G) and syringyl (S) units of lignin (Higuchi, 1990; Ralph et al., 1998; Boerjan et al., 2003). The methylation reactions at the C3 and C5 hydroxyl functions of the lignin precursors were thought to occur mainly at the cinnamic acid level via a bifunctional COMT. However, the association of *CCoAOMT* expression with lignification (Pakusch et al., 1991; Ye et al., 1994; Ye & Varner, 1995; Ye, 1997; Martz et al., 1998; Chen et al., 2000) and the observation that down-regulation of *COMT* preferentially affected the amount of S units suggested the existence of an alternative pathway for the methylation of the lignin precursors at the hydroxycinnamoyl CoA level and specific O-methyltransferase *i.e.* CCoAOMT converts caffeoyl CoA into feruloyl CoA and 5-hydroxyferuloyl CoA into sinapyl CoA (Martz et al., 1998).

#### **2.1.1.7 Cinnamoyl CoA reductase (CCR)**

The reduction of cinnamoyl CoA esters to cinnamaldehydes is the first metabolic step committed to monolignol formation (Lacombe & Hawkins, 1997). This first reductive step in

the 4–*O*-glucoside of *p*-coumaric acid are good substrates (Franke et al., 2002; Nair et al., 2002; Schoch et al., 2001). *p*-Coumarate is first converted to *p*-coumaroyl-CoA by 4CL, with subsequent conversion to *p*-coumaroyl-shikimate and *p*-coumaroyl-quinate, the substrates for C3H, by *p*-hydroxycinnamoyl-CoA:quinate-(CQT) or *p*-hydroxycinnamoyl-CoA:shikimate *p*-hydroxycinnamoyltransferase (CST) (Schoch et al., 2001). These enzymes, described as reversible enzymes, can convert caffeoyl-shikimate or caffeoyl-quinate (chlorogenic acid) into caffeoyl-CoA, the substrate for CCoAOMT. A reversible acyltransferase with both CQT and CST activity, designated HCT, has been purified and the corresponding gene cloned from tobacco (Hoffmann et al., 2002). Silencing of *HCT* through RNA interference (RNAi) lead to reduction in lignin, hyper accumulation of flavonoids and growth inhibition in

Genetic and biochemical functions of 4-Coumarate Coenzyme A ligase (4CL; EC: 6.2.1.12) genes have been clearly demonstrated in association with monolignol biosynthesis (Lewis and Yamamoto, 1990; Lee et al., 1997; Hu et al., 1998, 1999; Harding et al., 2002). *4CL* genes usually exist as a multi-gene family. Four *4CL* genes were detected in the *Arabidopsis* genome and the expression of each member was regulated differentially in tissues and development stages (Raes et al., 2003). In aspen trees, two *4CL* genes were cloned and their expression were clearly distinct, with one in epidermal & leaf tissue and the other specifically in developing xylem tissue (Harding et al., 2002; Hu et al., 1998). Furthermore, the enzymatic activities of *4CL* members from aspen, loblolly pine, tobacco, soybean, *Arabidopsis*, and many other species were found to have distinct substrate specificities (Hu et al., 1998; Lindermayr et al., 2003; Voo et al., 1995). Whether the substrate specificity of the *4CL* members relates to different metabolic pathways is unknown. As the 4CL catalytic kinetics vary among species, it is also likely that the mainstream pathway mediated by 4CL may not be exactly the same in all plant species or tissues. Nevertheless, monolignol

S-adenosyl-L-methionine methyltransferases are key enzymes in the phenylpropanoid, flavanoid and many other metabolic pathways in plants. The enzymes Caffeate 3-O methyltransferase (COMT; EC: 2.1.1.68) and Caffeoyl CoA 3-O methyltransferase (CCoAOMT; EC: 2.1.1.104) control the degree of methoxylation in lignin precursors *i.e. p*- hydroxyphenyl (H), guaiacyl (G) and syringyl (S) units of lignin (Higuchi, 1990; Ralph et al., 1998; Boerjan et al., 2003). The methylation reactions at the C3 and C5 hydroxyl functions of the lignin precursors were thought to occur mainly at the cinnamic acid level via a bifunctional COMT. However, the association of *CCoAOMT* expression with lignification (Pakusch et al., 1991; Ye et al., 1994; Ye & Varner, 1995; Ye, 1997; Martz et al., 1998; Chen et al., 2000) and the observation that down-regulation of *COMT* preferentially affected the amount of S units suggested the existence of an alternative pathway for the methylation of the lignin precursors at the hydroxycinnamoyl CoA level and specific O-methyltransferase *i.e.* CCoAOMT converts caffeoyl CoA into feruloyl CoA and 5-hydroxyferuloyl CoA into

The reduction of cinnamoyl CoA esters to cinnamaldehydes is the first metabolic step committed to monolignol formation (Lacombe & Hawkins, 1997). This first reductive step in

*Arabidopsis* (Chapple, 2010; Hoffmann et al., 2004, 2007)

**2.1.1.5 Coumarate Coenzyme-A ligase (4CL)** 

biosynthesis is tightly controlled by *4CL*. **2.1.1.6 O-methyltransferases (O-MT)** 

sinapyl CoA (Martz et al., 1998).

**2.1.1.7 Cinnamoyl CoA reductase (CCR)** 

lignin biosynthetic pathway is performed by Cinnamoyl CoA reductase (EC: 1.2.1.44) and it controls the over-all carbon flux towards lignin. CCR activity is found to be generally low in plants so it is hypothesized that it may play a crucial role as a rate limiting step in regulation of lignin biosynthesis (Ma & Tian, 2005). CCR is apparently encoded by a single gene per haploid genome in *Eucalyptus* (Lacombe & Hawkins, 1997), poplar (Leple et al*.*, 1998), ryegrass (Larsen, 2004; McInnes et al., 2002), *Triticum* (Ma, 2007) and tobacco (Piquemal & Lapierre, 1998) and by two genes in maize (Pichon, Courbou et al., 1998), and *Arabidopsis*  (Lauvergeat & Lacomme, 2001). The *CCR* genes in various species appear as a multiple member family. In the *Populus* genome, there exist 8 *CCR*-homolog or *CCR*-like gene sequences (Li & Cheng, 2005). *Triticum* (Ma & Tian, 2005; Ma, 2007), maize (Pichon & Courbou, 1998), switchgrass (Escamilla-Trevino & Shen, 2010), *Medicago* (Zhou & Jackson, 2010) and *Arabidopsis* (Lauvergeat & Lacomme, 2001) have been shown to possess two or more than two isoforms (*CCR1* and *CCR2*) which are involved in mutually exclusive or redundant functions like, constitutive lignifications and defense. Several other *CCR* gene sequences have been deposited in the GenBank database, but their functions have still not been demonstrated. It is proposed that all CCR enzymes have a similar catalyzing mechanism for converting the CoA esters to aldehydes in monolignol biosynthesis.

#### **2.1.1.8 Coniferaldehyde 5-hydroxylase (CAld5H)**

CAld5H enzyme like C4H belongs to cytochrome P450 monoxygenase family. The hydroxylation reaction in the biosynthesis of S-unit (syringyl) was first considered to occur at the ferulate level (Grand, 1984), and hence, the enzyme was called Ferulate 5-hydroxylase (F5H). However, studies later have revealed that F5H can also function at later steps in the pathway, mainly at the coniferyl aldehyde or coniferyl alcohol level (Humphreys et al., 1999; Li et al., 2000). This enzyme was therefore alternatively renamed coniferaldehyde- 5 hydroxylase (CAld5H) (Osakabe et al., 1999). F5H/CAld5H is unusual in that it is a multifunctional plant P450 with three physiologically relevant substrates. The *K*m for the substrates such as coniferaldehyde, coniferyl alcohol and for the ferulic acid are 1 μM, 3 μM and 1000 μM respectively. This study demonstrates that the coniferaldehyde is the most preferred substrate for the enzymes (Humphrey et al*.,* 1999). Considerable evidence is now available that shows that in angiosperm trees, the syringyl monolignol pathway branches out from guaiacyl pathway through coniferaldehyde and is regulated in sequence by three genes encoding coniferaldehyde 5-hydroxylase (*CAld5H*), 5-hydroxyconiferaldehyde *O*-methyltransferase (*COMT*) and sinapyl alcohol dehydrogenase (*SAD*).

#### **2.1.1.9 Cinnamyl/Sinapyl alcohol dehydrogenases (CAD/SAD)**

In gymnosperm wood, coniferyl alcohol is the major monolignol units while both coniferyl and sinapyl alcohols are present in angiosperm wood. CAD (E.C: 1.1.1.195), depicts a class of NADPH dependent oxidoreductase, suggested to catalyze multiple cinnamyl alcohol formations from their corresponding cinnamaldehydes (Lewis & Yamamoto, 1990; Whetten & Sederoff, 1995; Whetten et al., 1998). This reduction of aldehydes to corresponding alcohols has been considered to be an indicator of lignin biosynthesis because of its specific role at the end of the monolignol biosynthesis pathway (Baucher et al., 1996). When the *Populus* tree was studied for monolignol biosynthesis in wood forming tissue, in addition to *CAD*, it was found in aspen that another gene, its sequence similar to but distinct from *CAD*, is also associated with lignin biosynthesis (Li et al., 2001). The biochemical characterization of the recombinant protein encoded by this gene indicated that the enzymatic activity has specific affinity toward sinapaldehyde, therefore it was named SAD. Compared with SAD enzyme kinetics, CAD showed a catalytic specificity towards coniferaldehyde instead. The catalytic specificities of the two enzymes have been further verified in protein structure analysis (Bomati & Noel, 2005). Furthermore, it was demonstrated that the expression of *CAD* is associated with G-lignin accumulation while *SAD* was associated with S-lignin formation during xylem differentiation (Li et al., 2001). The evidence from molecular, biochemical and cellular characterizations strongly suggest that *CAD* is involved in G-monolignol biosynthesis and *SAD* in S-monolignol biosynthesis in aspen wood formation.

#### **2.1.2 Transport of monolignols**

After the synthesis, the lignin precursors or monolignols are transported to the cell wall where they are oxidized and polymerized. The monolignols formed are insoluble and toxic to the plant cell and hence are converted to their respective glucosides by the action of UDPglycosyltransferases (UDP-GT). This conversion renders the monolignols, soluble and less toxic to the plant cells, which can be stored in plant vacuoles, and transported to the cell wall as the need arises. It has been hypothesized that these monolignol glucosides are storage or transport forms of the monolignols (Steeves et al., 2001).

#### **2.1.3 Dehydrogenation and polymerization**

After transport of the monolignols to the cell wall, lignin is formed through dehydrogenative polymerization of the monolignols (Christensen et al., 2000). The dehydrogenation of monolignol radicals has been attributed to different class of enzymes, such as peroxidases (POX), laccases (LAC), polyphenol oxidases, and coniferyl alcohol oxidase. Lignin is a hydrophobic and optically inactive polymer, which is highly complex and heterogeneous in nature. Lignin polymerization is a radical coupling reaction, where the monolignols are first activated into phenoxy radicals in an enzyme catalyzed dehydrogenation reaction. These radicals couple to form dimers, oligomers and eventually the lignin polymer (Freudenberg, 1968). Peroxidases are heme-containing oxidoreductases that use H2O2 as the ultimate electron acceptor. The natural electron donor molecules in a peroxidase catalyzed reaction vary and include, monolignols, hydroxycinnamic acids (Zimmerlin et al., 1994), tyrosine residues in extensions (Brownleader et al., 1995) and auxin (Hinman & Lang, 1965). Several reports on peroxidase activity or gene expression in ligninforming tissues have appeared, but only a few isoenzymes or genes have been specifically associated with lignification (Christensen et al., 2001; Marjamaa et al., 2006; Quiroga et al., 2000; Sato et al., 1993).

#### **2.2 Regulation of monolignol biosynthesis**

Developmental program of lignification associated with certain types of plant cells, such as xylem and fibers, require coordinated regulation of different lignin biosynthesis genes, as well as with genes controlling other aspects of plant growth and development. Different transcription factors such as R2R3-MYB, KNOX, MADS, LIM have been found to be regulating lignin biosynthesis in many plants, although the understanding of the molecular mechanism of pathway regulation is still limiting (Campbell & Rogers, 2004; Zhou et al., 2006, 2008). Lignification can be modified in a more efficient and precise way by understanding the regulation of these pathways via altering the expression of relevant transcription factors.

#### **3. Lignin as barrier for paper production**

100 Genetic Engineering – Basics, New Applications and Responsibilities

enzymatic activity has specific affinity toward sinapaldehyde, therefore it was named SAD. Compared with SAD enzyme kinetics, CAD showed a catalytic specificity towards coniferaldehyde instead. The catalytic specificities of the two enzymes have been further verified in protein structure analysis (Bomati & Noel, 2005). Furthermore, it was demonstrated that the expression of *CAD* is associated with G-lignin accumulation while *SAD* was associated with S-lignin formation during xylem differentiation (Li et al., 2001). The evidence from molecular, biochemical and cellular characterizations strongly suggest that *CAD* is involved in G-monolignol biosynthesis and *SAD* in S-monolignol biosynthesis

After the synthesis, the lignin precursors or monolignols are transported to the cell wall where they are oxidized and polymerized. The monolignols formed are insoluble and toxic to the plant cell and hence are converted to their respective glucosides by the action of UDPglycosyltransferases (UDP-GT). This conversion renders the monolignols, soluble and less toxic to the plant cells, which can be stored in plant vacuoles, and transported to the cell wall as the need arises. It has been hypothesized that these monolignol glucosides are

After transport of the monolignols to the cell wall, lignin is formed through dehydrogenative polymerization of the monolignols (Christensen et al., 2000). The dehydrogenation of monolignol radicals has been attributed to different class of enzymes, such as peroxidases (POX), laccases (LAC), polyphenol oxidases, and coniferyl alcohol oxidase. Lignin is a hydrophobic and optically inactive polymer, which is highly complex and heterogeneous in nature. Lignin polymerization is a radical coupling reaction, where the monolignols are first activated into phenoxy radicals in an enzyme catalyzed dehydrogenation reaction. These radicals couple to form dimers, oligomers and eventually the lignin polymer (Freudenberg, 1968). Peroxidases are heme-containing oxidoreductases that use H2O2 as the ultimate electron acceptor. The natural electron donor molecules in a peroxidase catalyzed reaction vary and include, monolignols, hydroxycinnamic acids (Zimmerlin et al., 1994), tyrosine residues in extensions (Brownleader et al., 1995) and auxin (Hinman & Lang, 1965). Several reports on peroxidase activity or gene expression in ligninforming tissues have appeared, but only a few isoenzymes or genes have been specifically associated with lignification (Christensen et al., 2001; Marjamaa et al., 2006; Quiroga et al.,

Developmental program of lignification associated with certain types of plant cells, such as xylem and fibers, require coordinated regulation of different lignin biosynthesis genes, as well as with genes controlling other aspects of plant growth and development. Different transcription factors such as R2R3-MYB, KNOX, MADS, LIM have been found to be regulating lignin biosynthesis in many plants, although the understanding of the molecular mechanism of pathway regulation is still limiting (Campbell & Rogers, 2004; Zhou et al., 2006, 2008). Lignification can be modified in a more efficient and precise way by

storage or transport forms of the monolignols (Steeves et al., 2001).

in aspen wood formation.

2000; Sato et al., 1993).

**2.1.2 Transport of monolignols** 

**2.1.3 Dehydrogenation and polymerization** 

**2.2 Regulation of monolignol biosynthesis** 

From an agro-economical point of view, lignin is considered to have a negative impact because it affects the paper manufacture and limits digestibility of forage crops. High quantity and low Syringyl (S) to Guaiacyl (G) lignin ratio plays a detrimental role in economy and ecology of paper production. Every unit increase in S/G ratio decreases the cost of paper production by two and half times. Both lignin content and composition are known to have impact on pulp & paper because residual lignin in the wood fibers causes a discoloration and a low brightness level of the pulp (Chaing et al., 1988). Consequently, for the production of high quality paper, lignin has to be removed from cellulose during the pulping process without damaging the polysaccharide component of wood. During chemical (Kraft) pulping, a large amount of Sodium hydroxide (NaOH) and Sodium sulfide (Na2S) are required to extract lignin from the pulp (Axegard et al., 1992). Subsequently, the residual lignin is further removed with bleaching agents, such as Chlorine dioxide (ClO2), Hydrogen peroxide (H2O2), Sodium hypochlorite(NaOCl), Oxygen(O2), or Ozone (O3) (Axegard et al.,1992; Biermann, 1993; Christensen et al., 2000).

These lignin extraction & bleaching procedures can partly degrade cellulose and consequently, reduce pulp quality and paper strength. Lignin extraction consumes large quantities of chemicals and energy leading to poor environmental image for these industries (Biermann, 1996; Higuchi, 1985; Odendahl, 1994). For this reason, engineering of plants with cell wall structures that are more susceptible to the krafting, and thus, more amenable to hydrolysis, or are sufficiently altered so as to shunt the above processes is an attractive approach to improve pulping efficiency and potentially alleviate some of the negative environmental impacts of the paper making industry. Apart from the great deal of work in the lignin field for improving the pulping process, many examples can be found based on research aimed at altering the lignin content for improving bio-fuel production (Chen, 2006; Chen & Dixion, 2007; Davision, 2006; Franke et al., 2002), as well as for improving forage crop digestibility (Table 1).

#### **4. Genetic engineering of phenylpropanoid biosynthetic pathway**

Despite the extensive literature on genetic modifications of lignin biosynthesis in a variety of plants, only a few studies have reported the impact of modified lignin content and composition on pulping and bleaching processes. Nevertheless, significant progress has been made in this field, as summarized in Table 1.

Emerging genetic engineering strategies *in planta* including manipulation of lignin biosynthesis at regulatory level, controlling monolignol polymerization enzymes, and modifications of lignin polymer structure, together with exploration of lignin degrading enzymes from other organisms provide us the necessary tools for producing designer plant species with reduced/altered lignin traits, so as to meet the needs of paper, livestock industries, etc. However, traditional genetic engineering strategies such as upregulation and downregulation of monolignol biosynthetic genes are still applied and have been successful in facilitating lignin decomposition by altering both lignin content and composition. One such example based on genetic modifications in *Leucaena* is described here, providing insights into the reactions, and regulation of genes involved in lignin biosynthesis, and its impact on determining lignin quality for paper industries.


Table 1. Genetic engineering of different lignin biosynthetic genes, and their effects on lignin contents and composition

Fig. 3. Levels of control for manipulation of lignin biosynthesis. The control level 1 involves manipulation of lignin biosynthetic genes; level 2 and 3 involves manipulation of genes involved in transport and polymerization of monolignols; while level 4 deals with manipulations of different regulatory factors

#### **4.1** *Leucaena leucocephala* **as a source of pulp**

A great deal of knowledge on the molecular biology and regulation of phenylpropanoid biosynthesis has been derived from investigations in plants such as *Arabidopsis*, Alfalfa etc.

such example based on genetic modifications in *Leucaena* is described here, providing insights into the reactions, and regulation of genes involved in lignin biosynthesis, and its

Table 1. Genetic engineering of different lignin biosynthetic genes, and their effects on lignin

Fig. 3. Levels of control for manipulation of lignin biosynthesis. The control level 1 involves manipulation of lignin biosynthetic genes; level 2 and 3 involves manipulation of genes involved in transport and polymerization of monolignols; while level 4 deals with

A great deal of knowledge on the molecular biology and regulation of phenylpropanoid biosynthesis has been derived from investigations in plants such as *Arabidopsis*, Alfalfa etc.

Lignin Composition

S/G Ratio↓ - H/G↑ - - - S/G Ratio↑ S/G Ratio↑ Aldehyde units↑ S/G Ratio↓ S/G Ratio↑ - S/G Ratio↓ High H " S/G Ratio↑ S/G Ratio↓ " " S/G Ratio↑ Aldehyde units↑ free phenolics ↑ S/G Ratio↓ H/G Ratio↓ - S/G Ratio↓ "

Phenotype References

Baucher et al., 1999 Shadle et al., 2007 Ziebell et al., 2010 Reddy et al., 2005 Marita et al., 2003 Guo et al., 2001 Sewalt et al., 1997 O'Connell et al., 2002 Halpin et al., 1994 Vailhe et al., 1998 Kavousi et al., 2010 Goujon et al., 2003 Reddy et al., 2005 Shadle et al., 2007 Reddy et al., 2005 Guo et al., 2001a, b Reddy et al., 2005 Baucher et al., 1999 Jouanin et al., 2000 Petit, Conil et al., 1999 Lapierre et al., 2004 Voelker et al., 2010 Wadenback et al., 2007 Chen et al., 2003 He et al., 2003 Day et al., 2009

Forage Digestibility↑ " " " Cellulose content↑ Forage Digestibility↑ ' Pulping Efficiency↑ " Forage Digestibility↑ Saccharification↑ Forage Digestibility↑ " " " " Unchanged Forage Digestibility↑ Pulping Efficiency↓ Pulping Efficiency↑ " Cellulose content↑ Pulping Efficiency↑ Forage Digestibility↑ " -

impact on determining lignin quality for paper industries.

Content

Reduced " " " " " " " Unchanged " Reduced " " " " " Unchanged " Reduced " Unchanged Reduced " " " "

Plant Gene Lignin

CAD HCT C3H/ HCT CP 450 enzymes COMT CCoAOMT PAL/CCoAOMT

CCR CAD

POX CCR C4H HCT C3H CCoAOMT Cald5H CAD COMT CCoAOMT CAD 4CL CCR CAD COMT CCoAOMT

CAD/CCoAOMT

*Medicago* 

*Nicotiana* 

*Arabidopsis*  Alfalfa

*Populus* 

*Picea Festuca*  Maize *Linum* 

contents and composition

manipulations of different regulatory factors

**4.1** *Leucaena leucocephala* **as a source of pulp** 

While these models will continue to serve as platforms for studying lignifications, a number of other plant species, e.g., *Leucaena leucocephala* have recently been selected for such studies. The paper industry in India mainly uses bamboos, *Eucalyptus* sp., *Casuarina* sp. and *Leuacaena* sp. as a source for paper pulp. Selection of the species depends upon availability, price and acceptability by any given industrial unit. However *Leucaena* sp. is extensively used in India and nearly a quarter of raw materials for paper and pulp industry comes from this plant (Srivastava et al., 2011). *Leucaena* is also valued as an excellent source of nutritious forage.

It has been estimated that dried leaves of *Leucaena* contain nearly 28-35% of protein content of high nutritional quality. Besides this, *Leuceana* is also an excellent source of firewood, industrial fuel, organic fertilizer, timber and gum (Cottom et al., 1977). A native of Central America, *Leucaena* has been naturalized pan-tropically, with members of its genera being vigorous, drought tolerant, highly palatable, high yielding & rich in protein and grow in wide range of soils (Hughes, 1998; Jones, 1979). However, these attributes are limited by the occurrence of anti-nutritive factors in the fodder, such as tannins and mimosine (Hammond et al., 1989; Hegarty et al., 1964; Jones, 1979).

#### **4.2 Phenylpropanoid biosynthesis genes from** *Leucaena leucocephala*

Different genes involved in phenylpropanoid biosynthesis were studied in detail from *Leucaena*. Table 2 summarizes the details of such genes isolated from *Leucaena* along with their GenBank accession numbers.

#### **4.3 Regeneration system of** *Leucaena leucocephala*

Genetic transformation of plants needs reproducible robust regeneration system. Regeneration of complete plants through tissue culture has made it possible to introduce foreign genes in to plant genome and recover transgenic plants. The limited success rate for


Table 2. Phenylpropanoid biosynthesis genes from *Leucaena*: C4H: Cinnamate 4-Hydroxylase; 4CL: 4 Coumarate CoA Ligase; CCoAOMT: Caffeoyl CoA O-Methyl Transferase; COMT: Caffeate *O*-Methyl Transferase; CCR: Cinnamoyl CoA Reductase; CAld5H: Coniferaldehyde 5-Hydroxylase; CAD: Cinnamyl Alcohol Dehydrogenase; POX: Peroxidase; \*Partial sequence

regeneration of leguminous trees has been reported in few research works. The complete *in vitro* plantlet regeneration of *Leucaena* (Cultivar, K-67) from lateral bud explants has been optimized with the maximum shoot multiplication rate of 22 shoots per explants. These regenerated plantlets were transplanted *ex vitro* with 80% survival rates (Goyal et al., 1985). In addition to the regeneration from lateral bud explants, an alternative (both direct and callus mediated/indirect) plantlet regeneration system has been successfully demonstrated with 100% regeneration frequency using cotyledon explants from 3-4 days old plants (Cultivar, K-636). It is interesting to note that the plantlets regenerated from cotyledonary explants rooted without any requirements of growth regulators on basal media (Saafi et al., 2002). Addition of Thidiazuron (TDZ) to the shoot induction medium has substantially improved the number of *in vitro* shoots per explants as compared to the basal shoot induction medium with N6-Benzyladenine (BA). Liquid pulse treatment of the induced shoots with TDZ resulted in the improvement in the subsequent rooting. The plantlets regenerated in this manner showed more than 90% survival rate *ex vitro* when grown in coco-peat mixture (Shaik et al., 2009). In order to improve overall *in vitro* plantlet regeneration efficiency, attempts to propagate elite (cultivar K-8, K-636) and wild type varieties of *Leucaena* were made by supplementing the basal shoot induction media with puterscin. It has been observed that putriscine (9.3 µM) significantly enhanced the number of regenerated shoots from hypocotyls explants when compared to the induction medium containg only BA (22.2 µM). The incidence of yellowing and leaf abscission was successfully

PAL JN540043\* -- -- *-- Ll-PAL* 

CAld5H EU041752 1.826 511 57.42 *Ll-Cald5H*  CAD EU870436 1.178 357 38.87 *Ll-CAD* 

Glucosylhydrolase EU328158 1.524 507 57.57 *Ll-GH Family 1*  R2R3 MYB factor GU901209 0.911 235 26.50 *Ll\_R2R3\_MYB2\_SSM* 

Table 2. Phenylpropanoid biosynthesis genes from *Leucaena*: C4H: Cinnamate 4-Hydroxylase; 4CL: 4 Coumarate CoA Ligase; CCoAOMT: Caffeoyl CoA O-Methyl Transferase; COMT: Caffeate *O*-Methyl Transferase; CCR: Cinnamoyl CoA Reductase; CAld5H: Coniferaldehyde

regeneration of leguminous trees has been reported in few research works. The complete *in vitro* plantlet regeneration of *Leucaena* (Cultivar, K-67) from lateral bud explants has been optimized with the maximum shoot multiplication rate of 22 shoots per explants. These regenerated plantlets were transplanted *ex vitro* with 80% survival rates (Goyal et al., 1985). In addition to the regeneration from lateral bud explants, an alternative (both direct and callus mediated/indirect) plantlet regeneration system has been successfully demonstrated with 100% regeneration frequency using cotyledon explants from 3-4 days old plants (Cultivar, K-636). It is interesting to note that the plantlets regenerated from cotyledonary explants rooted without any requirements of growth regulators on basal media (Saafi et al., 2002). Addition of Thidiazuron (TDZ) to the shoot induction medium has substantially improved the number of *in vitro* shoots per explants as compared to the basal shoot induction medium with N6-Benzyladenine (BA). Liquid pulse treatment of the induced shoots with TDZ resulted in the improvement in the subsequent rooting. The plantlets regenerated in this manner showed more than 90% survival rate *ex vitro* when grown in coco-peat mixture (Shaik et al., 2009). In order to improve overall *in vitro* plantlet regeneration efficiency, attempts to propagate elite (cultivar K-8, K-636) and wild type varieties of *Leucaena* were made by supplementing the basal shoot induction media with puterscin. It has been observed that putriscine (9.3 µM) significantly enhanced the number of regenerated shoots from hypocotyls explants when compared to the induction medium containg only BA (22.2 µM). The incidence of yellowing and leaf abscission was successfully

**Amino acid no.** 

> 505 505 505

> 542 519

> 244 244

> 334 336

> 316 316 316

1075 1073 **Mm, kDa** 

57.94 57.93 57.99

58.87 56.74

*27.55 27.57* 

36.30 36.52

33.94 34.03 34.20

123.30 119.97 **Annotations** 

*LlC4H1 LlC4H2 LlC4H3*

> *4CL 1 4CL2*

*CCoAOMT2 CCoAOMT1* 

*Ll-CCRH2 Ll-CCRH1* 

> *POX1 POX2 Ll-POX*

*Ll-7CesA Ll-8CesA* 

**cDNA, kb** 

> 1.836 1.761 1.760

> 1.935 1.831

> 0.735 0.735

> 1.005 1.011

> 0.951 0.951 0.951

> 3.391 3.368

5-Hydroxylase; CAD: Cinnamyl Alcohol Dehydrogenase; POX: Peroxidase; \*Partial

**Name of Genes Accession** 

C4H GU183363

4CL FJ205490

CCR EU195224

CCoAOMT DQ431234

Lignin POX GU143879

Cellulose Synthase(CesA)

sequence

**Nos.** 

HQ191221 HQ191222

FJ205491\*

DQ431233

DQ986907

GU143878 EU649680

FJ871987 GQ267555 abridged by addition of glutamine (685 µM) or adenine (540 µM) which indirectly added *ex vitro* survival of the plants. All the regenerated plantlets from hypocotyls explants exhibited 100% *in vitro* rooting and were subsequently transplanted *ex vitro* (Sirisha et al., 2008). *In vitro* regeneration system for some other cultivars of *Leucaena* (K-8, K-29, K-68 and K-850) from mature trees derived nodal explants as well as seedlings derived cotyledonary node explants have also been reported, where cultivar K-29 gave the best response *in vitro*. Indirect (through callus phase) somatic embryogenesis of cv. K-29 using 40.28 μM NAA and 12.24 μM were also established. These somatic embryos were further matured in full strength medium (Rastogi et al., 2008).

Considering all above discussed reports, it can be suggested that our reports (Shaik et al., 2009) of improved method also have produced a consistent regeneration system for *Leucaena* which will be beneficial for the mass propagation and genetic transformation of *Leucaena*  species. Lignin content in wood pulp adversely influences the quality of paper produced. In *Leucaena* which is an important paper pulp wood crop, it becomes important to identify and multiply elite clones having naturally low lignin content. In addition to this approach there is a need to develop transgenic plants with altered or reduced lignin content for its efficient and eco-friendly removal from pulp. The above mentioned multiple regeneration pathways are an excellent tool to introducing foreign genes. Out of all these methods shoot regeneration from cotyledonary node explants are more responsive to multiple shoot induction (Hussain et al., 2007). Genetic transformation procedures particularly particle bombardment which is considered as most effective means of gene delivery can be applied to the transformation of these shoot meristems. Cotyledonary explant derived multiple shoots form most suitable tissue for genetic transformation due to their higher regeneration frequency. Therefore, our recent study dealt with multiple shoot induction from the cotyledonary nodes of *Leucaena* in response to cytokinins, thidiazuron (TDZ) and N6 benzyladenine (BA) supplemented in half strength MS (½-MS) medium and also their effect on *in vitro* rooting of the regenerated shoots (Fig. 5). The addition of cytokinins to the medium was found essential for multiple shoot induction. *Leucaena* cotyledonary nodes carried a high potential for rapid multiple shoot regeneration on medium containing lower concentrations of TDZ (0.05 or 0.23 µM) (Shaik et al., 2009). As multiple shoots originated from the mass of closely placed shoot initials of axillary meristems (Fig. 5), this system could be efficiently used for particle bombardment mediated transformation. This efficient and high frequency *in vitro* regeneration system is highly reproducible and can be used for mass propagation and genetic transformation of *Leucaena*.

#### **4.4 Genetic transformation of** *Leucaena*

The genetic transformation protocols based on *Agrobacterium*-mediated and/or direct gene transfers by biolistic bombardment have been successfully applied to numerous woody angiosperm species (Merkle & Nairn, 2005), including *Populus* and *Betula.* The introduction of transgenes have included both sense and antisense strategies (referring to the orientation of the introduced gene into the plant genome) (Strauss et al*.,* 1995; Baucher et al*.,* 1998) and RNAi technology (Merkle & Nairn, 2005). In the antisense strategy, duplex formation between the antisense transgene and the endogenous gene transcripts is proposed to induce the degradation of duplexes and, correspondingly, lead to suppressed gene expression (Strauss et al*.,* 1995). Regeneration system for *Leucaena* has already been established in our previous works (Shaik et al., 2009; Sirisha et al., 2008). To exploit this to produce transgenic *Leucaena* plants for reduced/altered lignin content, various phenylpropanoid pathway genes (C4H, 4CL, CCoAoMT, CCR, CAld5H, CAD and POX) were cloned and used for transformation experiments*.*

Fig. 5. *In vitro* shoot regeneration in *L. leucocephala* (cultivar K-636). A: Cotyledonary nodes, B-C: Multiple shoot induction in cotyledonary nodes on ½-MS + TDZ (0.23 µM), D: Shortened and fasciated shoots of *Leucaena* on ½-MS + TDZ (0.45 µM), E: Rooted shoots of *Leucaena* on ½-MS + NAA (0.54 µM) and F: Hardened *in vitro* propagated plant of *L. leucocephala* in Sand: soil mixture. (Shaik et al., 2009)

One day old embryo axes without cotyledons were used as explants for transformation. It was carried out by three methods: 1) Particle bombardment; 2) Particle bombardment followed by co-cultivation and 3) Agro-infusion method. The transformation efficiencies with various gene constructs are summarized in Table 3. It can be observed that maximum efficiency (100%) was noted with CAD using particle bombardment followed by cocultivation as a means of transformation. However, CCR with the same procedure gave only 10% efficiency. In general it was concluded that a combination of particle bombardment method followed by co-cultivation was most effective in transforming the shoot meristems of *Leucaena*.


Table 3. Transformation efficiency of *Leucaena* downregulated with phenylpropanoid pathway genes using different methods. PB, Particle Bombardment; CC, Co-Cultivation; AI, Agro Infusion. \*Measured after 3 rounds on selection, #Calculated as per the number of plants screened

#### **4.4.1 Peroxidase (***LlPOX***): A case study**

106 Genetic Engineering – Basics, New Applications and Responsibilities

*Leucaena* plants for reduced/altered lignin content, various phenylpropanoid pathway genes (C4H, 4CL, CCoAoMT, CCR, CAld5H, CAD and POX) were cloned and used for

Fig. 5. *In vitro* shoot regeneration in *L. leucocephala* (cultivar K-636). A: Cotyledonary nodes,

One day old embryo axes without cotyledons were used as explants for transformation. It was carried out by three methods: 1) Particle bombardment; 2) Particle bombardment followed by co-cultivation and 3) Agro-infusion method. The transformation efficiencies with various gene constructs are summarized in Table 3. It can be observed that maximum efficiency (100%) was noted with CAD using particle bombardment followed by cocultivation as a means of transformation. However, CCR with the same procedure gave only 10% efficiency. In general it was concluded that a combination of particle bombardment method followed by co-cultivation was most effective in transforming the shoot meristems

B-C: Multiple shoot induction in cotyledonary nodes on ½-MS + TDZ (0.23 µM), D: Shortened and fasciated shoots of *Leucaena* on ½-MS + TDZ (0.45 µM), E: Rooted shoots of *Leucaena* on ½-MS + NAA (0.54 µM) and F: Hardened *in vitro* propagated plant of *L.* 

*leucocephala* in Sand: soil mixture. (Shaik et al., 2009)

of *Leucaena*.

transformation experiments*.*

Numerous reports on peroxidase activity in lignin forming tissues have been reported, but only a few isoenzymes have been specifically associated with lignification (Sato et al.,1993; Quiroga et al., 2000; Christensen et al., 2001; Marjamaa et al., 2006). It is likely that the control of the whole lignification process requires a mechanism for the co-ordinated expression and/or activation of the monolignol biosynthetic genes/enzymes and the radical forming peroxidases. Data from transgenic plants down regulated for peroxidase activity has confirmed the role of some POX isoforms in lignin polymerization in tobacco and *Populus sieboldii* (Miq.)X *Populus grandidentata* (Michx.) (Talas-Ogras et al., 2001; Blee et al., 2003; Li et al., 2003b). Both quantitative (up to 50% reduction) and qualitative changes were reported, but no obvious growth phenotypes, other than larger xylem elements were found.

Recombinant pCAMBIA1301 binary vector harboring partial sequence of *LlPOX* in antisense orientation was used to transform the embryos isolated from aseptically germinated seeds of *Leucaena* (Fig. 6). Two fragments (one from conserved region and another from nonconserved region) of *LlPOX* cloned in anti-sense orientation were used for transformation. The *Leucaena* embryo axes were bombarded with microcarriers coated with recombinant pCAMBIA vectors using PDS-1000/He Biolistic Particle Delivery System. After growing the embryos on regeneration media without selection for one week, these embryos were subjected to three rounds of selections. The plants, which survived were shifted to ½-MS with Cytokinin, 2-ip (2-isopentenyl adenine) 0.5 mg/ L to enhance elongation of transformed shoots. In all the above cases, the bombarded explants were subjected to transient GUS assay 48 hrs after second bombardment and the putative transgenic plants, which survived three rounds of selection, were analyzed for the gene integration into the plant genome.

As an alternative strategy, *Leucaena* embryo axes were also transformed by a combination of particle bombardment followed by co-cultivation with *Agrobacterium* (GV2260) harboring

Fig. 6. Genetic transformations in *Leucaena leucocephala* by particle bombardment method using *Anti-LlPOX* construct. A: Embryos arranged for bombardment, B-C: Transient GUS expression, D. Bombarded embryos on selection, D1a and D1b. Regenerating embryos on selection, D2a and D2b. Necrosis and dying of untransformed embryos on selection, E-F: Regeneration and hardening of putative transformants

Fig. 7. a. Untransformed plant not subjected to genetic transformation, b. putative transformants using construct Anti*-LlPOX (NC)* of *POX* gene and c. putative transformants using constructs *Anti-LlPOX (C)* of *POX* gene. NC-Non conserved; C-Conserved

respective recombinant pCAMBIA vectors. After particle bombardment, the embryo axes were then transferred onto the respective regeneration medium, co-cultivated in the dark at 25 ± 2 °C for 3 days. After co-cultivation, the embryo axes were washed thoroughly with Cefotaxime 250 mg/ L in sterile distilled water and transferred onto the regeneration medium. Higher levels of transient GUS expression confirmed the transformation efficiency. The present study was performed using, two different antisense constructs of *Leucaena*  peroxidase gene. We observed severe stunted or retarded growth in plants when transformed using constructs having conserved domain. These plants were found to grow barely up to 0.5 cm, soon followed by the death of the apical meristem and rise of a fresh axillary bud from its axis, which again dies and this process was found to be repeating. As a

Fig. 6. Genetic transformations in *Leucaena leucocephala* by particle bombardment method using *Anti-LlPOX* construct. A: Embryos arranged for bombardment, B-C: Transient GUS expression, D. Bombarded embryos on selection, D1a and D1b. Regenerating embryos on selection, D2a and D2b. Necrosis and dying of untransformed embryos on selection, E-F:

Fig. 7. a. Untransformed plant not subjected to genetic transformation, b. putative

using constructs *Anti-LlPOX (C)* of *POX* gene. NC-Non conserved; C-Conserved

transformants using construct Anti*-LlPOX (NC)* of *POX* gene and c. putative transformants

respective recombinant pCAMBIA vectors. After particle bombardment, the embryo axes were then transferred onto the respective regeneration medium, co-cultivated in the dark at 25 ± 2 °C for 3 days. After co-cultivation, the embryo axes were washed thoroughly with Cefotaxime 250 mg/ L in sterile distilled water and transferred onto the regeneration medium. Higher levels of transient GUS expression confirmed the transformation efficiency. The present study was performed using, two different antisense constructs of *Leucaena*  peroxidase gene. We observed severe stunted or retarded growth in plants when transformed using constructs having conserved domain. These plants were found to grow barely up to 0.5 cm, soon followed by the death of the apical meristem and rise of a fresh axillary bud from its axis, which again dies and this process was found to be repeating. As a

Regeneration and hardening of putative transformants

result, the plant attained a height of 2.5 cm on an average and even failed to produce roots when transferred to rooting medium. When non-conserved *AntiPOX* construct was used in *Leucaena* transformation, normal regeneration was noticed but the plants were thin and slow growing compared to the untransformed control plants. Comparative growth pattern of *Leucaena* are shown in Fig. 7.

