**Meet the editor**

Dr Eddy C. Agbo, DVM, PhD is an Entrepreneurial Scientist and Chairman/CEO of Fyodor Biotechnologies Corporation. Prior to founding Fyodor, he held several research and senior management academic and industry positions, with over 16 years of experience. He is the lead inventor on pending patents, and author of numerous scientific publications. Dr Agbo is an acclaimed international

biotechnology expert and has spoken at such fora as the Corporate Council on Africa, Washington DC, Africa Business Conference, Harvard Business School, Boston and, African Technology Policy Network, Cairo, among others. He is a member of the Greater Baltimore Committee USA and of its Bioscience Strategy Sub-committee, and profiled in the SmartCEO Magazine and Baltimore Business Journal. Dr. Agbo earned his PhD in Molecular Genetics from Utrecht University, MS in Biotechnology from Wageningen University (both in The Netherlands), and Doctor of Veterinary Medicine (DVM) degree from the University of Ibadan, Nigeria.

### Contents

#### **Preface XIII**

**Part 1 Plant Biotechnology 1**  Chapter 1 **Applications of Biotechnology in Kiwifruit (***Actinidia***) 3**  Tianchi Wang and Andrew P. Gleave Chapter 2 **Biotechnological Tools for Garlic Propagation and Improvement 31**  Alejandrina Robledo-Paz and Héctor Manuel Tovar-Soto Chapter 3 **Plant Beneficial Microbes and Their Application in Plant Biotechnology 57**  Anna Russo, Gian Pietro Carrozza, Lorenzo Vettori, Cristiana Felici, Fabrizio Cinelli and Annita Toffanin **Part 2 Medical Biotechnology 73**  Chapter 4 *In Vivo* **Circular RNA Expression by the Permuted Intron-Exon Method 75**  So Umekage, Tomoe Uehara, Yoshinobu Fujita, Hiromichi Suzuki and Yo Kikuchi Chapter 5 **DNA Mimicry by Antirestriction and Pentapeptide Repeat (PPR) Proteins 91**  Gennadii Zavilgelsky and Vera Kotova Chapter 6 **Platelet Rich Plasma (PRP) Biotechnology: Concepts and Therapeutic Applications in Orthopedics and Sports Medicine 113**  Mikel Sánchez, Isabel Andia,

Eduardo Anitua and Pello Sánchez

X Contents


	- **Part 3 Microbial Biotechnology 209**
	- **Part 4 Animal Biotechnology 275**

Chapter 17 **Gender, Knowledge, Scientific Expertise, and Attitudes Toward Biotechnology: Technological Salience and the Use of Knowledge to Generate Attitudes 399**  Richard M. Simon

VI Contents

Chapter 7 **Polymers in the Pharmaceutical Applications -** 

**Polyesters and Polycarbonates 139**  Ewa Oledzka and Marcin Sobczak

Chapter 9 **Controlling Cell Migration with Micropatterns 187** 

Chapter 10 **Microbial Expression Systems and Manufacturing** 

Chapter 11 **Exogenous Catalase Gene Expression as a Tool** 

Chapter 12 **Acupuncture for the Treatment of Simple Obesity: Basic and Clinical Aspects 277**  Wei Shougang and Xie Xincai

Chapter 13 **Spermatogonial Stem Cells and Animal Transgenesis 303**  Flavia Regina Oliveira de Barros, Mariana Ianello Giassetti

Chapter 14 **Gene Expression Microarrays in Microgravity Research:** 

Chapter 15 **Biotechnology Patents: Safeguarding Human Health 349** 

Chapter 16 **Biotechnology Virtual Labs: Facilitating Laboratory Access Anytime-Anywhere for Classroom Education 379**

> Shyam Diwakar, Krishnashree Achuthan, Prema Nedungadi and Bipin Nair

**Toward the Identification of Major Space Genes 319**

**from a Market and Economic Perspective 211** Hans-Peter Meyer and Diego R. Schmidhalter

**for Enhancing Metabolic Activity and Production of Biomaterials in Host Microorganisms 251**

Ahmad Iskandar Bin Haji Mohd Taha, Hidetoshi Okuyama, Takuji Ohwada, Isao Yumoto and Yoshitake Orikasa

