**2. In vitro manipulation of plants**

Plant cell and tissue culture or in vitro manipulation of plant is the key of modern plant biotechnology. Whole plant can be regenerated under aseptic conditions (in glass vessels) using tissues and even cell when provided balanced nutritional conditions. This technology successfully lead to production of elite cultivars, conservation of endangered plant, production of virus free plant, safeguarding of germplasm and production of secondary metabolites. Beside all these, establishment of culturing protocol is main principle in near about all transgenic plant production strategies. Ability of cell to generate into whole organism is attributed to totipotency and plant cells are unique in this case. However, understanding culture conditions with regard to plant species and explant type is critical for development of reliable system. The physiology of explant is more important because stage and age of explant respond differentially under same conditions. While, some plant species can be easily propagated and some species demand variability in growth regulator(s) concentration(s).

The development of successful tissue culture procedure demand appropriate physiological and chemical conditions. Physiological settings include temperature, pH, light and humidity. As a matter of concern, plant cells and tissues have capability to accommodate minor variations in these parameters. However, regarding chemical environment, that include growth medium and hormone, a little variation may wrench the ability of

Soybean: Plant Manipulation to *Agrobacterium* Mediated Transformation 299

embryos at high concentration of auxin (Naphthalene acetic acid; NAA), however, the germination of soybean somatic embryos usually do not require exogenous growth regulators and young immature cotyledons have great tendency to give rise somatic embryos. After embryo development and in secondary stage of plantlet formation desiccation perform positive role for better recovery (Parrott et al., 1988; Finer, 1988). Lazzeri et a*l*. (1987) furhter reported that embryo initiation in soybean system is predominantly multicellular and 2,4-D plays a major role in it. However, efficiency of process can be enhanced by NAA and these induced embryos were closely related to zygotic embryos. Subculturing also influence frequency of normal embryo development during somatic embryogenesis. Although, complete cotyledon is considered to produce embryos, Hartweck et al. (1988) reported that epidermal and sub-epidermal cells at distal periphery of cotyledon and geterogeneous embryogenic tissues in central region of cotyledons can produce embryos in the presence of NAA and 2,4-D, respectively. Later on Liu et al. (1992) stated that epidermal cells produce somatic embryos without intervening callus phase (direct organogenesis) and presence of 2,4-D and NAA play major role in this histo-differentiation. Different developmental stages of somatic embryo formation initiate from proembryo while secondary embryogenesis and chimeric embryo development occur during differentiation (Gyulai et al., 1993). The differentiation process takes place in 4-6 weeks, initiated by three and four cell embryo leading to development of globular and heart shape embryo. Abaxial side of explant facing the medium resulted in faster formation of somatic embryos from subepidermal tissue in the presence of silver nitrate, irrespective to pH conditions and high light intensity causes faster production of somatic embryos (Santarem et al., 1997; Hofmann et al., 2004). Meurer et al. (2001) & Fermando et al. (2002) worked on soybean somatic embryogenesis from immature zygotic cotyledons from different locations. They found that genotype and location strongly affect soybean primary embryo development and to develop somatic embryos one should be able to realize acceptable level of embryo initiation of each cultivar. Influence of genetic variations in soybean on embryo initiation from immature cotyledons has been well established but upturn in weight, volume, embryo developmental stages and plant recovery can partially be overcome by modifying protocols. The use of ethylene inhibitor, low concentration of nitrogen and sucrose, desiccation, spermidine and alteration in nitrogen source, polyethylene glycol and sorbitol, reported by different researchers, significantly enhanced embryoid formation and their maturation to plant. Conclusively, in addition to above mentioned factors, breeding line; immature embryo age, quality and appropriate choice and concentration of hormone is essential for significant

Besides using immature cotyledons and embryos, a lot of work has also been carried out using cotyledonary node explants from seeds or plantlets after few days of germination. First report of plant regeneration from soybean cotyledonary node segment of seedlings grown in the presence of BAP was by Cheng et al., 1980. They obtained multiple shoot bud formation on medium containing high concentration of BAP but better bud growth was noticed when cultures were transferred to low concentration of BAP. Wright and co-workers also reported that BAP is an essential component of media for shoot induction from cotyledonary node explants. Carbon source (sucrose or fructose) and salt concentration (full MS, ½ MS or 1/4MS) have different effects even hormone concentration is kept constant. They further reported that seedlings germinated on water agar medium were not so responsive for shoot induction (Wright et al., 1986). BAP treatment to embryonic axes does not allow the cell to remain quiescent and cells are reprogrammed to produce multiple somatic foci (Buising et al., 1994). Presence of cytokinins (BAP) interrupts chromosomal

results.