LlPOX was immuno-cytolocalized in the transformants generated following the above mentioned protocols. Control and transformed plants of same age group were selected. The control plants showed better growth and bio-metric parameters (height, growth and rooting) over the transformants. POX was immuno-cytolocalized in stem tissues of control untransformed plants (Fig. 8 A, B, C) and putative transformants (Fig. 8 D, E, F), with a view to find whether there exists reduction in peroxidase expression in lignifying tissues (*i.e*  vascular bundle and xylem fibres). It was observed that the transformants showed reduced levels of POX near the sites of lignifications. It was also noted that *Leucaena* transformed by *AntiLlPOX* from conserved region resulted in discontinuity in vascular bundle assemblies.

Fig. 8. Immuno-cytolocalization of POX in *Leucaena*. A, B & C stem sections of control plants showing higher levels of POX protein on xylem tissues over the transformed plants D, E & F. Control plants show a well developed vascular bundles (continuous ring) over transformants (discontinuous ring)


Table 4. Lignin estimation of transgenic *Leucaena* plants. NC-Nonconserved; C-conserved

Likewise, rest of the antisense constructs (*4CL*, *CAld5*, *CCR*, *CAD*, and *C4H*) were successfully utilized for genetic transformation of *Leucaena* and were subsequently characterized for transformation efficiency and lignin content (Table 4). Plants having antisense construct of *C4H*, *CCR*, *CAD* and *POX* showing stunted growth. But in case of *4CL* transformants no such morphological appearance were observed.

### **5. Conclusions**

Thanks to years of painstaking research in to the chemistry of lignin, it is now seen as a potential target for genetic engineering of plants, mostly aggravated by its industrial and agricultural applications. However, much of our understanding of lignin biochemistry comes from studies of model plants like *Arabidopsis*, Tobacco, Poplar, etc. Furthermore, this technology needs to be transferred to other plant species. *Leucaena,* a multiple utility leguminous tree, is targeted for ongoing research to alter its lignin content due to its importance in paper and pulp industry in India. Keeping this in mind, attempts were made to improve pulp yielding properties by genetically engineering lignin metabolism so as to gratify the demand of such industries. The results presented here highlight the challenges and limitations of lignin down-regulation approaches: it is essential but difficult to find a level of lignin reduction that is sufficient to be advantageous but not so severe as to affect normal growth and development of plants.

These findings may contribute in the development of *Leucaena* with altered lignin composition/content having higher lignin extractability, making the paper & pulp industry more economic and eco-friendly. The multi-purpose benefits of lignin down regulation in this plant can also be extrapolated to improved saccharification efficiency for biofuel production and forage digestibility, apart from enhanced pulping efficiency. Although genetic engineering promises to increase lignin extraction and degradability during the pulping processes, the potential problems associated with these techniques, like increased pathogen susceptibility, phenotypic abnormalities, undesirable metabolic activities, etc. must be addressed before its large scale application. In order to overcome such barriers, significant progress must be made in understanding lignin metabolism, and its effects on different aspects of plant biology.

Nevertheless, the current genetic engineering technology provides the necessary tools for a comprehensive investigation for understanding lignin chemistry, which were hardly possible using classical breeding methods.

#### **6. Acknowledgements**

Authors would like to thank the research grant funded by Council of Scientific and Industrial Research (CSIR) NIMTLI, India. The project was conceived by SKR and BMK. SS and AKY acknowledges UGC-CSIR; MA, SKG, NMS, PSK, AOU, RKV, SK, SS, RJSK, PS, PP, KC and SA acknowledges CSIR, and SO acknowledges Dept. of Biotechnology (DBT) India for their fellowship grants. Valuable suggestions and feedback provided by Dr. V.S.S. Prasad for preparing this manuscript is duly acknowledged. Authors would also like to thank the Director, National Chemical Laboratory Pune, India.

#### **7. References**

Axegard, P.; Jacobson, B.; Ljunggren, S. & Nilvebrant, N.O. (1992). Bleaching of kraft pulps – A research perspective. *Papier*, 46, V16–V25. ISSN 0031-1340

characterized for transformation efficiency and lignin content (Table 4). Plants having antisense construct of *C4H*, *CCR*, *CAD* and *POX* showing stunted growth. But in case of *4CL*

Thanks to years of painstaking research in to the chemistry of lignin, it is now seen as a potential target for genetic engineering of plants, mostly aggravated by its industrial and agricultural applications. However, much of our understanding of lignin biochemistry comes from studies of model plants like *Arabidopsis*, Tobacco, Poplar, etc. Furthermore, this technology needs to be transferred to other plant species. *Leucaena,* a multiple utility leguminous tree, is targeted for ongoing research to alter its lignin content due to its importance in paper and pulp industry in India. Keeping this in mind, attempts were made to improve pulp yielding properties by genetically engineering lignin metabolism so as to gratify the demand of such industries. The results presented here highlight the challenges and limitations of lignin down-regulation approaches: it is essential but difficult to find a level of lignin reduction that is sufficient to be advantageous but not so severe as to affect

These findings may contribute in the development of *Leucaena* with altered lignin composition/content having higher lignin extractability, making the paper & pulp industry more economic and eco-friendly. The multi-purpose benefits of lignin down regulation in this plant can also be extrapolated to improved saccharification efficiency for biofuel production and forage digestibility, apart from enhanced pulping efficiency. Although genetic engineering promises to increase lignin extraction and degradability during the pulping processes, the potential problems associated with these techniques, like increased pathogen susceptibility, phenotypic abnormalities, undesirable metabolic activities, etc. must be addressed before its large scale application. In order to overcome such barriers, significant progress must be made in understanding lignin metabolism, and its effects on

Nevertheless, the current genetic engineering technology provides the necessary tools for a comprehensive investigation for understanding lignin chemistry, which were hardly

Authors would like to thank the research grant funded by Council of Scientific and Industrial Research (CSIR) NIMTLI, India. The project was conceived by SKR and BMK. SS and AKY acknowledges UGC-CSIR; MA, SKG, NMS, PSK, AOU, RKV, SK, SS, RJSK, PS, PP, KC and SA acknowledges CSIR, and SO acknowledges Dept. of Biotechnology (DBT) India for their fellowship grants. Valuable suggestions and feedback provided by Dr. V.S.S. Prasad for preparing this manuscript is duly acknowledged. Authors would also like to

Axegard, P.; Jacobson, B.; Ljunggren, S. & Nilvebrant, N.O. (1992). Bleaching of kraft pulps –

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## **Genetic Engineering of Plants for Resistance to Viruses**

Richard Mundembe1,2, Richard F. Allison3 and Idah Sithole-Niang1 *1Department of Biochemistry, University of Zimbabwe, Mount Pleasant, Harare, 2Department of Microbiology, School of Molecular and Cell Biology, University of the Witwatersrand, Private Bag 3, Johannesburg, 3Department of Plant Pathology, Michigan State University, East Lansing, 1Zimbabwe 2South Africa 3USA* 

#### **1. Introduction**

120 Genetic Engineering – Basics, New Applications and Responsibilities

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> Genetic engineering has been identified as one key approach to increasing agricultural production and reducing losses due to biotic and abiotic stresses in the field and in storage (Sairam and Prakash, 2005; Yuan *et. al*., 2011). This chapter primarily deals with resistance to viral diseases. It is therefore very important that anyone embarking on a research project to genetically engineer plants fully understands the variety of plant transformation methods that are available, the various forms of (plasmid) constructs that can be used, and their potential implications on the safety of the final product.

> The methods that can be used for plant transformation include *Agrobacterium*-mediated transformation, microprojectile bombardment/ biolistics, direct protoplast transformation, electroporation of cells and tissues, electro-transformation, the pollen tube pathway method, and other methods such as infiltration, microinjection, silicon carbide mediated transformation and liposome mediated transformation (Rakoczy-Trojanowska, 2002). Each of these methods, as will be discussed in this chapter, utilizes a different approach to deliver DNA into the vicinity of chromosomes into which the DNA may then integrate. The markers and reporter genes that may be used in conjunction with the different approaches, and additional sequences meant to facilitate integration may have some biosafety implications.

> The aim of this chapter is to evaluate the different methods that are used for plant transformation, and to discuss specific results obtained after plant transformation for virus resistance using two of the methods: *Agrobacterium*-mediated transformation and electrotransformation. Implications on biosafety will be discussed as well.

#### **2. Plant transformation**

Figure 1 shows the generalized structure of a plant cell. For stable genetic transformation, the desired DNA fragment must be delivered across the cell wall if not removed by pretreatment, the cell membrane, across the cytoplasm, the nuclear membrane into the nucleus. Similarly, for organelle transformation, the DNA must be transported across the organelle membrane to reach the organelle's matrix. Once inside the nucleus, the desired DNA fragment must undergo recombination with the host chromosome so that it becomes integrated into the host chromosome, and its inheritance pattern becomes the same as that of the host chromosome. To date, the mechanisms of integration are not well understood, and there is no targeting of particular chromosomes. Also, a lot still needs to be done in terms of organelle transformation. These topics are reviewed in detail in Tinland 1996; Ow, 2002; Tzfira *et al*., 2004; Maliga 2004 and Kumar *et al*., 2006.

Fig. 1. Diagram to illustrate the structure of a plant cell

Genetic engineering will result in plants that carry additional genes from the same or other species, and are thus referred to as transgenic plants. Such plants may also be referred to as transformed plants, because their genotype and phenotype may have changed from one state to another, for example from disease-susceptible to disease-resistant. The term 'transformed plant' also relates to the original method of *Agrobacterium*-mediated transformation, where, after the bacterium transfers the T-DNA, the recipient plant cells become 'cancerous', and result in cankers that characterize the crown gall disease.

The term 'genetically modified plant' is much broader than 'transformed plant'. While a strict definition of 'plant transformation' may not be practical because of the varying genetics of the plants, it is generally accepted that the plant must be confirmed as transformed based on Southern DNA hybridization evidence of multiple independent transformation events showing different sized fragments correlating to different profiles of

Similarly, for organelle transformation, the DNA must be transported across the organelle membrane to reach the organelle's matrix. Once inside the nucleus, the desired DNA fragment must undergo recombination with the host chromosome so that it becomes integrated into the host chromosome, and its inheritance pattern becomes the same as that of the host chromosome. To date, the mechanisms of integration are not well understood, and there is no targeting of particular chromosomes. Also, a lot still needs to be done in terms of organelle transformation. These topics are reviewed in detail in Tinland 1996; Ow, 2002;

Genetic engineering will result in plants that carry additional genes from the same or other species, and are thus referred to as transgenic plants. Such plants may also be referred to as transformed plants, because their genotype and phenotype may have changed from one state to another, for example from disease-susceptible to disease-resistant. The term 'transformed plant' also relates to the original method of *Agrobacterium*-mediated transformation, where, after the bacterium transfers the T-DNA, the recipient plant cells

Cell wall (cellulose, lignin, pectic substances

Tonoplast (lipid bilayer)

Vacuole

Cell membrane (lipid bilayer)

Nucleus, surrounded by nuclear envelop (double membrane), enclosing nucleoplasm, in which chromosomes

are located

The term 'genetically modified plant' is much broader than 'transformed plant'. While a strict definition of 'plant transformation' may not be practical because of the varying genetics of the plants, it is generally accepted that the plant must be confirmed as transformed based on Southern DNA hybridization evidence of multiple independent transformation events showing different sized fragments correlating to different profiles of

become 'cancerous', and result in cankers that characterize the crown gall disease.

Tzfira *et al*., 2004; Maliga 2004 and Kumar *et al*., 2006.

Cytoplasm, with endoplasmic reticulum, ribosomes etc, in addition to the large membranebound organelles

Chloroplast, a double membrane organelle, enclosing grana

Mitochondrion, a double membrane organelle

Fig. 1. Diagram to illustrate the structure of a plant cell

the restriction endonucleases used, and appropriate sustained phenotypic expression of the transgene exclusively in the transformed plants (Potrykus, 1991, Birch 2002).

In plant pathology, the concept of resistance and susceptibility genes is widespread. In the gene-for-gene model of pathogen incompatibility, resistance (R) genes and associated avirulence (Avr) genes have been well studied (reviewed in Belkhadir *et al*., 2004). But one aspect that has not been well elucidated is the concept of susceptible genes. Very few susceptibility genes have been identified. However one example is the Os8N3, a host disease-susceptibility gene for bacterial blight of rice which is a vascular disease caused by *Xanthomonas oryzae* pv. *oryzae* (Yang *et al*., 2006). Deletion of Os8N3 in rice plants by genetic engineering approaches is postulated to result in genetically engineered plants resistant to *Xanthomonas oryzae* pv. *Oryzae*. One may ask if these plants will be considered transgenic. Most susceptibility genes, however, are thought to be essential for plant growth and development, such that their deletion or mutation will result in non-viable plants.

It must be noted that 'transgenic', 'transformed' and 'genetically modified' are not equivalent terms. The definition of transformed plants should be broad enough to encompass deletions. Southern hybridization probes targeting the deletion junctions may be used to confirm the deletion event, and absence of susceptibility gene product can be demonstrated.

Conventional breeding also results in re-assortment of genes from the two genomes that are crossed, and is therefore some form of genetic modification as well. However, no genetic engineering is involved in the process, and the crosses usually involve closely related species. Genetic engineering is particularly useful when the gene/trait of interest is not present in closely related species, making conventional breeding impossible. Furthermore, conventional breeding is not precise, since extensive re-assortment of genes occurs when two species are crossed, and takes a very long time. Genetic engineering therefore becomes the approach of choice especially when there are no Biosafety issues to grapple with. The most common approach in genetic engineering involves excising the gene of interest using restriction enzymes, and cloning it into a plant transformation vector before transfer into the cells of the target species where the gene will integrate into the chromosome. This process is usually more precise and faster. In this case the resulting plants are transgenic, because they carry a gene from another species, introduced by genetic engineering.

Many transgenic plants resistant to diseases have been produced. Collinge and co-workers list the most common genes used for transgenic disease-resistant crops that have been fieldtested (Collinge *et al*., 2010). Against fungal diseases, these are the polygalacturonse inhibitor protein (grape, raspberry, tomato), proteinase (soybean), R-gene (Rpg-1, Pi9, RB2, Rps1-k) (barley, festuca, potato, soybean), cell death regulator (wheat), toxin detoxifier (barley, wheat) pathogenesis-related proteins (barley, wheat, grape, cotton, peanut, potato, rice, sweet potato, sorghum, tobacco), chitinases (alfalfa, apple, cotton, melon, onion, papaya, squash, carrot, peanut, rice, tobacco, wheat, tomato), oxalate oxidases (bean, cowpea, lettuce, sunflower, peanut, potato, soybean, tobacco), thionin (barley, potato, rice), antimicrobial peptides (cotton, grape, plum, poplar, tobacco, wheat), cecropin (cotton, maize, papaya), stilbene synthase (potato, tobacco), and antimicrobial metabolites (grape, potato, strawberry, tobacco). Against bacterial diseases, attacin (apple), cecropin (apple, papaya, pear, potato, sugarcane), hordothionin (rice, tomato), indolicidin (tobacco), lysozyme (citrus, potato, sugarcane), megainin (grape), proteinase K (rice, tomato), R-gene

of pepper, tomato, rice (tomato), and transcription factors (tomato) have been field-tested. Against plant viruses, single-stranded DNA binding G5 protein (cassava), viral movement proteins (raspberry, tomato), ribonuclease (pea, potato, wheat), replicase (cassava, papaya, potato, tomato), nuclear inclusion protein (melon, potato, squash, wheat), coat protein (alfalfa, barley, beet, grape, lettuce, maize, melon, papaya, pea, peanut, pepper, pineapple, plum, potato, raspberry, soybean, squash, sugarcane, tobacco, tomato, wheat). Virus resistance will be discussed further in section 2.1.

Despite performing well in field tests, most of the transgenic plants have not been commercialized. For instance, coat protein transgenic plants make up three quarters of commercialized virus resistant plants. However, the newer and more sophisticated approaches such as RNA interference are set to become more predominant on the market.

There still remain many challenges to plant transformation. Most methods are not effective for all plant species, but are species- or even cultivar specific. Usually the target for transformation is a small group of cells or an organ, which should then grow and regenerate a whole plant. Regeneration of whole plants *in vitro* is not routine for some agriculturally important species. Thus, there are some very important crops for which no routine, reliable reproducible transformation procedure exists. Therefore the efforts to develop more and better transformation methods continue.

The methods that are available for plant transformation include *Agrobacterium*-mediated transformation, microprojectile bombardment/ biolistics, direct protoplast transformation, electroporation of cells and tissues, electro-transformation, and other methods such as microinjection, silicon carbide mediated transformation and liposome mediated transformation. Each of these methods, as will be discussed in this chapter, utilizes a different approach to deliver DNA into the vicinity of chromosomes into which the DNA may then integrate.

#### **2.1 Plant viruses**

#### **2.1.1 Plant viral diseases**

Biotechnology, through genetic engineering, has the potential to contribute to increased agricultural production by making crops better able to cope with both biotic and abiotic stress. Different research groups are working on different aspects of both biotic and abiotic constraints to increase agricultural production. However, the scope of this chapter will only cover biotic stress and plant viruses in particular. Plant viruses significantly reduce yields in all cultivated crops. By the turn of the millennium, there are as many as 675 plant viruses known and yet annual crop losses due to viruses are valued at US\$60 billion (Fields 1996).

There are various ways of controlling viral diseases such as:


of pepper, tomato, rice (tomato), and transcription factors (tomato) have been field-tested. Against plant viruses, single-stranded DNA binding G5 protein (cassava), viral movement proteins (raspberry, tomato), ribonuclease (pea, potato, wheat), replicase (cassava, papaya, potato, tomato), nuclear inclusion protein (melon, potato, squash, wheat), coat protein (alfalfa, barley, beet, grape, lettuce, maize, melon, papaya, pea, peanut, pepper, pineapple, plum, potato, raspberry, soybean, squash, sugarcane, tobacco, tomato, wheat). Virus

Despite performing well in field tests, most of the transgenic plants have not been commercialized. For instance, coat protein transgenic plants make up three quarters of commercialized virus resistant plants. However, the newer and more sophisticated approaches such as RNA interference are set to become more predominant on the market. There still remain many challenges to plant transformation. Most methods are not effective for all plant species, but are species- or even cultivar specific. Usually the target for transformation is a small group of cells or an organ, which should then grow and regenerate a whole plant. Regeneration of whole plants *in vitro* is not routine for some agriculturally important species. Thus, there are some very important crops for which no routine, reliable reproducible transformation procedure exists. Therefore the efforts to develop more and

The methods that are available for plant transformation include *Agrobacterium*-mediated transformation, microprojectile bombardment/ biolistics, direct protoplast transformation, electroporation of cells and tissues, electro-transformation, and other methods such as microinjection, silicon carbide mediated transformation and liposome mediated transformation. Each of these methods, as will be discussed in this chapter, utilizes a different approach to deliver DNA into the vicinity of chromosomes into which the DNA

Biotechnology, through genetic engineering, has the potential to contribute to increased agricultural production by making crops better able to cope with both biotic and abiotic stress. Different research groups are working on different aspects of both biotic and abiotic constraints to increase agricultural production. However, the scope of this chapter will only cover biotic stress and plant viruses in particular. Plant viruses significantly reduce yields in all cultivated crops. By the turn of the millennium, there are as many as 675 plant viruses known and yet annual crop losses due to viruses are valued at US\$60 billion (Fields 1996).

• The use of disease-free planting material. Virus-free stocks are obtained by virus elimination through heat therapy and/or meristem tissue culture. This approach is effective for seed-borne viruses, but is ineffective for viral diseases transmitted by

• Adopting cultural practices that minimize epidemics, for example by crop rotation, quarantine, rouging diseased plants and using clean implements. Pesticides may also be

There are various ways of controlling viral diseases such as:

resistance will be discussed further in section 2.1.

better transformation methods continue.

may then integrate.

**2.1 Plant viruses** 

vectors.

**2.1.1 Plant viral diseases** 

used to control viral vectors, but the virus may be transmitted to the plant before the vector is killed.


Of all the methods of controlling viral diseases listed above, engineered cross protection seems to have a lot of potential that is only now beginning to be exploited. Before genetic engineering techniques were more widely accepted and applied, natural disease resistance genes bred into target cultivars by classical breeding methods constituted the major focus for introducing disease resistance into plants.

There are 139 monogenic and 40 polygenic virus resistance traits that have been described (Khetapal *et al*., 1998; Hull 2001), but very few have been cloned, and in most cases the mechanism of resistance has not been elucidated (Ellis *et al*., 2000; Dinesh-Kumar *et al*., 2000). Virus-resistant crops that have been obtained by classical breeding include sugarcane resistant to Sugarcane mosaic potyvirus (SCMV) and gerkins (cucumber) resistant to Cucumber mosaic virus (CMV). The N-gene of *Nicotiana glutinosa* that is responsible for the necrotic local lesion reaction of TMV, has also been bred into some *N. tabacum* lines, resulting in the hypersensitive reaction and no systemic infection. Classical breeding has also been used to convey polygenic traits.

#### **2.1.2 Non-viral genes**

One approach to protect plants against a viral infection is by the expression of a single chain variable fragment (scFv) antibody directed against that particular virus (Tavladoraki *et al*., 1993; Voss *et al*., 1995). This has been demonstrated for the icosahedral Artichoke mottle crinkled tombusvirus (AMCV) and the rod-shaped Tobacco mosaic tobamovirus (TMV). However, the resistance obtained this way is not broad-spectrum resistance.

An approach that can yield broad-spectrum resistance to viral diseases is to target the inhibition of production of a product that is essential for the establishment of infection in the cell. An example is S-adenosylhomocysteine hydrolase (SAHH), an enzyme involved in the transmethylation reactions that use S- adenosyl methionine as a methyl donor (Masuta *et al*., 1995). Lowering expression of the enzyme suppresses the 5'-capping of mRNA that is required for efficient translation. Overexpression of cytokinin in crops results in stunting. This phenotype may be due to induction of acquired resistance (Masuta *et al*., 1995).

Expression of the pokeweed (*Phytolacca americana)* antiviral protein (PAP), a ribosome inhibiting protein (RIP), in plants protects the plants against infection by viruses (Ready *et al*., 1986; Lodge *et al*., 1993). In this case, expression of this single gene in the plant results in protection against a wide range of plant viruses.

#### **2.1.3 Pathogen-derived resistance**

#### **Definition**

Pathogen-derived resistance (PDR), also called parasite-derived protection is the resistance conveyed to a host organism as a result of the presence of a transgene of pathogen origin in the target host organism (Sanford & Johnson, 1985). The concept of pathogen-derived resistance predicts that a 'normal' host-pathogen relationship can be disrupted if the host organism expresses essential pathogen-derived genes. The initial hypothesis was that host organisms expressing pathogen gene products at incorrect levels, at the wrong developmental stage or in dysfunctional forms, may disrupt the normal replication cycle of the pathogen and result in an attenuated or aborted infection.

#### **Classical cross protection**

Pathogen derived resistance is an extension of the phenomenon of "cross protection" in which inoculation of a host plant with a milder strain of a pathogen can protect the plant from superinfection by more severe strains of the same or a very closely related pathogen (Wilson 1993). An example of cross protection is in tobacco where infecting tobacco plants with the U1 strain of tobacco mosaic tobamovirus (TMV) protects the plants against future infections with a more virulent strain of TMV.

In practice, the protected plants usually become superinfected, and so the definition given above is not practical. For practical purposes, cross protection is still defined by an earlier definition as "the use of a virus to protect against the economic damage by severe strains of the same virus" (Gonsalves & Garnsey, 1989). Classical cross protection, according to this practical definition, has been evaluated in the field in some countries outside Africa for the control of Citrus tristeza closterovirus (CTV), Papaya ringspot potyvirus (PRSV), Zucchini yellow mosaic potyvirus (ZYMV) and Cucumber mosaic cucumovirus (CMV) (ibid).

#### **Engineered protection**

The genetic engineering approach to cross protection was first demonstrated by Powell-Abel and co-workers who expressed the TMV coat protein gene in transgenic plants and obtained some degree of resistance against TMV (Powell-Abel *et al*., 1986). Many viral genes and gene products have since been shown to be effective in conveying engineered PDR. Engineered PDR can be divided into protein-based PDR (coat protein-, replicase- and movement protein-mediated resistances, using these proteins in their wild type or defective forms) and nucleic acid-based PDR (antisense, sense and satellite RNA-mediated resistances, defective interfering RNA or DNA and antiviral ribozymes).

In general, when classical cross protection is incomplete, smaller lesions than in control nonprotected plants are formed, indicating reduced movement and maybe reduced replication as well. On the other hand, transgenic plants engineered to confer protection to TMV show no reduction in movement or replication. However, the local lesions for PDR against PVX indicate a reduction in virus replication and movement (Hemenway *et al*., 1988). This demonstrates the similarity between classical and engineered protection.

The phenotype of PDR varies from delay in symptom development, through partial inhibition of virus replication, to complete immunity to challenge virus or inoculated viral RNA (Wilson, 1993; Baulcombe, 1996). Even a simple delay in symptom development could be useful if it allows plant biomass, seed or fruit development to outpace disease development.

#### **Coat protein-mediated resistance**

126 Genetic Engineering – Basics, New Applications and Responsibilities

Pathogen-derived resistance (PDR), also called parasite-derived protection is the resistance conveyed to a host organism as a result of the presence of a transgene of pathogen origin in the target host organism (Sanford & Johnson, 1985). The concept of pathogen-derived resistance predicts that a 'normal' host-pathogen relationship can be disrupted if the host organism expresses essential pathogen-derived genes. The initial hypothesis was that host organisms expressing pathogen gene products at incorrect levels, at the wrong developmental stage or in dysfunctional forms, may disrupt the normal replication cycle of

Pathogen derived resistance is an extension of the phenomenon of "cross protection" in which inoculation of a host plant with a milder strain of a pathogen can protect the plant from superinfection by more severe strains of the same or a very closely related pathogen (Wilson 1993). An example of cross protection is in tobacco where infecting tobacco plants with the U1 strain of tobacco mosaic tobamovirus (TMV) protects the plants against future

In practice, the protected plants usually become superinfected, and so the definition given above is not practical. For practical purposes, cross protection is still defined by an earlier definition as "the use of a virus to protect against the economic damage by severe strains of the same virus" (Gonsalves & Garnsey, 1989). Classical cross protection, according to this practical definition, has been evaluated in the field in some countries outside Africa for the control of Citrus tristeza closterovirus (CTV), Papaya ringspot potyvirus (PRSV), Zucchini

The genetic engineering approach to cross protection was first demonstrated by Powell-Abel and co-workers who expressed the TMV coat protein gene in transgenic plants and obtained some degree of resistance against TMV (Powell-Abel *et al*., 1986). Many viral genes and gene products have since been shown to be effective in conveying engineered PDR. Engineered PDR can be divided into protein-based PDR (coat protein-, replicase- and movement protein-mediated resistances, using these proteins in their wild type or defective forms) and nucleic acid-based PDR (antisense, sense and satellite RNA-mediated resistances, defective

In general, when classical cross protection is incomplete, smaller lesions than in control nonprotected plants are formed, indicating reduced movement and maybe reduced replication as well. On the other hand, transgenic plants engineered to confer protection to TMV show no reduction in movement or replication. However, the local lesions for PDR against PVX indicate a reduction in virus replication and movement (Hemenway *et al*., 1988). This

The phenotype of PDR varies from delay in symptom development, through partial inhibition of virus replication, to complete immunity to challenge virus or inoculated viral RNA (Wilson, 1993; Baulcombe, 1996). Even a simple delay in symptom development could

demonstrates the similarity between classical and engineered protection.

yellow mosaic potyvirus (ZYMV) and Cucumber mosaic cucumovirus (CMV) (ibid).

**2.1.3 Pathogen-derived resistance** 

**Classical cross protection** 

**Engineered protection** 

the pathogen and result in an attenuated or aborted infection.

infections with a more virulent strain of TMV.

interfering RNA or DNA and antiviral ribozymes).

**Definition** 

Coat protein-mediated resistance (CP-MR) is the phenomenon by which transgenic plants expressing a plant virus coat protein (CP) gene can resist infection by the same or a homologous virus. The level of protection conferred by CP genes in transgenic plants varies from immunity to delay and attenuation of symptoms. CP-MR has been reported for more than 35 viruses representing more than 15 different taxonomic groups including the tobamo-, potex-, cucumo-, tobra-, carla-, poty-, luteo-, and alfamo- virus groups. The resistance requires that the CP transgene be transcribed and translated. Hemenway and coworkers (1998) have demonstrated direct correlation between CP expression level and the level of resistance obtained. The case of CP-MR to TMV is is important because most of the earlier and more detailed work on CP-MR was done with TMV (Bevan *et al*., 1985; Beachy *et al*., 1986; Powell- Abel *et al*., 1986; Register 1988 and Powell *et al*., 1990).

#### **2.1.4 RNA interference (RNAi)**

RNA interference is the process that depends on small RNAs (sRNAs) to regulate the expression of the eukaryotic genome (Hohn and Vazquez, 2011). This newly elucidated mechanism opens up many possibilities for genetic engineering interventions due to the simplicity of the molecules involved. Small RNAs regulate many biological processes in plants, including maintenance of genome integrity, development, metabolism, abiotic stress responses and immunity to pathogens (Hohn and Vazquez, 2011; Katiya-Agarwal, 2011). The RNA molecules involved are small and of two types, micro RNAs (miRNAs) and small interfering RNAs (siRNAs). miRNAs are transcribed from miRNA genes by RNA polymerase II, as primary miRNA (pri-miRNA) that then folds into a stem loop structure (imperfectly base-paired) that is then processed in a very specific manner by a number of proteins to result in 22-24mer RNA molecules. These RNA molecules are then incorporated into AGO1 or AGO10 and guide the complex to target mRNA for cleavage or translational inhibition on the basis of sequence complementarity. siRNAs on the other hand, are derived from perfectly paired double stranded RNA (dsRNA) precursors, that are derived either from antisense or are a result of RNA-dependent RNA polymerase (RDR) transcription. Details of types of siRNAs, their origins and processing, and how this approach is used to convey virus resistance in transgenic plants are presented in Hohn and Vazquez (2011) and Katiya-Agarwal (2011).

#### **3.** *Agrobacterium***-mediated transformation**

The structure of the Ti plasmid and the requirement for transfer has been established, and the natural host range of the bacterium expanded (Cheng 2004). The first reports of *in vitro* plant transformation utilised the ability of *Agrobacterium tumefaciens* to transfer a specific region of its Ti plasmid DNA into plant cells where they subsequently become integrated into the plant cell genome (Marton *et al*., 1979; Barton *et al*., 1983; Herrera-Estrella *et al*., 1983). This application is based on the observation that in natural diseases of dicotyledonous plants, crown gall disease caused by *Agrobacterium tumefaciens* and hairy root disease caused by *Agrobacterium rhizogenes*, the bacterium transfers part of the DNA of its Ti or Ri plasmid DNA respectively into the host plant where it becomes integrated into the host genome (Herrera-Estrella *et al*., 1983). The plant host cells are referred to as transformed. The transferred DNA is referred to as the T-DNA and is demarcated by conserved left and right border sequences (ibid). The integrated genes are passed on to the progeny of the initially infected cell, and their expression (using the host's transcription and translation machinery) results in the cancerous growth that characterise the crown gall or hairy root diseases that results. The tumours produce specific amino acid derivatives called opines that are utilized by the *Agrobacterium* as a carbon source (Zupan and Zambryski, 1997). Within the T-DNA is a 35 kb virulence (vir) region that includes the genes virA to virR (Zhu *et al*. 2000), flanked by imperfect 25 bp direct repeat sequences known as the left and right borders. A number of virulence genes (chv) located on the *Agrobacterium* chromosome mediate chemotaxis and attachment of the bacterium to the plant cell wall (Zupan & Zambryski, 1997).

In adapting the *Agrobacterium* system to genetic engineering, only the sequences that are essential for transfer and integration into the host genome have been retained, and DNA sequences of interest are inserted into the transferred DNA region. The first generation plasmids for *Agrobacterium*-mediated plant transformation were the disarmed Ti-plasmids. The oncogenes within the left and right borders of the naturally occurring plasmid pTiC58 were replaced with pBR322 sequences, to give pGV3850 (Zambryski *et al*., 1983), and further improved by the addition of a selectable marker (Bevan *et al*., 1983). Use of intermediate vectors enabled use of smaller plasmids with unique cloning sites for initial cloning experiments in *E. coli* (Matzke & Chilton 1981). The intermediate vector could be transferred from *E. coli* to *Agrobacterium* by conjugation, utilizing a helper plasmid, e.g. RK2013, to supply the requirements for conjugation (ibid). Homologous recombination between the intermediate plasmid and a resident disarmed Ti-plasmid of the *Agrobacterium* (e.g. pGV3850) resulted in a larger plasmid known as a cointegrate disarmed Ti-plasmid.

In a different approach, the virulence genes were placed in a separate plasmid such as pAL4404 where these functions would be provided in *trans* for the transfer of DNA on another smaller plasmid with only the left and right borders, markers and other sequences of interest that need to be transferred such as pBin19 in the same *Agrobacterium* cell (Zupan & Zambryski, 1997). This system is known as the binary vector system. The vectors carry a broad host range replication origin, e.g. *ori* V of pBin 19, which allows replication in *E. coli* and *Agrobacterium*. The *A. tumefaciens* is used most extensively in plant transformation because of the belief that the DNA transfer is discreet, with high proportion of integration events with single or low T-DNA copy number, compared to other methods of plant transformation (Zupan & Zambryski, 1997).

Plasmid origin of replication may encourage rearrangements and recombination, leading to silencing and deletion of transgene in subsequent generations. Gene disruption may occur at the site of insertion, resulting in loss of some essential functions (Birch, 1997). It is therefore important to obtain as many transformants as possible so as to be able to disregard all abnormal regenerants resulting from this or other phenomena. T-DNA transfer occurs sequentially but not always completely from the right border to the left border (Wang *et al*., 1984).

Recently, it has also been realized that some sequences outside the borders also get transferred, and integrate into the host genome (Parmyakova *et al*., 2008). This is undesirable in genetically modified plants for commercial release. Current efforts are to reduce or even

DNA respectively into the host plant where it becomes integrated into the host genome (Herrera-Estrella *et al*., 1983). The plant host cells are referred to as transformed. The transferred DNA is referred to as the T-DNA and is demarcated by conserved left and right border sequences (ibid). The integrated genes are passed on to the progeny of the initially infected cell, and their expression (using the host's transcription and translation machinery) results in the cancerous growth that characterise the crown gall or hairy root diseases that results. The tumours produce specific amino acid derivatives called opines that are utilized by the *Agrobacterium* as a carbon source (Zupan and Zambryski, 1997). Within the T-DNA is a 35 kb virulence (vir) region that includes the genes virA to virR (Zhu *et al*. 2000), flanked by imperfect 25 bp direct repeat sequences known as the left and right borders. A number of virulence genes (chv) located on the *Agrobacterium* chromosome mediate chemotaxis and

In adapting the *Agrobacterium* system to genetic engineering, only the sequences that are essential for transfer and integration into the host genome have been retained, and DNA sequences of interest are inserted into the transferred DNA region. The first generation plasmids for *Agrobacterium*-mediated plant transformation were the disarmed Ti-plasmids. The oncogenes within the left and right borders of the naturally occurring plasmid pTiC58 were replaced with pBR322 sequences, to give pGV3850 (Zambryski *et al*., 1983), and further improved by the addition of a selectable marker (Bevan *et al*., 1983). Use of intermediate vectors enabled use of smaller plasmids with unique cloning sites for initial cloning experiments in *E. coli* (Matzke & Chilton 1981). The intermediate vector could be transferred from *E. coli* to *Agrobacterium* by conjugation, utilizing a helper plasmid, e.g. RK2013, to supply the requirements for conjugation (ibid). Homologous recombination between the intermediate plasmid and a resident disarmed Ti-plasmid of the *Agrobacterium* (e.g.

attachment of the bacterium to the plant cell wall (Zupan & Zambryski, 1997).

pGV3850) resulted in a larger plasmid known as a cointegrate disarmed Ti-plasmid.

transformation (Zupan & Zambryski, 1997).

1984).

In a different approach, the virulence genes were placed in a separate plasmid such as pAL4404 where these functions would be provided in *trans* for the transfer of DNA on another smaller plasmid with only the left and right borders, markers and other sequences of interest that need to be transferred such as pBin19 in the same *Agrobacterium* cell (Zupan & Zambryski, 1997). This system is known as the binary vector system. The vectors carry a broad host range replication origin, e.g. *ori* V of pBin 19, which allows replication in *E. coli* and *Agrobacterium*. The *A. tumefaciens* is used most extensively in plant transformation because of the belief that the DNA transfer is discreet, with high proportion of integration events with single or low T-DNA copy number, compared to other methods of plant

Plasmid origin of replication may encourage rearrangements and recombination, leading to silencing and deletion of transgene in subsequent generations. Gene disruption may occur at the site of insertion, resulting in loss of some essential functions (Birch, 1997). It is therefore important to obtain as many transformants as possible so as to be able to disregard all abnormal regenerants resulting from this or other phenomena. T-DNA transfer occurs sequentially but not always completely from the right border to the left border (Wang *et al*.,

Recently, it has also been realized that some sequences outside the borders also get transferred, and integrate into the host genome (Parmyakova *et al*., 2008). This is undesirable in genetically modified plants for commercial release. Current efforts are to reduce or even eliminate these undesirable effects through using special vector constructs that prevent integration of vector sequences. It is thought that integration of sequences outside the borders is a result of erroneous recognition of either right or left border sequences, and Vir D proteins are central to this event. However, the transfer always starts at or adjacent to the left right borders. The reduction can be achieved by using vectors that have positive or negative selection markers, or easily identifiable markers, outside the T-DNA, or using vectors with increased numbers of terminal repeats, or with left terminal repeats surrounded by native DNA regions that serve as termination enhancers, or the so-called 'green vectors' in which the sequences outside the T DNA have been removed (Parmyakova *et al*., 2008) Alternatively, one can use vectors in which the undesirable sequences can be removed by mechanisms such as site-specific recombination, or use vectors with sequences of plant origin only. But there still are problems associated with each approach.

Fig. 2. Illustration of the binary plasmids used for tobacco transformation by *Agrobacterium*mediated transformation

Despite these limitations, *Agrobacterium*-mediated transformation is still a very useful tool in plant molecular virology. In our laboratory, *Agrobacterium*-mediated transformation was used as a tool to evaluate mechanisms of resistance to Cowpea aphid-borne mosaic virus (CABMV) in *Nicotiana benthamiana*, an experimental host of the virus. CABMV is a positive sense RNA virus that is a member of the genus *Potyvirus* (Sithole-Niang *et al*., 1996; Mundembe *et al*., 2009). In an experiment to evaluate the mechanisms of pathogen-derived resistance, *N. benthamiana* was transformed with recombinant pBI 121 carrying various forms of the CABMV coat protein gene, following the method of co-cultivation of leaf explants with *A. tumefaciens* described by An *et al.* (1987). The constructs used were pBI121- CPk which results in an expressed CABMV coat protein, pBI121-PC which results in antisense CP, pBI121-CPstop which results in a form of the CP mRNA that cannot be translated and CPcore which results in only the core region of the CP, together with a pBI121 control.

Evaluation of the responses of transgenic plants obtained indicate that coat proteinmediated resistance only results in delayed symptom development, while RNA mediated approaches may result in recovery or immunity. Out of 68 CP expressing transgenic plants challenged with CABMV, 19 expressed delayed symptom development; and none displayed immunity. Out of 26 CP stop lines, 3 displayed delayed symptom development, 4 tolerance, and 3 recovery phonotypes. Out of 49 antisense lines, 1 displayed delayed symptom delayed symptom development and 3 lines showed modified symptoms.

At the time of carrying out these experiments cowpea could not be transformed in a reliable, reproducible manner, and many research groups were working towards developing a suitable transformation procedure. However, the experiments with transgenic tobacco served the purpose of evaluating the effectiveness of the different approaches. Coat protein mediated resistance would only result in delayed symptom development, RNA mediated approaches are likely to give higher levels of resistance, maybe even immunity.