Chapter 8 **Translating 2A Research into Practice 161** 

Taro Toyota, Yuichi Wakamoto, Kumiko Hayashi and Kiyoshi Ohnuma

**Part 3 Microbial Biotechnology 209** 

and José Antônio Visintin

Jade Q. Clement

Rajendra K. Bera

**Part 4 Animal Biotechnology 275** 

Garry A. Luke

**Natural and Bioactive Initiators and Catalysts** 

**in the Synthesis of Biodegradable and Bioresorbable** 


### Preface

This book represents a crystallization of some of the leading-edge research and development topics evolving in the field of biotechnology. It comprises 19 Chapters from an extensive background of leading authors, covering topics ranging from Plant, Medical, Microbial, Animal to General Biotechnology. The key idea was to bring multiple cutting-edge topics in biotechnology into a single text, as a handy tool for students, scholars and practitioners interested in related topics.

All of the material in this book was developed under rigorous peer review, with appeal to a broad range of readers ranging from social scientists to students and researchers. A substantial proportion of the material is original, and has been prepared specifically for this book; part was put together from published articles.

The publishing process was considerably longer than usual partly due to the novelty of the papers and partially due to the fact that the referees were relatively more cautious with several of the papers, which were substantially innovative.

> **Eddy C. Agbo, DVM, PhD**  Chairman & CEO Fyodor Biotechnologies Corp Baltimore, Maryland USA

## **Part 1**

**Plant Biotechnology** 

## **Applications of Biotechnology in Kiwifruit (***Actinidia***)**

Tianchi Wang and Andrew P. Gleave *The New Zealand Institute for Plant & Food Research Limited New Zealand* 

#### **1. Introduction**

*Actinidia* is a genus of 55 species and about 76 taxa native to central China and with a wide geographic distribution throughout China and South Eastern Asia (X. Li et al., 2009). Palaeobiological studies estimate *Actinidia* to be at least 20–26 million years old (Qian & Yu, 1991). *Actinidia* species are vigorous and long-lived perennial vines, producing oblong or spherical berries that vary considerably in shape and colour (Fig. 1). *Actinidia* are normally dioecious, but occasional plants have perfect flowers (A. R. Ferguson, 1984). The basic chromosome number in *Actinidia* is *X*=29, with a diploid number of 58. During evolution a chromosome may have duplicated (McNeilage & Considine, 1989), followed by an aneuploid event, such as breakage of a centromere, to give an additional chromosome (He et al., 2005). The genus has a reticulate polyploidy structure, with diploids, tetraploids, hexaploids and octaploids occurring in diminishing frequency (A. R. Ferguson et al., 1997). The genus has unusual inter- and intra-taxal variation in ploidy (A. R. Ferguson & Huang, 2007; A. R. Ferguson et al., 1997), with, for example, *A. chinensis* found as both diploid and tetraploid and *A. arguta* as usually tetraploid, but also found as diploid, hexaploid or octaploid. In this chapter, we will describe advances in *Actinidia* plant tissue culture and molecular biology and the present and future applications of these biotechnology techniques in kiwifruit breeding and germplasm improvement.

#### **2. Global significance of kiwifruit**

*Actinidia* species were introduced to Europe, the U.S.A., and New Zealand in the late 19th and early 20th century (A.R. Ferguson & Bollard, 1990). New Zealand was largely responsible for the initial development and commercial growing of kiwifruit, with the first commercial orchards established in the 1930s. Domestication and breeding of firstly A*ctinidia deliciosa,* and more recently, *A. chinensis,* from wild germplasm has resulted in varieties now cultivated commercially in a number of continents. The inherent qualities of novel appearance, attractive flesh colour, texture and flavour, high vitamin C content and favourable handling and storage characteristics make kiwifruit a widely acceptable and popular fruit crop for producers and consumers.