regeneration. Growth medium consists of appropriate level of essential minerals (major, minor and trace elements), vitamins, carbon source (monosaccharide or disaccharide) and in some specific cases additives such as charcoal, amino acids, specific chemical etc. Now a number of media are commercially available for plant tissue culture such as MS (Murashige & Skoog, 1962); B5 (Gmaborg et al., 1968); SH (Schenk & Hildebrandt, 1972); LS (Linsmaier & Skoog, 1965); White, 1963 and many more. The choice of suitable media depends upon a number of factors such as plant specie, explant type, explant age, geographical distribution of plant and even season if explant is picked from in vivo condition. These basal medium are designed to keep the plant tissue alive and somewhat proliferative. However, for callus induction, shoot and root differentiation, plant growth regulators are required for these developmental programs. Most common classes of growth regulators include auxins, cytokinins and gibberellins either natural or synthetic. For all the stages of development from a cell to whole plant, appropriate type and concentration of these hormones is required that is selected only on hit and trial basis.

### **2.1 Callogenesis to organogenesis**

Callus is mass of undifferentiated cells that develop when explant is grown on appropriate medium. Callogenesis is basically absence of organogenesis. Callus often produces organs and in this situation callus proliferation is halted. Organ production is dependent upon level of cytokinin in the medium. Such differentiation that lead to bud or shoot formation is also termed as direct organogenesis. However, depending upon hormone type and concentration, callus may undergo different developmental stages that lead to somatic embryogenesis (indirect organogenesis). Organ formation is hooked on the balance of auxin and cytokinin and even ability of cell to develop shoot or root. During culture in the presence of suitable phytohormones, cell become competent that leads to differentiation and lastly morphogenesis occurs.

Sometime cell irrespective to plant tissue or callus may undergo embryo formation. These somatic embryos like zygotic embryos pass through different developmental stages as bipolar, globular, torpedo and cotyledonary. These somatic embryos can be successfully bred into whole plant even in the absence of growth hormones.

### **2.2 Soybean tissue culture strategies**

Meristemic tissue formation from cotyledons of immature embryos of *Glycine max* through somatic embryogenesis first time was observed by Lippmann & Lippmann, 1984. Age of explant and concentration of auxin in the medium strongly affect the development of somatic embryos. However, addition of cytokinin along with 2,4-dichloro phenoxy acetic acid (2,4-D) and higher concentration of sugar inhibited embryo formation. Li and coworkers (Li et al., 1985) obtained thousands of plantlets and somatic embryoids from single cell of young embryo when cultured on Murashige and Skoog (MS) medium containing 6 benzyl amino purine (BAP) and indole acetic acid (IAA) under low light conditions. Single cells obtained in this case converted into proembyos in liquid medium leading to somatic embryos formation and hence plantlet on agar containing medium. Further, Lazzeri et al. (1985) presented a reliable system for the regeneration from somatic tissues of soybean. They predicted that formation of somatic embryos from immature cotyledons of soybean looks imitated process that occurs over a range of culture conditions; and the efficiency of embryogenesis depends upon physiological and chemical conditions mostly plant growth regulators. Surface and subsurface cells of cotyledons can be converted into somatic

regeneration. Growth medium consists of appropriate level of essential minerals (major, minor and trace elements), vitamins, carbon source (monosaccharide or disaccharide) and in some specific cases additives such as charcoal, amino acids, specific chemical etc. Now a number of media are commercially available for plant tissue culture such as MS (Murashige & Skoog, 1962); B5 (Gmaborg et al., 1968); SH (Schenk & Hildebrandt, 1972); LS (Linsmaier & Skoog, 1965); White, 1963 and many more. The choice of suitable media depends upon a number of factors such as plant specie, explant type, explant age, geographical distribution of plant and even season if explant is picked from in vivo condition. These basal medium are designed to keep the plant tissue alive and somewhat proliferative. However, for callus induction, shoot and root differentiation, plant growth regulators are required for these developmental programs. Most common classes of growth regulators include auxins, cytokinins and gibberellins either natural or synthetic. For all the stages of development from a cell to whole plant, appropriate type and concentration of these hormones is required