Therefore, as the method for cowpea transformation become available one would know which particular constructs to use to get the desired levels of resistance.

#### **4. Microprojectile bombardment/ biolistics**

Microprojectile bombardment, also known as biolistics, is the most commonly used method falling into the category of direct gene transfer methods. In direct gene transfer methods a plasmid in which the sequences of interest are cloned is delivered across the various plant cell barriers by physical means to enter the cell where integration into the plant genome may occur. The vectors used in direct plant transformation methods usually include the gene of interest cloned between a promoter and a terminator, and the plasmid components of an origin of replication, an antibiotic resistance gene, a selectable marker for use in plants (e.g. herbicide or antibiotic resistance) or reporter gene (e.g. GUS, luciferase genes). The whole plasmid may be transferred into the plant cell and may be integrated into the plant genome as a whole or as fragments. The barriers to be crossed by the DNA in direct DNA transfer methods are the cell wall and the cell membrane before it can cross the cytoplasm and the nuclear envelop to enter the nucleoplasm where the DNA may integrate into the plant genome (Figure 1). Some direct DNA transfer procedures utilize whole plasmids, supercoiled or linear, which may ultimately integrate as a whole, or at least large parts thereof, including the gene of interest (Smith *et al*., 2001).

Direct gene transfer methods were developed in an effort to transform economically important crops that remained recalcitrant to *Agrobacterium*-mediated transformation because of limitations such as genotype and host cell specificity. Some direct gene transfer methods may also circumvent difficult tissue culture methods.

Mundembe *et al*., 2009). In an experiment to evaluate the mechanisms of pathogen-derived resistance, *N. benthamiana* was transformed with recombinant pBI 121 carrying various forms of the CABMV coat protein gene, following the method of co-cultivation of leaf explants with *A. tumefaciens* described by An *et al.* (1987). The constructs used were pBI121- CPk which results in an expressed CABMV coat protein, pBI121-PC which results in antisense CP, pBI121-CPstop which results in a form of the CP mRNA that cannot be translated and CPcore which results in only the core region of the CP, together with a pBI121

Evaluation of the responses of transgenic plants obtained indicate that coat proteinmediated resistance only results in delayed symptom development, while RNA mediated approaches may result in recovery or immunity. Out of 68 CP expressing transgenic plants challenged with CABMV, 19 expressed delayed symptom development; and none displayed immunity. Out of 26 CP stop lines, 3 displayed delayed symptom development, 4 tolerance, and 3 recovery phonotypes. Out of 49 antisense lines, 1 displayed delayed symptom delayed

At the time of carrying out these experiments cowpea could not be transformed in a reliable, reproducible manner, and many research groups were working towards developing a suitable transformation procedure. However, the experiments with transgenic tobacco served the purpose of evaluating the effectiveness of the different approaches. Coat protein mediated resistance would only result in delayed symptom development, RNA mediated

Therefore, as the method for cowpea transformation become available one would know

Microprojectile bombardment, also known as biolistics, is the most commonly used method falling into the category of direct gene transfer methods. In direct gene transfer methods a plasmid in which the sequences of interest are cloned is delivered across the various plant cell barriers by physical means to enter the cell where integration into the plant genome may occur. The vectors used in direct plant transformation methods usually include the gene of interest cloned between a promoter and a terminator, and the plasmid components of an origin of replication, an antibiotic resistance gene, a selectable marker for use in plants (e.g. herbicide or antibiotic resistance) or reporter gene (e.g. GUS, luciferase genes). The whole plasmid may be transferred into the plant cell and may be integrated into the plant genome as a whole or as fragments. The barriers to be crossed by the DNA in direct DNA transfer methods are the cell wall and the cell membrane before it can cross the cytoplasm and the nuclear envelop to enter the nucleoplasm where the DNA may integrate into the plant genome (Figure 1). Some direct DNA transfer procedures utilize whole plasmids, supercoiled or linear, which may ultimately integrate as a whole, or at least large parts

Direct gene transfer methods were developed in an effort to transform economically important crops that remained recalcitrant to *Agrobacterium*-mediated transformation because of limitations such as genotype and host cell specificity. Some direct gene transfer

approaches are likely to give higher levels of resistance, maybe even immunity.

which particular constructs to use to get the desired levels of resistance.

**4. Microprojectile bombardment/ biolistics** 

thereof, including the gene of interest (Smith *et al*., 2001).

methods may also circumvent difficult tissue culture methods.

symptom development and 3 lines showed modified symptoms.

control.

Sanford and co-workers (1987) were the first to report of plant transformation by microprojectile bombardment. Gold or tungsten particles coated with DNA are propelled at high speed toward the plant tissue where they may penetrate the plant cell walls to introduce the DNA into the cytoplasm, vacuoles, nucleus or other structures of intact cells. A modified bullet gun or electric discharge gun is used to propel the particles (Klein *et al*., 1987; Christou *et al*., 1988). Inside the cell, the DNA may be expressed transiently for two or three days before being degraded, or may become integrated into the nuclear or chloroplast genome, and considered stably integrated if it is passed faithfully to subsequent generations. DNA-coated particles delivered into the nucleus are 45 times more likely to be transiently expressed than those delivered to the cytosol, and 900 times more likely to be expressed than those delivered to the vacuole (Yamashita *et al*., 1991). Efficiency of transformation is influenced by the stage of the cell cycle (Iida *et al*., 1991; Kartzke *et al*., 1990). The DNA is also likely to be expressed if it is delivered to the cell close to the time the nuclear membrane disappears at mitosis (Bower & Birch, 1990; Vasil *et al*., 1991).

Direct DNA transfer methods seem to result in transformants with higher copy numbers than *Agrobacterium*-mediated transformation methods (Hadi *et al*., 1996; Christou *et al*., 1989). The multiple copies may be integrated at the same or tightly linked loci, most likely in relation to replication forks or integration hot spots resulting from initial integration events (Cooley *et al*., 1995, Kohli *et al*., 1998). Increasing the amount of DNA entering the cell in bombardment increases the copy number (Smith *et al*., 2001). The DNA may undergo rearrangements (deletions, direct repetitions, inverted repetitions, ligation, concatamerization) prior to, or during integration (Cooley *et al*., 1995). The site of integration is thought to be random. Ninety percent of T-DNA integrations are into random sites within transcriptionally active regions (Lindsey *et al*., 1993).

Like *Agrobacterium*-mediated transformation, microprojectile bombardment also results in integration of vector sequences if they are part of the DNA molecule bombarded into the plant cell (Kohli *et al*., 1999). However, microprojectile bombardment provides an opportunity for the introduction of minimal gene cassettes into the cells. In this approach, only the required gene expression cassettes (promoter, coding region of interest, terminator) is bombarded into the plant cells, or can be co-transformed together with marker genes to be removed before commercialization (Yao *et al*., 2007; Zhao *et al*., 2007). While the screening and selection might be more difficult, probably depending on detection of the gene sequence or gene product of interest, the approach is very attractive since reporter genes and selection markers are completely avoided (Zhao *et al*., 2007).

Marker genes are unnecessary in established transgenic plants, and also limit options when additional transgenes are to be added (stacking) to the original transgenic line. Herbicide resistance genes may potentially be transferred to weeds by outcrossing. Consumers may also worry about the possibility of antibiotic resistance genes spreading to gut microflora, even though there is no scientific evidence for this.

A variation of the microprojectile bombardment method designed to increase the chances of integration is the **Agrolistic transformation method**. In this method, the transforming plasmid is transferred to the plant cell by a direct mechanism together with a second plasmid coding for *A. tumefaciens* proteins involved in the integration process (Zupan & Zambryski, 1997). Transient expression of the *A. tumefaciens* proteins will direct integration of the plasmid into the plant cell genome. As a result, entry of the plasmid into the cell is by a direct/physical mechanism, but integration into the genome is by a mechanism similar to *Agrobacterium*-mediated transformation. The agrolistic transformation method was expected to address one of the main drawbacks of the microprojectile bombardment method which is that there seem to be a high incidence of high copy number. However, a second drawback that the gene gun accessories are very expensive is still valid.

#### **5. Electroporation and PEG-mediated transformation of protoplasts**

Plant cell walls can be removed by enzymatic degradation to produce protoplasts. Polyethylene glycol (PEG) causes permeabilization of the plasma membrane, allowing the passage of macromolecules into the cell. Pazkowski and co-workers were the first to produce transgenic plants after PEG transformation of protoplasts, and many more monocotyledonous and dicotyledonous species have now been transformed using this method (Pazkowski *et al*. 1984). In electroporation, the protoplasts are subjected to an electric pulse that renders the plasma membrane of the protoplasts permeable to macromolecules. The cell wall and whole plants can be regenerated, if procedures exist.

The transgenic plants generated using these methods seem to have characteristics similar to those of plants derived from all other direct transformation methods. However, it is important to note that carrier DNA (usually ~500 bp fragments of calf thymus DNA) is usually included in the transformation mixture to increase transformation efficiency. This may have some consequences in terms of prevalence of transgene rearrangements and integration of superfluous sequences (Smith *et al*., 2001).

The cell cycle stage of the protoplasts at the time of transformation influence the transgene integration pattern. Non-synchronized protoplasts produce predominantly non-rearranged single copy transgenes in contrast to M phase protoplasts that give multiple copies usually at separate loci (Kartzke *et al*., 1990). The S phase protoplasts give high copy numbers, usually with rearrangements. Irradiation of protoplasts shortly before or after addition of DNA in direct transformation procedures increases both the frequency of transformation and number of integration sites (Koehler *et al*., 1989, 1990, Gharti-Chhertri *et al*., 1990). This is consistent with a mechanism of integration that is partly mediated by DNA repair mechanisms.

The main drawbacks of these methods are that protoplast cultures are not easy to establish and maintain, and regeneration of whole plants from the protoplasts is often unreliable for some important species.

#### **6. Electroporation of intact cells and tissues**

DNA can be introduced into intact cells and tissues in a manner similar to electroporation of protoplasts. Thus pollen, microspores, leaf fragments, embryos, callus, seeds and buds can be used as targets for transformation (Rakoczy-Trojanowska 2002). Protocols for efficient electroporation of cell suspensions of tobacco, rice and wheat (Abdul-Baki, *et al*., 1990; De la Pena, *et al*., 1987; Zaghmout and Trolinder, 1993), and protocols for regeneration of transgenic plants are available. For maize in particular, the transformation efficiencies are comparable to those obtained by bombardment (Dashayes *et al*., 1985; D'Halluin *et al*., 1992).

#### **7. Electro-transformation**

132 Genetic Engineering – Basics, New Applications and Responsibilities

a direct/physical mechanism, but integration into the genome is by a mechanism similar to *Agrobacterium*-mediated transformation. The agrolistic transformation method was expected to address one of the main drawbacks of the microprojectile bombardment method which is that there seem to be a high incidence of high copy number. However, a second drawback

Plant cell walls can be removed by enzymatic degradation to produce protoplasts. Polyethylene glycol (PEG) causes permeabilization of the plasma membrane, allowing the passage of macromolecules into the cell. Pazkowski and co-workers were the first to produce transgenic plants after PEG transformation of protoplasts, and many more monocotyledonous and dicotyledonous species have now been transformed using this method (Pazkowski *et al*. 1984). In electroporation, the protoplasts are subjected to an electric pulse that renders the plasma membrane of the protoplasts permeable to macromolecules. The cell wall and whole plants can be regenerated, if procedures exist.

The transgenic plants generated using these methods seem to have characteristics similar to those of plants derived from all other direct transformation methods. However, it is important to note that carrier DNA (usually ~500 bp fragments of calf thymus DNA) is usually included in the transformation mixture to increase transformation efficiency. This may have some consequences in terms of prevalence of transgene rearrangements and

The cell cycle stage of the protoplasts at the time of transformation influence the transgene integration pattern. Non-synchronized protoplasts produce predominantly non-rearranged single copy transgenes in contrast to M phase protoplasts that give multiple copies usually at separate loci (Kartzke *et al*., 1990). The S phase protoplasts give high copy numbers, usually with rearrangements. Irradiation of protoplasts shortly before or after addition of DNA in direct transformation procedures increases both the frequency of transformation and number of integration sites (Koehler *et al*., 1989, 1990, Gharti-Chhertri *et al*., 1990). This is consistent with a mechanism of integration that is partly mediated by DNA repair

The main drawbacks of these methods are that protoplast cultures are not easy to establish and maintain, and regeneration of whole plants from the protoplasts is often unreliable for

DNA can be introduced into intact cells and tissues in a manner similar to electroporation of protoplasts. Thus pollen, microspores, leaf fragments, embryos, callus, seeds and buds can be used as targets for transformation (Rakoczy-Trojanowska 2002). Protocols for efficient electroporation of cell suspensions of tobacco, rice and wheat (Abdul-Baki, *et al*., 1990; De la Pena, *et al*., 1987; Zaghmout and Trolinder, 1993), and protocols for regeneration of transgenic plants are available. For maize in particular, the transformation efficiencies are comparable to those obtained by bombardment (Dashayes *et al*., 1985;

**5. Electroporation and PEG-mediated transformation of protoplasts** 

that the gene gun accessories are very expensive is still valid.

integration of superfluous sequences (Smith *et al*., 2001).

**6. Electroporation of intact cells and tissues** 

mechanisms.

some important species.

D'Halluin *et al*., 1992).

DNA can also be delivered into cells, tissues and organs by electrophoresis (Ahokas 1989; Griesbach and Hammond, 1994; Songstad *et al*., 1995). This method is known as transformation by electrophoresis or electro-transformation. The tissue to be transformed is placed between the cathode and anode. The anode is placed in a pipette tip containing agarose mixed with the DNA to be used for transformation. The assembly is illustrated in Figure 3.

Fig. 3. Diagrammatic illustration of the electro-transformation equipment and experimental set-up

We used this method of transformation on cowpea seedlings, at a time when there was no efficient, reliable, reproducible method for cowpea transformation. The main obstacles to cowpea transformation were that the tissues into which DNA could be introduced failed to regenerate whole plants. We therefore decided to target apical meristems for transformation. In the event of successful transformation, the seeds from transgenic branches of the cowpea plants would be transgenic, and could be screened for desired transformation events.

We had previously made constructs based on CABMV coat protein gene designed to confer various levels of resistance to the virus in transgenic plants (Figure 2). Circular or linearised binary plasmid constructs were electrophoresed into the apical meristematic region of cowpea seedling of various ages and lengths, untreated or pre-treated with acid or alkali, under various conditions of current and voltage as summarized in Table 1.

#### **7.1 Electrotransformation of cowpea**

Cowpea (*Vigna unguiculata* variety 475/89) seeds were sterilized by shaking in 10% (v/v) bleach for 10 min at room temperature, and washed with double distilled water for 5 min. The seeds were then rolled on a moistened paper towel and placed in a beaker with water and incubated in the growth room at 28C until the seeds germinated (7 – 12 d).

For each transformation attempt, a seedling was removed from the paper towel, pre-treated (where applicable) and placed in the transformation tube. About 1 μl of DNA (0.5μg/μl, circular, or linearized by *Nhe*I or *Nh*eI/*Nde*I digestion) was mixed with about 9 μl of 2% (v/v) low melting point agarose (made up in transformation buffer) and allowed to set at the tip of a 200 μl pipette whose tip had been widened by cutting. Both the pipette tip and the transformation tube (Figure 2) were filled with transformation buffer (0.12 M LiCl, 1 mM Hepes, 0.54 mM MgCl2, 0.005% L-ascorbic acid, pH 7.2). The setup (Figure 3) was connected to a power source and allowed to run under the various current and voltage settings.

The aspects of the seedlings that were noted include the height and age of the plant on the day of manipulation, whether the cotyledons were still attached to the plant or had fallen off, and whether the first true leaves were open or closed. The pretreatments were: none, punched meristem, seedling were exposed to temperatures of 35 °C for 1 hour before manipulation, the manipulations were carried out at increased temperatures of >30 °C, meristems and leaves pretreated with 0.1M HCl, or 0.1 M CaCl2, or 2,4-D + kinetin, NAA + BAP. The voltage settings used were DC or AC, at 30, 40, 125 or 250 V; the current was either 1.0 or 0.15 mA), the duration was kept constant at 15 min. The distance between the electrodes varied with the length of the seedling, and was recorded.


Table 1. below summarizes the potentially transgenic events that were obtained in the experiment

A common feature of the GUS positive plants in Table 1 is that the manipulations were carried out on plants that had straight stems, first true leaves open and cotyledons still attached to the seedling. No pre-treatment other than maybe punching the meristem appear to be necessary. The pre-treatments except punching the meristem do not seem to increase transformation efficiency. Both DC and AC are effective in delivery DNA to the plant cells.

Cowpea (*Vigna unguiculata* variety 475/89) seeds were sterilized by shaking in 10% (v/v) bleach for 10 min at room temperature, and washed with double distilled water for 5 min. The seeds were then rolled on a moistened paper towel and placed in a beaker with water

For each transformation attempt, a seedling was removed from the paper towel, pre-treated (where applicable) and placed in the transformation tube. About 1 μl of DNA (0.5μg/μl, circular, or linearized by *Nhe*I or *Nh*eI/*Nde*I digestion) was mixed with about 9 μl of 2% (v/v) low melting point agarose (made up in transformation buffer) and allowed to set at the tip of a 200 μl pipette whose tip had been widened by cutting. Both the pipette tip and the transformation tube (Figure 2) were filled with transformation buffer (0.12 M LiCl, 1 mM Hepes, 0.54 mM MgCl2, 0.005% L-ascorbic acid, pH 7.2). The setup (Figure 3) was connected

and incubated in the growth room at 28C until the seeds germinated (7 – 12 d).

to a power source and allowed to run under the various current and voltage settings.

electrodes varied with the length of the seedling, and was recorded.

**Age (days)/ Size (cm)** 

7 d 8 cm

8 d 6 cm

3 d 5 cm

8 d 9 cm

Table 1. below summarizes the potentially transgenic events that were obtained in the

A common feature of the GUS positive plants in Table 1 is that the manipulations were carried out on plants that had straight stems, first true leaves open and cotyledons still attached to the seedling. No pre-treatment other than maybe punching the meristem appear to be necessary. The pre-treatments except punching the meristem do not seem to increase transformation efficiency. Both DC and AC are effective in delivery DNA to the plant cells.

**Current/ Time/ Distance between electrodes** 

0.15 V 15 min 7 cm

0.15 V 15 min 1.5 cm

0.15 V 15 min 7 cm

0.15 V 15 min 6 cm

The aspects of the seedlings that were noted include the height and age of the plant on the day of manipulation, whether the cotyledons were still attached to the plant or had fallen off, and whether the first true leaves were open or closed. The pretreatments were: none, punched meristem, seedling were exposed to temperatures of 35 °C for 1 hour before manipulation, the manipulations were carried out at increased temperatures of >30 °C, meristems and leaves pretreated with 0.1M HCl, or 0.1 M CaCl2, or 2,4-D + kinetin, NAA + BAP. The voltage settings used were DC or AC, at 30, 40, 125 or 250 V; the current was either 1.0 or 0.15 mA), the duration was kept constant at 15 min. The distance between the

**Stem First** 

**true leaves** 

Straight Open On No

Straight Open On No

Straight Open On No

Straight Open On Punched

**Cotyledons Notes** 

pretreatment

pretreatment, AC 30 sec

pretreatment

meristem

**7.1 Electrotransformation of cowpea** 

**Plant ID at screening** 

**217** pBI121-

**301** pBI121-

**309** pBI121-

**398** pBI121-

experiment

**DNA construct** 

CPcore, circular

CPk, *Nhe*I linearized

CPk, *Nhe*I linearized

CPk, *Nhe*I linearized The leaves of GUS positive plants had a sectored appearance; this was not unexpected since the transformation procedure targets the general apical meristem area of the cowpea seedling. As a result, both meristematic and somatic cells may become transformed, to result in a chimeric plant. Such a chimeric plant appears as a mosaic of transformed and nontransformed sectors, and poses a challenge in terms of sampling especially in this particular case where a destructive GUS assay was used. Since PCR is very sensitive and amplifies any signal present, the CP transgene could be detected in some GUS positive plants. However, the signal detected by both the GUS assay and PCR could be transient, and Southern analysis is the standard way of determining whether integration has occurred. Southern and other analyses of these lines through subsequent generations, if fertile, would be necessary. There is need to ensure that the germline is transformed to enable the transgene to be passed to subsequent generations.

GUS positive sectors were obtained only from plants that had cotyledons attached, open first true leaves and had developed straight stems at the time of manipulation. The electrotransformation procedure stresses the seedling, and only those seedlings that have developed sufficiently will take up exogenous DNA, survive and develop using the food reserve of cotyledons as well as the photosynthate from first true leaves. The pBI121 binary constructs used in this experiment have a gene for kanamycin resistance.

However, kanamycin resistance is not an effective assay against germinating cowpea seedlings since the germinating cowpea seedlings were not affected by kanamycin. This is probably because of the large food reserves of the seedlings.

The various seedling pre-treatments except punching the meristem did not appear to improve transformation efficiency. Punching the meristem wounds the seedling and may make the meristematic cells more accessible to the exogenous DNA since the epidermal cells will have been removed. Acid and calcium chloride pretreatments were expected to make the cell wall and cell membrane respectively more permeable to DNA. Besides chemically weakening the cell wall, acid pretreatment may also induce the production of expansins that may result in further weakening of cell walls (Cosgrove, 2001). The heat and plant growth substance pretreatments were expected to induce other chemical messengers and heat shock proteins that may increase the chances of integration events in the cell (Hong & Verling, 2001). However, no improvement in transformation efficiency was observed.

The mechanism of DNA integration after uptake by electrophoresis is not known, but is likely to occur by non-homologous recombination into sites on the genome that are undergoing repair or replication, as is the case for other direct DNA transfer methods (Smith *et al*., 2001). Not all GUS-positive lines tested CP-positive possibly because of incomplete transfer. This also means that it is possible that some transformants were GUS-negative but CP-positive, and these would not detected in this screening procedure.

Transformation by electrophoresis, if successful, is a procedure that can be used to avert one of the major concerns of GMOs. The procedure does not necessarily require the use of selectable markers such as antibiotic or herbicide resistance genes, and only the exact sequence required for a particular characteristic in the transgene may be used. It is not understood how integration would occur, but T-DNA borders do not seem to be required. DNA integration by direct transformation methods appears to be random. In this experiment, transformation is not enhanced by pre-treatment with high temperature, hydrochloric acid, calcium chloride, kinetin, BAP or NAA. Both circular and linearised DNA seemed to be effective. However, the seedling must have developed a straight stem with the first true leaves open, but the cotyledons must be intact. This may be important in ensuring survival of the seedling after the rather harsh handling and subjection to electrophoresis that stresses the plant.

#### **8. Other methods of plant transformation**

#### **8.1 Microinjection**

DNA can also be delivered to the plant cell nucleus or cytoplasm by microinjection. This approach is more widely used for large animal cells such as frog egg cells or cells of mammalian embryo. Animal cells are usually immobilised with a holding pipette and gentle suction. For plant cells, the cell wall which contains a thick layer of cellulose and lignins is a barrier to the glass microtools. Removal of the cell wall to form protoplasts might allow use of the microtools, but the plant cells might release hydrolases and other toxic compounds from the vacuole, leading to rapid death of the cells (Lorz *et al*., 1981). Protoplasts may also be attached to glass slides by coating with polyL- lysine, or by or agarose. Poly-L-lysine is toxic to some cells. Agarose reduces visibility around the cells to be manipulated. Microinjection has been used for the transformation of tobacco (Schnorf *et al.,* 1991), petunia (Griesbach, 1987), rape (Neuhaus *et al*., 1987) and barley (Holm *et al*., 2000), with the transgenic plants being recovered at very low frequencies. Microinjection therefore remains of limited use for plant transformation, even though it would be very attractive for introduction of whole chromosomes into plant cells.

#### **8.2 Silicon carbide whisker-mediated transformation**

In this method of plant transformation, silicon carbide crystals (average dimensions of 0.6 μm diameter, 10 – 80 μm long) are mixed with DNA and plant cells by vortexing, enabling the crystals to pierce the cell walls (Kaeppler *et al*., 1990, Songstad *et al*., 1995). The method appears to be widely adaptable, and can be used with as little as 0.1 μg DNA. It appears as if there is a lot of scope for further development of this method of plant transformation (Thompson *et al*., 1995).

The method is simple and easy to adapt to new crops, but the transformation efficiencies are low, and the fibres must be handled with care since they pose a health risk to the experimenter. Success has however been reported with maize (Bullock *et al*., 2001; Frame *et al*., 1994; Kaepler *et al*., 1992; Petolino *et al*., 2000; Wang *et al*., 1995), rice (Nagatani, 1997), wheat (Brisibe, *et al*., 2000; Serik, *et al*., 1996), tobacco (Kaeppler *et al*., 1990), *Lolium multiflorum*, *L. perenne*, *Festuca arundinacea*, and *Agrostis stolonifera* (Dalton *et al*., 1998).

#### **8.3 The pollen tube pathway**

DNA is applied to the cut styles shortly after pollination, and flows down the pollen tube to reach the ovules. This approach has been used to transform rice (Luo an Wa, 1988), wheat (Mu *et al*., 1999), soybean (Hu and Wang 1999), *Petunia hybrida* (Tjokrokusumo *et al*., 2000) and watermelon (Chen *et al*., 1998). Relatively high transformation efficiencies have been reported.

hydrochloric acid, calcium chloride, kinetin, BAP or NAA. Both circular and linearised DNA seemed to be effective. However, the seedling must have developed a straight stem with the first true leaves open, but the cotyledons must be intact. This may be important in ensuring survival of the seedling after the rather harsh handling and subjection to electrophoresis that

DNA can also be delivered to the plant cell nucleus or cytoplasm by microinjection. This approach is more widely used for large animal cells such as frog egg cells or cells of mammalian embryo. Animal cells are usually immobilised with a holding pipette and gentle suction. For plant cells, the cell wall which contains a thick layer of cellulose and lignins is a barrier to the glass microtools. Removal of the cell wall to form protoplasts might allow use of the microtools, but the plant cells might release hydrolases and other toxic compounds from the vacuole, leading to rapid death of the cells (Lorz *et al*., 1981). Protoplasts may also be attached to glass slides by coating with polyL- lysine, or by or agarose. Poly-L-lysine is toxic to some cells. Agarose reduces visibility around the cells to be manipulated. Microinjection has been used for the transformation of tobacco (Schnorf *et al.,* 1991), petunia (Griesbach, 1987), rape (Neuhaus *et al*., 1987) and barley (Holm *et al*., 2000), with the transgenic plants being recovered at very low frequencies. Microinjection therefore remains of limited use for plant transformation, even though it would be very attractive for

In this method of plant transformation, silicon carbide crystals (average dimensions of 0.6 μm diameter, 10 – 80 μm long) are mixed with DNA and plant cells by vortexing, enabling the crystals to pierce the cell walls (Kaeppler *et al*., 1990, Songstad *et al*., 1995). The method appears to be widely adaptable, and can be used with as little as 0.1 μg DNA. It appears as if there is a lot of scope for further development of this method of plant transformation

The method is simple and easy to adapt to new crops, but the transformation efficiencies are low, and the fibres must be handled with care since they pose a health risk to the experimenter. Success has however been reported with maize (Bullock *et al*., 2001; Frame *et al*., 1994; Kaepler *et al*., 1992; Petolino *et al*., 2000; Wang *et al*., 1995), rice (Nagatani, 1997), wheat (Brisibe, *et al*., 2000; Serik, *et al*., 1996), tobacco (Kaeppler *et al*., 1990), *Lolium multiflorum*, *L. perenne*, *Festuca arundinacea*, and *Agrostis stolonifera* (Dalton *et al*., 1998).

DNA is applied to the cut styles shortly after pollination, and flows down the pollen tube to reach the ovules. This approach has been used to transform rice (Luo an Wa, 1988), wheat (Mu *et al*., 1999), soybean (Hu and Wang 1999), *Petunia hybrida* (Tjokrokusumo *et al*., 2000) and watermelon (Chen *et al*., 1998). Relatively high transformation efficiencies have been

stresses the plant.

**8.1 Microinjection** 

(Thompson *et al*., 1995).

**8.3 The pollen tube pathway** 

reported.

**8. Other methods of plant transformation** 

introduction of whole chromosomes into plant cells.

**8.2 Silicon carbide whisker-mediated transformation** 


Table 2. Summary of plant transformation methods

A modification of the procedure is to inject plasmid DNA or *A. tumefaciens* carrying the plasmid DNA into inflorescences in the premeiotic stage, without removing the stigma, as was done for rye (De la Pena *et al*., 1987), to result in high transformation efficiencies.

#### **8.4 Liposome mediated transformation**

Liposomes are microscopic spherical vesicles that form when phospholipids are hydrated. They can be loaded with a variety of molecules, including DNA. Liposomes loaded with DNA can be made to fuse with protoplast membrane and deliver their contents into the cytoplasm by endocytosis. Liposomes can also be carried through the pores of pollen grains to fuse with the membrane of the pollen grain. Transgenic plants have been reported by liposome-mediated transformation only from tobacco (Dekeyser *et al*., 1990) and wheat (Zhu *et al*., 1993). The process is inexpensive, but is laborious and inefficient, and so has not been widely adopted. It might be worthwhile to consider delivering the liposomes through the pollen tube pathway.

#### **8.5 Infiltration**

Infiltration (vacuum infiltration) is a method for plant transformation almost exclusively used for the transformation of *Arabidopsis*. Inflorescences of plants in early generative phase (5 – 15 cm) are immersed in *A. tumefaciens* and 5% sucrose. The inflorescences are then placed under vacuum for several minutes. Typically 0.5 to 4% of the seeds harvested from the inflorescences will be transgenic (Chung *et al*., 2000; Clough *et al*., 1998; Ye *et al*., 1999). This method is highly optimized and works well for *Arabidopsis*.

#### **9. Summary and conclusions**

There now exists a wide variety of methods of plant transformation that can be used to produce virus-resistant plants (Table 2). *Agrobacterium*-mediated transformation and microprojectile bombardment have been used to produce virus resistant plants that have been field-tested, or even been commercialized. These transgenic plants are also important as study material to further understand the methods of plant transformation. However, consumer demands require continuous improvement of these methods, and it is hoped that some of these methods will evolve to become marker-free, vector-free plant transformation methods.

#### **10. Acknowledgements**

We acknowledge The French Ministry of Foreign Affairs for funding the tobacco transformation experiments and The Rockefeller Foundation for funding the cowpea transformation experiments.

#### **11. References**

Abdul-Baki, A.A., Saunders, J.A., Matthews, B.F. and Pittarelli, G.W. (1990). DNA uptake during electroporation of germinating pollen grains. Plant Sci. 70:181-190.

Ahokas, H. (1989). Transfection of germinating barley seed electrophoretically with exogenous DNA. *Theor. Appl. Genet*. 77: 469-472.

A modification of the procedure is to inject plasmid DNA or *A. tumefaciens* carrying the plasmid DNA into inflorescences in the premeiotic stage, without removing the stigma, as

Liposomes are microscopic spherical vesicles that form when phospholipids are hydrated. They can be loaded with a variety of molecules, including DNA. Liposomes loaded with DNA can be made to fuse with protoplast membrane and deliver their contents into the cytoplasm by endocytosis. Liposomes can also be carried through the pores of pollen grains to fuse with the membrane of the pollen grain. Transgenic plants have been reported by liposome-mediated transformation only from tobacco (Dekeyser *et al*., 1990) and wheat (Zhu *et al*., 1993). The process is inexpensive, but is laborious and inefficient, and so has not been widely adopted. It might be worthwhile to consider delivering the liposomes through the

Infiltration (vacuum infiltration) is a method for plant transformation almost exclusively used for the transformation of *Arabidopsis*. Inflorescences of plants in early generative phase (5 – 15 cm) are immersed in *A. tumefaciens* and 5% sucrose. The inflorescences are then placed under vacuum for several minutes. Typically 0.5 to 4% of the seeds harvested from the inflorescences will be transgenic (Chung *et al*., 2000; Clough *et al*., 1998; Ye *et al*., 1999).

There now exists a wide variety of methods of plant transformation that can be used to produce virus-resistant plants (Table 2). *Agrobacterium*-mediated transformation and microprojectile bombardment have been used to produce virus resistant plants that have been field-tested, or even been commercialized. These transgenic plants are also important as study material to further understand the methods of plant transformation. However, consumer demands require continuous improvement of these methods, and it is hoped that some of these methods will evolve to become marker-free, vector-free plant transformation

We acknowledge The French Ministry of Foreign Affairs for funding the tobacco transformation experiments and The Rockefeller Foundation for funding the cowpea

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This method is highly optimized and works well for *Arabidopsis*.

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**8.4 Liposome mediated transformation** 

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**9. Summary and conclusions** 

**10. Acknowledgements** 

transformation experiments.

**11. References** 

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## **Strategies for Improvement of Soybean Regeneration via Somatic Embryogenesis and Genetic Transformation**

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#### **1. Introduction**

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mediated by cationic liposome and regeneration of transgenic plantlets. *Chin. J.* 

The seed, which contains the embryo, is the primary entity of reproduction in angiosperms. In flowering plants, as in other eukaryotes, the embryo develops from the zygote formed by gametic fusion. However, during the course of evolution many plant species have evolved different methods of asexual embryogenesis to overcome various environmental and genetic factors that prevent fertilization (Sharma & Thorpe, 1995; Raghavan, 1997).

Somatic embryogenesis (SE), starting from somatic or gametic (microspore) cells without fusion of gametes (Williams & Maheswaran, 1986), is one form of asexual reproduction. This process occurs either naturally or *in vitro* after experimental induction (Dodemam et al., 1997), and is a remarkable phenomenon unique to plants. The process is feasible because plants possess cellular totipotency, whereby individual somatic cells can regenerate into a whole plant (Reinert, 1959).

SE has been observed in tissue cultures of several angiosperm and gymnosperm plant species, and involves a series of morphological changes that are similar, in several aspects, to those associated to the development of zygotic embryos. In soybean (*Glycine max* (L.) Merrill), histological sections of embryogenic structures can be found in some reports (Barwale et al., 1986; Finer & McMullen, 1991; Kiss et al., 1991; Liu et al., 1992; Sato et al., 1993). A characterization of the developmental stages of soybean somatic embryos was performed by Christou & Yang (1989), Fernando et al. (2002), Rodrigues et al. (2005), and Santos et al. (2006). The pro-embryo, globular, heart-shaped, torpedo and cotyledonary embryo stages were found, closely resembling the ontogeny of zygotic embryos. However, the absence of a characteristic suspensor, as well as the delay in the establishment of inner organization were the main differences between zygotic and somatic embryogenic processes (Santos et al., 2006).

### **2. Soybean somatic embryogenic process**

In general, the *in vitro* soybean somatic embryogenic process can be divided into different phases: induction, proliferation, histodifferentiation, maturation, germination and conversion into plants.

#### **2.1 Somatic embryo induction**

According to Sharp et al. (1982), the induction of somatic embryogenesis (Fig. 1 A1, B1, C1) can be considered as termination of the existing gene expression pattern in the explant tissue, and its replacement for an embryogenic gene expression program in those cells of the explant tissue which will give rise to somatic embryos. These authors used the term "induced embryogenic determined cell" (IEDC) to describe an embryogenic cell that has been originated from a non-embryogenic cell. Cells from very immature zygotic embryos, which already have their embryogenic gene expression program activated, were termed "pre-embryogenic determined cells" (PEDCs). For the purposes of regeneration, both terms may be referred to simply as "embryogenic cells" (ECs) (Carman, 1990; Merkle et al., 1995).

There is a major developmental difference among explants with respect to the ontogeny of somatic embryos. The obtainment of somatic embryogenesis in legumes depends on whether the explant tissue consists of PEDCs (for example, very immature zygotic embryos) or non-ECs (for example, differentiated plant tissues). In the first case, a stimulus to the explant may be sufficient to induce cell division for the formation of somatic embryos, which appear to arise directly from the explant tissue in a process referred to as **direct embryogenesis** (Fig. 1 A1, B1). In contrast, non-EC tissue must undergo several mitotic divisions in the presence of an exogenous auxin for induction of the ECs. Cells resulting from these mitotic divisions are manifested as a callus, and the term **indirect embryogenesis** is used to indicate that a callus phase intervenes between the original explant and the appearance of somatic embryos (Fig. 1 C1) (Merkle et al., 1995).

Thus, the somatic embryo induction process can be achieved using different approaches, as illustrated in Figure 1. Somatic embryos induced from very immature zygotic embryos (torpedo-stage) upon exposure to cytokinins were only obtained in clovers (*Trifolium* ssp.) (Maheswaran & Williams, 1984) (Fig. 1 A). In soybean, somatic embryos can be induced in response to auxins, and regenerated directly from cotyledonary-stage zygotic immature embryos without an intervening callus phase (Lazzeri et al., 1985; Finer, 1988; Bailey et al., 1993; Santarém et al., 1997) (Fig. 1 B). Finally, some legumes, notably alfalfa (*Medicago sativa*), can be regenerated from leaf-derived callus (Bingham et al., 1988). In this case, the tissue responds to combinations of auxins and cytokinins (Fig. 1 C).

The type of growth regulator and explant, as well as genotype ability to respond to *in vitro* stimulus, are the main factors affecting somatic embryogenesis induction. The role of exogenous cytokinins during the induction phase depends on whether somatic embryogenesis is direct or indirect. When SE is originated from callus, the frequency of somatic embryo formation is enhanced by cytokinins. However, in direct systems, such as in soybean, in which somatic embryos are formed directly from immature zygotic embryos, addition of a cytokinin reduces the frequency of embryo formation (Merkle et al., 1995). Soybean SE is induced by two auxins: α-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D), but the most commonly used is 2,4-D. The exact

In general, the *in vitro* soybean somatic embryogenic process can be divided into different phases: induction, proliferation, histodifferentiation, maturation, germination and conversion

According to Sharp et al. (1982), the induction of somatic embryogenesis (Fig. 1 A1, B1, C1) can be considered as termination of the existing gene expression pattern in the explant tissue, and its replacement for an embryogenic gene expression program in those cells of the explant tissue which will give rise to somatic embryos. These authors used the term "induced embryogenic determined cell" (IEDC) to describe an embryogenic cell that has been originated from a non-embryogenic cell. Cells from very immature zygotic embryos, which already have their embryogenic gene expression program activated, were termed "pre-embryogenic determined cells" (PEDCs). For the purposes of regeneration, both terms may be referred to simply as "embryogenic cells" (ECs) (Carman, 1990; Merkle et al., 1995). There is a major developmental difference among explants with respect to the ontogeny of somatic embryos. The obtainment of somatic embryogenesis in legumes depends on whether the explant tissue consists of PEDCs (for example, very immature zygotic embryos) or non-ECs (for example, differentiated plant tissues). In the first case, a stimulus to the explant may be sufficient to induce cell division for the formation of somatic embryos, which appear to arise directly from the explant tissue in a process referred to as **direct embryogenesis** (Fig. 1 A1, B1). In contrast, non-EC tissue must undergo several mitotic divisions in the presence of an exogenous auxin for induction of the ECs. Cells resulting from these mitotic divisions are manifested as a callus, and the term **indirect embryogenesis** is used to indicate that a callus phase intervenes between the original explant and the

Thus, the somatic embryo induction process can be achieved using different approaches, as illustrated in Figure 1. Somatic embryos induced from very immature zygotic embryos (torpedo-stage) upon exposure to cytokinins were only obtained in clovers (*Trifolium* ssp.) (Maheswaran & Williams, 1984) (Fig. 1 A). In soybean, somatic embryos can be induced in response to auxins, and regenerated directly from cotyledonary-stage zygotic immature embryos without an intervening callus phase (Lazzeri et al., 1985; Finer, 1988; Bailey et al., 1993; Santarém et al., 1997) (Fig. 1 B). Finally, some legumes, notably alfalfa (*Medicago sativa*), can be regenerated from leaf-derived callus (Bingham et al., 1988). In this case, the tissue

The type of growth regulator and explant, as well as genotype ability to respond to *in vitro* stimulus, are the main factors affecting somatic embryogenesis induction. The role of exogenous cytokinins during the induction phase depends on whether somatic embryogenesis is direct or indirect. When SE is originated from callus, the frequency of somatic embryo formation is enhanced by cytokinins. However, in direct systems, such as in soybean, in which somatic embryos are formed directly from immature zygotic embryos, addition of a cytokinin reduces the frequency of embryo formation (Merkle et al., 1995). Soybean SE is induced by two auxins: α-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D), but the most commonly used is 2,4-D. The exact

**2. Soybean somatic embryogenic process** 

appearance of somatic embryos (Fig. 1 C1) (Merkle et al., 1995).

responds to combinations of auxins and cytokinins (Fig. 1 C).

into plants.