Commercial kiwifruit growing areas have expanded rapidly and consistently since the 1990s. By 2010, the global kiwifruit planting area had reached over 150,000 ha. China (70,000 ha), Italy (27,000 ha), New Zealand (14,000 ha) and Chile (14,000 ha) account for about 83% of world kiwifruit plantings, and global kiwifruit production represents about 0.22% of total production for major fruit crops, with the majority of kiwifruit consumed as fresh fruit. Science has made a significant contribution to the success of the New Zealand kiwifruit industry, particularly in developing excellent breeding programmes and technologies for optimal plant growth, orchard management, fruit handling and storage, and transport to the global market, to ensure high quality premium fruit reach the consumer.

Fig. 1. Fruit of the *Actinidia* genus showing variation in flesh colour, size and shape

Kiwifruit have a reputation for being a highly nutritious food. A typical commercial *A. deliciosa* 'Hayward' kiwifruit contains about 85 mg/100 g fresh weight of vitamin C, which is 50% more than an orange, or 10 times that of an apple (A. R. Ferguson & Ferguson, 2003). The fruit of some *Actinidia* species, such as *A. latifolia, A. eriantha* and *A. kolomikta,* have in excess of 1000 mg of vitamin C per 100 g fresh weight (A. R. Ferguson, 1990; A. R. Ferguson & MacRae, 1992). Kiwifruit are also an excellent source of potassium, folate and vitamin E (Ferguson & Ferguson, 2003), and are high amongst fruit for their antioxidant capacity (H. Wang et al., 1996).

#### **2.1 Breeding and commercial cultivars**

The extensive *Actinidia* germplasm resources, with tremendous genetic and phenotypic diversity at both the inter- and intra-specific levels, offer kiwifruit breeders infinite opportunities for developing new products. Since its development in the 1920s, *A. deliciosa* 'Hayward' has continued to perform extraordinarily well on the global market in terms of production and sales; it remains the dominant commercial kiwifruit cultivar. Advances in *Actinidia* breeding have seen the appearance of a number of new commercial kiwifruit varieties. In 1999 an *A. chinensis* cultivar named 'Hort16A', developed in New Zealand by HortResearch (now Plant & Food Research), entered the international market, with fruit sold under the name of ZESPRI® GOLD Kiwifruit, reflecting the distinctive golden-yellow fruit flesh. 'Hort16A' fruit are sweet tasting and the vine is more subtropical than 'Hayward'. Subsequently, a range of new cultivars were commercialised in China and Japan, some of which have become significant internationally. Jintao®, a yellow-fleshed cultivar selected in

of world kiwifruit plantings, and global kiwifruit production represents about 0.22% of total production for major fruit crops, with the majority of kiwifruit consumed as fresh fruit. Science has made a significant contribution to the success of the New Zealand kiwifruit industry, particularly in developing excellent breeding programmes and technologies for optimal plant growth, orchard management, fruit handling and storage, and transport to the

global market, to ensure high quality premium fruit reach the consumer.

Fig. 1. Fruit of the *Actinidia* genus showing variation in flesh colour, size and shape

Wang et al., 1996).

**2.1 Breeding and commercial cultivars** 

Kiwifruit have a reputation for being a highly nutritious food. A typical commercial *A. deliciosa* 'Hayward' kiwifruit contains about 85 mg/100 g fresh weight of vitamin C, which is 50% more than an orange, or 10 times that of an apple (A. R. Ferguson & Ferguson, 2003). The fruit of some *Actinidia* species, such as *A. latifolia, A. eriantha* and *A. kolomikta,* have in excess of 1000 mg of vitamin C per 100 g fresh weight (A. R. Ferguson, 1990; A. R. Ferguson & MacRae, 1992). Kiwifruit are also an excellent source of potassium, folate and vitamin E (Ferguson & Ferguson, 2003), and are high amongst fruit for their antioxidant capacity (H.