Callus is mass of undifferentiated cells that develop when explant is grown on appropriate medium. Callogenesis is basically absence of organogenesis. Callus often produces organs and in this situation callus proliferation is halted. Organ production is dependent upon level of cytokinin in the medium. Such differentiation that lead to bud or shoot formation is also termed as direct organogenesis. However, depending upon hormone type and concentration, callus may undergo different developmental stages that lead to somatic embryogenesis (indirect organogenesis). Organ formation is hooked on the balance of auxin and cytokinin and even ability of cell to develop shoot or root. During culture in the presence of suitable phytohormones, cell become competent that leads to differentiation and

Sometime cell irrespective to plant tissue or callus may undergo embryo formation. These somatic embryos like zygotic embryos pass through different developmental stages as bipolar, globular, torpedo and cotyledonary. These somatic embryos can be successfully

Meristemic tissue formation from cotyledons of immature embryos of *Glycine max* through somatic embryogenesis first time was observed by Lippmann & Lippmann, 1984. Age of explant and concentration of auxin in the medium strongly affect the development of somatic embryos. However, addition of cytokinin along with 2,4-dichloro phenoxy acetic acid (2,4-D) and higher concentration of sugar inhibited embryo formation. Li and coworkers (Li et al., 1985) obtained thousands of plantlets and somatic embryoids from single cell of young embryo when cultured on Murashige and Skoog (MS) medium containing 6 benzyl amino purine (BAP) and indole acetic acid (IAA) under low light conditions. Single cells obtained in this case converted into proembyos in liquid medium leading to somatic embryos formation and hence plantlet on agar containing medium. Further, Lazzeri et al. (1985) presented a reliable system for the regeneration from somatic tissues of soybean. They predicted that formation of somatic embryos from immature cotyledons of soybean looks imitated process that occurs over a range of culture conditions; and the efficiency of embryogenesis depends upon physiological and chemical conditions mostly plant growth regulators. Surface and subsurface cells of cotyledons can be converted into somatic

bred into whole plant even in the absence of growth hormones.

that is selected only on hit and trial basis.

**2.1 Callogenesis to organogenesis** 

lastly morphogenesis occurs.

**2.2 Soybean tissue culture strategies** 

embryos at high concentration of auxin (Naphthalene acetic acid; NAA), however, the germination of soybean somatic embryos usually do not require exogenous growth regulators and young immature cotyledons have great tendency to give rise somatic embryos. After embryo development and in secondary stage of plantlet formation desiccation perform positive role for better recovery (Parrott et al., 1988; Finer, 1988). Lazzeri et a*l*. (1987) furhter reported that embryo initiation in soybean system is predominantly multicellular and 2,4-D plays a major role in it. However, efficiency of process can be enhanced by NAA and these induced embryos were closely related to zygotic embryos. Subculturing also influence frequency of normal embryo development during somatic embryogenesis. Although, complete cotyledon is considered to produce embryos, Hartweck et al. (1988) reported that epidermal and sub-epidermal cells at distal periphery of cotyledon and geterogeneous embryogenic tissues in central region of cotyledons can produce embryos in the presence of NAA and 2,4-D, respectively. Later on Liu et al. (1992) stated that epidermal cells produce somatic embryos without intervening callus phase (direct organogenesis) and presence of 2,4-D and NAA play major role in this histo-differentiation. Different developmental stages of somatic embryo formation initiate from proembryo while secondary embryogenesis and chimeric embryo development occur during differentiation (Gyulai et al., 1993). The differentiation process takes place in 4-6 weeks, initiated by three and four cell embryo leading to development of globular and heart shape embryo. Abaxial side of explant facing the medium resulted in faster formation of somatic embryos from subepidermal tissue in the presence of silver nitrate, irrespective to pH conditions and high light intensity causes faster production of somatic embryos (Santarem et al., 1997; Hofmann et al., 2004). Meurer et al. (2001) & Fermando et al. (2002) worked on soybean somatic embryogenesis from immature zygotic cotyledons from different locations. They found that genotype and location strongly affect soybean primary embryo development and to develop somatic embryos one should be able to realize acceptable level of embryo initiation of each cultivar. Influence of genetic variations in soybean on embryo initiation from immature cotyledons has been well established but upturn in weight, volume, embryo developmental stages and plant recovery can partially be overcome by modifying protocols. The use of ethylene inhibitor, low concentration of nitrogen and sucrose, desiccation, spermidine and alteration in nitrogen source, polyethylene glycol and sorbitol, reported by different researchers, significantly enhanced embryoid formation and their maturation to plant. Conclusively, in addition to above mentioned factors, breeding line; immature embryo age, quality and appropriate choice and concentration of hormone is essential for significant results.