**2.1 Somatic embryo induction** 

mechanism underlying the auxin-induced somatic embryo formation is not understood, but some studies with other legumes suggested certain auxin-induced cellular processes such as embryo-specific DNA methylation (Vergara et al., 1990), disruption of tissue integrity by interrupting cell–cell interaction (Smith & Krikorian, 1989) and establishment of cell polarity (Merkle et al., 1995). However, auxins are not the only substances able to induce embryogenesis. Several other factors that alter gene expression programs (e.g., stress) or disrupt cell-cell interaction (physical disruption of the tissue) can also direct this transition (Gharyal & Maheshwari, 1983; Dhanalakshmi & Lakshmanan, 1992).

Fig. 1. Embryogenic processes in legumes. Somatic embryos may be induced (1), histodifferentiated/matured (3), desiccated (4), germinated (5), and converted into plants (6). Alternatively, auxin can be used to maintain repetitive embryogenesis – embryo proliferation (2), which continues until auxin is withdrawn from the medium, allowing somatic embryos to resume their development. (A) The youngest zygotic embryos respond to cytokinin; (B) older zygotic embryos respond to auxin, and (C) differentiated plant tissues respond to combination of auxin and cytokinins by forming callus. (Adapted from Parrott et al., 1995. Drawing by S. N. C. Richter)

The choice of explant is a critical factor that determines the success of most tissue culture experiments. Immature, meristematic tissues proved to be the most suitable explant for somatic embryogenesis in legumes (Lakshmanan & Taji, 2000). For instance, cotyledons of immature zygotic embryos have been the most used explants for the induction of SE in soybean (Lazzeri et al., 1985; Finer, 1988; Bailey et al., 1993; Santarém et al., 1997; Droste et al., 2002). However, in this species, somatic embryos have also been obtained from leaf and stem (Ghazi et al., 1986), cotyledonary node (Kerns et al., 1986), anther (Santos et al., 1997; Rodrigues et al., 2005) and embryonic axes (Kumari et al., 2006).

The last but not least important factor affecting somatic embryo induction is plant genotype (Merkle et al., 1995). In soybean, considerable variation in embryogenic capacity was found to exist between individual genotypes (Komatsuda et al., 1991; Bailey et al., 1993a, b; Santos et al., 1997; Droste et al., 2001; Meurer et al., 2001; Tomlin et al., 2002; Hiraga et al., 2007; Yang et al., 2009; Droste et al., 2010) as will be discussed below (Genotype-dependent response and screening of highly responsive cultivars section).

#### **2.2 Embryo proliferation**

A common characteristic of embryogenic tissue is that it can remain embryogenic indefinitely. This proliferative process has been variously termed secondary, recurrent or repetitive embryogenesis (Fig. 1 B2). In soybean, the primary somatic embryos can have multicellular origins, while secondary somatic embryos (i.e. originating from another somatic embryo) tend to have unicellular origins (Merkle et al., 1995). Hartweck et al. (1988) found somatic embryos originating from groups of cells in soybean zygotic cotyledons, while Sato et al. (1993) found embryos proliferating from globular-stage soybean somatic embryos that originate from single cells.

Proliferation of embryogenic cells is apparently influenced by a variety of factors, some of which are controlled during the culture process, and some of which are yet undefined. Some of the factors that have been investigated are also associated with induction phase, such as plant genotype and growth regulators (Merkle et al., 1995).

The most broadly documented factor associated with continuous proliferation of embryogenic cells is auxin. For soybean, secondary somatic embryo proliferation is possible if it is maintained in a medium containing the auxin 2,4-D (Finer & Nagasawa, 1988). Single epidermal cells have been shown to initiate soybean secondary somatic embryos (Sato et al., 1993). The exact role of auxin in triggering proliferation is unknown. Furthermore, the level of auxin required to maintain repetitive embryogenesis depends on the culture protocol adopted.

#### **2.3 Embryo histodifferentiation and maturation**

After induction, somatic embryos start an ontogenetic development process similar to that of their zygotic counterparts (Merkle et al., 1995). The process of organ formation through which a globular-stage embryo develops into a cotyledon-stage embryo has been termed histodifferentiation (Fig. 1 B3) (Carman, 1990).

In general, continued embryo histodifferentiation beyond the globular stage and subsequent maturation requires the removal of growth regulators from the medium - or at least a

The choice of explant is a critical factor that determines the success of most tissue culture experiments. Immature, meristematic tissues proved to be the most suitable explant for somatic embryogenesis in legumes (Lakshmanan & Taji, 2000). For instance, cotyledons of immature zygotic embryos have been the most used explants for the induction of SE in soybean (Lazzeri et al., 1985; Finer, 1988; Bailey et al., 1993; Santarém et al., 1997; Droste et al., 2002). However, in this species, somatic embryos have also been obtained from leaf and stem (Ghazi et al., 1986), cotyledonary node (Kerns et al., 1986), anther (Santos et al., 1997;

The last but not least important factor affecting somatic embryo induction is plant genotype (Merkle et al., 1995). In soybean, considerable variation in embryogenic capacity was found to exist between individual genotypes (Komatsuda et al., 1991; Bailey et al., 1993a, b; Santos et al., 1997; Droste et al., 2001; Meurer et al., 2001; Tomlin et al., 2002; Hiraga et al., 2007; Yang et al., 2009; Droste et al., 2010) as will be discussed below (Genotype-dependent

A common characteristic of embryogenic tissue is that it can remain embryogenic indefinitely. This proliferative process has been variously termed secondary, recurrent or repetitive embryogenesis (Fig. 1 B2). In soybean, the primary somatic embryos can have multicellular origins, while secondary somatic embryos (i.e. originating from another somatic embryo) tend to have unicellular origins (Merkle et al., 1995). Hartweck et al. (1988) found somatic embryos originating from groups of cells in soybean zygotic cotyledons, while Sato et al. (1993) found embryos proliferating from globular-stage soybean somatic

Proliferation of embryogenic cells is apparently influenced by a variety of factors, some of which are controlled during the culture process, and some of which are yet undefined. Some of the factors that have been investigated are also associated with induction phase, such as

The most broadly documented factor associated with continuous proliferation of embryogenic cells is auxin. For soybean, secondary somatic embryo proliferation is possible if it is maintained in a medium containing the auxin 2,4-D (Finer & Nagasawa, 1988). Single epidermal cells have been shown to initiate soybean secondary somatic embryos (Sato et al., 1993). The exact role of auxin in triggering proliferation is unknown. Furthermore, the level of auxin required to maintain repetitive embryogenesis depends on the culture protocol

After induction, somatic embryos start an ontogenetic development process similar to that of their zygotic counterparts (Merkle et al., 1995). The process of organ formation through which a globular-stage embryo develops into a cotyledon-stage embryo has been termed

In general, continued embryo histodifferentiation beyond the globular stage and subsequent maturation requires the removal of growth regulators from the medium - or at least a

Rodrigues et al., 2005) and embryonic axes (Kumari et al., 2006).

response and screening of highly responsive cultivars section).

plant genotype and growth regulators (Merkle et al., 1995).

**2.3 Embryo histodifferentiation and maturation** 

histodifferentiation (Fig. 1 B3) (Carman, 1990).

**2.2 Embryo proliferation** 

adopted.

embryos that originate from single cells.

decrease in growth regulator concentrations associated with induction and proliferation down to levels that enable proper embryo development (Merkle et al., 1995). Auxins suppress the development of the apical meristem, probably governed by the same mechanism involved in the establishment of apical dominance (Parrott et al., 1995).

The role of cytokinins during the histodifferentiation is more difficult to assess. Aung et al. (1982) observed a decrease in endogenous cytokinin levels during soybean zygotic embryo development. On the other hand, the inclusion of a cytokinin during the histodifferentiation phase can compensate for auxin-induced detrimental effects on meristem development. Globular-stage soybean somatic embryos, when exposed to a cytokinin, decrease the development rates while the apical meristem elongates so as to form multiple shoots (Wright et al., 1991).

Due to the fact that traditional embryogenesis protocols have typically used the same medium for histodifferentiation and the subsequent maturation phases, it is difficult to review the literature and determine if a given treatment affects the histodifferentiation stage or the maturation stage (Merkle et al., 1995).

Following histodifferentiation, the period of embryo development in which cell expansion and reserve deposition occur is considered the maturation phase (Fig. 1 B3) (Bewley et al., 1985). The time required for somatic embryos to achieve physiological maturity is speciesspecific, mirroring the maturation period of zygotic embryos *in planta* (Parrott et al., 1995). Analyses of reserve accumulations in developing somatic embryos have revealed both similarities and differences in comparison to zygotic embryos. These differences may primarily be attributed to the *in vitro* maturation conditions used. Many reports on somatic embryos reserve accumulations have not been carried out with optimal maturation protocols. Hence, manipulation of culture conditions to prolong and improve embryo maturation, and to prevent precocious germination, will probably add to the similarities observed between zygotic and somatic embryos (Merkle et al., 1995).

Currently, a trend is observed in the specification of numerous protocols towards providing various growth regulators in the medium during embryo maturation. Yet, there is ample information suggesting that neither exogenous auxin nor cytokinin is actually required for normal embryo maturation, as evidenced by the normal development of soybean zygotic (Hsu & Obendorf, 1982) or somatic embryos (Parrott et al., 1988) in media devoid of any growth regulators. Indeed, poorly developed meristem or swollen hypocotyls may be an undesired outcome of the application of exogenous auxins and cytokinins, respectively. Consequently, a treatment that binds and removes auxin, by adding activated charcoal to the maturation medium (Ebert & Taylor, 1990), for instance, may improve embryo development and enhance germination (Buchheim et al., 1989). Differently from auxins and cytokinins, abscisic acid (ABA) may be necessary during embryogenesis to initiate the synthesis of storage proteins and proteins involved in desiccation tolerance (Galau et al., 1990).

#### **2.4 Embryo germination and conversion into plants**

Embryos removed from maturation conditions for further development often display poor or aberrant subsequent germination, growth and vigor (Parrott et al., 1988). These observations suggest that further post-maturation treatments are required. One of the fundamental aspects of zygotic embryo development not normally encountered during somatic embryo development is desiccation (Fig. 1 B4), which leads to embryo quiescence. It has been proposed that desiccation is required for the correct transition from an embryo maturation program to a germination program (Kermode, 1990). The desiccation period has been linked with the synthesis of proteins associated with the ability to germinate (Rosenberg & Rinne, 1986, 1988). Partial desiccation has been shown to enhance conversion of soybean somatic embryos (Hammatt & Davey, 1987; Parrott et al., 1988; Buchheim et al., 1989).

While most studies report the development of roots and the germination of somatic embryos, little distinction is made between germination and conversion. According to Ranch et al. (1985), soybean germination refers to both root and shoot development in an embryo with intact hypocotyl (Fig. 1 B5). Walker & Parrott (2001) described soybean apparent conversion as the development of expanded trifoliolates and branched roots under *in vitro* conditions (Fig. 1 B6). However, according to the authors, the definition of actual conversion refers to survival following transfer to soil.

While germination capacity may be affected by several culture medium components or environmental manipulations, one aspect that cannot be manipulated so readily is the genetic background of the embryo (Merkle et al., 1995). Studies with soybean plants indicated that germination/conversion capacity is also greatly influenced by genotype (Komatsuda & Ohyama, 1988; Bailey et al., 1993; Santos et al., 1997; Droste et al., 2001, 2010).

#### **3. Genotype-dependent response and screening of highly responsive cultivars**

The more closely the pattern of somatic embryo gene expression matches that of zygotic embryos, the greater the chance of obtaining highly efficient regeneration systems. Such normalization of gene expression patterns will be achieved through the optimization of media and culture protocols for each individual stage of embryo development. According to Meurer et al*.* (2001), there are two ways to optimize soybean regeneration via embryogenic cultures. The first is screening a large number of new cultivars in order to identify those with embryogenic potential. Since SE is a heritable trait (Parrott et al., 1989), the potential for embryogenesis can be also improved through conventional crossing between nonresponsive cultivars and highly competent cultivars (Kita et al., 2007). An alternative and additive approach is to optimize embryogenesis protocols for improve results of an interesting cultivar.

Several studies have revealed the differences among soybean genotypes in their capacity to respond to the different steps of somatic embryogenesis (Bailey et al., 1993; Santos et al., 1997; Simmonds & Donaldson, 2000; Droste et al., 2001; Meurer et al., 2001; Tomlin et al., 2002; Hiraga et al., 2007; Yang et al., 2009; Droste et al., 2010). The efficiencies were shown to be different among cultivars at each SE phase: induction, proliferation, histodifferentiation/maturation, germination and conversion (Bailey et al., 1993; Santos et al., 1997). As each phase must be under independent genetic control, the best cultivar performance in one stage not necessarily is the best in another one. In this context, Bailey et al. (1993a) reported that PI 417138 was among the least inducible genotypes studied, though it did have the highest germination and conversion capacity. In the same way, IAS-5 cultivar

fundamental aspects of zygotic embryo development not normally encountered during somatic embryo development is desiccation (Fig. 1 B4), which leads to embryo quiescence. It has been proposed that desiccation is required for the correct transition from an embryo maturation program to a germination program (Kermode, 1990). The desiccation period has been linked with the synthesis of proteins associated with the ability to germinate (Rosenberg & Rinne, 1986, 1988). Partial desiccation has been shown to enhance conversion of soybean somatic embryos (Hammatt & Davey, 1987; Parrott et al., 1988; Buchheim et al.,

While most studies report the development of roots and the germination of somatic embryos, little distinction is made between germination and conversion. According to Ranch et al. (1985), soybean germination refers to both root and shoot development in an embryo with intact hypocotyl (Fig. 1 B5). Walker & Parrott (2001) described soybean apparent conversion as the development of expanded trifoliolates and branched roots under *in vitro* conditions (Fig. 1 B6). However, according to the authors, the definition of actual

While germination capacity may be affected by several culture medium components or environmental manipulations, one aspect that cannot be manipulated so readily is the genetic background of the embryo (Merkle et al., 1995). Studies with soybean plants indicated that germination/conversion capacity is also greatly influenced by genotype (Komatsuda & Ohyama, 1988; Bailey et al., 1993; Santos et al., 1997; Droste et al., 2001, 2010).

The more closely the pattern of somatic embryo gene expression matches that of zygotic embryos, the greater the chance of obtaining highly efficient regeneration systems. Such normalization of gene expression patterns will be achieved through the optimization of media and culture protocols for each individual stage of embryo development. According to Meurer et al*.* (2001), there are two ways to optimize soybean regeneration via embryogenic cultures. The first is screening a large number of new cultivars in order to identify those with embryogenic potential. Since SE is a heritable trait (Parrott et al., 1989), the potential for embryogenesis can be also improved through conventional crossing between nonresponsive cultivars and highly competent cultivars (Kita et al., 2007). An alternative and additive approach is to optimize embryogenesis protocols for improve results of an

Several studies have revealed the differences among soybean genotypes in their capacity to respond to the different steps of somatic embryogenesis (Bailey et al., 1993; Santos et al., 1997; Simmonds & Donaldson, 2000; Droste et al., 2001; Meurer et al., 2001; Tomlin et al., 2002; Hiraga et al., 2007; Yang et al., 2009; Droste et al., 2010). The efficiencies were shown to be different among cultivars at each SE phase: induction, proliferation, histodifferentiation/maturation, germination and conversion (Bailey et al., 1993; Santos et al., 1997). As each phase must be under independent genetic control, the best cultivar performance in one stage not necessarily is the best in another one. In this context, Bailey et al. (1993a) reported that PI 417138 was among the least inducible genotypes studied, though it did have the highest germination and conversion capacity. In the same way, IAS-5 cultivar

**3. Genotype-dependent response and screening of highly responsive** 

conversion refers to survival following transfer to soil.

1989).

**cultivars** 

interesting cultivar.

had the lowest embryo yield but the highest conversion capacity (Santos et al., 1997). Tomlin et al. (2002) and Hiraga et al. (2007) suggested that differences in somatic embryo induction efficiency among soybean cultivars are likely to be attributed to differences in endogenous auxin levels or in the auxin sensitivity.

One of the major challenges in soybean is to identify genotypes highly responsive to SE induction, embryo proliferation and conversion into plants. A direct comparison among previous reports is difficult because each research group has adopted different protocols for SE and evaluation methods. In some studies only the first phases such as induction and/or proliferation were analyzed, while in other studies genotypes were screened upon plant regeneration.

Simmonds & Donaldson (2002) screened 18 of 20 short-season soybean genotypes in Canada for proliferative embryogenic capacity. Only five genotypes produced embryogenic cultures which were proliferative for at least six months. Nine soybean cultivars representing different US growing regions were evaluated at each of three locations using uniform embryogenic induction and proliferation protocols. Several cultivars were identified as uniformly embryogenic at the primary induction phase at all locations, among which Jack was the best (Meurer et al., 2001).

Twenty-six genotypes including soybean wild relatives and Japanese cultivars were screened for differences of competence in both somatic embryogenesis and subsequent shoot formation. All genotypes were able to induce somatic embryos but with wide variation. *Glycine gracilis*, *G. gracilis* T34, Masshokutou (kou 502) and Masshokutou (kou 503) varieties presented high competence of germination from somatic embryos (Komatsuda & Ohyama, 1988). Hiraga et al. (2007) examined the capacity for plant regeneration through somatic embryogenesis in Japanese soybean cultivars. Induction of somatic embryos from immature cotyledons, embryo proliferation in liquid medium and differentiation into cotyledon-stage embryos were evaluated. Yuuzuru and Yumeyutaka cultivars were found to have high potentials of plant regeneration through SE, being superior or comparable to North American cultivar Jack.

From a preliminary experiment using 98 Chinese soybean genotypes, 12 varieties were selected for further study in order to enhance the efficiency of somatic embryogenesis and plant regeneration. Significant differences in somatic embryogenesis were found among genotypes. N25281, N25263, and N06499 varieties were shown to have the highest somatic embryogenic capacities. The greatest average number of plantlets regenerated per explant was observed in N25281 (Yang et al., 2009).

Regarding Brazilian genotypes, Bonacin et al. (2000) demonstrated the influence of genotype influence in somatic embryogenic capability of five cultivars, of which BR-16, FT-Cometa and IAS-5 were the most embryogenic ones. That study only assessed somatic embryo induction stage. Other studies also reported the high capacity of IAS-5 to produce somatic embryos (Santos et al., 1997; Di Mauro et al., 2000; Droste et al., 2001). More recently, Droste et al. (2010) identified Brazilian soybean genotypes with potential to respond to *in vitro* culture stimuli for somatic embryo induction, embryo proliferation and plant regeneration. Somatic embryos were induced in all eight tested genotypes, but differences were observed at each stage. IAS-5 and BRSMG 68 Vencedora had high embryo induction frequencies, repetitive embryogenic proliferation, low precocious embryo germination, better embryo differentiation and plant regeneration. Thus, this work identified BRSMG 68 Vencedora and confirmed IAS-5 as genotypes with high potential for somatic embryogenesis and plant regeneration.

#### **4. Optimization of soybean somatic embryogenesis protocol**

Although somatic embryogenesis was described long ago and may be considered a routine procedure for other plant species, the first record of the event in soybean was made by Beversdorf & Bingham (1977), when no more than a few embryos were produced. It was only in 1983 that Christianson et al*.* (1983) regenerated, for the first time, soybean plants via SE. Subsequently, several papers have reported the development of somatic embryos from cotyledons of immature embryos. But until today the measured frequencies at which these embryos convert into plants are significantly lower than that expected (Parrott et al., 1988; Meurer et al., 2001; Walker & Parrott, 2001; Droste et al., 2001, 2010). Therefore, efforts have been directed towards developing and rening protocols for initiation, proliferation, and histodifferentiation/maturation of soybean somatic embryogenesis.

#### **4.1 Induction and proliferation of somatic embryos**

Most protocols described in the early studies failed to promote the satisfactory induction of somatic embryos. This limitation was overcome when embryos were induced from immature soybean cotyledons by placing the explant on high levels of 2,4-D (40 mg/l) (Finer, 1988). Comparing the capacity to induce soybean SE, the mean number of embryos produced on 2,4-D was signicantly higher than that produced in the presence of NAA (Hoffmann et al., 2004). It is noteworthy to mention that the type of auxin used in the medium also inuences culture morphology. Somatic embryos induced on 2,4-D are friable, translucent, yellowish-green in color, and globular to torpedo in shape. Somatic embryos induced on NAA are compact, opaque, pale-green in color, with an advanced morphology, forming cotyledon-like structures (Lazzeri et al., 1987; Hoffmann et al., 2004). Furthermore, Lazzeri et al. (1987) described that somatic embryos initiated on NAA had more normal embryo morphology.

The synergistic effect of pH, solidifying agent, 2,4-D concentration, explants orientation and wounding have also been reported to improve efficiency of somatic embryo induction (Santarém et al., 1997). Embryo initiation was higher when explants were cultured with the abaxial side facing the induction medium containing high concentration of 2,4-D, pH adjusted to 7.0 and solidified with GelriteTM. Effect of pH was not observed or was slighter when somatic embryos were induced in the presence of other growth regulators (Lazzeri et al., 1987; Komatsuda & Ko, 1990; Hoffman et al., 2004; Bonacin et al., 2000). It has been suggested that the effect of pH on somatic embryo initiation may be related to auxin uptake into cultured explants, and that a pH of 7.0 may facilitate slower and more gradual uptake of 2,4-D at relatively high level. Wounding treatment did not increase the number of embryos, although in wounded explants somatic embryos were induced earlier than in nonwounded counterparts (Santarém et al., 1997). In addition, efficiency on SE induction was highest when in a medium containing 2-3% sucrose. Cultures initiated on lower sucrose concentrations tended to produce higher amount of friable embryos, while increased concentrations of this sugar impaired embryo induction (Lippmann & Lippmann, 1984; Lazzeri et al., 1987, 1988; Hoffmann et al., 2004).

differentiation and plant regeneration. Thus, this work identified BRSMG 68 Vencedora and confirmed IAS-5 as genotypes with high potential for somatic embryogenesis and plant

Although somatic embryogenesis was described long ago and may be considered a routine procedure for other plant species, the first record of the event in soybean was made by Beversdorf & Bingham (1977), when no more than a few embryos were produced. It was only in 1983 that Christianson et al*.* (1983) regenerated, for the first time, soybean plants via SE. Subsequently, several papers have reported the development of somatic embryos from cotyledons of immature embryos. But until today the measured frequencies at which these embryos convert into plants are significantly lower than that expected (Parrott et al., 1988; Meurer et al., 2001; Walker & Parrott, 2001; Droste et al., 2001, 2010). Therefore, efforts have been directed towards developing and rening protocols for initiation, proliferation, and

Most protocols described in the early studies failed to promote the satisfactory induction of somatic embryos. This limitation was overcome when embryos were induced from immature soybean cotyledons by placing the explant on high levels of 2,4-D (40 mg/l) (Finer, 1988). Comparing the capacity to induce soybean SE, the mean number of embryos produced on 2,4-D was signicantly higher than that produced in the presence of NAA (Hoffmann et al., 2004). It is noteworthy to mention that the type of auxin used in the medium also inuences culture morphology. Somatic embryos induced on 2,4-D are friable, translucent, yellowish-green in color, and globular to torpedo in shape. Somatic embryos induced on NAA are compact, opaque, pale-green in color, with an advanced morphology, forming cotyledon-like structures (Lazzeri et al., 1987; Hoffmann et al., 2004). Furthermore, Lazzeri et al. (1987) described that somatic embryos initiated on NAA had more normal

The synergistic effect of pH, solidifying agent, 2,4-D concentration, explants orientation and wounding have also been reported to improve efficiency of somatic embryo induction (Santarém et al., 1997). Embryo initiation was higher when explants were cultured with the abaxial side facing the induction medium containing high concentration of 2,4-D, pH adjusted to 7.0 and solidified with GelriteTM. Effect of pH was not observed or was slighter when somatic embryos were induced in the presence of other growth regulators (Lazzeri et al., 1987; Komatsuda & Ko, 1990; Hoffman et al., 2004; Bonacin et al., 2000). It has been suggested that the effect of pH on somatic embryo initiation may be related to auxin uptake into cultured explants, and that a pH of 7.0 may facilitate slower and more gradual uptake of 2,4-D at relatively high level. Wounding treatment did not increase the number of embryos, although in wounded explants somatic embryos were induced earlier than in nonwounded counterparts (Santarém et al., 1997). In addition, efficiency on SE induction was highest when in a medium containing 2-3% sucrose. Cultures initiated on lower sucrose concentrations tended to produce higher amount of friable embryos, while increased concentrations of this sugar impaired embryo induction (Lippmann & Lippmann, 1984;

**4. Optimization of soybean somatic embryogenesis protocol** 

histodifferentiation/maturation of soybean somatic embryogenesis.

**4.1 Induction and proliferation of somatic embryos** 

Lazzeri et al., 1987, 1988; Hoffmann et al., 2004).

regeneration.

embryo morphology.

After induced, the early-staged somatic embryos can be maintained and proliferated by subculturing the tissue on either semi-solid medium (Finer, 1988) or liquid suspension culture medium (Finer & Nagasawa, 1988). As liquid medium allows greater contact of plant tissue with medium components, proliferation in liquid is usually more efficient than on a semi-solid medium (Samoylov et al., 1998a). On the other hand, soybean embryogenic suspension cultures can be very difficult to establish and maintain (Santarém et al., 1999). Although maintenance of soybean embryogenic cultures in liquid medium was facilitated by the development of FN medium (Finer & Nagasawa, 1988), its efficiency remains low. Changes in individual medium components, such as carbohydrate type and concentration, total nitrogen, ammonium, nitrate, and other macronutrients, improved proliferation of soybean suspension cultures in liquid medium and resulted in the development of an optimized medium, referred to as FN Lite (Samoylov et al., 1998a).

Somatic embryos incubated in a medium containing NAA do not proliferate so well as those produced on a medium containing 2,4-D (Liu et al., 1992). As discussed above, somatic embryos initiated on NAA are more advanced in embryo morphology than those induced on 2,4-D. As a consequence, they are also not suitable for use in establishing repetitive cultures (Lazzeri et al., 1987; Hoffmann et al., 2004). Embryogenic cultures induced from immature cotyledons on a medium containing 40 mg/l required lower levels of 2,4-D for efficient proliferation (Bailey et al., 1993; Santarém & Finer, 1999). Twenty mg/l 2,4-D are necessary to efficiently maintain repetitive embryogenesis on semi-solid medium (Santarém & Finer, 1999), while 5 mg/l 2,4-D are sufficient on liquid suspension culture medium (Samoylov et al., 1998a). Furthermore, in contrast to the effect of pH on embryo initiation, cultures maintained on semisolid medium at pH 5.8 or 7.0 showed no difference in proliferation rates, suggesting that once embryogenesis has been induced, the tissue does not necessary have to be maintained under the same conditions (Santarém et al., 1997).

#### **4.2 Histodifferentiation/maturation of somatic embryos and recovery of plants**

Even after three decades of intense research, soybean regeneration via somatic embryogenesis remains low if compared to other crops. Developmental limitations in somatic embryos are usually related to inadequate culture media composition (Merkle et al., 1994). So, efforts to better mimic the developmental environment of zygotic embryos were made to further improve SE. Deficiency in maturation stage has been identified as the main obstacle to somatic embryo conversion into plant. Somatic and zygotic embryos have been shown to diverge in sugars, protein, and total lipid accumulation, indicating that the somatic embryos did not develop properly and, as consequence, bring about difficulties for plant regeneration (Chanprame et al., 1998). In addition to embryo nutrition, appropriate sugar accumulation has been shown to be involved in soybean seed desiccation tolerance (Blackman et al., 1992).

Histodifferentiation and maturation of soybean somatic embryos has been basically achieved through the use of two protocols. The first one is a two-step process, whereby embryos are rst histodifferentiated on MSM6AC medium (Bailey et al., 1993), which consists of solidied MS basal salts supplemented with 6% maltose and 0.5% activated charcoal. After 30 days, embryos are transferred to the same medium, but without charcoal, in order to allow growth and maturation to proceed. The second protocol is based on a liquid medium termed FNLS3, which consists of basal Finer & Nagasawa "Lite" (FNL) salts supplemented with 3% sucrose (Samoylov et al., 1998b). The main advantage of the liquid FNLS3 medium over the semi-solid MSM6AC/MSM6 medium is its ability to produce larger numbers of mature somatic embryos in a short period of time. On the other hand, liquid based protocols require greater care during handling.

Successful plant recovery has been reported using both protocols, though numerous studies suggested modifications to these basic media in order to optimize histodifferentiation and maturation of soybean somatic embryos, with the final goal of obtaining higher conversion rates. The main adaptations were: the type and concentration of carbon source, addition of osmotic agents, growth regulator agents and/or amino acids, type of basal salts, as well as the medium supplementation with activated charcoal (Tables 1 and 2). Modifications in other culture media are not considered in the present review.

#### **4.2.1 Carbon source and osmotic agents**

Carbon source is critical for embryo nutritional health and improves somatic embryo maturation. Carbohydrates are commonly used as carbon sources for *in vitro* development of tissues, and maltose and sucrose have been usually added to soybean tissue culture media in an optimal concentration range of 3% to 6% (Samoylov et al., 1998b; Körbes & Droste, 2005; Schmidt et al., 2005). Carbon source type and concentration required for histodifferentiation/maturation appear to differ between solid and liquid media.

The effect of carbohydrates on embryo histodifferentiation and maturation on liquid medium was analyzed by Samoylov et al. (1998b). FNL medium supplemented with 3% sucrose (FNL0**S3**) or 3% maltose (FNL0**M3**) were compared. Data indicated that sucrose promotes embryo growth and significantly increases the number of cotyledon-stage embryos recovered during histodifferentiation and maturation. However, the percentages of plants recovered from embryos differentiated and matured in FNL0**S3** was lower than those grown in FNL0**M3** (Samoylov et al., 1998b). This limitation was partially solved by adding 3% sorbitol to the medium, which resulted in an increment in germination and conversion frequencies (Walker & Parrott, 2001). The modified medium was named FNLS3S3 medium. It was suggested that sorbitol acts as an osmotic agent and/or promotes accumulation of triglycerides in somatic embryos. After addition of sorbitol, the effects of using 3% sucrose (FNL0**S3**S3) or 3% maltose (FNL0**M3**S3) were compared again (Schmidt et al., 2005). Conversion rate of embryos differentiated on maltose was higher when compared to those obtained in sucrose. However, sucrose was considered the most appropriate carbon source, since its use in liquid maturation media resulted in a higher number of larger embryos, which required less time to reach physiological maturity.

The media that have been used in the two-step histodifferentiation/maturation process (MS**M6**AC/MS**M6**) contain 6% maltose (Bailey et al., 1993). Körbes & Droste (2005) compared conversion frequencies when 6% maltose was replaced by 3% maltose (MS**M3**) or 6% sucrose (MS**S6**). Results showed that maturation in MSS6 medium leads to an increment in rates of histodifferentiated embryos with normal morphology, as well as in plant recovery.

The quality of somatic embryos can be positively influenced by a low osmotic potential in maturation medium (Walker & Parrott, 2001; Körbes & Droste, 2005). Carbohydrates can act as an osmotic agent (Li et al., 1998). Since molecular weights of maltose and sucrose are very similar, no signicant effect on the osmotic potential of the medium could be expected (Schmidt et al., 2005). Nevertheless, differences observed in embryos matured in media supplemented with maltose or sucrose have been related to these compounds' osmotic potentials (Samoylov et al., 1998b; Körbes & Droste, 2005). Influence of other osmotic agents (sorbitol, mannitol and polyethylene glycol) has also been tested in soybean somatic embryogenesis (Walker & Parrott, 2001; Körbes & Droste, 2005; Schmidt et al., 2005), but positive effects were only reported for sorbitol (Walker & Parrott, 2001), as described above.

#### **4.2.2 Amino acids**

154 Genetic Engineering – Basics, New Applications and Responsibilities

supplemented with 3% sucrose (Samoylov et al., 1998b). The main advantage of the liquid FNLS3 medium over the semi-solid MSM6AC/MSM6 medium is its ability to produce larger numbers of mature somatic embryos in a short period of time. On the other hand,

Successful plant recovery has been reported using both protocols, though numerous studies suggested modifications to these basic media in order to optimize histodifferentiation and maturation of soybean somatic embryos, with the final goal of obtaining higher conversion rates. The main adaptations were: the type and concentration of carbon source, addition of osmotic agents, growth regulator agents and/or amino acids, type of basal salts, as well as the medium supplementation with activated charcoal (Tables 1 and 2). Modifications in

Carbon source is critical for embryo nutritional health and improves somatic embryo maturation. Carbohydrates are commonly used as carbon sources for *in vitro* development of tissues, and maltose and sucrose have been usually added to soybean tissue culture media in an optimal concentration range of 3% to 6% (Samoylov et al., 1998b; Körbes & Droste, 2005; Schmidt et al., 2005). Carbon source type and concentration required for

The effect of carbohydrates on embryo histodifferentiation and maturation on liquid medium was analyzed by Samoylov et al. (1998b). FNL medium supplemented with 3% sucrose (FNL0**S3**) or 3% maltose (FNL0**M3**) were compared. Data indicated that sucrose promotes embryo growth and significantly increases the number of cotyledon-stage embryos recovered during histodifferentiation and maturation. However, the percentages of plants recovered from embryos differentiated and matured in FNL0**S3** was lower than those grown in FNL0**M3** (Samoylov et al., 1998b). This limitation was partially solved by adding 3% sorbitol to the medium, which resulted in an increment in germination and conversion frequencies (Walker & Parrott, 2001). The modified medium was named FNLS3S3 medium. It was suggested that sorbitol acts as an osmotic agent and/or promotes accumulation of triglycerides in somatic embryos. After addition of sorbitol, the effects of using 3% sucrose (FNL0**S3**S3) or 3% maltose (FNL0**M3**S3) were compared again (Schmidt et al., 2005). Conversion rate of embryos differentiated on maltose was higher when compared to those obtained in sucrose. However, sucrose was considered the most appropriate carbon source, since its use in liquid maturation media resulted in a higher number of larger embryos,

The media that have been used in the two-step histodifferentiation/maturation process (MS**M6**AC/MS**M6**) contain 6% maltose (Bailey et al., 1993). Körbes & Droste (2005) compared conversion frequencies when 6% maltose was replaced by 3% maltose (MS**M3**) or 6% sucrose (MS**S6**). Results showed that maturation in MSS6 medium leads to an increment in rates of histodifferentiated embryos with normal morphology, as well as in plant

The quality of somatic embryos can be positively influenced by a low osmotic potential in maturation medium (Walker & Parrott, 2001; Körbes & Droste, 2005). Carbohydrates can act as an osmotic agent (Li et al., 1998). Since molecular weights of maltose and sucrose are very

histodifferentiation/maturation appear to differ between solid and liquid media.

liquid based protocols require greater care during handling.

other culture media are not considered in the present review.

which required less time to reach physiological maturity.

recovery.

**4.2.1 Carbon source and osmotic agents** 

The nitrogen source is also a critical component for proper embryo maturation. With a view to soybean embryo proliferation, 1 g/l asparagine was included in the formulation of the original FN liquid medium (Finer & Nagasawa, 1988), and it was also used in the FN Lite proliferation medium (Samoylov et al., 1998a) and FNL histodifferentiation/maturation medium (Samoylov et al., 1998b). On the other hand, culture media supplemented with glutamine were shown to be beneficial to zygotic soybean embryos (Thompson et al., 1977), by increasing embryo size (Lippmann & Lippmann, 1993; Dyer et al., 1987), and inducing storage oil and protein synthesis (Saravitz & Raper, 1995). Schmidt et al. (2005) compared the effect of FNLS3S3 supplemented with asparagine or glutamine on embryo histodifferentiation/maturation. The results showed that cotyledonary-stage embryos obtained on 30 mM lter-sterilized glutamine were larger and exhibited an overall higher quality. The modified medium was named FNLS3S3G30 medium.

Methionine supplementation has also been reported to be helpful for growth stimulation (Coker et al., 1987). Thus, addition of 1 mM methionine to FNL0S3S3 medium was tested and a clear positive effect on soybean somatic embryo histodifferentiation and maturation, manifested mainly by conversion percentages, was demonstrated (Schmidt et al., 2005). Again, this medium was renamed FNLS3S3G30M1 medium.

Improvements in the efficiency of solid MSM6 histodifferentiation/maturation medium were also obtained by using the ingredients from the optimized FNLS3S3G30M1 liquid medium recipe, specifically glutamine and methionine (Schmidt et al., 2005).

#### **4.2.3 Basal salts**

A comparison of embryo development showed that embryos differentiated into yellowgreen cotyledon-stage faster when cultured on FNLS3 than when maintained on MSM3 liquid media (Samoylov et al., 1998b). On the other hand, the number of histodifferentiated embryos and the frequency of germinated embryos recovered from MSM3 were higher than those obtained on FNLS3. Further studies showed that germination rates were increased when embryos histodifferentiated on FNLS3 supplemented with sorbitol, glutamine and methionine (Walker & Parrott, 2001; Schmidt et al., 2005).

#### **4.2.4 Abscisic acid**

Abscisic acid (ABA) is a growth regulator involved in plant development, especially during embryo development and maturation and in response to abiotic stresses. In seeds, ABA induces storage protein synthesis and affects dormancy induction and maintenance (Rock & Quatrano, 1995). ABA prevents precocious seed germination and is thought to play a major


Table 1. Optimization of MSM6AC medium. D20 - MS salts (Murashige & Skoog, 1962), B5 vitamins (Gamborg et al., 1968), 3% sucrose, 20 mg/l 2,4-D, and 0.2% Gelrite or 0.3% Phytagel (pH 5.8) (Wright et al., 1991). MSM6AC - MS salts, B5 vitamins, 6% maltose, 0.5% activated charcoal and 0.25% Gelrite or 0.3% Phytagel (pH 5.8) (Bailey et al., 1993). MSM6 - MS salts, B5 vitamins, 6% maltose and 0.25% Gelrite or 0.3% Phytagel (pH 5.8) (Finer & McMullen, 1991). MSS6 - MS salts, B5 vitamins, 6% sucrose, 0.3% Phytagel (pH 5.8) (Körbes & Droste, 2005). MSO - MS basal salts, B5 vitamins, 3% sucrose, and 0.2% Gelrite, at pH 5.8

role in the sequence of events leading to desiccation tolerance (Hoekstra et al., 2001). ABA supplementation in culture media was shown to affect somatic embryogenesis of a variety of plants. Having this in mind, it was suggested that medium containing low ABA concentrations (0.38 or 1 µM) helps soybean immature somatic embryos to achieve maturity and further develop apical meristems (Ranch et al., 1885; Lazzeri et al., 1987). However, independently of the protocol used, no significant effect was observed when ABA was added to histodifferentiation or maturation medium (Tian & Brown, 2000; Schmidt et al.,

rentiation Maturation Regeneration

D20 MSS6 MSS6 MSO • increase

/ conversion

MSM6 MSO • increase embryo

• increase the

MSS6 MSO • increase the

MSM6AC MSM6AC MSO • promote embryo

Beneficial effects

histodifferentiation of embryo with normal morphology • improve embryo viability • enhance embryo germination

conversion into plants

histodifferentiation of embryo with normal morphology • increase embryo

conversion into plants

conversion into plants

number of histodifferentiated

embryos • increase embryo

> number of histodifferentiated

embryos

growth • increase

Culture tissue stage

Proliferation Histodiffe-

FN + 50 µM ABA

> D20 + 50 µM ABA

MSM6 + 50 µM ABA

 MSM6 + 30 mM glutamine + 1 mM Methionine

D20 MSS6 +

0.5 % AC

Table 1. Optimization of MSM6AC medium. D20 - MS salts (Murashige & Skoog, 1962), B5 vitamins (Gamborg et al., 1968), 3% sucrose, 20 mg/l 2,4-D, and 0.2% Gelrite or 0.3% Phytagel (pH 5.8) (Wright et al., 1991). MSM6AC - MS salts, B5 vitamins, 6% maltose, 0.5% activated charcoal and 0.25% Gelrite or 0.3% Phytagel (pH 5.8) (Bailey et al., 1993). MSM6 - MS salts, B5 vitamins, 6% maltose and 0.25% Gelrite or 0.3% Phytagel (pH 5.8) (Finer & McMullen, 1991). MSS6 - MS salts, B5 vitamins, 6% sucrose, 0.3% Phytagel (pH 5.8) (Körbes & Droste, 2005). MSO - MS basal salts, B5 vitamins, 3% sucrose, and 0.2% Gelrite, at pH 5.8 role in the sequence of events leading to desiccation tolerance (Hoekstra et al., 2001). ABA supplementation in culture media was shown to affect somatic embryogenesis of a variety of plants. Having this in mind, it was suggested that medium containing low ABA concentrations (0.38 or 1 µM) helps soybean immature somatic embryos to achieve maturity and further develop apical meristems (Ranch et al., 1885; Lazzeri et al., 1987). However, independently of the protocol used, no significant effect was observed when ABA was added to histodifferentiation or maturation medium (Tian & Brown, 2000; Schmidt et al.,

Reference Main

Tian & Brown, 2000

Weber et. al., 2007

Körbes & Droste, 2005

Schmidt et al., 2005

Droste et al., 2010

modification

Abscisic acid (ABA) addition

> ABA addition

Maltose replacement with sucrose

Glutamine and methionine addition

Activated charcoal (AC) addition

2005; Weber et al., 2007). Tian & Brown (2000) investigated the effect of ABA addition to culture media in different embryogenic stages: proliferation, histodifferentiation and maturation. The positive effects of ABA were observed only when embryos at a globular stage (proliferation) were treated prior to histodifferentiation induction. Addition of 50 µM ABA promoted growth of histodifferentiated embryos, increased proportion of morphological normal histodifferentiated embryos, improved embryo viability after desiccation and increased germination frequency. In agreement, Weber et al. (2007) demonstrated that presence of ABA during proliferation or during both proliferation and maturation stages increased percentage of converted plants.


Table 2. Optimization of FNLS3 medium. FN Lite - FN Lite macro salts (Samoylov et al., 1998a), MS micro salts (Murashige & Skoog, 1962), B5 vitamins (Gamborg et al., 1968), 6.7 mM L-asparagine, 1% sucrose , 146.1 mM mannitol and 0.5 mg/l 2,4-D, at pH 5.8 (Samoylov et al., 1998a). FNL0S3 – FN Lite macro salts, MS micro salts, B5 vitamins, 6.7 mM Lasparagine and 3% sucrose, at pH 5.8 (Samoylov et al., 1998b). MSO - MS basal salts, B5 vitamins, 1.5% sucrose, and 0.2% Gelrite, at pH 5.8.

#### **4.2.5 Activated charcoal**

Activated charcoal (AC) is a porous material composed of carbon. AC has a unique adsorption capacity and is often used in plant tissue culture to improve cell growth and development. Applicability of AC is credited mainly to its capacity as adsorbent of inhibitory substances in the culture medium (Thomas, 2008). As previously discussed (Induction and proliferation of somatic embryos section), high concentrations of 2,4-D are necessary to stimulate soybean somatic embryo induction and proliferation (Finer, 1988; Samoylov et al., 1998a; Santarém & Finer, 1999). On the other hand, this growth regulator can lead to abnormal embryo histodifferentiation or development of apical meristem, especially in long-term cultures under exposure to 2,4-D. AC is presumably able to adsorb 2,4-D or other auxins released from developing tissues, promoting a more normal embryo morphology and increasing germination ability (Merkle et al., 1995).

AC was present in a medium originally described by Bailey et al. (1993) for embryo histodifferentiation/maturation, but it has not been applied in further studies (Samoylov et al., 1998b; Walker & Parrott, 2001; Körbes & Droste, 2005; Schmidt et al., 2005; Weber et. al., 2007). Recently, it has been observed that addition of activated charcoal and ABA to the first step histodifferentiation/maturation medium increased the number of histodifferentiated embryos (Droste et al., 2010). Since positive effects were not identified when ABA was added in histodifferentiation/maturation medium (Schmidt et al., 2005; Weber et. al., 2007) (discussed in the section about abscisic acid), the benefits described by Droste et al. (2010) must be related to AC supplementation.

It is important to stress that studies were carried out with different genotypes and that soybean response to *in vitro* culture conditions is genotype-dependent. Further studies, specially focusing on embryo histodifferentiation and maturation, are important and necessary to improve soybean embryogenesis of highly responsive cultivars.

After appropriate maturation, a desiccation stage is required. Conversion of partialdesiccated soybean somatic embryos was shown to proceed more vigorously than nondesiccated ones (Buchheim et al., 1989). The partial desiccation has been adopted by most research teams (Bailey et al., 1993; Samoylov et al., 1998b; Tian & Brown, 2000; Walker & Parrott, 2001; Droste et al., 2002, 2010; Körbes & Droste, 2005; Schmidt et al., 2005; Yang et al., 2009; Wiebke-Strohm et al., 2011). Although there is a broad number of media available for histodifferentiation/maturation, MSO (MS basal salts, B5 vitamins, 3% sucrose, and 0.2% Gelrite™, at pH 5.8) medium has been almost exclusively used for soybean somatic embryos germination/conversion stage (Finer & McMullen, 1991; Bailey et al., 1993; Samoylov et al., 1998b; Tian & Brown, 2000; Walker & Parrott, 2001; Droste et al., 2002, 2010; Körbes & Droste, 2005; Schmidt et al., 2005; Wiebke-Strohm et al., 2011).

#### **5. Genetic transformation of soybean somatic embryos via particle bombardment and bombardment/***Agrobacterium* **integrated system**

Plant genetic transformation is described as the introduction of recombinant DNA in plant cells using genetic engineering methods. Transgenic plants represent a priceless tool for molecular, genetic, biochemical and physiological studies. Plant genetic transformation also offers a significant advancement for soybean breeding programs, in terms of allowing the production of novel and genetically diverse plant materials.

Activated charcoal (AC) is a porous material composed of carbon. AC has a unique adsorption capacity and is often used in plant tissue culture to improve cell growth and development. Applicability of AC is credited mainly to its capacity as adsorbent of inhibitory substances in the culture medium (Thomas, 2008). As previously discussed (Induction and proliferation of somatic embryos section), high concentrations of 2,4-D are necessary to stimulate soybean somatic embryo induction and proliferation (Finer, 1988; Samoylov et al., 1998a; Santarém & Finer, 1999). On the other hand, this growth regulator can lead to abnormal embryo histodifferentiation or development of apical meristem, especially in long-term cultures under exposure to 2,4-D. AC is presumably able to adsorb 2,4-D or other auxins released from developing tissues, promoting a more normal embryo

AC was present in a medium originally described by Bailey et al. (1993) for embryo histodifferentiation/maturation, but it has not been applied in further studies (Samoylov et al., 1998b; Walker & Parrott, 2001; Körbes & Droste, 2005; Schmidt et al., 2005; Weber et. al., 2007). Recently, it has been observed that addition of activated charcoal and ABA to the first step histodifferentiation/maturation medium increased the number of histodifferentiated embryos (Droste et al., 2010). Since positive effects were not identified when ABA was added in histodifferentiation/maturation medium (Schmidt et al., 2005; Weber et. al., 2007) (discussed in the section about abscisic acid), the benefits described by Droste et al. (2010)

It is important to stress that studies were carried out with different genotypes and that soybean response to *in vitro* culture conditions is genotype-dependent. Further studies, specially focusing on embryo histodifferentiation and maturation, are important and

After appropriate maturation, a desiccation stage is required. Conversion of partialdesiccated soybean somatic embryos was shown to proceed more vigorously than nondesiccated ones (Buchheim et al., 1989). The partial desiccation has been adopted by most research teams (Bailey et al., 1993; Samoylov et al., 1998b; Tian & Brown, 2000; Walker & Parrott, 2001; Droste et al., 2002, 2010; Körbes & Droste, 2005; Schmidt et al., 2005; Yang et al., 2009; Wiebke-Strohm et al., 2011). Although there is a broad number of media available for histodifferentiation/maturation, MSO (MS basal salts, B5 vitamins, 3% sucrose, and 0.2% Gelrite™, at pH 5.8) medium has been almost exclusively used for soybean somatic embryos germination/conversion stage (Finer & McMullen, 1991; Bailey et al., 1993; Samoylov et al., 1998b; Tian & Brown, 2000; Walker & Parrott, 2001; Droste et al., 2002, 2010; Körbes &

necessary to improve soybean embryogenesis of highly responsive cultivars.

**5. Genetic transformation of soybean somatic embryos via particle bombardment and bombardment/***Agrobacterium* **integrated system** 

Plant genetic transformation is described as the introduction of recombinant DNA in plant cells using genetic engineering methods. Transgenic plants represent a priceless tool for molecular, genetic, biochemical and physiological studies. Plant genetic transformation also offers a significant advancement for soybean breeding programs, in terms of allowing the

Droste, 2005; Schmidt et al., 2005; Wiebke-Strohm et al., 2011).

production of novel and genetically diverse plant materials.

morphology and increasing germination ability (Merkle et al., 1995).

**4.2.5 Activated charcoal** 

must be related to AC supplementation.

Advancements in the use of plant transgenesis have been reported for a large number of species. Such progress entails the adoption of different protocols, which include the genetic transformation mediated by polyethyleneglycol (PEG) and liposomes (known under the heading of *chemical methods*), microinjection, electroporation and particle bombardment (called *physical methods*), as well as the use of viral and/or bacterial vectors, as in agroinfection and the *Agrobacterium* system (named *biological methods*).

Soybean transformation was first reported in 1988 by two independent groups using different methods (Hinchee et al., 1988; Christou et al., 1988). Even after more than two decades, the stable transformation of soybean cannot yet be considered as routine because it depends on the ability to bring together efficient transformation and regeneration techniques. Two methods have been successfuly used: **particle bombardment** (McCabe et al., 1988; Christou et al., 1989; Finer & McMullen, 1991; Christou & McCabe, 1992; Finer et al., 1992; Stewart et al., 1996; Aragão et al., 2000; Droste et al., 2002; Homrich et al*.,* 2008; Wu et al., 2008; Li et al., 2009; Hernandez-Garcia et al., 2009; Xing et al., 2010; Viana et al., 2011) and *Agrobacterium tumefaciens* **system** (Parrott et al., 1989; Trick et al., 1997; Trick & Finer, 1998; Aragão et al., 2000; Yan et al., 2000; Ko et al., 2003, 2004; Paz et al., 2006; Hong et al., 2007; Miklos et al., 2007; Liu et al., 2008; Wang & Xu, 2008; Wiebke-Strohm et al., 2011).

Regardless of the method used, the unicellular origin of soybean secondary somatic embryos makes them a useful target tissue for transformation, allowing the production of fully transformed plants. The first target used for transformation was primary somatic embryos, but chimerical plants were obtained (Parrott et al., 1989) due to the multicellular nature of primary embryos (Sato et al., 1993). Finer (1988) showed that the secondary somatic embryos proliferated directly from the apical or terminal portions of the older primary somatic embryos. Sato et al. (1993) proved that somatic embryo proliferation occurred from single epidermal cells of existing somatic embryos. Using proliferative embryos as target tissue many studies succeed in regenerating completely transformed plants (Finer & McMullen, 1991; Finer et al., 1992; Stewart et al., 1996; Trick et al., 1997; Trick & Finer, 1998; Droste et al., 2002; Homrich et al., 2008; Schmidt et al., 2008; Wu et al., 2008; Li et al., 2009; Hernandez-Garcia et al., 2009; Xing et al., 2010; Wiebke-Strohm et al., 2011).

#### **5.1** *Agrobacterium* **system**

*Agrobacterium tumefaciens* is a soil-borne Gram-negative phytopatogenic bacterium that naturally infects different plants causing the crown gall disease (DeCleene & DeLay, 1976). The origin of these sicknesses is interkingdom horizontal gene transfer. When virulent strains of *Agrobacterium* infect plant cells, they transfer one or more segments of DNA (transferred DNA or T-DNA) from Ti (**T**umor **i**nducing) plasmids into host plant cells (recently reviewed by Gelvin, 2010 a,b; Pitzschke & Hirt, 2010). In recent decades, disarmed (non-tumorigenic) *A. tumefaciens* strains have also provided a means to produce genetically modied plants. In order to obtain engineered binary vectors derived from Ti plasmids, oncogenes present in T-DNA region are replaced by any foreign DNA of interest (Gelvin, 2010b).

The advantages of *Agrobacterium*-mediated gene transfer include the possibility of transferring relatively large segments of DNA, lower number of transgene copies integration into plant genomes, rare transgene rearrangement, lower frequency of genomic DNA interspersion and reduced abnormal transgene expression (Gelvin, 2003; Kohli et al., 2003). Moreover, this system involves low operating cost and simplicity of transformation protocols. On the other hand, plants differ greatly in their susceptibility to *Agrobacterium*mediated transformation. These differences occur among species, cultivars or tissues (Droste et al., 1994; Gelvin, 2010b). In addition, in our laboratory this transformation system usually results in lower transformation rates, if compared to particle bombardment.

*Agrobacterium*-mediated transformation system is a growing trend in crop transformation programs (Somers et al., 2003). Soybean has long been considered recalcitrant to *Agrobacterium* (DeCleene & DeLey, 1976), especially due to the low success rate in recovering transgenic plants. Studies with tumorigenic *Agrobacterium* strains (Pedersen et al., 1983; Droste et al., 1994; Mauro et al., 1995) and transient assays (Meurer et al., 1998; Droste et al., 2000) showed that soybean can be readily transformed by this bacterium. Currently, it is known that addition of acetosyringone during bacterial infection, combination of appropriate *Agrobacterium* strain and soybean cultivar, as well as development of super virulent bacterium strain and suitable plasmids increase efficiency of soybean transformation (Somers et al., 2003; Ko et al., 2003, 2004; Wiebke-Strohm et al., 2011).

Transgenic plants regenerated via somatic embryogenesis were obtained using two different target tissues: immature zygotic cotyledons and secondary somatic embryos. In the first case, wounded immature zygotic cotyledons are co-cultivated with *Agrobacterium* suspension, after which embryogenic tissue formation is induced from the surface of these cotyledons. In the second case, proliferative secondary somatic embryos are first obtained and then submitted to transformation experiments.

Transformation of zygotic cotyledons by *A. tumefaciens* and subsequent regeneration of transformed soybean plants was first reported by Parrott et al. (1989). Plants were regenerated from primary somatic embryos and, due to embryo multi-cellular origin, plants were chimeric. As a consequence, transgenes were not present in the germ line and transmitted to the progeny. Still using zygotic cotyledons as target, a new approach was developed in which formation of secondary somatic embryos was allowed under continued selection system. The unicellular origin of secondary somatic embryos permitted recovery of complete, stable and fertile transgenic plants, whose progeny also displayed these characteristics (Yan et al., 2000; Ko et al., 2003).

Transformation of proliferative secondary somatic embryos via *A. tumefaciens* has proven to be challenging and only succeeded when combined to physical methods that generate an entry point to bacteria penetration. Instead of the conventional transformation system, two alternative methods have been proposed for this target tissue: the Sonication-Assisted *Agrobacterium*-mediated Transformation (SAAT) (Trick et al., 1997; Trick & Finer, 1998), and the combined DNA-free particle bombardment and *Agrobacterium* system (bombardment/*Agrobacterium* integrated system) (Droste et al., 2000; Wiebke et al., 2006, Wiebke-Strohm et al., 2011). The difference between these methods lies in the technique used to induce tissue wounding: while the first one used sonication, the second relied upon bombardment. Although these methods proved to be feasible, both systems are timeconsuming, laborious, and depend on the availability of specific equipments for routine application.

It is important to stress that the success in soybean *Agrobacterium*-mediated transformation and regeneration via somatic embryogenesis depends on the availability of cultivars with superior response to *in vitro* culture stimuli and high susceptibility to this bacterium. So far, regeneration of transgenic plants was achieved using immature zygotic cotyledons of Jack, Williams and Dwight cultivars (Yan et al., 2000; Ko et al., 2003) or proliferative secondary somatic embryos of Chapman, Bragg, IAS5 & BRMG 68 Vencedora cultivars (Trick et al., 1997; Trick & Finer, 1998; Droste et al., 2000; Wiebke-Strohm et al., 2011).

#### **5.2 Particle bombardment**

160 Genetic Engineering – Basics, New Applications and Responsibilities

2003). Moreover, this system involves low operating cost and simplicity of transformation protocols. On the other hand, plants differ greatly in their susceptibility to *Agrobacterium*mediated transformation. These differences occur among species, cultivars or tissues (Droste et al., 1994; Gelvin, 2010b). In addition, in our laboratory this transformation system usually

*Agrobacterium*-mediated transformation system is a growing trend in crop transformation programs (Somers et al., 2003). Soybean has long been considered recalcitrant to *Agrobacterium* (DeCleene & DeLey, 1976), especially due to the low success rate in recovering transgenic plants. Studies with tumorigenic *Agrobacterium* strains (Pedersen et al., 1983; Droste et al., 1994; Mauro et al., 1995) and transient assays (Meurer et al., 1998; Droste et al., 2000) showed that soybean can be readily transformed by this bacterium. Currently, it is known that addition of acetosyringone during bacterial infection, combination of appropriate *Agrobacterium* strain and soybean cultivar, as well as development of super virulent bacterium strain and suitable plasmids increase efficiency of soybean transformation

Transgenic plants regenerated via somatic embryogenesis were obtained using two different target tissues: immature zygotic cotyledons and secondary somatic embryos. In the first case, wounded immature zygotic cotyledons are co-cultivated with *Agrobacterium* suspension, after which embryogenic tissue formation is induced from the surface of these cotyledons. In the second case, proliferative secondary somatic embryos are first obtained

Transformation of zygotic cotyledons by *A. tumefaciens* and subsequent regeneration of transformed soybean plants was first reported by Parrott et al. (1989). Plants were regenerated from primary somatic embryos and, due to embryo multi-cellular origin, plants were chimeric. As a consequence, transgenes were not present in the germ line and transmitted to the progeny. Still using zygotic cotyledons as target, a new approach was developed in which formation of secondary somatic embryos was allowed under continued selection system. The unicellular origin of secondary somatic embryos permitted recovery of complete, stable and fertile transgenic plants, whose progeny also displayed these

Transformation of proliferative secondary somatic embryos via *A. tumefaciens* has proven to be challenging and only succeeded when combined to physical methods that generate an entry point to bacteria penetration. Instead of the conventional transformation system, two alternative methods have been proposed for this target tissue: the Sonication-Assisted *Agrobacterium*-mediated Transformation (SAAT) (Trick et al., 1997; Trick & Finer, 1998), and the combined DNA-free particle bombardment and *Agrobacterium* system (bombardment/*Agrobacterium* integrated system) (Droste et al., 2000; Wiebke et al., 2006, Wiebke-Strohm et al., 2011). The difference between these methods lies in the technique used to induce tissue wounding: while the first one used sonication, the second relied upon bombardment. Although these methods proved to be feasible, both systems are timeconsuming, laborious, and depend on the availability of specific equipments for routine

It is important to stress that the success in soybean *Agrobacterium*-mediated transformation and regeneration via somatic embryogenesis depends on the availability of cultivars with superior response to *in vitro* culture stimuli and high susceptibility to this bacterium. So far,

results in lower transformation rates, if compared to particle bombardment.

(Somers et al., 2003; Ko et al., 2003, 2004; Wiebke-Strohm et al., 2011).

and then submitted to transformation experiments.

characteristics (Yan et al., 2000; Ko et al., 2003).

application.

Genetic transformation by particle bombardment (Sanford, 1988), also called particle or projectile acceleration, biolistics or biobalistics, consists of the introduction of DNA in intact cells and tissues by accelerated microprojectiles driven at high speeds. These projectiles are able to cross the wall and the membranes of the cell and of the nucleus, where DNA fragments are liberated (Trick & Finer, 1997). In this organelle, exogenous DNA may then be integrated to chromosomal DNA through processes of illegitimate or homologous recombination that depends exclusively on cell components (Sanford, 1990; Kohli et al., 2003). Particle bombardment affords the introduction of DNA in plant cells by means of plasmids (Hadi et al., 1996; Homrich et al., 2008) or gene cassettes (Fu et al., 2000; Breitler et al., 2002). In this method, DNA is adhered to metal particles called *microcarriers*. The metals used in the process have to be inert, like gold or tungsten, so as to prevent particles from reacting with DNA or cell components (Christou et al., 1990). The DNA-particle complex is accelerated towards the target cells using different apparatuses based on diverse acceleration mechanisms.

Particle bombardment can be achieved through high or low helium pressure gene guns. So, penetration in the target tissue can be controlled very accurately, directing the majority of the particles carrying the DNA to a specific cell layer. This is an extremely important feature, because different explants may require different acceleration conditions for optimum particle penetration (Christou et al., 1990). As single epidermal cells are responsible for the initiation of secondary somatic embryos (Sato et al., 1993), a shallow penetration resulting from bombardment of low helium pressure ensures efficient transformation (Sato et al., 1993). Finer et al. (1992) developed a low helium pressure particle accelerator called *Particle Inflow Gun* (PIG).

The main advantage of particle bombardment lies in the possibility to transfer genes to any cell or tissue type independently of genotype and without having to consider the compatibility between host and bacterium, as required by the *Agrobacterium* system. On the other hand, using this technique, multiple DNA copies are introduced which may recombine or be fragmented (Hadi et al., 1996; Kohli et al., 2003).

Several studies about soybean transformation via particle bombardment using embryogenic tissues have been published (Finer & McMullen, 1991; Finer et al., 1992; Stewart et al., 1996; Droste et al., 2002; Homrich et al., 2008; Schmidt et al., 2008; Wu et al., 2008; Li et al., 2009; Hernandez-Garcia et al., 2009; Xing et al., 2010).

#### **6. Conclusion**

Proliferative somatic embryos are one of the most suitable and convenient targets for soybean genetic transformation. However, after two decades, the stable transformation of somatic embryos cannot yet be considered as routine. The absence of a highly efficient regeneration procedure is the main limiting factor. Advances have been made in the identification of cultivars with high potential for embryogenesis, as well as in the optimization of media and culture protocols. Large efforts are being made to render more efficient soybean plant regeneration via somatic embryogenesis. By recognizing the critical factors, the protocols of each individual stage of the somatic embryogenic process can be improved to more closely simulate zygotic embryo development *in planta*.

#### **7. Acknowledgment**

Authors are grateful to Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq, Brazil for the Postdoctoral Fellowships.

#### **8. References**


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cotyledons of soya bean (*Glycine max* L. Merr.). *Annals of Botany*, Vol.41, No.1,

and maturation by abscisic acid treatment. *Canadian Journal of Plant Science*, Vol.80,

Screening of soybean, *Glycine max* (L.) Merrill, lines for somatic embryo induction and maturation capability from immature cotyledons. *In Vitro Cellular and Developmental Biology-Plant*, Vol.38, (November-December 2002), pp. 543–548, ISSN


## **Genetic Engineering and Biotechnology of Growth Hormones**

Jorge Angel Ascacio-Martínez and Hugo Alberto Barrera-Saldaña *Department of Biochemistry and Molecular Medicine, School of Medicine, Autonomous University of Nuevo León, Monterrey Nuevo León, Av. Madero Pte. s/n Col. Mitras Centro, Monterrey, N.L., México* 

#### **1. Introduction**

172 Genetic Engineering – Basics, New Applications and Responsibilities

Xing, A.; Moon, B.P.; Mills, K.M.; Falco, S.C. & Li, Z. (2010). Revealing frequent alternative

Yan, B.; Reddy, M.S.S.; Collins, G.B. & Dinkins, R.D. (2000). *Agrobacterium tumefaciens*

Yang, C.; Zhao, T.; Yu, D. & Gai J. (2009). Somatic embryogenesis and plant regeneration in

2010), pp. 772–782, ISSN 1467-7644

2009), pp. 180-181, ISSN 1054-5476

ISSN 0721-7714

polyadenylation and widespread low-level transcription read-through of novel plant transcription terminators. *Plant Biotechnology Journal*, Vol.8, No.7, (September

mediated transformation of soybean [*Glycine max* (L) Merrill] using immature zygotic cotyledon explants. *Plant Cell Reports*, Vol.19, No.11, (2000), pp. 1090-1097,

Chinese soybean (*Glycine max* (L.) Merr.)-impacts of mannitol, abscisic acid, and explants age. *In Vitro Cellular and Developmental Biology-Plant*, Vol. 45, No. 2, (April

> In its modern conception, biotechnology is the use of genetic engineering techniques to manipulate microorganisms, plants, and animals in order to produce commercial products and processes that benefit man. These techniques, which are the backbone of the biotechnological revolution that began in the mid 1970s, have permitted the isolation and manipulation of specific genes and the development of transgenic microorganisms that produce mainly eukaryotic proteins of therapeutic use, such as vaccines, enzymes, and hormones.

> Biotechnology is present in diverse areas such as food production, degradation of industrial waste, mining, and medicine. Recent achievements include drug production in transgenic animals and plants, as well as the commercial exploitation of gene sequences generated by the human genome project and similar projects of plants and animals of commercial interest that are and will be in process.

> Human growth hormone was, after insulin, the second product of this new technology. This product was developed and commercialized initially by Genentech, and was used clinically for treating growth problems and dwarfism (1). Furthermore, growth hormones from different animal species have also been produced in transgenic organisms and these have been used in different examples in the aquatic animal and livestock sectors.

#### **2. The growth hormone (GH) family**

GHs belong to a family of proteins with structural similarity and certain common functions that include prolactin (Prl), somatolactin (SL), chorionic somatomammotropin (CS), proliferin (PLF) and proteins related to Prl (PLP) (2). This family represents one of the most physiologically diverse protein groups that have evolved by gene duplication. The two most studied members of this family have been GH and Prl, which have been described from primitive fish to mammals; however, other members of the family are not so amply distributed or studied.

#### **2.1 Structure of growth hormones**

GHs (see Figure 1), in general, have a molecular weight of around 22,000 Daltons (22 kDa or simply 22k) and do not require post-translational modifications. They are synthesized in somatotrophs in the hypophysis, intervening as an important endocrine factor in postnatal somatic growth and lactation.

Fig. 1. Growth hormones' consensus tridimensional structure. The GHs have in general 190 aminoacidic residues, four alpha helixes, and two sulphide bonds

#### **2.2 Hormones of the human growth hormone family**

#### **HGH22k**

HGH22k (or HGHN) is the main product of the GH gene (hGH-N) active in the hypophysis and it is responsible for postnatal growth as well as being an important modulator of carbohydrate, lipid, nitrogen and mineral metabolism. It is the best known hormone and the only one of the HGH family that has been commercialized.

As mentioned, besides being the cure for hypophyseal dwarfism, HGH22k postulated benefits are as an anabolic in athletics and for the treatment of trauma because of its postulated regenerative properties (3).

#### **HGH20k**

In addition to the mRNA of HGH22k, an alternative processing pathway of the primary transcript of the hGH-N gene generates a second mRNA that is responsible for the production of the 20k isoform of HGH or HGH20k. Its smaller size is due to elimination of the first 45 nucleotides of the third exon of the mRNA and of the amino acids that correspond to positions 32-46 of the hormone, producing a protein with 176 amino acid residues (4).

This isoform comprises approximately 10% of all the GH produced in the hypophysis and although it has not been shown to be the etiological agent of any known disease, it is known that its levels are significantly higher in patients with active acromegaly and in those with anorexia nervosa (5).

The administration of exogenous HGH20k suppresses endogenous secretion of HGH22k in healthy subjects, which suggests that the regulation of secretion of both hormones is physiologically similar (6). In vitro findings suggest that both hormones can equally stimulate bone remodeling and allow anabolic effects on skeletal tissue when they are administered in vivo to laboratory animals (7).

#### **HGHV**

174 Genetic Engineering – Basics, New Applications and Responsibilities

GHs (see Figure 1), in general, have a molecular weight of around 22,000 Daltons (22 kDa or simply 22k) and do not require post-translational modifications. They are synthesized in somatotrophs in the hypophysis, intervening as an important endocrine factor in postnatal

Fig. 1. Growth hormones' consensus tridimensional structure. The GHs have in general 190

HGH22k (or HGHN) is the main product of the GH gene (hGH-N) active in the hypophysis and it is responsible for postnatal growth as well as being an important modulator of carbohydrate, lipid, nitrogen and mineral metabolism. It is the best known hormone and the

As mentioned, besides being the cure for hypophyseal dwarfism, HGH22k postulated benefits are as an anabolic in athletics and for the treatment of trauma because of its

In addition to the mRNA of HGH22k, an alternative processing pathway of the primary transcript of the hGH-N gene generates a second mRNA that is responsible for the production of the 20k isoform of HGH or HGH20k. Its smaller size is due to elimination of the first 45 nucleotides of the third exon of the mRNA and of the amino acids that correspond to positions 32-46 of the hormone, producing a protein with 176 amino acid

aminoacidic residues, four alpha helixes, and two sulphide bonds

**2.2 Hormones of the human growth hormone family** 

only one of the HGH family that has been commercialized.

postulated regenerative properties (3).

**HGH22k** 

**HGH20k** 

residues (4).

**2.1 Structure of growth hormones** 

somatic growth and lactation.

Several isoforms also derive from the GH gene expressed in the placenta (hGH-V)(Table 1). The most abundant mRNA from this gene in the placenta at terminus also codifies for a 22 kDa isoform. A less abundant isoform (HGHV2) originate from a species of mRNA that retains the fourth intron and due to this, it codifies for a 26 kDa protein that anchors to the membrane and which could have a local action (8). A 25 kDa protein is also derived by glycosylation of residue 140 of asparagine from the 22 kDa isoform (9, 10). Finally, two new transcripts of this gene have recently been identified: one already known that as in the case of the HGH20k also produces a 20 kDa protein, and another novel splicing variation that results in a mRNA known as hGHV3, that traduces into a 24 kDa isoform (11).


\*Only the mRNAs that codify each have been identified.

Table 1. HGH-V isoforms generated by alternative splicing and processing

During pregnancy, while hypophyseal HGHN progressively disappears from the maternal circulation until undetectable values are reached at weeks 24 to 25, HGHV progressively increases until birth, suggesting that it has a key role during human gestation (12). It has also been found that in cases of intrauterine growth restriction, circulating levels of HGHV measured between week 31 and birth are lower than those reported in normal pregnancy (13, 14, 15).

Finally, although low levels of this hormone have been associated with intrauterine growth retardation, cases of hGH-V gene deletion have also been reported, but without an apparent pathology (16).

#### **2.3 Human chorionic somatomammotropin (HCS)**

HCS is detected in maternal serum from the fourth week of gestation, increasing throughout the pregnancy in a linear fashion, and reaching high production levels of a couple of grams per day at the end of gestation. These actions result in both elevation of glucose and amino acids in the maternal circulation. These are in turn used by the fetus for his/her development. It also generates free fatty acids (by lipolytic effect), which are used as an energy source by the fetus (17, 18). Little is known about the HCS physiological role, and still is not known its action mechanism. Producing rHCS by biotechnology will help to advance these investigations.

#### **2.4 In vitro bioassays for GHs and CSHs**

As stated above except for HGH22k, the functions of the rest of hormones of the human GH family have been not completely defined. Their biological activities are being studied, classifying them into at least two general categories:


#### **2.5 The human GH locus**

Besides the two hGH genes (normal and variant), three HCSs complement the multigenic HGH family from the human genome and these are arranged in the following order: HGH-N, HCS-1, HCS-2, HGH-V y HCS-3 (21, 22) (Figure 2). While HCS-1 appears to be a pseudogene, HCS-2 and HCS-3 are very active in the placenta and interestingly; mature versions of the hormones that they codify are identical (23).

In the last few years, in our laboratory, all the hGH and HCS genes have been cloned and expressed in cell culture, and the factors that affect their levels of expression have been particularly studied (24).

In the same way, and using polymerase chain reaction (PCR) with consensus primers, several new genes and complementary DNAs (cDNAs) to the mRNA of numerous GHs have been isolated in our laboratory, mainly from mammals (unpublished results).

#### **3. Growth hormone of animal origin**

#### **3.1 Bovine growth hormone (BGH)**

Bovine growth hormone (BGH) or bovine somatotropin improves the efficiency of milk production (per unit of food consumed) (25), and the production (body weight) and composition (muscle: fat ratio) of meat (26). In the case of milk cows, this permits a reduction in the number of animals needed for milk production and a subsequent savings in maintenance, feeding, water, drugs, etc. It also reduces the production of manure, and nitrogen from urine and methane (27).

per day at the end of gestation. These actions result in both elevation of glucose and amino acids in the maternal circulation. These are in turn used by the fetus for his/her development. It also generates free fatty acids (by lipolytic effect), which are used as an energy source by the fetus (17, 18). Little is known about the HCS physiological role, and still is not known its action mechanism. Producing rHCS by biotechnology will help to

As stated above except for HGH22k, the functions of the rest of hormones of the human GH family have been not completely defined. Their biological activities are being studied,

a. Somatogenic activities. These involve linear bone growth and alterations in carbohydrate metabolism; effects that are in part mediated by local and hepatic generation of insulinlike growth factor-I (IGF-I). The somatogenic activity of HGHV has been studied by stimulating body weight increase in hypophysectomized rats, reporting a linear

b. Lactogenic activities. These include stimulation of lactation and reproductive functions (20). The lactogenic activities of this hormone have been studied using a cell model (by mitogenic response to Nb2 cells) and a response that is parallel to HGH22k has been

Besides the two hGH genes (normal and variant), three HCSs complement the multigenic HGH family from the human genome and these are arranged in the following order: HGH-N, HCS-1, HCS-2, HGH-V y HCS-3 (21, 22) (Figure 2). While HCS-1 appears to be a pseudogene, HCS-2 and HCS-3 are very active in the placenta and interestingly; mature

In the last few years, in our laboratory, all the hGH and HCS genes have been cloned and expressed in cell culture, and the factors that affect their levels of expression have been

In the same way, and using polymerase chain reaction (PCR) with consensus primers, several new genes and complementary DNAs (cDNAs) to the mRNA of numerous GHs

Bovine growth hormone (BGH) or bovine somatotropin improves the efficiency of milk production (per unit of food consumed) (25), and the production (body weight) and composition (muscle: fat ratio) of meat (26). In the case of milk cows, this permits a reduction in the number of animals needed for milk production and a subsequent savings in maintenance, feeding, water, drugs, etc. It also reduces the production of manure, and

have been isolated in our laboratory, mainly from mammals (unpublished results).

advance these investigations.

**2.5 The human GH locus** 

particularly studied (24).

**3. Growth hormone of animal origin** 

**3.1 Bovine growth hormone (BGH)** 

nitrogen from urine and methane (27).

**2.4 In vitro bioassays for GHs and CSHs** 

classifying them into at least two general categories:

reported, although it is significantly less (19).

versions of the hormones that they codify are identical (23).

increase comparable to that produced by HGH22k (19).

Fig. 2. HGH-HCS multigenic complex. Located on Chromosome 17, every gene is indicated; the tissue where they are expressed and the proteic isoforms that are produced are shown

Milk from cows treated with rBGH, does not differ from that of untreated cows (28, 29). The characteristics that have been evaluated include the freezing point, pH, thermal properties, susceptibility to oxygenation, and sensory characteristics, including taste; in fact all organoleptic properties are conserved. Also, differences have not been found in the properties necessary for producing cheese, including initial growth of the culture, coagulation, acidification, production and composition (29).

rBGH is administered subcutaneously and is dispensed as a long-acting suspension that is applied in a determined period of time. The taste of bovine meat and milk treated with rBGH is not altered, but the fat content is less.

#### **3.2 Caprine growth hormone (CHGH)**

For small ruminants there are studies in lactating goats in which the administration of rBGH increased milk production 23% and stimulated mammary gland growth more than in those that were frequently milked, with it being similar to prolactin (30). However, the production of recombinant CHGH, which is identical to ovine and thus can be used in both animals, had not been reported, until we achieve its expression on the methylotrophic yeast *Pichia pastoris*. (See section 7.2).

#### **3.3 Equine growth hormone (ECGH)**

With regard to horses, GH is used in the prevention of muscle wasting, in the repair of tendons and fractured bones, as well as for the treatment of anovulation in mares. Besides this, it is also used for repairing muscle tissue, to tonify and invigorate race horses, and for improving physical conditions in older horses by restoring nitrogen balance. It can also stimulate growth and early maturity in young horses, increase milk production in lactating mares and promote wound healing, especially of bone and cartilage (31, 32), as occurred in the case shown in Figure 3.

Fig. 3. Uses of equine GH. The race horse "Might and Power" (right) became the winner of the Melbourne Cup in 1997. But in 1999, a tendon from one of its hooves was severely damaged. The horse was treated with ECGH, recovered and in 2000 was able to return to horse racing (32)

#### **3.4 Canine growth hormone (CFGH)**

With regard to the dog (*Canis familiaris*), each day there is more evidence of the role that its GH (CFGH) plays in bone fracture treatment, in which the hormone helps reduce the bone restoration period (33).

It is no less important in the treatment of obesity in dogs, thanks to the metabolism activation produced by the hormone in removing fatty acids, and in general, in counteracting symptoms related to the presence or absence of the same GH. Also, since this hormone is identical to pig GH (PGH) (33), its virtues are valid for the application of CFGH in the porcine industry, where it generates leaner meat (34), which is of greater value.

#### **3.5 Feline growth hormone (FCGH)**

Although there is very little literature on cat GH (FCGH), the benefits identified in other GHs apply to this feline species, since these animals present the symptoms mentioned before for dogs, which are caused by the absence or low concentration of FCGH (dwarfism and alopecia, among others). Also, as referred to in the literature, biological tests of adipogenic activity in culture cells use cat serum (which contains FCGH) instead of bovine serum, because FCGH lacks adipogenicity (17).

Therefore, recombinant production of this GH would be useful in the mentioned tests. It is important to point out the usefulness that recombinant FCGH would have in future research on the metabolic study and role of this hormone in this and other feline species, including of course, large wild cats in captivity.

#### **4. Biological potential of GHs**

178 Genetic Engineering – Basics, New Applications and Responsibilities

improving physical conditions in older horses by restoring nitrogen balance. It can also stimulate growth and early maturity in young horses, increase milk production in lactating mares and promote wound healing, especially of bone and cartilage (31, 32), as occurred in

Fig. 3. Uses of equine GH. The race horse "Might and Power" (right) became the winner of the Melbourne Cup in 1997. But in 1999, a tendon from one of its hooves was severely damaged. The horse was treated with ECGH, recovered and in 2000 was able to return to

With regard to the dog (*Canis familiaris*), each day there is more evidence of the role that its GH (CFGH) plays in bone fracture treatment, in which the hormone helps reduce the bone

It is no less important in the treatment of obesity in dogs, thanks to the metabolism activation produced by the hormone in removing fatty acids, and in general, in counteracting symptoms related to the presence or absence of the same GH. Also, since this hormone is identical to pig GH (PGH) (33), its virtues are valid for the application of CFGH in the porcine industry, where it generates leaner meat (34), which is of greater value.

Although there is very little literature on cat GH (FCGH), the benefits identified in other GHs apply to this feline species, since these animals present the symptoms mentioned before for dogs, which are caused by the absence or low concentration of FCGH (dwarfism and alopecia, among others). Also, as referred to in the literature, biological tests of adipogenic activity in culture cells use cat serum (which contains FCGH) instead of bovine

the case shown in Figure 3.

horse racing (32)

restoration period (33).

**3.4 Canine growth hormone (CFGH)** 

**3.5 Feline growth hormone (FCGH)** 

serum, because FCGH lacks adipogenicity (17).