Besides using immature cotyledons and embryos, a lot of work has also been carried out using cotyledonary node explants from seeds or plantlets after few days of germination. First report of plant regeneration from soybean cotyledonary node segment of seedlings grown in the presence of BAP was by Cheng et al., 1980. They obtained multiple shoot bud formation on medium containing high concentration of BAP but better bud growth was noticed when cultures were transferred to low concentration of BAP. Wright and co-workers also reported that BAP is an essential component of media for shoot induction from cotyledonary node explants. Carbon source (sucrose or fructose) and salt concentration (full MS, ½ MS or 1/4MS) have different effects even hormone concentration is kept constant. They further reported that seedlings germinated on water agar medium were not so responsive for shoot induction (Wright et al., 1986). BAP treatment to embryonic axes does not allow the cell to remain quiescent and cells are reprogrammed to produce multiple somatic foci (Buising et al., 1994). Presence of cytokinins (BAP) interrupts chromosomal

Soybean: Plant Manipulation to *Agrobacterium* Mediated Transformation 301

plant recovery from protoplast derived calli. However, composition of medium varies embryogenic calli initiation and then somatic embryo differentiation (Zhang & Komatsuada 1993). Zhao et al. (1998) reported that TDZ plays an important role in embryo induction and germination during soybean anther culture but plant differentiation rate was quite low. Addition of 2,4-D in the medium and culturing in light significantly increased the morphogenic response of anther walls and connective tissues. No androgenic response was observed in anther culture of four soybean genotypes but somatic embryogenesis was observed from the epidermis and the middle layer (Rodrigues et al., 2004, 2005). Higher concentration of 2,4-D during anther culture results in plasmolysis of microspores. Time of culture was also found effective for induction of somatic embryos derived from anther culture. Frequencies of binucleate symmetrical grains and multinucleate / multicellular structure formation were also found significant in the day of culture and cultivar interaction

Transformation is the alteration in genetic makeup of a cell due to incorporation of a foreign DNA fragment that expresses in the cell resulting variation in physiochemical properties. Plant transformation is now a routine practice and carried out through different approaches including *Agrobacterium* mediated, gene gun, electroporation, microinjection and few more. More than 120 diverse plant species have been transformed. Now in most of the developed countries transgenic crops are cultivated with improved nutritional quality and tolerance to biotic and abiotic stresses. This not only improved food quality and quantity for humans and animals but also somewhat has positive influence on environment. Even after a lot of advancement in transformation technologies, many plant species including soybean is

*Agrobacterium* mediated transformation of soybean has shown significant improvement and enabled public and private sector for production of commercial cultivars with transgenic traits. A number of reports describe condition standardization for T-DNA delivery, effect of *Agrobacterium* strain and choice of cultivar and conditions to produce high yield of transformants. Beside all above mentioned conditions, soybean cultivar susceptibility to *Agrobacterium* can not be overlooked. Although, protocols for production of transgenic plant have been standardized but all seems ineffective. We are far away from getting transformants from a single experiment especially in case of soybean that is still considered

Nature has offered *Agrobacterium* the ability to transfer some part of DNA from plasmid to plant cell. This T-DNA (transfer DNA) naturally causes callus formation on plant's parts termed as crown gall disease. However, this is multifarious procedure that involves two biological systems; bacteria and plant cell and success is subjected to compatibility. Unsurprisingly virulence story of *Agrobacterium* is the key for tumor induction. This virulence provokes by simple carbohydrates and phenolic compounds that are released by injured plant tissue. After this initiative, vir genes activate and produce proteins. These proteins hold the charge of transformation that include scratch of T-DNA, carry, direct towards the plant cell and finally integrate into plant genome. Naturally this T-DNA contains genes that are involved in biosynthesis of plant hormones that are involved in

(Cardoso et al., 2007).