#### **4.1 Growth hormones of human origin**

Although HGH22k is widely commercialized and more functions now have been recognized to it (Table 2), the same does not occur with the other proteins and isoforms from this family; essentially the 20 kDa isoform of HGH, HGHV, also the isoform of 20 kDa of the latter (HGHV20k), and lastly, HCS. Partly because of this, many of their functions and mechanisms of action are still unknown.


(Taken from Elian y cols., 1999), (3).

Table 2. New functions atributed to HGH22k

It is believed that some of the hormone's less abundant natural variants, such as HGH20k, could retain desirable properties of the principal hormone and lack some of its other undesirable effects, such as its diabetogenic effect, which occurs with prolonged use (35).

#### **4.2 Growth hormones of animal origin**

The biotechnological potential of GHs could be enormous, since besides its use in species of the same origin, it has been demonstrated that the GHs of mammals have activity in phylogenetically lower animals.

For example, BGH and porcine GH (PGH) have been used experimentally for the treatment of hypophyseal dwarfism in dogs (36) and cats (37).

Regarding farm animals, porcine, bovine, caprine and ovine livestock have been treated with exogenous GH to improve production, since it increases food conversion efficiency, growth rate, weight gain, and milk and meat production. What is surprising is the finding that BGH stimulates salmon growth, and even more interesting that bovine chorionic somatomammotropin (BCS) works even better (38).

#### **5. Expression systems for growth hormones**

#### **5.1 The history of human recombinant GH**

As previously mentioned, among the first cDNAs cloned and expressed in the bacteria *Escherichia coli* is precisely HGH (1). This expression system has been used since 1985 for the production of recombinant HGH by Genentech (protropin), which was later followed by Lily (humatrope), Biotech (biotropin), Novo Nordisk (norditropin), Serono (serostim), and others.

#### **5.2 Different biotechnological hosts**

Since the recombinant protein is frequently recovered from *E. coli* with undesirable modifications (extra methionine, incorrect folding, aggregated forms, etc.) and contaminated with highly pyrogenic substances, toilsome purification schemes are needed to obtain it with the desired purity, structure and biological activity. For this, subsequent efforts have focused on the search for better expression systems, with production being attempted with *Saccharomyces cerevisiae* (39), *Bacillus subtilis* (40), mammal cell cultures (41), as well as transgenic animals (42). Unfortunately, these expression systems do not offer a production level greater than that of *E. coli* and therefore in most cases they are not profitable.

In our laboratory, we succeeded in producing HGH22k in *E. coli* by fusing it with maltose binding protein (rHGH-MBP) in 1994 (unpublished results). However, due to the fact that to recover the hormone, whether from the periplasm or the cytoplasm, complicated strategies were needed, together with the limitations of the bacterial systems for folding and processing foreign proteins correctly, we proposed searching for an expression system that allows synthesizing the protein, purifying it with greater ease while retaining functionality. Thus, the evaluation of different expression systems was started in our laboratories, considering the methylotrophic yeast *Pichia pastoris* as the best (43).

It is believed that some of the hormone's less abundant natural variants, such as HGH20k, could retain desirable properties of the principal hormone and lack some of its other undesirable effects, such as its diabetogenic effect, which occurs with prolonged use (35).

The biotechnological potential of GHs could be enormous, since besides its use in species of the same origin, it has been demonstrated that the GHs of mammals have activity in

For example, BGH and porcine GH (PGH) have been used experimentally for the treatment

Regarding farm animals, porcine, bovine, caprine and ovine livestock have been treated with exogenous GH to improve production, since it increases food conversion efficiency, growth rate, weight gain, and milk and meat production. What is surprising is the finding that BGH stimulates salmon growth, and even more interesting that bovine chorionic

As previously mentioned, among the first cDNAs cloned and expressed in the bacteria *Escherichia coli* is precisely HGH (1). This expression system has been used since 1985 for the production of recombinant HGH by Genentech (protropin), which was later followed by Lily (humatrope), Biotech (biotropin), Novo Nordisk (norditropin), Serono (serostim), and

Since the recombinant protein is frequently recovered from *E. coli* with undesirable modifications (extra methionine, incorrect folding, aggregated forms, etc.) and contaminated with highly pyrogenic substances, toilsome purification schemes are needed to obtain it with the desired purity, structure and biological activity. For this, subsequent efforts have focused on the search for better expression systems, with production being attempted with *Saccharomyces cerevisiae* (39), *Bacillus subtilis* (40), mammal cell cultures (41), as well as transgenic animals (42). Unfortunately, these expression systems do not offer a production level greater than that of *E. coli* and

In our laboratory, we succeeded in producing HGH22k in *E. coli* by fusing it with maltose binding protein (rHGH-MBP) in 1994 (unpublished results). However, due to the fact that to recover the hormone, whether from the periplasm or the cytoplasm, complicated strategies were needed, together with the limitations of the bacterial systems for folding and processing foreign proteins correctly, we proposed searching for an expression system that allows synthesizing the protein, purifying it with greater ease while retaining functionality. Thus, the evaluation of different expression systems was started in our laboratories,

**4.2 Growth hormones of animal origin** 

of hypophyseal dwarfism in dogs (36) and cats (37).

somatomammotropin (BCS) works even better (38).

**5. Expression systems for growth hormones** 

**5.1 The history of human recombinant GH** 

**5.2 Different biotechnological hosts** 

therefore in most cases they are not profitable.

considering the methylotrophic yeast *Pichia pastoris* as the best (43).

others.

phylogenetically lower animals.

#### **5.3** *Pichia pastoris* **as a biotechnological host for GHs**

Yeasts offer the best of both prokaryotes and eukaryotes, since, in addition to performing some of the post-translational modifications that are common in superior organisms, they are easily grown in flasks and bioreactors, like bacteria, using simple and inexpensive culture media (44).

*P. pastoris* is a methylotrophic yeast (capable of growing in methanol as its only carbon source) that performs post-translational modifications, produces recombinant protein levels of one or two orders of magnitude above that of *Saccharomyces cerevisiae* (45), is capable of secreting heterologic proteins into the culture media (where the levels of native protein are very low), and in contrast with the latter, can be cultivated at cell densities of more than 100 g/L of dry weight (46).

#### **6. Recombinant growth hormones**

In our laboratories, we identified as a scientific objective and a technological advantage, the construction and evaluation of GH protein producing *P. pastoris* strains. This as a first step in evaluating its potential in medicine as well as in animal health and productivity; searching to develop both infrastructure and experience in producing, purifying, and testing its biological activity.

Also, as previously mentioned, mammalian GHs have activity in phylogenetically inferior animals, nevertheless potentially adverse reactions to heterologic GHs can be triggered, which is why having a GH specific-species would avoid these undesirable side effects.

Regarding human hormones, we proposed constructing productive strains for the HGH22k, the HGH20k, the HGHV, and the HCS. With regard to animal GHs, we channeled our efforts into building strains to produce GHs from bovines (BGH), caprines (CHGH), ovines (OGH), equines (ECGH), canines (CFGH), porcines (PGH) and felines (FCGH); all based on the *Pichia pastoris* yeast expression system.

For this, the following experimental strategy was proposed:


As a result of this experimental work, we achieved the followings:


v. The data from the physicochemical and biological characterizations showed that the methodology described herein generates heterologous proteins that are identical to their natural counterparts and biologically active.

### **7. Technological platform for the production of recombinant GHs**

#### **7.1 Overall strategy**

As depicted in figures 4, the following are the two stages of the overall strategy in which the work was divided:


#### **7.2 Construction of propagating GH cDNA plasmids (pBS-XGHs)**

Oligonucleotides for GHs cDNAs amplification by PCR were designed based on consensus nucleotide sequences of GHs (mature region) of related mammals. Extra restriction sites were added on their flanks (*Xho*I and *Avr*II) to facilitate insertion of the amplicon into the expression vector. With these, each of the hormones' cDNAs was amplified from plasmids previously constructed in our laboratory carrying the respective nucleotide sequences. Each amplicon was cloned into propagating plasmid such as the pBS II KS plasmid (+) and subsequently subcloned into the yeast expression vector pPIC9 at its multicloning site, between the restriction sites *Xho*I and *Avr*II (after previous purification of the corresponding fragment and vector), thereby giving rise to each of the pPIC9-XGH expression plasmids.

In CHGH's case, which differs from BGH in a single aa residue, a different strategy was implemented. Site-directed mutagenesis was used relying on a primer to convert codon 130 of BGH cDNA into one corresponding to CHGH. A 345 bp region containing the mutated GH cDNA was thus amplified, which was cloned in pBS and later transferred into pPIC9- BGH to converted it into pPIC9-CHGH (49).

#### **7.3 Construction of expression plasmids (pPIC9-XGHs) for each hormone**

Preparative digestions of pBS-XGH and pPIC9 with the enzymes *Xho*I and *Avr*II were performed for all GHs except for CHGH. For CHGH, *Apa*I and *Xma*I (natural site) enzymes, which release a 133 bp fragment containing the mutagenized codon for CHGH, were used. This was purified and linked into the previously digested pPIC9-BGH vector in the same sites, replacing the fragment to originate the pPIC9-CHGH expressor vector The ligation reactions between pPIC9 and each cDNA were used to transform competent Ca++ cells of XL1-Blue *Escherichia coli*. PCR was used to verify that the resulting tranformants indeed carried each pPIC9-XGH, where "x" corresponds to each of the sequences of the hormone in question. The candidate clones produced by PCR with AOX1 primers for an amplicon of 1050 bp, since the expression cassette for each hormone is flanked by long regions of the AOX1 gene. While strains that were not integrated into the "cassette" gave rise to an amplicon of only 500 bp.

v. The data from the physicochemical and biological characterizations showed that the methodology described herein generates heterologous proteins that are identical to

As depicted in figures 4, the following are the two stages of the overall strategy in which the

a. Construction of *P. pastoris* strains carrying the hormones´ expression cassettes producing

Oligonucleotides for GHs cDNAs amplification by PCR were designed based on consensus nucleotide sequences of GHs (mature region) of related mammals. Extra restriction sites were added on their flanks (*Xho*I and *Avr*II) to facilitate insertion of the amplicon into the expression vector. With these, each of the hormones' cDNAs was amplified from plasmids previously constructed in our laboratory carrying the respective nucleotide sequences. Each amplicon was cloned into propagating plasmid such as the pBS II KS plasmid (+) and subsequently subcloned into the yeast expression vector pPIC9 at its multicloning site, between the restriction sites *Xho*I and *Avr*II (after previous purification of the corresponding fragment and vector), thereby giving rise to each of the

In CHGH's case, which differs from BGH in a single aa residue, a different strategy was implemented. Site-directed mutagenesis was used relying on a primer to convert codon 130 of BGH cDNA into one corresponding to CHGH. A 345 bp region containing the mutated GH cDNA was thus amplified, which was cloned in pBS and later transferred into pPIC9-

Preparative digestions of pBS-XGH and pPIC9 with the enzymes *Xho*I and *Avr*II were performed for all GHs except for CHGH. For CHGH, *Apa*I and *Xma*I (natural site) enzymes, which release a 133 bp fragment containing the mutagenized codon for CHGH, were used. This was purified and linked into the previously digested pPIC9-BGH vector in the same sites, replacing the fragment to originate the pPIC9-CHGH expressor vector The ligation reactions between pPIC9 and each cDNA were used to transform competent Ca++ cells of XL1-Blue *Escherichia coli*. PCR was used to verify that the resulting tranformants indeed carried each pPIC9-XGH, where "x" corresponds to each of the sequences of the hormone in question. The candidate clones produced by PCR with AOX1 primers for an amplicon of 1050 bp, since the expression cassette for each hormone is flanked by long regions of the AOX1 gene. While strains that were not integrated into the "cassette" gave rise to an

**7.3 Construction of expression plasmids (pPIC9-XGHs) for each hormone** 

**7. Technological platform for the production of recombinant GHs** 

b. Production and characterization of the recombinant hormones.

**7.2 Construction of propagating GH cDNA plasmids (pBS-XGHs)** 

their natural counterparts and biologically active.

**7.1 Overall strategy** 

work was divided:

rGHs/HCSs.

pPIC9-XGH expression plasmids.

amplicon of only 500 bp.

BGH to converted it into pPIC9-CHGH (49).

Fig. 4. General strategy for strain construction and recombinant hormones production. (A) Genetic engineering phase. The steps followed to construct and characterize new strains of GHs and HCSs producing *Pichia pastoris* are shown. Protocols followed were based in different techniques (47, 48). (B) Biotechnology phase. The steps followed for the production and scaling, semipurification and bioassay of each of the recombinant hormones are shown

Fig. 5. Detection of the expression "cassettes" of cfGH, ecGH, fcGH and hCS in P. pastoris genome. Analysis by PCR with AOX1 primer yeast strains transfected. In each case, the 1050 bp corresponds to the expression cassette of the recombinant hormone in question, while the 2105 bp to the AOX1 gene of the yeast itself. A) The diagram shows the linearized "cassette" of XGH and the gel products (which transfected into *Pichia pastoris* integrate the "cassette" into the genome) with *Sac*I enzyme: cfGH = dog GH, ecGH = horse GH, fcGH = cat GH and HCS = human CS; in lane 1 NC-GH = uncut plasmid pPIC9 and in lane 6 NC = uncut pPIC9 plasmid. B) CF (1 and 2) = dog GH lanes 2 and 3 respectively, and CD (1 and 2) = horse GH lanes 4 and 5, respectively. C) (-) = negative PCR lane 1, Plas= amplification positive control lane 2, Lev = *Pichia pastoris* genomic DNA lane 3, M= pb marker lane 4 and fc (1 and 2) = cat GH lanes 5 and 6 respectively. D) Lanes 1 to 4 correspond to strains with the HCS "cassette". M = marker-bp λ *Bste*II. The gels correspond to 1% agarose

#### **8. Construction of GHs producing** *Pichia pastoris* **strains**

The *Pichia pastoris* GS115 strain has a mutation in the histidinol dehydrogenase (his4) gene, which prevents it from synthesizing histidine. The class of plasmids used to transform it contains this gene (his4). The transformants are selected for their ability to restore growth in

**1 2 3 4 5 6 M 1 2 3 4** 

**Kpb 3.7 2.3 1.9 1.4 1.3**

**0.7**

 **aox1 2105bp fcGH 1050bp**

Fig. 5. Detection of the expression "cassettes" of cfGH, ecGH, fcGH and hCS in P. pastoris genome. Analysis by PCR with AOX1 primer yeast strains transfected. In each case, the 1050 bp corresponds to the expression cassette of the recombinant hormone in question, while the 2105 bp to the AOX1 gene of the yeast itself. A) The diagram shows the linearized "cassette" of XGH and the gel products (which transfected into *Pichia pastoris* integrate the "cassette" into the genome) with *Sac*I enzyme: cfGH = dog GH, ecGH = horse GH, fcGH = cat GH and HCS = human CS; in lane 1 NC-GH = uncut plasmid pPIC9 and in lane 6 NC = uncut pPIC9 plasmid. B) CF (1 and 2) = dog GH lanes 2 and 3 respectively, and CD (1 and 2) = horse GH lanes 4 and 5, respectively. C) (-) = negative PCR lane 1, Plas= amplification positive control lane 2, Lev = *Pichia pastoris* genomic DNA lane 3, M= pb marker lane 4 and fc (1 and 2) = cat GH lanes 5 and 6 respectively. D) Lanes 1 to 4 correspond to strains with the HCS "cassette".

The *Pichia pastoris* GS115 strain has a mutation in the histidinol dehydrogenase (his4) gene, which prevents it from synthesizing histidine. The class of plasmids used to transform it contains this gene (his4). The transformants are selected for their ability to restore growth in

**Kpb 3.7 2.3 1.9 1.4 1.3 cfGH 1050bp** **1 2 3 4 5**

**M CF1 CF2 CD1 CD2**

**M 1 2 3 4** 

 **aox1 2105bp**

 **ecGH 1050bp**

 **HCS 1050bp**

 **aox1 2105bp**

 **pPIC9 8.0 Kpb**

**NC cfGH ecGH fcGH HCS NC**

**Linearization**

**A B**

**xGH cassette**

 **C D**

**(-) Plas Lev M fc1 fc2**

M = marker-bp λ *Bste*II. The gels correspond to 1% agarose

**8. Construction of GHs producing** *Pichia pastoris* **strains** 

**pPIC9-GH 8.6 Kpb**

a medium lacking histidine. The plasmid vectors of the pPIC series and those constructed to express the GHs are of this class.

#### **8.1 Insertion of GHs expression cassettes into the genome of** *P. pastoris*

Each pPIC-XGH vector was linearized with the enzyme *Sac*I, transformed into the yeast previously made competent for transformation and left exposed to the homologous regions in the yeast genome necessary for recombination.

After incubating the DNA with competent cells, transformation reactions were plated to recover clones needing no histidine to grow (HIS+ transformants). Then transformants were analyzed on their genomic DNAs by PCR using AOX1 primers to verify the presence of the transgenic hormone expressing cassette.

Verification of integration into the yeast genome of the expression cassette of the hormones was achieved in agarose gel by confirming that the amplification reaction rendered a prominent band of 1050 bp, which corresponds to the expression cassette of the hormone involved in each case and another of 2105 bp corresponding to the endogenous gene AOX1 of the yeast genome (Figure 5). In addition, each hormone "cassette" was subjected to nucleotide sequencing to verify that all they corresponded to the expected growth hormones.

#### **8.2 Analysis of new** *Pichia pastoris* **strains´ phenotypes**

*Pichia pastoris* strains were grown and the biomass was adjusted to low cell density (0.5 u at 600 nm). These were transferred to induction medium with 0.5% methanol and grown for

Fig. 6. Mut phenotype characterization in *Pichia pastoris* strains. Growth kinetics in minimal medium using methanol as sole carbon source. Induction was started at the density of 0.5 U and ended after about 100 hrs. (A) Plot of the samples P3-1 and 2 = dog GH 1 and 2 strains, CS3 CS-2 = 2 and strain human strains pPIC9 = "mock" with the pPIC9 plasmid. (B) Graph of the C6-5T samples = horse GH, P3-1T =dog GH, C6-4T = horse GH, GH P3-Q2 = P3-dog and dog-2B = GH

100 hours with the addition of methanol every 24 hours to compensate for its evaporation. Biomass growth was analyzed under methanol as the sole carbon source. The Mut+ phenotype strains metabolize methanol more rapidly, achieving significantly higher cell densities than their Muts counterparts that metabolize more slowly, appreciating a slight increase in biomass under the same fermentation conditions.

An analysis of the growth of the strains after 100-hour fermentation with 0.5% methanol identified the Mut phenotype of each strain.

After fermentation, the strains found to be Mut+ reached about 15 optical units at 600 nm, while those that were Muts did not exceed 2 units (Fig. 6). In the strains that had been built previously, their Mut phenotype was inferred when these were fermented in the bioreactor.

### **9. Production and analysis of recombinant hormones in the flask**

To test the fermentation of strains, a biomass was generated in a flask. This was inoculated with a colony of each strain in 25 ml of biomass producing culture medium (BMGY) (50). This was incubated at 30°C at 250 rpm for 24 to 48 hours for the first stage of growth until a biomass with an OD of 600 nm of 10 was reached.

For the second stage, which is the induction of the recombinant hormone production, yeasts were harvested by centrifugation and the packed cells were washed with 30 mL sterile water, then these were pelleted and resuspended in fresh cassette induction medium (with methanol) (BMMY) (50). The induction was maintained by adding methanol every 24 hours to a final concentration of 1% to compensate for loss by evaporation. The experiment lasted 96 hrs. Figure 7 shows the process that was followed.

When analyzing the polyacrylamide gels of proteins from the culture media of each strain, we observed that all constructions produced and directed the secretion into the medium of the recombinant hormone in question to a greater or lesser extent. For the particular case of CFGH it was observed that a strain of Muts phenotype displayed better production of recombinant protein than its counterpart Mut+. Strain of HGH-V proved to be the least productive (Fig. 8).

Figure 9 shows the gel with the results of the production of all recombinant strains generated in *Pichia pastoris*. They all produced different amounts of their respective hormone at the level of 22 kDa, except for the HGH20k, which, migrated below the rest of the recombinant hormones.

The percentage of each recombinant hormone in the culture medium was estimated by densitometry of each gel. For this we used the Gel-Doc software by BIO-RAD (Hercules, CA. EUA) and the ImageJ program (51). The results of estimation of the percentage of each hormone in relation to background proteins from *Pichia pastoris* were: HCS = 65%, CFGH = 60%, HGH22k = 30%, ECGH = 30%, BGH = 25%, FCGH = 25%, CHGH = 25%, HGH20k = 12% and HGHV = 8%.

Production kinetics was carried out for CHGH strain in a flask with a volume of 50 ml of rich medium. Samples were taken at 24, 48, 72, 96 and 120 hours of induction with methanol with restitution every 24 hours of 1% methanol. Bradford protein determination showed that the production of total protein secreted into the culture medium was 20μg/mL by densitometry and 60% represented CHGH giving us 12μg/mL of production of the recombinant hormone.

100 hours with the addition of methanol every 24 hours to compensate for its evaporation. Biomass growth was analyzed under methanol as the sole carbon source. The Mut+ phenotype strains metabolize methanol more rapidly, achieving significantly higher cell densities than their Muts counterparts that metabolize more slowly, appreciating a slight

An analysis of the growth of the strains after 100-hour fermentation with 0.5% methanol

After fermentation, the strains found to be Mut+ reached about 15 optical units at 600 nm, while those that were Muts did not exceed 2 units (Fig. 6). In the strains that had been built previously, their Mut phenotype was inferred when these were fermented in the bioreactor.

To test the fermentation of strains, a biomass was generated in a flask. This was inoculated with a colony of each strain in 25 ml of biomass producing culture medium (BMGY) (50). This was incubated at 30°C at 250 rpm for 24 to 48 hours for the first stage of growth until a

For the second stage, which is the induction of the recombinant hormone production, yeasts were harvested by centrifugation and the packed cells were washed with 30 mL sterile water, then these were pelleted and resuspended in fresh cassette induction medium (with methanol) (BMMY) (50). The induction was maintained by adding methanol every 24 hours to a final concentration of 1% to compensate for loss by evaporation. The experiment lasted

When analyzing the polyacrylamide gels of proteins from the culture media of each strain, we observed that all constructions produced and directed the secretion into the medium of the recombinant hormone in question to a greater or lesser extent. For the particular case of CFGH it was observed that a strain of Muts phenotype displayed better production of recombinant protein than its counterpart Mut+. Strain of HGH-V proved to be the least

Figure 9 shows the gel with the results of the production of all recombinant strains generated in *Pichia pastoris*. They all produced different amounts of their respective hormone at the level of 22 kDa, except for the HGH20k, which, migrated below the rest of

The percentage of each recombinant hormone in the culture medium was estimated by densitometry of each gel. For this we used the Gel-Doc software by BIO-RAD (Hercules, CA. EUA) and the ImageJ program (51). The results of estimation of the percentage of each hormone in relation to background proteins from *Pichia pastoris* were: HCS = 65%, CFGH = 60%, HGH22k = 30%, ECGH = 30%, BGH = 25%, FCGH = 25%, CHGH = 25%,

Production kinetics was carried out for CHGH strain in a flask with a volume of 50 ml of rich medium. Samples were taken at 24, 48, 72, 96 and 120 hours of induction with methanol with restitution every 24 hours of 1% methanol. Bradford protein determination showed that the production of total protein secreted into the culture medium was 20μg/mL by densitometry and 60% represented CHGH giving us 12μg/mL of production of the recombinant hormone.

**9. Production and analysis of recombinant hormones in the flask** 

increase in biomass under the same fermentation conditions.

identified the Mut phenotype of each strain.

biomass with an OD of 600 nm of 10 was reached.

96 hrs. Figure 7 shows the process that was followed.

productive (Fig. 8).

the recombinant hormones.

HGH20k = 12% and HGHV = 8%.

Fig. 7. Outline of the fermentation process. General procedure for the biotechnological production of recombinant hormones by fermentation of each strain. Strains were plated to activate them, incubated in liquid medium to generate a biomass flask, and the induced transgene expression by adding methanol. The culture medium was analyzed by SDS-PAGE in search of the hormones that are migrating around 22 kDa

#### **10. Production scaling in the bioreactor**

When passing to a bioreactor and increasing the scale, it is possible to obtain protein concentrations 20 to 200 times greater than in flasks. In the fermentor *Pichia pastoris* reaches high cell densities greater than 100 g/L of dry weight (46).

The fermentor was Bioflo 3000 (1 liter) of New Brunswick Scientific (NBSC) (NJ. EUA). The type of fermentation conducted was in fed-batch. The parameters monitored were scheduled addition of substrates to the fermentor, pH, percentage of dissolved oxygen, agitation, temperature, and aeration. The process involved three basic steps: 1) obtaining high densities of biomass, 2) induction of the cassette expression of each hormone with methanol and 3) harvest of biomass and culture medium containing the recombinant protein. Figure 10 shows the steps followed for the recombinant production of each hormone.

Fig. 8. Production in *Pichia pastoris* of recombinant GHs (CFGH, HGH, HGH-V and ECGH) at flask level. The bands correspond to the GHs proteins resolved at the level of 22 kDa that come from the culture media induced with methanol. The lanes are: M = molecular weight marker, HGHV1 = HGH variant strain 1; HGHV2 = HGH variant strain 2; HGH = normal pituitary HGH of 22 kDa; DGH-1 = Dog GH strain 1; DGH-2 = dog or *Canis familiaris* GH strain 2; ECGH-2 = horse GH strain 2, ECGH-4 = horse GH strain 4 and the last lane identified as (-) = negative control of PCR. Mut+= methanol utilization plus; Muts = "methanol utilization slow". Note the prominent band of the DGH-2 corresponding to the Muts phenotype, compared to the lower intensity of Mut+. The samples correspond to 500 µL concentrates of the original media. Gel corresponds to one of 15% polyacrylamide-SDS stained with Coomassie blue

Fig. 9. Flask production in all strains yielding recombinant hormones. In all cases a prominent band is seen (except for HGH20k) at the 22 kDa level for each hormone. They are seen in their respective lane in each case indicated by their name. The lanes of the left side gel show the proteins from the culture media with recombinant hormones: horse = ECGH, goat = CHGH, HCS (6 and 2) = Human chorionic somatomammotropin clones 6 and 2, dog = CFGH and HGH (1 and 2) = cloned human GH 1 and 2. In the right gel lanes: cat (1 and 2) = GH 1 and 2 from cat; cow = BGH; HGHv = HGH placental variant and HGH20k = isoform of 20 kDa of hGH. The pPIC9 lane refers to the "mock" strain of *Pichia pastoris*. SDS-PAGE 15% gels are silver stained

**Mut Mut + S**

**GH**

**M HGHV1 HGHV2 HGH-N DGH-1 DGH-2 ECGH2 ECGH4 (-)**

Fig. 8. Production in *Pichia pastoris* of recombinant GHs (CFGH, HGH, HGH-V and ECGH) at flask level. The bands correspond to the GHs proteins resolved at the level of 22 kDa that come from the culture media induced with methanol. The lanes are: M = molecular weight marker, HGHV1 = HGH variant strain 1; HGHV2 = HGH variant strain 2; HGH = normal pituitary HGH of 22 kDa; DGH-1 = Dog GH strain 1; DGH-2 = dog or *Canis familiaris* GH strain 2; ECGH-2 = horse GH strain 2, ECGH-4 = horse GH strain 4 and the last lane identified as (-) = negative control of PCR. Mut+= methanol utilization plus; Muts = "methanol utilization slow". Note the prominent band of the DGH-2 corresponding to the Muts phenotype, compared to the lower intensity of Mut+. The samples correspond to 500 µL concentrates of the original media. Gel corresponds to one of 15% polyacrylamide-SDS stained with Coomassie blue

**pPIC9 M Horse Goat HCS6 HCS2 Dog HGH1 HGH2 pPIC9 M Cat1 Cat2 Cow GHGv HGH20k**

**24**

**20 GH**

Fig. 9. Flask production in all strains yielding recombinant hormones. In all cases a

prominent band is seen (except for HGH20k) at the 22 kDa level for each hormone. They are seen in their respective lane in each case indicated by their name. The lanes of the left side gel show the proteins from the culture media with recombinant hormones: horse = ECGH, goat = CHGH, HCS (6 and 2) = Human chorionic somatomammotropin clones 6 and 2, dog = CFGH and HGH (1 and 2) = cloned human GH 1 and 2. In the right gel lanes: cat (1 and 2) = GH 1 and 2 from cat; cow = BGH; HGHv = HGH placental variant and HGH20k = isoform of 20 kDa of hGH. The pPIC9 lane refers to the "mock" strain of *Pichia pastoris*. SDS-PAGE

**KD 66**

> **45 36**

**24**

**20**

**14**

**24**

**20**

15% gels are silver stained

Fig. 10. Recombinant hormone production bioreactor. This illustrates the stages of the biotechnological process of production, from the preparation of the fermentor and the medium, to the analysis of proteins in the fermented culture medium

Fig. 11. "DownStream" Process for recombinant hormones. The medium was separated from the biomass by centrifugation. This was pre-filtered with a 0.45µm membrane, and ultra-dialyzed in a cutoff membrane of 10 kDa. It was then passed through an anion exchange column (FF-QS) and/or in an affinity column by immobilized metal (IMAC). Purity was analyzed in PAGE-SDS, quantified, and finally lyophilized and stored for later use

### **11. Semipurification of recombinant hormones**

Figure 11 shows the process of purification or "downstream" that was followed for each recombinant hormone produced.

Each culture medium containing the recombinant hormone was ultra-dialyzed and ultraconcentrated. The pore membrane used was 14 kDa. At the same time it was ultradyalized. The ultraconcentrate obtained was lyophilized to preserve the samples and all the powder was recovered in 50 mL plastic tubes and weighed. Total protein was quantified in each lyophilized culture medium with the Bradford method (52). Samples were stored at -20°C until use.

Each sample was prepared for loading into a chromatographic column. Five mg of the total protein from each sample was adjusted to the conditions of the loading buffer. A column was loaded with Q-Sepharose fast flow anion exchange resin; column filling with resin was carried out by gravity flow. After passing the sample, washing was done with 10 mL of loading buffer. To recover the proteins from the column, 15 mL of elution buffer (loading buffer plus NaCl) was passed with an ionic strength increased sequentially with NaCl. Total protein was measured by the Bradford method (52) for each of the fractions recovered to see what percentage of the total they represented.

The collected fractions were visualized on discontinuous polyacrylamide gel concentrations of 4-15% under denaturing conditions (SDS-PAGE), and stained with Coomassie and silver techniques (53). The fractions were subjected to lyophilization, the powder was recovered and weighted, total protein was measured by the Bradford method. In addition, using PAGE-SDS the percentage of purity of the monomer was determined with ImageJ software. The samples were stored at -20°C until used for further analysis or to determine their biological activity.

#### **12. Testing the biological activity of recombinant hormones**

#### **12.1 Lactogenic activity bioassay**

The biological activity of hormones produced in *Pichia pastoris* recombinant was determined by their ability to promote proliferation of the Nb2 cell line, which comes from rat lymphoma (54, 55). GHs were also tested for their somatogenic activity in the adipocyte differentiation model based on the glyceraldehyde 3-phosphate dehydrogenase (GPDH) assay.

The cell-free culture medium was dialyzed and each hormone was quantified by gel densitometry. Dilutions of each recombinant hormones in the culture medium dialyzed were tested. Cell proliferation was determined by tetrazolium salt assay (MTT) (56) and was expressed as the average of three repetitions, in comparison with the positive control recombinant rat prolactin (rRPRL) and the negative control (culture medium without hormone). The concentrations tested were 0.001, 0.01, 0.1, 1, 10 and 50 nM of the following hormones, CFGH, ECGH, FCGH, HGH, HCS and RPRL. Upon completion of the testing time, we proceeded to measure the effect of hormones on cell proliferation by MTT assay (56). The activity was evaluated by color generation based on the reduction of tetrazolium salt (methyl 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyl tetrazolium) from yellow to purple forming crystals by Nb2 cell metabolism. An increase of living cells is reflected by increased metabolic activity. This increase directly correlates with the formation of absorbance monitored formazan crystals; i.e., the greater the number of cells, the greater the increase in cell metabolism with greater formazan formation and greater biological activity.

The bioassay was carried out in triplicate in a humid atmosphere with 5% CO2-95% air at 37°C. After the incubation period of 3 days, we added 10µL of MTT (to a final concentration of 0.5 mg/mL) to each well. Samples were incubated for 4 hrs under the atmospheric conditions mentioned above. After this time, we added 100 uL of formazan solubilizing solution (10% SDS in 0.01 M HCl) to each of the wells. These were left incubating overnight under the same atmospheric conditions. It was verified that the precipitate of formazan purple crystals had completely dissolved and absorbance was measured at 590 nm using an ELISA plate reader.

#### **12.2 Somatogenic bioassay**

190 Genetic Engineering – Basics, New Applications and Responsibilities

Figure 11 shows the process of purification or "downstream" that was followed for each

Each culture medium containing the recombinant hormone was ultra-dialyzed and ultraconcentrated. The pore membrane used was 14 kDa. At the same time it was ultradyalized. The ultraconcentrate obtained was lyophilized to preserve the samples and all the powder was recovered in 50 mL plastic tubes and weighed. Total protein was quantified in each lyophilized culture medium with the Bradford method (52). Samples

Each sample was prepared for loading into a chromatographic column. Five mg of the total protein from each sample was adjusted to the conditions of the loading buffer. A column was loaded with Q-Sepharose fast flow anion exchange resin; column filling with resin was carried out by gravity flow. After passing the sample, washing was done with 10 mL of loading buffer. To recover the proteins from the column, 15 mL of elution buffer (loading buffer plus NaCl) was passed with an ionic strength increased sequentially with NaCl. Total protein was measured by the Bradford method (52) for each of the fractions recovered to see

The collected fractions were visualized on discontinuous polyacrylamide gel concentrations of 4-15% under denaturing conditions (SDS-PAGE), and stained with Coomassie and silver techniques (53). The fractions were subjected to lyophilization, the powder was recovered and weighted, total protein was measured by the Bradford method. In addition, using PAGE-SDS the percentage of purity of the monomer was determined with ImageJ software. The samples were stored at -20°C until used for further analysis or to determine their

The biological activity of hormones produced in *Pichia pastoris* recombinant was determined by their ability to promote proliferation of the Nb2 cell line, which comes from rat lymphoma (54, 55). GHs were also tested for their somatogenic activity in the adipocyte differentiation model based on the glyceraldehyde 3-phosphate dehydrogenase (GPDH)

The cell-free culture medium was dialyzed and each hormone was quantified by gel densitometry. Dilutions of each recombinant hormones in the culture medium dialyzed were tested. Cell proliferation was determined by tetrazolium salt assay (MTT) (56) and was expressed as the average of three repetitions, in comparison with the positive control recombinant rat prolactin (rRPRL) and the negative control (culture medium without hormone). The concentrations tested were 0.001, 0.01, 0.1, 1, 10 and 50 nM of the following hormones, CFGH, ECGH, FCGH, HGH, HCS and RPRL. Upon completion of the testing time, we proceeded to measure the effect of hormones on cell proliferation by MTT assay (56). The activity was evaluated by color generation based on the reduction of tetrazolium salt (methyl 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyl tetrazolium) from yellow to purple forming crystals by Nb2 cell metabolism. An increase of living cells is reflected by increased

**12. Testing the biological activity of recombinant hormones** 

**11. Semipurification of recombinant hormones** 

recombinant hormone produced.

were stored at -20°C until use.

biological activity.

assay.

**12.1 Lactogenic activity bioassay** 

what percentage of the total they represented.

To test the biological activity of goat GH, we assayed fibroblasts from cell line 3T3-F442A (pre-adipocytes) and with the glyceraldehyde 3-phosphate dehydrogenase (GPDH) assay as described in (18, 57). Cells were exposed to different concentrations of CHGH in the medium. The positive control was 10% fetal bovine serum (FBS) (v/v) the negative control was 0.25% FBS (v/v). The cells were incubated for 12 days, were harvested and proteins extracted; the supernatants were frozen in aliquots for subsequent tests. Specific activity of GPDH was measured in cell extracts by NADH oxidation measured in a spectrophotometer at 340 nm; 40 µg of protein was used. CHGH demonstrated biological activity in the essay. The same was doing with the others hormones showing biological activity.

#### **13. Contributions**

These are the first reports in the literature of the production by biotechnology of the recombinant GHs described here using the *Pichia pastoris* methylotrophic system. The new strains of *Pichia pastoris* constructed with GH cassettes such as canine-porcine, equine, feline, and caprine, complemented our strains previously constructed for GHs of 22 y 20 kDa, HCS and bovine GH (unpublished results), making our collections of GH clones the largest one for this protein family in the world as far as we know.

All hormones were efficiently produced, processed, and secreted into the culture media. Each hormone constituted the main proteic band among proteins secreted by *Pichia pastoris* analyzed by SDS-PAGE. The phenotypes Muts resulted the best for producing GHs, as was the case for CFGH (58) and for HCS (Ascacio-Martínez y Barrera-Saldaña, unpublished results).

All our constructed strains had correct processing of their heterologous secretion *Saccharomyces cerevisiae* alpha mating factor signal peptide in the maturation of the recombinant GHs. They were secreted into the culture media in their native and bioactive form (49, 59, 60).

Using a bioreactor increased the production of the recombinant proteins 10 to 20 times compared to an Erlenmeyer flask. Ionic chromatography was a good option in all cases for semipurification of rGHs and HCS in this system. All hormones showed biological activity in the Nb2 essay, showing that human GH had more activity than animal GHs. The same happened in the pre-adypocite system (3T3), concluding that *Pichia pastoris* produces, processes, and secretes rGHs in the bioactive form.

Biotechnological platforms were developed that made possible to move from the construction of the producer clones to the bioassay of the semipurified recombinant protein. With the technology here developed we have acquired the capacity to advance in scaling the production of veterinary and livestock rGHs for their field tests.

#### **14. Perspectives**

We have developed an efficient expression system and laboratory fermentor-scale production biotechnological platform with which to partner with the productive sector to produce virtually any GH of human and animal origin with acceptable quantity, quality and activity to start field evaluations. Doing that, would allow us investigate their full potential in animal biotechnology, to then offer them as an option in veterinary treatments and to stimulate cattle production and the health of competition animals. In addition to the obvious veterinary or livestock application, their availability will also allow the discovery of unexpected biological activities for animal wellness.

#### **15. Acknowledgements**

The authors want to thank J.M. Reyes, J.P. Palma, C.N. Sanchez, L.L. Escamilla, H.L. Gallardo, E.L. Cab, R.G. Padilla and M. Guerrero for their support, experiments during their thesis and valuable contributions to the information here reviewed. Authors thank Sergio Lozano for his critical reading of the manuscript.[

#### **16. References**


happened in the pre-adypocite system (3T3), concluding that *Pichia pastoris* produces,

Biotechnological platforms were developed that made possible to move from the construction of the producer clones to the bioassay of the semipurified recombinant protein. With the technology here developed we have acquired the capacity to advance in scaling the

We have developed an efficient expression system and laboratory fermentor-scale production biotechnological platform with which to partner with the productive sector to produce virtually any GH of human and animal origin with acceptable quantity, quality and activity to start field evaluations. Doing that, would allow us investigate their full potential in animal biotechnology, to then offer them as an option in veterinary treatments and to stimulate cattle production and the health of competition animals. In addition to the obvious veterinary or livestock application, their availability will also allow the discovery of

The authors want to thank J.M. Reyes, J.P. Palma, C.N. Sanchez, L.L. Escamilla, H.L. Gallardo, E.L. Cab, R.G. Padilla and M. Guerrero for their support, experiments during their thesis and valuable contributions to the information here reviewed. Authors thank Sergio

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**14. Perspectives** 

**15. Acknowledgements** 

**16. References** 

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**Part 3** 

**Biosafety** 

196 Genetic Engineering – Basics, New Applications and Responsibilities

[57] Castro-Munozledo, F., Beltran-Langarica, A., Kuri-Harcuch, W., Commitment of 3T3-

[58] Ascacio-Martínez, J.A., Barrera-Saldaña H.A. Production and secretion of biologically

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H.A. Production of recombinant human placental variant growth hormone in

peptide in the CHH/MIH/GIH family of Penaeus monodon, in Pichia pastoris is directed by secretion signal of the alpha-mating factor from Saccharomyces

### **Genetically Engineered Virus-Vectored Vaccines – Environmental Risk Assessment and Management Challenges**

Anne Ingeborg Myhr and Terje Traavik

*Genøk- Centre of Biosafety, and Faculty of Health Sciences, University of Tromsø, Tromsø Norway* 

#### **1. Introduction**

Genetically engineered or modified viruses (GMVs) are being increasingly used as live vaccine vectors and their applications may have environmental implications that must be taken into account in risk assessment and management processes. In most legislative frameworks GMVs are treated as GMOs (genetically modified organisms), which require ERA (environmental risk assessment) in addition to the evaluation of the quality, safety and efficacy of the product before marketing authorization or clinical trial applications are submitted. The ERA is performed in order to identify the potential risks for public health and the environment that may arise due to the use and release of GMVs. If risks are identified and considered as not acceptable, the ERA process should go on to propose appropriate risk management strategies capable to reduce these risks (Anliker et al., 2010; Kühler et al, 2009).