**3. Plant transformation: a prospective to revolution** 

considered recalcitrant to transformation.

**3.1 Biological way to introduce DNA into plant cell** 

uncontrolled proliferation of plant cell leading to callus formation.

obstinate to transformation.

DNA replication in large number of cells in shoot apex that ultimately leads to formation of multi cell loci leading to shoot development. Thidiazuron (TDZ) induce adventitious shoots more efficiently than BAP and hypocotyls proved better than cotyledonary nodes for multiple bud formation while plating method (hypocotyls ending in contact to media) and cutting of explant also effects adventitious shoot formation from mature soybean seed hypocotyl (Zia et al., 2010a). However, after shoot bud induction, placement of explant on zeatin riboside containing medium allow the shoots to increase in length more as compared with other cytokinins. Sairam et al. (2003) developed an efficient protocol for callogenesis and embryogenesis from cotyledonary node explant on MS medium containing 2,4-D and BAP. According to them regeneration efficiency was genotype dependent and the best choice of carbon source might be sorbitol for callus induction and maltose for organogenesis. Addition of other growth regulators such as TDZ and Kinetin in MS or B5 medium varied embryo or shoot formation in different soybean genotypes from mature half seed's nodal segment. However, different stages of proliferation and regeneration also vary depending upon genotype. Such variability's can partially be overcome by some modifications in embryogenesis and regeneration protocols (Bailey et al., 1993). Recently Loganathan et al. (2010) reported the somatic embryogenesis from immature embryonic shoot tips on MS medium containing 6% sucrose, 2,4-D and amino acids. The embryos efficiently regenerated into shoots on hormone free MS medium containing charcoal. While, 72-96hr desiccation positively influenced on plantlet formation.

There are very few reports of soybean regeneration from other explants. In 1977 Beversdorf & Bingham reported callogenic response from hypocotyls and ovaries as explant on semi solid and liquid medium. They failed to regenerate shoots; however, they observed structures similar in appearance to embryos in liquid medium. Primary leaf explant turned into callus when cultured on B5 medium. Indirect organogenesis was successfully achieved when callus was further cultured on modified medium containing pyroglutamic acid that greatly enhanced regeneration capability (Wright et al., 1987). Kim et al. (1994) stated that addition of proline in the medium increased the number of shoots but decreased the length of generated shoots. They also reported that cobalt and zinc also play an effective role in shoot induction from primary leaf nodes. Droste et al. (1993) cultured primary leaf less meristem on organic enriched medium and find microscopic bud like structure within two weeks; however, very few plants were developed from these buds. Reichert et al. (2003) and Tripathi & Tiwari (2003) demonstrated that regeneration efficiency from hypocotyls, epicotyl and primary leaf explants is also genotype maturity dependent. The shoots regenerate from acropetal end and/or central region of cotyledonary node tissue. They further concluded that explant, inoculation medium and appropriate concentration/ combination of growth hormone are also essential for better regeneration efficiency. Stem node segments were also cultured on different basal medium for shoot bud formation (Saka et al., 1980). Combination of MS salts and B5 vitamins supplemented with BAP was found better choice to produce shoot buds. However, bud growth stimulated on medium containing low BAP concentration and replacement of sucrose with fructose.