To obtain marketing authorization within the EU, a GMV has to meet the criteria and requirements of the EU pharmaceutical legislation for both medical and veterinary applications, as well as the EU environmental legislation on the deliberate release of GMOs. Hence, although viruses are not organisms, it will be necessary to perform an ERA similar to the procedure under Directive 2001/18/EC on the deliberate release of GMOs into the environment. For the purpose of an ERA, an organism is defined as a biological entity capable of replication or of transferring genetic material, and this definition will then include viruses and also replication-incompetent viral vectors. A MAA (marketing authorization application) for a GMV submitted to the European Medicines Agency (EMEA) has to include an ERA in accordance with the principles set out in Annex II Directive 2001/18/EC and its supplementing Commission Decision 2002/623/EC. The further process is well described in recent reviews, e.g. by Anliker et al. (2010) and Kühler et al (2009). Briefly, the ERA should be based on the technical and scientific information about the GMV as required in the Directive Annexes III and IV. The continued procedure is somewhat different from the deliberate GMO release process of the Directive. But even so, difficulties in preparing ERAs for GMVs may arise from the fact that Directive 2001/18/EC has a nearly exclusive focus on GM plants and agricultural products. Therefore, the EMEA has developed two specific guidelines for the preparation of ERAs to facilitate adaptation of the requirements and the methodology of the Directive to GMO-containing medicinal products. In addition, the GMV ERA needs to be performed according to the national Member State requirements to obtain authorization of GMV clinical and of field trials. The individual Member State may have different requirements, e.g. dependent on whether the trial is considered "contained use" according to Directive 98/81/EC or a "deliberate release" according to Directive 2001/18/EC. The former is focusing on containment measures, i.e. implementation of physical, chemical and biological barriers to preclude the GMVenvironment interactions. In contrast, the deliberate release Directive is based on a thorough case-by-case assessment of the potential environmental risks arising from GMV release or escape, and the biosafety measures to be utilized in order to eliminate or minimize these risks.

The objective of an ERA in accordance with Directive 2001/18/EC is to identify and assess on a case-by-case basis the potential harmful effects of a GMO for humans, animals (domestic and wildlife), plants, microorganisms and the environment at large (Anliker et al., 2010). Potential adverse effects should be acknowledged and considered irrespective of whether they are direct or indirect and whether the emerging effects appear immediately or delayed. When such effects have been identified, appropriate measures for their reduction or elimination need to be defined. Such measures have to be based on the realization that transmission of GMVs to non-target individuals, species or the environment at large may allow the GMV to spread further. In its turn this may induce genetic or phenotypic changes, competition with existing species or horizontal gene transfer of hereditary materials between species. To fully conceive and evaluate the environmental risks associated with such, and other, potential scenarios, detailed case-by-case knowledge of all possible adverse effects of a given GMV is crucial, because the quality and relevance of the ERA, and the possibilities for efficient risk management, are totally dependent on the ability to anticipate, predict and reveal potential adverse effects. This again is dependent on the society's willingness to invest in "what-if?"-based and precautionary science and in acknowledging and weighing the values that are knowingly or unknowingly influencing the decision making process following a MAA for a GMV application.

The main focus of risk-related research has previously been on the functionality and the intended immunological mechanisms of GMVs, while work on safety aspects, particularly in relation to ecosystem effects, often have been put off until later in vaccine development. By then, making fundamental changes to the vaccine in order to improve its safety can be extremely costly and time-consuming. In some cases hazards and irreversible harms may have been initiated already. Hence, we will argue that risk assessment and management should not be considered as two separate processes.

Traditionally, risk assessment has been considered as a "scientific" process, while risk management and communication has included value judgments with regard to acceptability, the trade-off criteria and the adaptation of strategies for coping with uncertainty. However, risk assessments are influenced by scientific, ethical, economic, social and political information. For instance, risk assessments include value judgments both with regard to consequences that should be avoided and the process of risk characterization. Consequently, risk assessment and management strategies need to be connected from the very start of a vaccine development project in order to unveil the full spectrum of environmental impacts.

In this article we describe GMV applications and the environmental impact questions and challenges that are connected to them. We then proceed to discuss the relevance and shortcomings of the present risk assessment framework and how this framework needs to be better connected to risk management strategies. Such management strategies are developed within particular frameworks that need to include awareness to normative standards and preferences regarding human relation to the natural environment. Moreover, we will elaborate on how precautionary motivated research involves the need to advance hypotheses about GMV specific harm and hazard endpoints and that such endpoints are dependent on both the objectives of ERA and of the management strategies.

#### **2. Creation and applications of GMVs**

200 Genetic Engineering – Basics, New Applications and Responsibilities

the requirements and the methodology of the Directive to GMO-containing medicinal products. In addition, the GMV ERA needs to be performed according to the national Member State requirements to obtain authorization of GMV clinical and of field trials. The individual Member State may have different requirements, e.g. dependent on whether the trial is considered "contained use" according to Directive 98/81/EC or a "deliberate release" according to Directive 2001/18/EC. The former is focusing on containment measures, i.e. implementation of physical, chemical and biological barriers to preclude the GMVenvironment interactions. In contrast, the deliberate release Directive is based on a thorough case-by-case assessment of the potential environmental risks arising from GMV release or escape, and the biosafety measures to be utilized in order to eliminate or minimize these risks. The objective of an ERA in accordance with Directive 2001/18/EC is to identify and assess on a case-by-case basis the potential harmful effects of a GMO for humans, animals (domestic and wildlife), plants, microorganisms and the environment at large (Anliker et al., 2010). Potential adverse effects should be acknowledged and considered irrespective of whether they are direct or indirect and whether the emerging effects appear immediately or delayed. When such effects have been identified, appropriate measures for their reduction or elimination need to be defined. Such measures have to be based on the realization that transmission of GMVs to non-target individuals, species or the environment at large may allow the GMV to spread further. In its turn this may induce genetic or phenotypic changes, competition with existing species or horizontal gene transfer of hereditary materials between species. To fully conceive and evaluate the environmental risks associated with such, and other, potential scenarios, detailed case-by-case knowledge of all possible adverse effects of a given GMV is crucial, because the quality and relevance of the ERA, and the possibilities for efficient risk management, are totally dependent on the ability to anticipate, predict and reveal potential adverse effects. This again is dependent on the society's willingness to invest in "what-if?"-based and precautionary science and in acknowledging and weighing the values that are knowingly or unknowingly influencing the decision

The main focus of risk-related research has previously been on the functionality and the intended immunological mechanisms of GMVs, while work on safety aspects, particularly in relation to ecosystem effects, often have been put off until later in vaccine development. By then, making fundamental changes to the vaccine in order to improve its safety can be extremely costly and time-consuming. In some cases hazards and irreversible harms may have been initiated already. Hence, we will argue that risk assessment and management

Traditionally, risk assessment has been considered as a "scientific" process, while risk management and communication has included value judgments with regard to acceptability, the trade-off criteria and the adaptation of strategies for coping with uncertainty. However, risk assessments are influenced by scientific, ethical, economic, social and political information. For instance, risk assessments include value judgments both with regard to consequences that should be avoided and the process of risk characterization. Consequently, risk assessment and management strategies need to be connected from the very start of a vaccine development project in order to unveil the full spectrum of environmental impacts. In this article we describe GMV applications and the environmental impact questions and challenges that are connected to them. We then proceed to discuss the relevance and shortcomings of the present risk assessment framework and how this framework needs to

making process following a MAA for a GMV application.

should not be considered as two separate processes.

Genetically engineered or modified viruses (GMVs), from a number of taxons, are being increasingly used as live vaccine vectors. The so far approved veterinary vaccines have most commonly been based on replication-competent canarypoxvirus or herpesviruses, and EMEA has published a guideline for Live Recombinant Vector Vaccines for Veterinary Use.

There are 4 broad GMV application areas that may have environmental implications:


In all cases there may be circumstances that enable GMVs to jump species barriers directly, or following recombination with naturally occurring viruses. All the different applications may, to varying extents, represent release or unintended escape of GMVs into the highly varying ecosystems.

The different application areas call for different considerations and options with regard to choice of virus vectors and genetically engineering (GE) strategies. Generally spoken, there are two strategies: The first is represented by *gene-deleted viruses* to be used for homologous vaccination, i.e. to achieve protective immunity against the GMV itself. The engineered deletions most commonly target genes that are necessary for the virus to carry out a full multiplication cycle, or are implied in viral virulence. Furthermore, "non-essential" genes may be deleted in order to obtain markers for monitoring unintended vaccine virus spread. Lack of the deleted marker gene will indicate that a field virus isolate originate from a GMV. A number of gene-deletion GMVs for vaccination against human and livestock diseases have been marketed, or are in the final stages of clinical trials. Most of them belong to the herpesvirus or adenovirus families.

Recombinant virus vectors obtained by transgenesis represent the second strategy. Such viruses are created in cell cultures by simultaneous transfection with a plasmid carrying a gene from the virus/microbe that is to be immunologically targeted, and infection with the virus vector of choice. The plasmid construct is such that the transgene contains DNA sequences homologous to a viral gene in each end. Hence the transgene is transferred and integrated to a predetermined site in the virus vector genome by homologous recombination. The most commonly used vector viruses are members of the DNA virus families *Poxviridae* and *Adenoviridae*. In many cases the vectors have been engineered by both endogenous gene deletions and transgene insertions.

At present there are considerable research efforts going into designing *replication-incompetent* versions of the most attractive vector viruses. This is possible under the qualification that the non-replicating vector is able to express the vaccine transgene and raise protective immunological responses at the same levels as the parental replication-competent virus strain. The main purposes are to minimize the risk-prone possibilities of productive infections with shedding of virus in the excreta of GMV-exposed individuals, and reversal of attenuated or modified viruses to full virulence through reversion of attenuating mutations.

Diseases for which GMV-vectored vaccines have been developed or are in the process of being developed, include AIDS, severe acute respiratory syndrome (SARS), Epstein-Barr virus, cytomegalovirus, West Nile virus, tuberculosis, malaria, influenza (human and equine), Rinderpest, Rift Valley fever, borreliosis, trypanosomiasis, leishmaniasis, cervical cancer, breast cancer, colorectal cancer, mesothelioma and melanoma. Most of the GMV vectors employed are assumed replication-incompetent, and are derived from members of the poxvirus and adenovirus families.

#### **3. Vaccines and vaccination (modified from Traavik, 1999)**

All vaccines have in common the intention to prevent disease or limit the effects of disease. Both humoral (antibody-mediated) and cellular arms of the immune system can contribute to a pathogen-specific acquired response that distinguishes specific immune protection from the innate and more general protection mediated by phagocytes (i.e. macrophages neutrophils and dendritic cells), cytokines and physical barriers. Because vaccination against a threatening disease may take place many years before exposure to the pathogen, immunological memory is a critical element. A long-lived immune response, which may be mobilized and augmented rapidly when called for, is essential.

Vaccination may have different purposes and fields of application. The most important are:


An ideal vaccine provides an optimal mobilization of the adaptive immune system with no unwanted side effects, and with long-lasting immunological memory. The most universal purpose is to prevent disease in individuals and prohibit transmission of disease agents between individuals. Generally, vaccination must be carried out before the individual becomes infected, but for some diseases, e.g. rabies, disease may be prevented even if vaccination takes place after infection.

Some important human and domestic animal pathogens, e.g. rabies virus, hantaviruses and a number of arboviruses, have reservoirs in free-ranging wildlife animals. Human and animal disease may then be prevented by vaccination of reservoir animals. Likewise, some free-ranging mammalian species are considered «pests» in the context of human food, animal fodder or other kinds of production. Enforced infertility following vaccination is now becoming an alternative to culling ("stamping out") for control and reduction of such pest animal populations.

Cancer cells often express surface antigens not present on their normal counterparts. Such unique antigens may provide targets for vaccines that may induce immune reactions to prevent and combat cancer cells.

Depending on the species, target-organs, epidemiological considerations etc., the vaccine delivery method and route may differ. In practical terms, the vaccine may be delivered by:


202 Genetic Engineering – Basics, New Applications and Responsibilities

At present there are considerable research efforts going into designing *replication-incompetent* versions of the most attractive vector viruses. This is possible under the qualification that the non-replicating vector is able to express the vaccine transgene and raise protective immunological responses at the same levels as the parental replication-competent virus strain. The main purposes are to minimize the risk-prone possibilities of productive infections with shedding of virus in the excreta of GMV-exposed individuals, and reversal of attenuated

Diseases for which GMV-vectored vaccines have been developed or are in the process of being developed, include AIDS, severe acute respiratory syndrome (SARS), Epstein-Barr virus, cytomegalovirus, West Nile virus, tuberculosis, malaria, influenza (human and equine), Rinderpest, Rift Valley fever, borreliosis, trypanosomiasis, leishmaniasis, cervical cancer, breast cancer, colorectal cancer, mesothelioma and melanoma. Most of the GMV vectors employed are assumed replication-incompetent, and are derived from members of

All vaccines have in common the intention to prevent disease or limit the effects of disease. Both humoral (antibody-mediated) and cellular arms of the immune system can contribute to a pathogen-specific acquired response that distinguishes specific immune protection from the innate and more general protection mediated by phagocytes (i.e. macrophages neutrophils and dendritic cells), cytokines and physical barriers. Because vaccination against a threatening disease may take place many years before exposure to the pathogen, immunological memory is a critical element. A long-lived immune response, which may be

Vaccination may have different purposes and fields of application. The most important are:

An ideal vaccine provides an optimal mobilization of the adaptive immune system with no unwanted side effects, and with long-lasting immunological memory. The most universal purpose is to prevent disease in individuals and prohibit transmission of disease agents between individuals. Generally, vaccination must be carried out before the individual becomes infected, but for some diseases, e.g. rabies, disease may be prevented even if

Some important human and domestic animal pathogens, e.g. rabies virus, hantaviruses and a number of arboviruses, have reservoirs in free-ranging wildlife animals. Human and animal disease may then be prevented by vaccination of reservoir animals. Likewise, some free-ranging mammalian species are considered «pests» in the context of human food, animal fodder or other kinds of production. Enforced infertility following vaccination is now becoming an alternative to culling ("stamping out") for control and reduction of such

Cancer cells often express surface antigens not present on their normal counterparts. Such unique antigens may provide targets for vaccines that may induce immune reactions to

or modified viruses to full virulence through reversion of attenuating mutations.

**3. Vaccines and vaccination (modified from Traavik, 1999)** 

mobilized and augmented rapidly when called for, is essential.

• Protection against and treatment of infectious diseases

• Induced infertility in domestic animals and wildlife

• Protection against and treatment of cancer

vaccination takes place after infection.

pest animal populations.

prevent and combat cancer cells.

the poxvirus and adenovirus families.


To a varying extent, all vaccine delivery strategies imply that vaccine-containing materials may end up in unintended locations, and hence release or escape of biologically active macromolecules (i.e. DNA or RNA), viruses or microorganisms into the environment may take place.

#### **4. Viruses that are used as GMVs**

Generally spoken, the families most widely employed as GM vaccine vectors have been members of the *Poxviridae*, *Adenoviridae* and *Herpesviridae* families. The frontrunners have been GM versions of the poxvirus genera Orthopoxvirus and Avipoxvirus. Our own experimental and sustainability oriented research have focused on these genera, and we will in section 5, 6 and 7 proceed by using such GM vectors as examples of ERA and risk management challenges.

There is now a tendency that GM viral vaccines are based on vectors that are replicationincompetent in the target species of choice. Such vectors may be selected on the basis of natural host species barriers, e.g. avipoxviruses with birds as natural hosts, and assumed replication-deficient in mammalian species. Other commonly used vectors have been made replication-incompetent by cell cultivation procedures (e.g. MVA, see below) or targeted genetic engineering, e.g. adenovirus vectors.

Veterinary vaccines against infectious diseases containing replication-competent GMVs have been on the market in the EU since the beginning of year 2000 (Kühler et al., 2009). Several vaccines for human use are currently in various stages of clinical trials, and a few of them, mostly based on adenovirus or poxvirus vectors, have already been approved for marketing.

#### **4.1 Poxviruses as GM vaccine vectors**

Our account will henceforth be directly relevant for poxvirus vectors, but at least in a general sense it will also be relevant for GMV vectors developed from other virus families.

Poxviruses are selected as potential vectors for several reasons (Liu, 2010): the extensive use of the smallpox vaccine (and the related modified vaccinia ankara), which provided knowledge of the human safety parameters; the large gene capacity for the insertion of a heterologous gene encoding the antigen of interest; the broad tropism of the virus for mammalian cells that could then result in a number of cells expressing the vaccine protein (heterologous antigen); the production of vaccine protein for a relatively short period of time (making the kinetics of the production of the heterologous protein more akin to antigen production from an acutely infecting pathogen, but less useful for gene therapy applications); and the location of the virus in the cytoplasm, thus avoiding integration risks that might occur with other alternatives, as a retroviral vector for example (Mastrangelo et al., 2000). The earliest vector for delivery of a heterologous antigen is a licensed veterinary vaccine employing a MVA (Modified Vaccinia Ankara) vector to deliver a rabies antigen (Mackowiak et al., 1999). The vaccine was developed for delivery as bait for wild animals. The success of using a vector to deliver an antigen that generated immunity sufficient to protect animals and curtail outbreaks in the wild, via oral delivery of bait (hence with imprecise dosing), in a variety of animal species, demonstrates many of the advantages of an ideal vector, in this case efficacy, safety, and even ease of distribution (oral delivery), which would not have been as facile with a non-vectored vaccine. Oral delivery may mobilize protective local immune responses on mucous membranes, a great asset for human, domestic animal and wildlife vaccines.

Various poxvirus vectors have undergone human clinical testing as HIV vaccines. In the largest clinical trial, a canarypox vector coding for HIV antigens was utilized as the first component of a prime-boost regimen in a clinical trial of an HIV vaccine involving 16,000 individuals (Rerks-Ngarm et al., 2009). In another prime-boost vaccination protocol for prophylaxis of HIV, the priming is being done with a DNA plasmid, followed by an MVA vector boost

(http://clinicaltrials.gov/ct2/show/NCT00820846?term=DNA+%2B+MVA+for+HIV&rank=5).

#### **4.2 Vaccine history, general characteristics and taxonomy of the family Poxviridae**

The bicentennial celebration of the first vaccination took place 15 years ago. In 1796 Edward Jenner injected cowpox virus into the boy James Phipps, and later on challenged him with fully virulent human smallpox (variola) virus. The boy survived, and Jenner had hence protected him against one of the most dreaded human diseases of all times. The smallpox vaccination story ended in a triumphant eradication, in 1979, of variola virus and smallpox disease, due to a world-wide vaccination campaign. (Fenner et al., 1988). In recognition of Edward Jenner´s contribution, procedures that aim at protection against disease by premobilization of the immune system were termed "Vaccination", derived from the Latin word *vacca,* hence honoring the cowpoxvirus. In that context it is well worth for present day scientists to reflect on the fact that the contemporaries of Jenner rejected his findings.

The family *Poxviridae* contains a vast number of complex DNA viruses that replicate in the cytoplasm of vertebrate and invertebrate hosts. The member viruses have been sorted into two subfamilies based on their host preferences, namely, the *Chordopoxvirinae* (infecting vertebrates) and *Entomopoxvirinae* (infecting invertebrates, more specifically arthropods). The *Chordopoxvirinae* consists of eight genera with different host ranges, namely, Orthopoxvirus, Parapoxvirus, Avipoxvirus, Capripoxvirus, Leporipoxvirus, Suipoxvirus, Molluscipoxvirus and Yatapoxvirus. The *Entomopoxvirinae* is divided into three genera based on the insect host of isolation. These are designated A, B, and C entomopoxviruses, respectively. Genetic information on the entomopoxviruses is very scanty.

The distinctive characteristics of members of the family *Poxviridae* include:

• A large and complex virion (larger than any other animal virus particles) with virionassociated transcriptase used for virus-specific mRNA synthesis. The virions are very

time (making the kinetics of the production of the heterologous protein more akin to antigen production from an acutely infecting pathogen, but less useful for gene therapy applications); and the location of the virus in the cytoplasm, thus avoiding integration risks that might occur with other alternatives, as a retroviral vector for example (Mastrangelo et al., 2000). The earliest vector for delivery of a heterologous antigen is a licensed veterinary vaccine employing a MVA (Modified Vaccinia Ankara) vector to deliver a rabies antigen (Mackowiak et al., 1999). The vaccine was developed for delivery as bait for wild animals. The success of using a vector to deliver an antigen that generated immunity sufficient to protect animals and curtail outbreaks in the wild, via oral delivery of bait (hence with imprecise dosing), in a variety of animal species, demonstrates many of the advantages of an ideal vector, in this case efficacy, safety, and even ease of distribution (oral delivery), which would not have been as facile with a non-vectored vaccine. Oral delivery may mobilize protective local immune responses on mucous membranes, a great asset for human,

Various poxvirus vectors have undergone human clinical testing as HIV vaccines. In the largest clinical trial, a canarypox vector coding for HIV antigens was utilized as the first component of a prime-boost regimen in a clinical trial of an HIV vaccine involving 16,000 individuals (Rerks-Ngarm et al., 2009). In another prime-boost vaccination protocol for prophylaxis of HIV, the priming is being done with a DNA plasmid, followed by an MVA

(http://clinicaltrials.gov/ct2/show/NCT00820846?term=DNA+%2B+MVA+for+HIV&rank=5).

**4.2 Vaccine history, general characteristics and taxonomy of the family Poxviridae**  The bicentennial celebration of the first vaccination took place 15 years ago. In 1796 Edward Jenner injected cowpox virus into the boy James Phipps, and later on challenged him with fully virulent human smallpox (variola) virus. The boy survived, and Jenner had hence protected him against one of the most dreaded human diseases of all times. The smallpox vaccination story ended in a triumphant eradication, in 1979, of variola virus and smallpox disease, due to a world-wide vaccination campaign. (Fenner et al., 1988). In recognition of Edward Jenner´s contribution, procedures that aim at protection against disease by premobilization of the immune system were termed "Vaccination", derived from the Latin word *vacca,* hence honoring the cowpoxvirus. In that context it is well worth for present day

scientists to reflect on the fact that the contemporaries of Jenner rejected his findings.

respectively. Genetic information on the entomopoxviruses is very scanty. The distinctive characteristics of members of the family *Poxviridae* include:

The family *Poxviridae* contains a vast number of complex DNA viruses that replicate in the cytoplasm of vertebrate and invertebrate hosts. The member viruses have been sorted into two subfamilies based on their host preferences, namely, the *Chordopoxvirinae* (infecting vertebrates) and *Entomopoxvirinae* (infecting invertebrates, more specifically arthropods). The *Chordopoxvirinae* consists of eight genera with different host ranges, namely, Orthopoxvirus, Parapoxvirus, Avipoxvirus, Capripoxvirus, Leporipoxvirus, Suipoxvirus, Molluscipoxvirus and Yatapoxvirus. The *Entomopoxvirinae* is divided into three genera based on the insect host of isolation. These are designated A, B, and C entomopoxviruses,

• A large and complex virion (larger than any other animal virus particles) with virionassociated transcriptase used for virus-specific mRNA synthesis. The virions are very

domestic animal and wildlife vaccines.

vector boost

resistant to environmental degradation. This is an advantage for the shelf life of vaccines, particularly in areas with unsatisfactory cooling conditions. But persistence in the environment may also enhance the chances of transboundary movements and nontarget infections.


The poxviruses are diverse in their structure, host range and host specificities. Members of each of the 8 genera of the subfamily *Chordopoxvirinae* are genetically and antigenically related and share similarities in morphology and host range within the genus. For example, similarities in the restriction endonuclease maps of several Orthopoxvirus genomes, such as the 90% sequence identity of the genes of Vaccinia and Variola viruses reflect genetic relatedness of members of each genus. It is, therefore, not surprising that intra-genus crosshybridization has been reported among these viruses.

Genetic divergence exists between members of each genus, particularly when such members come from geographically separated econiches. Indeed, Weli et al. (2004) even observed genetic heterogeneity among avipoxviruses isolated from different parts of Norway.

Sequence similarities of genomic repeats in various strains of each virus species suggests that these repeats evolved from unequal crossover events. Thus, genetic material of each virus bears a pointer to genetic recombination in its history. Further examination of the Orthopoxviruses shows evidence of genetic transpositions and deletions in the terminal hypervariable regions of the genome. The significance of these genetic interactions or phenomena in the ecology of poxviruses is at present a matter of conjecture but they makes risk assessment in the use and release of poxvirus-based GMVs absolutely necessary.

#### **4.3 Ecological distribution of** *Poxviridae*

The family *Poxviridae* contains a vast number of divergent viruses with equally divergent host specificities and host ranges. It seems justified to state that poxviruses will be found in any vertebrate species and geographical area where they are systematically looked for. However, very little is known about the characteristics and geographical distribution of most poxviruses occurring naturally in the field. This knowledge would be necessary if a meaningful risk assessment of poxvirus-based GM vaccines use or release in any target area should be undertaken. Without such knowledge there is no possibility of making meaningful ERA inclusions of adverse effects due to recombination events between GM poxvirus vaccines and naturally occurring poxviruses circulating within the actual area for application.

Many poxviruses are capable of zoonotically infecting man. It is likely that variola virus is derived from an ancient zoonotic virus that originated from a now extinct animal host species. In general, poxviruses show species specificities that range from narrow to broad, but *we still know little about the fundamental mechanisms that mediate the host tropism of individual poxviruses* (McFadden, 2005)*.* The unpredictability in a real world situation is illustrated by a macaque outbreak caused by a European orthopoxvirus strain carried by *Rattus norvegicus* in the Netherlands (Martina et al., 2006).

Variola virus (smallpox disease) has killed more members of the human population over the span of recorded history than all other infectious diseases combined. Variola virus may never again infect humans, but there are other poxviruses that can cause serious human disease. In 2003, an outbreak of human monkeypox occurred in the mid-western United States due to the inadvertent importation of monkeypox virus in a shipment of rodents from West Africa (Reed et al., 2004). Fortunately, the strain that caused this outbreak was more benign in humans than the more pathogenic variant that is found in central Africa, which results in mortality rates of 10–15%.

Variola virus, prior to its eradication, spread all over the world. Similar worldwide distribution seems to be true of VACV and Molluscum contagiosum virus. Man was the only known host and reservoir of Variola and Molluscum contagiosum viruses. Although the reservoir host of VACV is unknown, it has been shown, under natural conditions, to exhibit a wide host range among humans, rabbits, cows and buffaloes. Other poxviruses with worldwide distribution include numerous species of Avipoxvirus. Even with the relatively limited studies so far done, certain orthopoxviruses (parapoxviruses, capripoxviruses and yatapoxviruses) are notably associated with African domestic animals and wildlife, including rodents, squirrels and monkeys, which may serve as reservoirs of infection. Knowledge of the distribution of poxviruses that infect man and with host range which includes wildlife such as rodents, squirrels and monkeys, would be important in risk assessment of poxvirusbased GE vaccines and other poxvirus-based recombinant constructs prepared for use or release in ecosystems to which these hosts belong. Similarly, it is important to document the poxviruses carried by ectoparasites of animals in the target localities.

#### **4.4 Poxvirus vaccine vectors**

Poxviruses are being increasingly used as recombinant vectors for vaccination against numerous infectious diseases in humans, domestic animals, and wildlife (Pastoret & Vanderplasschen, 2003). For risk assessments and surveillance, information about the occurrence, distribution and ecology of poxviruses are hence important.

Poxviruses have several advantages for use as expression or vaccine vectors, including their large insertion capacity, their cytoplasmatic site of replication, their heat stability, the relatively high expression levels and proper post-translational modifications of foreign proteins as well as the ability to raise protective immune reactions locally on mucous membranes.

Vaccinia virus (VACV) strains are robust eukaryotic expression vectors that have been used for a number of different studies in biochemistry, cell biology, and immunology. Most vaccinia virus strains, such as the ones used in the smallpox eradication campaign, readily replicate in human cells and have been associated with a range of clinical complications in vaccinees. This fact is presently considered a major problem for the use of these strains as recombinant vaccines for mass vaccination. For laboratory use, in addition to mandatory biosafety level 2 procedures, proper precautions must be taken to prevent accidental exposure when managing replication-competent vaccinia strains.

#### **4.4.1 Non-replicating poxvirus vectors**

206 Genetic Engineering – Basics, New Applications and Responsibilities

species. In general, poxviruses show species specificities that range from narrow to broad, but *we still know little about the fundamental mechanisms that mediate the host tropism of individual poxviruses* (McFadden, 2005)*.* The unpredictability in a real world situation is illustrated by a macaque outbreak caused by a European orthopoxvirus strain carried by

Variola virus (smallpox disease) has killed more members of the human population over the span of recorded history than all other infectious diseases combined. Variola virus may never again infect humans, but there are other poxviruses that can cause serious human disease. In 2003, an outbreak of human monkeypox occurred in the mid-western United States due to the inadvertent importation of monkeypox virus in a shipment of rodents from West Africa (Reed et al., 2004). Fortunately, the strain that caused this outbreak was more benign in humans than the more pathogenic variant that is found in central Africa, which

Variola virus, prior to its eradication, spread all over the world. Similar worldwide distribution seems to be true of VACV and Molluscum contagiosum virus. Man was the only known host and reservoir of Variola and Molluscum contagiosum viruses. Although the reservoir host of VACV is unknown, it has been shown, under natural conditions, to exhibit a wide host range among humans, rabbits, cows and buffaloes. Other poxviruses with worldwide distribution include numerous species of Avipoxvirus. Even with the relatively limited studies so far done, certain orthopoxviruses (parapoxviruses, capripoxviruses and yatapoxviruses) are notably associated with African domestic animals and wildlife, including rodents, squirrels and monkeys, which may serve as reservoirs of infection. Knowledge of the distribution of poxviruses that infect man and with host range which includes wildlife such as rodents, squirrels and monkeys, would be important in risk assessment of poxvirusbased GE vaccines and other poxvirus-based recombinant constructs prepared for use or release in ecosystems to which these hosts belong. Similarly, it is important to document the

Poxviruses are being increasingly used as recombinant vectors for vaccination against numerous infectious diseases in humans, domestic animals, and wildlife (Pastoret & Vanderplasschen, 2003). For risk assessments and surveillance, information about the

Poxviruses have several advantages for use as expression or vaccine vectors, including their large insertion capacity, their cytoplasmatic site of replication, their heat stability, the relatively high expression levels and proper post-translational modifications of foreign proteins as well as the ability to raise protective immune reactions locally on mucous membranes.

Vaccinia virus (VACV) strains are robust eukaryotic expression vectors that have been used for a number of different studies in biochemistry, cell biology, and immunology. Most vaccinia virus strains, such as the ones used in the smallpox eradication campaign, readily replicate in human cells and have been associated with a range of clinical complications in vaccinees. This fact is presently considered a major problem for the use of these strains as recombinant vaccines for mass vaccination. For laboratory use, in addition to mandatory biosafety level 2 procedures, proper precautions must be taken to prevent accidental

*Rattus norvegicus* in the Netherlands (Martina et al., 2006).

poxviruses carried by ectoparasites of animals in the target localities.

occurrence, distribution and ecology of poxviruses are hence important.

exposure when managing replication-competent vaccinia strains.

results in mortality rates of 10–15%.

**4.4 Poxvirus vaccine vectors** 

To circumvent the problems associated with "classical" vaccinia virus strains, several laboratories have been increasingly involved in efforts to develop more attenuated, hostrestricted virus strains (for brief review, see McFadden, 2005). Generally, these efforts involve two related strategies: i). The isolation of chicken cell-culture adapted vaccinia virus variants that are replication-deficient in mammalian cells, for example MVA, or ii). The development of avipoxvirus platforms, such as canarypox (ALVAC) and fowlpox (TROVAC), supposed to be naturally non-permissive for mammalian cells. There is increasing evidence that such non-replicating vaccines are safer than the original vaccinia strains and are still comparably immunogenic.

#### **4.4.1.1 MVA (Modified vaccinia virus Ankara)**

Among the so-called "non-replicating poxvirus vectors", modified vaccinia Ankara (MVA) has elicited considerable interest because of its excellent safety record in humans, its ability to mobilize different protective arms of the immune system, and the possibility to raise local mucosal membrane immunity after oral delivery. In addition to being a promising vector for the construction of poxvirus-based recombinant vaccines, one appealing feature of MVA as an expression vector is that it can be used under biosafety level 1 laboratory conditions. MVA has in common with NYVAC and other adapted VACV strains that they are *supposedly not carrying out fully productive infections in "relevant" mammalian cell types.* Such VACV strains do, however, secure efficient expression and presentation to the immune system of protein products from transgenes inserted into different locations in the viral DNA genome.

MVA was derived from a Turkish smallpox vaccine strain (Ankara) (reviewed by Drexler et al., 2004). After more than 500 passages in chicken cells, it tested defective for replication in human cells and avirulent in lab animals. From 1968–1980, MVA was inoculated into more than 100,000 individuals in Germany with no reported secondary complications and it is now considered to be a suitable platform for the next generation of safer smallpox vaccines and recombinant poxvirus vectors. Genomic mapping and sequencing studies have revealed that MVA lost nearly 30 kb of genomic information during its extended passage in chicken cells. Furthermore, it has multiple deletions and mutations compared with the parental strain. Many of these genetic alterations were in host-response genes. It is assumed that these deletions render MVA unable to carry out productive infections in human cells.

MVA has been proven very efficient in induction of mucosal immunity. This is of course utterly important since a number of human and animal disease agents use mucosal surfaces as their portal of entrance to the organism.

#### **4.4.1.2 Avipoxvirus vectors**

Like MVA, canarypox virus (ALVAC) and fowlpox (TROVAC) vaccine vectors induce antibody and cytotoxic T-cell responses, critical in the immune defense against viruses, to vectored viral antigens in a range of mammalian species (Hel et al., 2002; Srinivasan et al., 2006). Replication of avipox virus vectors is regarded abortive in mammalian cells, eliminating the safety concerns that exist for replication competent vaccinia virus vectors. The avipox viruses infect mammalian cells and produce viral proteins, with the replication block occurring at the time of viral DNA synthesis. It should, however, be noted that the experimental data backing claims for total lack of avipoxvirus replication in mammalian cells are based on studies embracing very few viral species or strains, and also very few, and mostly virus-ecologically irrelevant, mammalian cell cultures. For some avipoxvirus/mammalian cell combinations fully productive infections have been demonstrated (Weli et al., 2004).

Licensed ALVAC-vectored vaccines for dogs, cats, horses, chicken and ferrets are commercially available, and an ALVAC-vectored HIV-1 vaccine has entered phase III clinical trials. Some additional ALVAC-vectored HIV-1 and some human cancer and malaria vaccines are in clinical phase I or II trials (Weli & Tryland, 2011).

#### **4.5 Current research on risk assessment of GMVs**

There is little peer-reviewed information available that relates to ERA of GMV releases. To our knowledge research related to environmental effects is only being performed for alphaherpesviruses (Thiry et al., 2006) and poxviruses (orthopox and avipoxviruses). Such environmental biosafety-related research has been performed for a number of years in Norway, but we have no present knowledge of other research groups with a similar focus. We have concentrated on biosafety issues of the orthopoxvirus strain MVA (Modified Vaccinia Ankara), considered to be a very safe vaccine vector because of high gene expression capacity and lack of viral replication in mammalian cells (Drexler et al., 2004).

The most relevant conclusions from our studies may be summed up as follows:


cells are based on studies embracing very few viral species or strains, and also very few, and mostly virus-ecologically irrelevant, mammalian cell cultures. For some avipoxvirus/mammalian cell combinations fully productive infections have been

Licensed ALVAC-vectored vaccines for dogs, cats, horses, chicken and ferrets are commercially available, and an ALVAC-vectored HIV-1 vaccine has entered phase III clinical trials. Some additional ALVAC-vectored HIV-1 and some human cancer and malaria

There is little peer-reviewed information available that relates to ERA of GMV releases. To our knowledge research related to environmental effects is only being performed for alphaherpesviruses (Thiry et al., 2006) and poxviruses (orthopox and avipoxviruses). Such environmental biosafety-related research has been performed for a number of years in Norway, but we have no present knowledge of other research groups with a similar focus. We have concentrated on biosafety issues of the orthopoxvirus strain MVA (Modified Vaccinia Ankara), considered to be a very safe vaccine vector because of high gene expression capacity and lack of viral replication in mammalian cells (Drexler et al., 2004).

• Orthopoxviruses, and hence potential recombination partners for orthopoxvirus vectored vaccines, are common in different small rodent species populations all over the country, and small rodent predator species are infected by and have antibodies to

• Recombination between an influenza-transgenic MVA and a naturally occurring orthopoxvirus is readily demonstrated in cell cultures. The recombinants may have phenotypic characteristics, some of which may point towards adverse effects, different from the parental viruses. Recombinants may be genetically unstable and "throw out" the influenza transgene. This will eliminate the most logical tag for vaccine monitoring, and will also diminish the ability of the vector to mobilize protective immune responses

• The absolute and relative permissivities for MVA multiplication and viral shedding have not been thoroughly studied. GM and unmodified MVA may, contrary to the general dogma, perform fully productive infections in highly relevant mammalian cell types, and other mammalian cell cultures that are semi-permissive to infection (Okeke

• DNA sequencing revealed that orthopoxviruses can be clearly separated into geographically distinct strains, and it was inferred that these strains have distinct evolutionary histories in different rodent lineages (Hansen et al., 2009). It is an open question whether these different virus strains have aberrant abilities to engage in

• Upon sequencing of an orthopoxvirus isolated from a human clinical case, it was established that this strain was a naturally occurring hybrid between two distinct orthopoxvirus species. This is the first proof of concept for orthopoxvirus recombinations

taking place under authentic ecological circumstances (Hansen et al., 2011).

The most relevant conclusions from our studies may be summed up as follows:

vaccines are in clinical phase I or II trials (Weli & Tryland, 2011).

such viruses (Sandvik et al., 1998, Tryland et al., 1998).

recombination events with GM vaccine vectors.

**4.5 Current research on risk assessment of GMVs** 

demonstrated (Weli et al., 2004).

(Hansen et al., 2004).

et al., 2006).


#### **5. Features by GMVs that are relevant for risk assessment and management frameworks**

Directive 2001/18 with annexes, the EMEA guidelines and CPB with annexes give a number of leads towards GMO characteristics that may indicate hazards or adverse effects. Characteristics that must be taken into consideration (Anliker et al., 2010) include the pathogenicity, virulence, infectivity, host range, tissue tropism, replication strategy, latency/reactivation, survival and stability of the GMO. Here we will point to two important features by risk assessment and management frameworks that may cause difficulties when preparing ERA and risk management strategies for GMVs.

#### **5.1 The distinction between chemicals and organisms**

Risk perspectives used on chemical pollutions have been influential when shaping risk concepts in biosafety related to genetically modified organisms (GMOs), and hence also GMVs. We will argue that, and discuss why, this starting point is of very doubtful relevance to self-replicating organisms and molecules.