Protoplast culture; isolated from immature cotyledons has also been reported. Dhir et al. (1991) cultured these protoplast in the liquid medium in the presence of combination of cytokinins (BAP, Kinetin, Zeatin) and observed 21% multiple shooting response from compact calli. The regeneration efficiency increased upto 30% when glutamine, aspragine and Gibberellic acid (GA3) were added in the medium. However, medium supplemented with different amino acids and their derivatives as nitrogen source was found better for

DNA replication in large number of cells in shoot apex that ultimately leads to formation of multi cell loci leading to shoot development. Thidiazuron (TDZ) induce adventitious shoots more efficiently than BAP and hypocotyls proved better than cotyledonary nodes for multiple bud formation while plating method (hypocotyls ending in contact to media) and cutting of explant also effects adventitious shoot formation from mature soybean seed hypocotyl (Zia et al., 2010a). However, after shoot bud induction, placement of explant on zeatin riboside containing medium allow the shoots to increase in length more as compared with other cytokinins. Sairam et al. (2003) developed an efficient protocol for callogenesis and embryogenesis from cotyledonary node explant on MS medium containing 2,4-D and BAP. According to them regeneration efficiency was genotype dependent and the best choice of carbon source might be sorbitol for callus induction and maltose for organogenesis. Addition of other growth regulators such as TDZ and Kinetin in MS or B5 medium varied embryo or shoot formation in different soybean genotypes from mature half seed's nodal segment. However, different stages of proliferation and regeneration also vary depending upon genotype. Such variability's can partially be overcome by some modifications in embryogenesis and regeneration protocols (Bailey et al., 1993). Recently Loganathan et al. (2010) reported the somatic embryogenesis from immature embryonic shoot tips on MS medium containing 6% sucrose, 2,4-D and amino acids. The embryos efficiently regenerated into shoots on hormone free MS medium containing charcoal. While,

There are very few reports of soybean regeneration from other explants. In 1977 Beversdorf & Bingham reported callogenic response from hypocotyls and ovaries as explant on semi solid and liquid medium. They failed to regenerate shoots; however, they observed structures similar in appearance to embryos in liquid medium. Primary leaf explant turned into callus when cultured on B5 medium. Indirect organogenesis was successfully achieved when callus was further cultured on modified medium containing pyroglutamic acid that greatly enhanced regeneration capability (Wright et al., 1987). Kim et al. (1994) stated that addition of proline in the medium increased the number of shoots but decreased the length of generated shoots. They also reported that cobalt and zinc also play an effective role in shoot induction from primary leaf nodes. Droste et al. (1993) cultured primary leaf less meristem on organic enriched medium and find microscopic bud like structure within two weeks; however, very few plants were developed from these buds. Reichert et al. (2003) and Tripathi & Tiwari (2003) demonstrated that regeneration efficiency from hypocotyls, epicotyl and primary leaf explants is also genotype maturity dependent. The shoots regenerate from acropetal end and/or central region of cotyledonary node tissue. They further concluded that explant, inoculation medium and appropriate concentration/ combination of growth hormone are also essential for better regeneration efficiency. Stem node segments were also cultured on different basal medium for shoot bud formation (Saka et al., 1980). Combination of MS salts and B5 vitamins supplemented with BAP was found better choice to produce shoot buds. However, bud growth stimulated on medium

72-96hr desiccation positively influenced on plantlet formation.

containing low BAP concentration and replacement of sucrose with fructose.

Protoplast culture; isolated from immature cotyledons has also been reported. Dhir et al. (1991) cultured these protoplast in the liquid medium in the presence of combination of cytokinins (BAP, Kinetin, Zeatin) and observed 21% multiple shooting response from compact calli. The regeneration efficiency increased upto 30% when glutamine, aspragine and Gibberellic acid (GA3) were added in the medium. However, medium supplemented with different amino acids and their derivatives as nitrogen source was found better for plant recovery from protoplast derived calli. However, composition of medium varies embryogenic calli initiation and then somatic embryo differentiation (Zhang & Komatsuada 1993). Zhao et al. (1998) reported that TDZ plays an important role in embryo induction and germination during soybean anther culture but plant differentiation rate was quite low. Addition of 2,4-D in the medium and culturing in light significantly increased the morphogenic response of anther walls and connective tissues. No androgenic response was observed in anther culture of four soybean genotypes but somatic embryogenesis was observed from the epidermis and the middle layer (Rodrigues et al., 2004, 2005). Higher concentration of 2,4-D during anther culture results in plasmolysis of microspores. Time of culture was also found effective for induction of somatic embryos derived from anther culture. Frequencies of binucleate symmetrical grains and multinucleate / multicellular structure formation were also found significant in the day of culture and cultivar interaction (Cardoso et al., 2007).