When the first genetically modified plants were approved and released the adequacy of the present framework for assessment of chemical substances i.e. ecotoxicological assessment, for GMOs was contested (Meyer, 2011). Chemicals have a release-dependent concentration decline with a given breakdown time in the environment, while GMOs follow a different environmental routes and degradation pathways than chemical pollutants. (Trans)Genes follow the path of the host genome, possibly eventually also the path of sexually compatible and some incompatible species (through vertical and horizontal gene transfer). Thus for GMOs exposure do not necessarily predict response, and accordingly risk models (based on the premise that exposure dose predicts response) have no or little utility in predicting the environmental behaviour of released transgenes. This was illustrated for example when Hilbeck et al. and Losey et al. reported adverse effects of Bt toxins and Bt maize pollen on non-target organisms in laboratory experiments (Hilbeck et al., 1998, Losey et al., 1999). To improve ecotoxicological testing, Andow and Hilbeck (2004) have suggested that investigation of non-target effects of GMOs may be done more efficiently by employment of a combination of two models; Ecotocixology testing of chemicals and risk assessment of non-indigenous species. However, the ecotoxicological approach still remains the recommended one in risk assessment procedures of GMOs although the new EU biosafety Directive 2001/18/EC partly supports the ecological approach since it prescribes a more detailed ERA.

#### **5.2 The distinction between viruses and organisms (modified from Traavik, 1999)**

It is important to keep up the distinction between viruses and organisms. Viruses are *not* organisms. Furthermore, the differences in genome strategies and life cycles between virus families are often more fundamental than between different mammalian or plant families.

Viruses multiply intracellularly in permissive host cells. One single virus particle (virion) infecting a permissive cell may give rise to billions of new particles during a short time (hours to days). The submicroscopic size of virions and the ability to spread over vast, even global, distances during short timespans are important, basic conceptions for ERA and risk management of any GMV. This is important in order to ask the relevant harm and hazard related questions, and hence to realize and conceive the risks connected with a given GM vaccine virus in a specific ecosystem and society context.

In addition to such fully productive infections, some virus/host cell combinations may result in persistent infections with virus shedding in the excreta for extended periods, while others lead to latent infection with viral DNA in a host chromosome-integrated or episomal state. Latent infections may be intermittently reactivated and accompanied by virus shedding. Integration of viral DNA into the host cell genome may by itself have harmful consequences, irrespective of viral gene expression or replication. The same virus strain may, under differently modulating conditions, display all these life cycle forms.

The host tropism, at the species-, organ- or cell type-level, is quite narrow for some viruses, while others have a much wider host-spectrum. For most viruses the molecular, genetic and epigenetic pathways determining host-cell specificity are not known in detail. Restrictions may be present at various steps during a virus multiplication cycle, from the lack of cell membrane receptors to subtle incompatibilities with host cell enzymes necessary for viral nucleic acid transcription and replication. Hence, minor genetic changes taking place during or after engineering of GM viruses may have profound effects on the host tropism and ability to spread to non-target host species.

For many virus/host cell combinations permissivity is a relative term, since it may be influenced to a considerable extent by the menu of genes expressed by the cell, and by the exact levels of gene expression. In culture, the permissivity of a given host cell may be manipulated experimentally by activation of intracellular signal transmission pathways, i.e. by hormones, growth factors, cytokines etc. Such procedures may also enhance persistent or reactivate latent infections. At the intra- as well as at the inter-species level of host animals this is illustrated by a vast variation in susceptibility for a given virus strain. Such variation may be related to sex, age, mating season, pregnancy, genetic differences, infection with other viruses or microorganisms, and environmental factors promoted by season, climatic changes or by pollution, e.g. EDCs (endocrine disrupting chemicals).

It is important to be aware of the distinction between viral infection and viral disease. An infected individual may shed virus and represent a transmission reservoir without showing clinical symptoms. Yet, other individuals within the same or other species may become clinically ill, or the viral infection may result in abortions, stillbirths, teratogenic or oncogenic effects. For persistent/latent infections, clinical symptoms may be present intermittently, only under special circumstances, or appear a long time after infection.

Different strains of the same viral species may have different virulence or pathogenicity, as well as host-cell or -species tropism. Even genetic differences at the single point mutation level may result in virus strains with aberrant phenotypic characteristics.

It is important to keep up the distinction between viruses and organisms. Viruses are *not* organisms. Furthermore, the differences in genome strategies and life cycles between virus families are often more fundamental than between different mammalian or plant families. Viruses multiply intracellularly in permissive host cells. One single virus particle (virion) infecting a permissive cell may give rise to billions of new particles during a short time (hours to days). The submicroscopic size of virions and the ability to spread over vast, even global, distances during short timespans are important, basic conceptions for ERA and risk management of any GMV. This is important in order to ask the relevant harm and hazard related questions, and hence to realize and conceive the risks connected with a given GM

In addition to such fully productive infections, some virus/host cell combinations may result in persistent infections with virus shedding in the excreta for extended periods, while others lead to latent infection with viral DNA in a host chromosome-integrated or episomal state. Latent infections may be intermittently reactivated and accompanied by virus shedding. Integration of viral DNA into the host cell genome may by itself have harmful consequences, irrespective of viral gene expression or replication. The same virus strain

The host tropism, at the species-, organ- or cell type-level, is quite narrow for some viruses, while others have a much wider host-spectrum. For most viruses the molecular, genetic and epigenetic pathways determining host-cell specificity are not known in detail. Restrictions may be present at various steps during a virus multiplication cycle, from the lack of cell membrane receptors to subtle incompatibilities with host cell enzymes necessary for viral nucleic acid transcription and replication. Hence, minor genetic changes taking place during or after engineering of GM viruses may have profound effects on the host tropism and

For many virus/host cell combinations permissivity is a relative term, since it may be influenced to a considerable extent by the menu of genes expressed by the cell, and by the exact levels of gene expression. In culture, the permissivity of a given host cell may be manipulated experimentally by activation of intracellular signal transmission pathways, i.e. by hormones, growth factors, cytokines etc. Such procedures may also enhance persistent or reactivate latent infections. At the intra- as well as at the inter-species level of host animals this is illustrated by a vast variation in susceptibility for a given virus strain. Such variation may be related to sex, age, mating season, pregnancy, genetic differences, infection with other viruses or microorganisms, and environmental factors promoted by season, climatic

It is important to be aware of the distinction between viral infection and viral disease. An infected individual may shed virus and represent a transmission reservoir without showing clinical symptoms. Yet, other individuals within the same or other species may become clinically ill, or the viral infection may result in abortions, stillbirths, teratogenic or oncogenic effects. For persistent/latent infections, clinical symptoms may be present intermittently, only under special circumstances, or appear a long time after infection.

Different strains of the same viral species may have different virulence or pathogenicity, as well as host-cell or -species tropism. Even genetic differences at the single point mutation

may, under differently modulating conditions, display all these life cycle forms.

changes or by pollution, e.g. EDCs (endocrine disrupting chemicals).

level may result in virus strains with aberrant phenotypic characteristics.

**5.2 The distinction between viruses and organisms (modified from Traavik, 1999)** 

vaccine virus in a specific ecosystem and society context.

ability to spread to non-target host species.

For GMVs it is hence conceivable that unintended phenotypic characteristics with unwanted ecological consequences may be established in addition to the intended modification(s). This may not become evident unless very comprehensive and carefully planned experiments and ecosystem surveillance/sampling programs are carried out. In many instances fully adequate studies are totally precluded by the complexity and the regular or occasional variations of the recipient ecosystem. Hence scientific uncertainty or ignorance is a state that must be accepted.

#### **6. The connection between risk assessment and risk management**

According to Anliker et al. (2010), an ERA based on Directive 2001/18/EC should follow four general principles:

"First, the GMO should be compared to the non-modified organism from which it is derived. Second, the ERA should be carried out on a scientifically sound premise and rely on known facts supported by data derived from specific testing of the GMO-containing medicinal product including its use in previous clinical trials. If necessary, this data can be substantiated by theoretical assumptions. Third, it is necessary to perform the ERA on a case-by-case basis, since the heterogeneity of the GMO-containing medicinal products and the differences in their clinical use make it difficult to apply standardised requirements or evaluations as part of the assessment. Finally, the ERA needs to be re-evaluated if new information on the GMO or its effects on human health or the environment becomes available."

We find it an interesting and elucidating statement that "data can be substantiated by theoretical assumptions". This opens the ERA gate for creation and inclusion, as well as theoretical, mathematic and experimental modeling, of hypotheses build on "Worst case scenarios", as pointed out by Kühler et al. (2009), and also for the use of stringent Precautionary principle versions in situations and processes dominated by scientific uncertainty and ignorance (see section 9).

The case-by-case and step-by-step approaches to GMV ERAs seem to be instituted in all relevant national and international legislations, including EU Directive 2001/18/EC and the Cartagena protocol on biosafety (CPB). Any ERA should include comparative data about characteristics of the GMV and its unmodified parental virus strain. Details about the genetic modification/engineering process, including data about effects, genomic location and DNA sequence of the transgenic (vaccine gene) insert must be present. Descriptions of the vaccination regime and release/possibilities for escape of the GMV must be considered. The receiving environment/ecosystem, including possible interactions between the GMV and the environment, have to be described. Plans for monitoring, control, waste treatment and emergency response plans have to be prepared and presented, and this is, of course, tasks that overlap with risk management activities.

Anliker et al. (2010) state: "Experience gained from the release of comparable GMOs into a similar environment can be used to support the ERA". In our view this is to some extent an unwanted premise for trustworthy ERAs since it opens up for subjective interpretations of "comparable GMOs" and "similar environment". Theoretical considerations and practical experience indicate that such parameters rarely exist for engineered organisms, and are even more rare for viruses. This should become evident while reading and reflecting on the scientific ignorance, uncertainty and lack of knowledge related to the nature, characteristics and ecology of GMVs.

The ERA procedure may be scientifically and operationally divided into five steps, whereby the ERA and risk management is integrated and coordinated (described in an excellent review by Anliker et al., 2010):


Through the outlined procedure each potentially harmful characteristic of a GMO should be turned into risks. The total process should form a basis for consideration of the overall risk to the ecosystem, animal and human health by deliberate release or marketing of a given GMO. The ERA should conclude on whether there is a need for a risk management plan. If necessary and possible the ERA should devise appropriate risk mitigation methods. And of primary importance: The ERA should reach a conclusion as to whether the overall environmental impact is at all acceptable or not (Kühler et al., 2009).

Step 1 of the ERA procedure is focusing on the identification of GMO characteristics that may result in adverse effects. This is the decisive step for whether, and to which extent, the ERA and the risk management plans will provide biosafety and make contributions to sustainable development as well as to good health of ecosystems, animals and humans.

In our opinion the creative conception and design of "What-if?"- and Worst-case-scenarioinspired ideas, questions and working hypotheses, must be encouraged and stimulated in order to enhance the chances of high precaution levels in ERA and risk management processes. Furthermore, we will argue that these goals will only be met if independent scientists and institutions carry out the relevant intellectual processes and research projects. Finally, although national legislations, and also the Cartagena protocol, require documented scientific evidence as a basis for ERAs and risk management plans, implementation of the Precautionary principle is always expressively required (Kûhler et al., 2009). The different versions, interpretations and possibilities for implementation of the precautionary principle are further discussed in section 9.

#### **6.1 Relevant risk assessment and management questions for GMVs**

The different virus families have their specific life cycles and host-preferences. Hence it is impossible to make risk assessment schemes that are valid for all potential virus vectors. Risk assessment must be performed on a case-by-case, step-by-step basis, taking into account the characteristics of the ecosystem into which the virus will be released, and the ability of the virus to engage in transboundary movements (Traavik, 1999; McFadden, 2005).

The most evident risk issues related to release of GMVs or unmodified viruses are the known and unknown unknowns related to (i) whether active multiplication with virus shedding in excreta takes place in target individuals, (ii) infection of non-target species, (iii)

The ERA procedure may be scientifically and operationally divided into five steps, whereby the ERA and risk management is integrated and coordinated (described in an excellent

3. Evaluation of the probability (likelihood) that each identified potential adverse effect

5. Application of risk management strategies related to marketing or deliberate release of

6. Evaluation of the overall risks of the GMO, based on the conclusions from the previous steps, taking into account the risk management strategies proposed in step 5, which

Through the outlined procedure each potentially harmful characteristic of a GMO should be turned into risks. The total process should form a basis for consideration of the overall risk to the ecosystem, animal and human health by deliberate release or marketing of a given GMO. The ERA should conclude on whether there is a need for a risk management plan. If necessary and possible the ERA should devise appropriate risk mitigation methods. And of primary importance: The ERA should reach a conclusion as to whether the overall

Step 1 of the ERA procedure is focusing on the identification of GMO characteristics that may result in adverse effects. This is the decisive step for whether, and to which extent, the ERA and the risk management plans will provide biosafety and make contributions to sustainable development as well as to good health of ecosystems, animals and humans.

In our opinion the creative conception and design of "What-if?"- and Worst-case-scenarioinspired ideas, questions and working hypotheses, must be encouraged and stimulated in order to enhance the chances of high precaution levels in ERA and risk management processes. Furthermore, we will argue that these goals will only be met if independent scientists and institutions carry out the relevant intellectual processes and research projects. Finally, although national legislations, and also the Cartagena protocol, require documented scientific evidence as a basis for ERAs and risk management plans, implementation of the Precautionary principle is always expressively required (Kûhler et al., 2009). The different versions, interpretations and possibilities for implementation of the precautionary principle

The different virus families have their specific life cycles and host-preferences. Hence it is impossible to make risk assessment schemes that are valid for all potential virus vectors. Risk assessment must be performed on a case-by-case, step-by-step basis, taking into account the characteristics of the ecosystem into which the virus will be released, and the ability of the virus to engage in transboundary movements (Traavik, 1999; McFadden, 2005). The most evident risk issues related to release of GMVs or unmodified viruses are the known and unknown unknowns related to (i) whether active multiplication with virus shedding in excreta takes place in target individuals, (ii) infection of non-target species, (iii)

2. Evaluation of the potential consequences of each adverse effect, if it occurs;

4. Estimation of the risk posed by each identified characteristic of the GMO;

were created to reduce or eliminate the risks identified in step 4.

environmental impact is at all acceptable or not (Kühler et al., 2009).

**6.1 Relevant risk assessment and management questions for GMVs** 

review by Anliker et al., 2010):

are further discussed in section 9.

should occur;

the GMO;

1. Identification of potential adverse effects;

recombinations with naturally occurring virus relatives and (iii) integration of GMV DNA into host cell chromosomes.

Ideally, before running the risk that any GMV becomes implanted into a species/population/new location/ecosystem a number of crucial questions should be answered (modified from Traavik, 1999), e.g.:


Some of these questions deal with the genetic and phenotypic characteristics of a supposedly genetically stable GMV. But the situation becomes even more complex and unpredictable if the GMV parental strain under certain conditions or circumstances is genetically unstable, giving rise to viral strains with altered characteristics (Traavik, 1999). At the present time it is strongly needed that questions and hypotheses related to the influence of climatic changes and xenobiotics, e.g. endocrine disrupting chemicals (EDCs) are included in ERAs and risk management plans.

Demanding ERA and risk management challenges connected to all the questions raised in this section are related to whether methods allowing detection of the phenomena listed, and still unlisted, have been developed, and whether the surveillance and monitoring programs that make their employment possible have been funded and operationalized.

#### **7. Biosafety Implications for environmental risk assessment and management frameworks**

We will argue that in order to harvest the potential benefits of any GMV, the approaches used in the Norwegian orthopoxvirus studies should be part of the regulatory risk governance frameworks all over the world (see section 4.5 as well as 6.1). In relation to ERA and risk management needs, some of the most urgent issues for targeted research will be treated in the following.

#### **7.1 Naturally occurring relatives**

With GMV use and release, it will be crucial to determine the occurrence, distribution and ecology of poxvirus relatives. In Norway rodents and other small mammals are considered to be natural reservoirs for different orthopoxviruses, and cowpoxvirus-like strains in particular (Sandvik et al., 1998). Approximately 20% of shrews and small rodents belonging to eight species carry orthopoxvirus DNA sequences in their organs, and 20% of such animals have specific serum antibodies as a sign of previous infections.

#### **7.2 Potential recombination events and their consequences**

If a GM orthopoxvirus infects an individual, animal or human, that already carries another orthopoxvirus, a recombination event, homologous or illegitimate, may follow. Foreign genes may hence be transferred from the GM donor virus to other GM poxvirus vaccines or to wild type orthopoxvirus recipient strains (Sandvik et al., 1998). The outcome may be hybrid viral progenies with unpredictable pathogenicity and altered host range. The probability and possible outcome of recombination is dependent on the characteristics of the viral vector used, and the occurrence of naturally occurring or genetically modified relatives.

In cell culture co-infection experiments with a MVA-based human influenza vaccine and a newly isolated Norwegian cowpox virus-like strain, a number of recombinant progeny virus strains were obtained (Hansen et al., 2004). One of the progeny strains displayed phenotypic characteristics different from both parents, and was genetically unstable upon cell culture passage, i.e. the influenza transgene was deleted at a high frequency. This is of paramount monitoring significance, since the inserted gene will always be the marker of choice for tracing and detection of vaccine-related effects.

Environmental release or escape of a GMV provides the opportunity for recombination with poxviruses present in target and non-target animals, including humans. In most parts of the world the occurrence and distribution of naturally occurring poxviruses are virtually unknown. But, in central-Africa it has been proposed that the vaccinia- rinderpest vaccine (RVFH) might recombine with a pox-relative, namely monkeypoxvirus. Since smallpox has been eradicated, the monkey pox is at present the most feared poxvirus (Reed, 2004), and has for instance caused virulent outbreaks in Zaire (Kaaden et al., 2002).

Under natural circumstances the probability of recombination may be low, but predictions and forecasts are made impossible by the fact that we lack knowledge about the natural occurrence and prevalence of poxviruses in all parts of the world. Furthermore, even when recombination events are rare, the consequences may be serious since one viral progeny particle may multiply into millions of identical particles in a matter of hours. In this context, it is disturbing that the present regulatory frameworks are based on a one time risk assessment of any given GMV, and does not take into account the putative consequences that may arise from successive releases of the same, or related, GMVs.

During the global smallpox eradication program (in the 1970s) transmission from vaccinated to unvaccinated persons was estimated to occur at a rate of 27 infected per million total vaccinated (Centre for Disease Control, 1991). It has been possible to isolate the vaccinia

and risk management needs, some of the most urgent issues for targeted research will be

With GMV use and release, it will be crucial to determine the occurrence, distribution and ecology of poxvirus relatives. In Norway rodents and other small mammals are considered to be natural reservoirs for different orthopoxviruses, and cowpoxvirus-like strains in particular (Sandvik et al., 1998). Approximately 20% of shrews and small rodents belonging to eight species carry orthopoxvirus DNA sequences in their organs, and 20% of such

If a GM orthopoxvirus infects an individual, animal or human, that already carries another orthopoxvirus, a recombination event, homologous or illegitimate, may follow. Foreign genes may hence be transferred from the GM donor virus to other GM poxvirus vaccines or to wild type orthopoxvirus recipient strains (Sandvik et al., 1998). The outcome may be hybrid viral progenies with unpredictable pathogenicity and altered host range. The probability and possible outcome of recombination is dependent on the characteristics of the viral vector used, and the occurrence of naturally occurring or genetically modified relatives.

In cell culture co-infection experiments with a MVA-based human influenza vaccine and a newly isolated Norwegian cowpox virus-like strain, a number of recombinant progeny virus strains were obtained (Hansen et al., 2004). One of the progeny strains displayed phenotypic characteristics different from both parents, and was genetically unstable upon cell culture passage, i.e. the influenza transgene was deleted at a high frequency. This is of paramount monitoring significance, since the inserted gene will always be the marker of choice for

Environmental release or escape of a GMV provides the opportunity for recombination with poxviruses present in target and non-target animals, including humans. In most parts of the world the occurrence and distribution of naturally occurring poxviruses are virtually unknown. But, in central-Africa it has been proposed that the vaccinia- rinderpest vaccine (RVFH) might recombine with a pox-relative, namely monkeypoxvirus. Since smallpox has been eradicated, the monkey pox is at present the most feared poxvirus (Reed, 2004), and

Under natural circumstances the probability of recombination may be low, but predictions and forecasts are made impossible by the fact that we lack knowledge about the natural occurrence and prevalence of poxviruses in all parts of the world. Furthermore, even when recombination events are rare, the consequences may be serious since one viral progeny particle may multiply into millions of identical particles in a matter of hours. In this context, it is disturbing that the present regulatory frameworks are based on a one time risk assessment of any given GMV, and does not take into account the putative consequences

During the global smallpox eradication program (in the 1970s) transmission from vaccinated to unvaccinated persons was estimated to occur at a rate of 27 infected per million total vaccinated (Centre for Disease Control, 1991). It has been possible to isolate the vaccinia

has for instance caused virulent outbreaks in Zaire (Kaaden et al., 2002).

that may arise from successive releases of the same, or related, GMVs.

animals have specific serum antibodies as a sign of previous infections.

**7.2 Potential recombination events and their consequences** 

tracing and detection of vaccine-related effects.

treated in the following.

**7.1 Naturally occurring relatives** 

virus from domesticated animals that have been in contact with recently vaccinated humans. The potential host spectrum of vaccinia virus is very broad and includes laboratory animals, pigs, cattle, camel, and monkey species (Fenner, 1996). In the past, vaccinia virus has for instance spread from vaccinated humans to domestic animals such as cattle (through milkers) and buffaloes, before spreading within the herd. Furthermore, it has been reported that during outbreaks of buffalopox, the buffalopox virus infected unvaccinated humans (Dumbell & Richardson, 1993). In Brazil, an emerging virus with similar characteristics as a smallpox vaccine virus caused disease both in cattle and their human caretakers (Damaso et al., 2000).

GMVs made for wildlife vaccination must be thermostable and have long environmental persistence, and VACs satisfy these criteria. The vaccinia-rabies vaccine (VRG), for instance, has been reported to persist 4 months under natural conditions without a significant loss of viral infectivity (Brochier et al., 1990). Environmental stability is a prerequisite for herd immunity in target animals, but by the same token it increases the risk of non-target effects, and for spread to non-target species and other ecosystems.

VACV has been shown, in cell cultures, to easily engage in genetic recombination with other orthopoxvirus species (Ball, 1987). A high number of closely related viral species are known, and high degrees of sequence homology across species borders have been demonstrated. It may, however, be safely assumed that a high number of unknown orthopoxviruses are circulating in many different types of ecosystems and biotopes all over the world. Biological (i.e. insects, migratory birds and animals, domestic and pet animal trade, infected individuals in an incubation period etc), as well as mechanical, (i.e. automobiles, airplanes) vectors may enable further dissemination and transmission of a virus to other ecosystems. Changed tropism for particular cells or tissues may result in divergent virulence in the target, or other known, host species for the GMV or its recombinant progenies compared to the parental, unmodified poxvirus (Traavik, 1999). In addition, the GMV or its recombinant progenies may have had their host restriction programs changed, so that the viruses can transfer to and infect formerly resistant host species. Other phenotypic changes may also happen. For instance, it has been reported that GE of a poxvirus created a GMV that was more virulent than the non-modified virus. As part of a strategy to develop pest animal contraceptive vaccines, the gene encoding human interleukin-4 (IL-4) was inserted into the Ectromelia virus (mousepox) genome (Jackson et al., 2001). Expression of IL-4 was intended to curb unwanted anti-viral effects. Unexpectedly, genetically mouse pox-resistant mice infected with the IL-4 expressing virus developed symptoms of acute mouse pox accompanied by high mortality.

#### **7.3 Non-target effects and transboundary spread**

GMV administration may have non-target effects. Baits containing GMVs may be eaten both by target and non-target animals. In the second instance, viral spread may also take place by direct/indirect human/animal contact, or by installation of the GMV into a food web. A non-target GMV transfer was illustrated by the account of a woman, with a predisposing skin disease, who, having removed a VRG bait from the mouth of her dog, became infected and developed pox lesions (Rupprecht et al., 2001).

The eradication of rabies by vaccination may have indirect consequences for the ecological balance; lack of disease favours an increase in the host animal population, in this case the European red fox. Therefore, it is crucial that monitoring of GMVs in the environment is initiated with the purpose to follow-up the performed risk assessment, to map the actual environmental effects and to identify unforeseen adverse ecological effects.

GMVs or their progenies resulting from mutations and/or recombination events may achieve new phenotypic traits of importance for their ecology, spread and host preferences. The opportunities for long distance spread and cross-species transfer of mammalian viruses have increased in recent years due to enhanced contact between humans and animal reservoirs. It is, however, difficult to predict when such events will take place since the viral adaptations that are needed are multifactorial and stochastic. Recent examples of viruses that have crossed species barriers are HIV, hantaviruses, haemorrhagic fever viruses, various arboviruses (e.g. West Nile Virus), avian and porcine influenza viruses, SARSassociated coronavirus, Nipah and Hendra viruses, and monkeypox virus.

The emergence of new viral infections often follows environmental, ecological and technological changes caused by human activities (Louz et al., 2005). Such activities may lead to an increased contact between humans and livestock on one hand, and animal hosts acting as reservoirs of zoonotic viruses on the other hand. Agricultural development, an increased exploitation of environmental resources, growth and increase in the mobility of the human population and trade and transportation of food and livestock, have been identified as important factors contributing to the introduction and spread of a number of new viruses in the human population.

Against this background the intensified use of viruses and their genetically modified variants as viral gene transfer vectors for biomedical research, experimental gene therapy and as live-vector vaccines is a cause for concern (reviewed by Louz et al., 2005).

#### **7.4 Future safety prospects**

There are now a number of examples that unwanted characteristics of poxvirus vectors can be modified or excluded by targeted mutagenesis, homologous recombination and reverse genetics (Najera et al., 2006; Chakroudi et al., 2005). But the safety benefits of these approaches can only be taken out when we have clarified putative GMV characteristics and adverse effect issues within the categories "known unknowns" as well as "unknown unknowns".

#### **8. The normative challenge by the concepts of harm and unwanted ecological consequences**

In previous sections we have described several potential harms to the environment and discussed the relevance for ERA and risk management strategies. We will here discuss concepts and definitions related to harms and hazards in the context of legislative frameworks, and we will argue that for descriptive as well as for normative purposes, biological, ecological and ethical terms are needed for identification of unwanted harm and unwanted ecological consequences..

Endpoints of any risk assessment and risk management are always connected to the regulative framework. Article 1 of the Cartagena Protocol specifies that the entire objective of the document is to protect and conserve biodiversity according to a precautionary

initiated with the purpose to follow-up the performed risk assessment, to map the actual

GMVs or their progenies resulting from mutations and/or recombination events may achieve new phenotypic traits of importance for their ecology, spread and host preferences. The opportunities for long distance spread and cross-species transfer of mammalian viruses have increased in recent years due to enhanced contact between humans and animal reservoirs. It is, however, difficult to predict when such events will take place since the viral adaptations that are needed are multifactorial and stochastic. Recent examples of viruses that have crossed species barriers are HIV, hantaviruses, haemorrhagic fever viruses, various arboviruses (e.g. West Nile Virus), avian and porcine influenza viruses, SARS-

The emergence of new viral infections often follows environmental, ecological and technological changes caused by human activities (Louz et al., 2005). Such activities may lead to an increased contact between humans and livestock on one hand, and animal hosts acting as reservoirs of zoonotic viruses on the other hand. Agricultural development, an increased exploitation of environmental resources, growth and increase in the mobility of the human population and trade and transportation of food and livestock, have been identified as important factors contributing to the introduction and spread of a number of

Against this background the intensified use of viruses and their genetically modified variants as viral gene transfer vectors for biomedical research, experimental gene therapy

There are now a number of examples that unwanted characteristics of poxvirus vectors can be modified or excluded by targeted mutagenesis, homologous recombination and reverse genetics (Najera et al., 2006; Chakroudi et al., 2005). But the safety benefits of these approaches can only be taken out when we have clarified putative GMV characteristics and adverse effect issues within the categories "known unknowns" as well as "unknown

**8. The normative challenge by the concepts of harm and unwanted ecological** 

In previous sections we have described several potential harms to the environment and discussed the relevance for ERA and risk management strategies. We will here discuss concepts and definitions related to harms and hazards in the context of legislative frameworks, and we will argue that for descriptive as well as for normative purposes, biological, ecological and ethical terms are needed for identification of unwanted harm and

Endpoints of any risk assessment and risk management are always connected to the regulative framework. Article 1 of the Cartagena Protocol specifies that the entire objective of the document is to protect and conserve biodiversity according to a precautionary

and as live-vector vaccines is a cause for concern (reviewed by Louz et al., 2005).

environmental effects and to identify unforeseen adverse ecological effects.

associated coronavirus, Nipah and Hendra viruses, and monkeypox virus.

new viruses in the human population.

**7.4 Future safety prospects** 

unwanted ecological consequences..

unknowns".

**consequences** 

approach. In the EU directive 2001/18/EC, it is stated that the applicant must submit a notification including an environmental risk assessment that considers direct and indirect effects, immediate and delayed effects, as well as potential cumulative and long term effects due to interaction with other GMOs and the environment. Harms to human and animal health are aspects that need to be characterised broadly. These include consideration of direct effects, e.g. development of disease, and indirect effects that may have a more complex nature; i.e. altered susceptibility to disease, welfare aspects and harm in social and ethical terms. With regard to harm to the environment, potential effects include the influence on and interactions with all organisms in the environment, and may be direct or indirect. Direct effects concern biological impacts on organisms, while indirect effects concern effects on animal health, contamination of wild gene pools or alterations in ecological relationships.

The legislative framework leaves many questions about what is to be treated as harm, which is a concept that both is descriptive and normative. For example, Raybould points to a central normative problem in the relationship between risk research and risk assessment of GM crops, and argues that the problem formulation (step 1) strongly depends on the respective stakeholder interests with regard to the environment and the goods and processes that need to be protected (Raybould, 2006). Moreover it is not only step 1 in environmental risk assessment but also step 5 that is a normative issue (Hill, 2005), hence risk assessments include value judgements both with regard to consequences that should be avoided and the process of risk characterisation. These choices are most often made before the risk assessment has been initiated. Accordingly, the question of harm is correlated to conception of human welfare and how to maintain and preserve nature and biodiversity. What is to count as serious harm to nature is based on contestable value judgments. There are for example distinct philosophical differences between giving priority to protection of human interests, i.e. anthropocentrism, versus preservation of ecosystems, i.e. biocentrism and ecocentrism (Dobson, 1998; Westra, 1998). In an anthropocentric context, the environment is protected to promote human well-being, as recreation purposes, or seen as a source for gaining new knowledge, assuming ecosystems to contain unknown information. Moreover, biodiversity centres represent valuable genetic pools for future agricultural and medicinal development. Hence, human interests provide a powerful set of motives for protecting the environment against activities that may have severe consequences (i.e. reduced biodiversity) for present and future generations.

Biocentrics and eccocentrics emphasize the need for a change from the anthropocentric exploitation of the environment towards a greater respect for the integrity of the animals and the environment. Biocentrics argue that as humans, we must provide rights to species and habitats and hence it is our duty to respect their integrity (Regan, 1980). An ecocentric approach involves more than consideration of impacts on human welfare and sustainability, and focuses on changes in both the biotic and the abiotic environment, as for instance effects on soil, water and air. Ecocentric approaches are not only aware of integrity of the animals and the environment, but do also acknowledge ecosystem integrity. In this context, preservation and protection of biological, ecological and genetic processes are necessary, irrespective of the instrumental value to humans. Application and release of GMVs can then be justified if it promotes the welfare of the ecosystem, or when it protects or adds to the diversity of the species in the community. This ideology differs with regard to value commitments and factual beliefs from anthropocentric GMV governance. Hence, how we approach the environment and the values we put on the environment may also affect the frames and approaches chosen in environmental risk assessment and management.

#### **8.1 Problem formulation processes for involvement of normative issues**

To take into account the normative issues Nelson and Banker (2007) have suggested that step 1 in the environmental risk assessment need to be developed into a "Problem Formulation". Scientists in support of the ecological approach to risk assessment of GM crops (see section 5.1) have been involved in developing the problem formulation and option assessment (PFOA) tool, that is based on stocktaking exercises, stakeholder consultation and broader public participation procedures. The PFOA has been used in developing countries with the intention to improve the ERA and as a technology assessment tool and entails involvement of not only scientists but also the public in identification of the problem to be analysed. Various types of knowledge held by the public in general (local knowledge) as well as knowledge held by different interest groups and affected parties are here seen as valuable insights that may help to critically broaden the scope of the risk assessment and are also considered important in identification of protection goals. Moreover, local knowledge is also highly relevant with regard to development of risk management strategies and can give valuable insight with regard to local conditions relevant for risk associated research and in monitoring activities.

By transforming ERA into a problem formulation process we would like to add that besides including local knowledge there is also a need for broader scientific expertise to ensure a thorough assessment of the GMV in question. By extending the scientific experts involved from molecular biologists and virologists, to include ecologists, biologists as well as ethicists and sociologists, more diverse approaches to problem formulation processes can be ensured. Involvement of different scientific disciplines will also be crucial in elaboration of normative issues, in identification of protection goals and in development of management strategies. The scientists involved should share their opinions with the public as well as relevant political authorities.

#### **9. Precautionary principle and precautionary approaches**

We have discussed how risk assessment and management strategies are developed within particular frameworks, including normative standards and preferences regarding our relation to the natural environment and the preservation/promotion of the environment (see section 5 and 8). In such cases, decision-makers have to make decisions that will include the challenging issue of how to handle uncertainties. We consider that the uncertainties involved with GMVs entail that the precautionary principle needs to be employed (Myhr & Traavik, 2007).

The precautionary principle is a normative principle for making practical decisions under conditions of scientific uncertainty, and provides a general approach to environmental and health protection (EC, 2000). The actual content of the precautionary principle and the

when it protects or adds to the diversity of the species in the community. This ideology differs with regard to value commitments and factual beliefs from anthropocentric GMV governance. Hence, how we approach the environment and the values we put on the environment may also affect the frames and approaches chosen in environmental risk

To take into account the normative issues Nelson and Banker (2007) have suggested that step 1 in the environmental risk assessment need to be developed into a "Problem Formulation". Scientists in support of the ecological approach to risk assessment of GM crops (see section 5.1) have been involved in developing the problem formulation and option assessment (PFOA) tool, that is based on stocktaking exercises, stakeholder consultation and broader public participation procedures. The PFOA has been used in developing countries with the intention to improve the ERA and as a technology assessment tool and entails involvement of not only scientists but also the public in identification of the problem to be analysed. Various types of knowledge held by the public in general (local knowledge) as well as knowledge held by different interest groups and affected parties are here seen as valuable insights that may help to critically broaden the scope of the risk assessment and are also considered important in identification of protection goals. Moreover, local knowledge is also highly relevant with regard to development of risk management strategies and can give valuable insight with regard to local conditions

By transforming ERA into a problem formulation process we would like to add that besides including local knowledge there is also a need for broader scientific expertise to ensure a thorough assessment of the GMV in question. By extending the scientific experts involved from molecular biologists and virologists, to include ecologists, biologists as well as ethicists and sociologists, more diverse approaches to problem formulation processes can be ensured. Involvement of different scientific disciplines will also be crucial in elaboration of normative issues, in identification of protection goals and in development of management strategies. The scientists involved should share their opinions with the public as well as relevant

We have discussed how risk assessment and management strategies are developed within particular frameworks, including normative standards and preferences regarding our relation to the natural environment and the preservation/promotion of the environment (see section 5 and 8). In such cases, decision-makers have to make decisions that will include the challenging issue of how to handle uncertainties. We consider that the uncertainties involved with GMVs entail that the precautionary principle needs to be employed (Myhr &

The precautionary principle is a normative principle for making practical decisions under conditions of scientific uncertainty, and provides a general approach to environmental and health protection (EC, 2000). The actual content of the precautionary principle and the

**8.1 Problem formulation processes for involvement of normative issues** 

relevant for risk associated research and in monitoring activities.

**9. Precautionary principle and precautionary approaches** 

assessment and management.

political authorities.

Traavik, 2007).

practical implications of its implementation in policy issues are however controversial (Foster et al., 2000). Several versions of the principle, ranging from ecocentric to anthropocentric, and from risk-adverse to risk-taking positions have been put forward (Raffensperger & Tickner, 1999). Here we would like to acknowledge the version of the principle that can be found in the 1990 Bergen Declaration on Sustainable Development:

"In order to achieve sustainable development, policies must be based on the precautionary principle. Environmental measures must anticipate, prevent and attack causes of environmental degradation. Where there are threats of serious or irreversible damage, lack of full scientific certainty should not be used as a reason for postponing measures to prevent environmental degradation."

What we appreciate with this version is the commitment to anticipate, prevent and attack causes of environmental degradation. We also acknowledge the passive voice in the following part of this version of the principle; "lack of full scientific certainty should not be used as a reason for postponing measures to prevent environmental degradation", and see that this entails lack of requirement of action. However, this version of the principle connects the importance of taking caution in innovation with the achievement of sustainable development, and is active in nature by stating that in practice its application demands anticipation and prevention of harms. For GMVs the present lack of knowledge and the uncertainties with regard to environmental effects as for example with regard to potential recombinational events, non-target effects and transboundary spread entails the necessity to initiate targeted research, in line with a precautionary approach to decision-making. In this sense, it can be argued that this version of the principle extends from being a formulaic decision-making rule to also include an approach to include scientific activity in decisionmaking

The important elements of what represents a precautionary approach to decision-making are (Wickson et al., 2011):


#### **10. Conclusion**

So far no GMVs have been thoroughly risk assessed from an environmental point of view. Risk assessments have focused on unintended effects of the vaccine arising in the vaccinated individuals, or in individuals of the same species that are infected by virus shed from vaccinated individuals. Here we have elaborated that risk assessment and management strategies need to be connected, and that a precautionary approach needs to be employed to GMVs.

A precautionary approach to GMVs includes initiation of risk-associated research with the intention to advance specific hypotheses about GMV specific harm and hazard assessment and management endpoints. Investigation of potential adverse effects and preventive measures needs to be initiated according to research and policy agendas encouraging broad and long-term thinking that supports precautionary actions. We consider that problem formulation processes represent a good approach for how to broaden the involvement of expertise and knowledge, and for identification of normative issues. As illustrated in section 8, risk management endpoints are dependent on both scientific and normative aspects, hence how nature, how harm and environmental damage are identified can influence the objectives of environmental risk management and thereby be used to identify specific targets for protection. These endpoints are also dependent on the objectives of environmental risk assessment research. After identification of endpoints, specific hypotheses for characterization of risk can be developed which enables targeted research to be carried out, where models are used and data are collected with the purpose of testing the risk hypotheses. We have in 4.5 briefly described some of the research that has been carried out in our own institution. We would like to stress that such approaches represent a good platform for research initiatives in other countries. Such risk-associated research can include questions presented in section 6.1. Accordingly, the assessment and management of potential adverse effects must include conception of the ecological background as well as normative discussions with regard to endpoints. Approaches that take this into account may secure that the final stages have a broad basis for decision-making: both with regard to representation and involvement of ecological aspects and ethical values.

#### **11. References**


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A precautionary approach to GMVs includes initiation of risk-associated research with the intention to advance specific hypotheses about GMV specific harm and hazard assessment and management endpoints. Investigation of potential adverse effects and preventive measures needs to be initiated according to research and policy agendas encouraging broad and long-term thinking that supports precautionary actions. We consider that problem formulation processes represent a good approach for how to broaden the involvement of expertise and knowledge, and for identification of normative issues. As illustrated in section 8, risk management endpoints are dependent on both scientific and normative aspects, hence how nature, how harm and environmental damage are identified can influence the objectives of environmental risk management and thereby be used to identify specific targets for protection. These endpoints are also dependent on the objectives of environmental risk assessment research. After identification of endpoints, specific hypotheses for characterization of risk can be developed which enables targeted research to be carried out, where models are used and data are collected with the purpose of testing the risk hypotheses. We have in 4.5 briefly described some of the research that has been carried out in our own institution. We would like to stress that such approaches represent a good platform for research initiatives in other countries. Such risk-associated research can include questions presented in section 6.1. Accordingly, the assessment and management of potential adverse effects must include conception of the ecological background as well as normative discussions with regard to endpoints. Approaches that take this into account may secure that the final stages have a broad basis for decision-making: both with regard to

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