The extensive *Actinidia* germplasm resources, with tremendous genetic and phenotypic diversity at both the inter- and intra-specific levels, offer kiwifruit breeders infinite opportunities for developing new products. Since its development in the 1920s, *A. deliciosa* 'Hayward' has continued to perform extraordinarily well on the global market in terms of production and sales; it remains the dominant commercial kiwifruit cultivar. Advances in *Actinidia* breeding have seen the appearance of a number of new commercial kiwifruit varieties. In 1999 an *A. chinensis* cultivar named 'Hort16A', developed in New Zealand by HortResearch (now Plant & Food Research), entered the international market, with fruit sold under the name of ZESPRI® GOLD Kiwifruit, reflecting the distinctive golden-yellow fruit flesh. 'Hort16A' fruit are sweet tasting and the vine is more subtropical than 'Hayward'. Subsequently, a range of new cultivars were commercialised in China and Japan, some of which have become significant internationally. Jintao®, a yellow-fleshed cultivar selected in Wuhan, China (H.W. Huang et al., 2002b), is now widely planted in Italy (Ferguson & Huang, 2007) and more recently, the *A. chinensis* cultivar 'Hongyang' selected in China, and with a distinctive yellow-fleshed fruit with brilliant red around the central core, is widely cultivated for the export market, particularly Japan (M. Wang et al., 2003). Most cultivars to date have been selected from *A. chinensis* and *A. deliciosa;* however*, A. arguta* are now commercially cultivated in USA, Chile and New Zealand (Ferguson & Huang, 2007). The fruit of *A. arguta* are small, smooth-skinned, with a rich and sweet flavour, and can be eaten whole (Williams et al., 2003). Internationally, kiwifruit breeding programmes are directed primarily at producing varieties mainly from *A. deliciosa* and *A. chinensis,* with large fruit size, good flavour, novel flesh colour, variations in harvest period, improved yield and growth habit, hermaphroditism, tolerance to adverse conditions and resistance to disease (A. R. Ferguson et al., 1996). Although kiwifruit cultivars currently on the commercial market have been developed using traditional breeding techniques (MacRae, 2007), the expansion of genetic, physiological and biochemical knowledge and the application of biotechnology tools are being used increasingly to assist breeders in the development of novel cultivars.

#### **3. Tissue culture and crop improvement**

Although the genetic diversity of *Actinidia* provides tremendous potential for cultivar improvement, there are features (including the vigorous nature of climbing vines, the 3- to 5-year juvenile period, the dioecious nature and the reticulate polyploidy structure) that make *Actinidia* less amenable to achieving certain breeding goals, compared with many other agronomic crops. Plant tissue culture, the *in vitro* manipulation of plant cells, tissues and organs, is an important technique for plant biotechnology, and a number of plant tissue culture techniques have been employed to overcome some of the limitations that *Actinidia* presents to classical breeding.

#### **3.1 Multiplications**

Plant tissue culture for kiwifruit propagation was first reported by Harada (1975), followed by numerous reports using a range of explant types and genotypes (Gui, 1979; M. Kim et al., 2007; Kumar & Sharma, 2002; Q.L. Lin et al., 1994; Monette, 1986). Murashige & Skoog (MS) basal salts are the most widely used media for shoot regeneration and callus formation. However, other media have been used successfully, including Gamborg B5 medium (Barbieri & Morini, 1987) and N6 medium (Q.L. Lin et al., 1994).

Multiplication protocols essentially follow three steps: (1) surface sterilization of explants with 0.5–1.5% sodium hypochlorite; (2) shoot multiplication from explants (e.g. buds, nodal sections or young leaves) on MS medium, supplemented with 2–3% sucrose, 0.1–1.0 mg/l zeatin and 0.01–0.1 mg/l naphthalene acetic acid (NAA), solidified with 0.7% agar, at pH 5.8; and (3) rooting on half strength MS medium containing 0.5–1.0 mg/l indole-3-butyric acid (IBA). Generally, cultures are incubated at 24±2ºC under a 16 h photoperiod (20–30 µmol/m2/s of light intensity applied). Shoot proliferation rates vary depending upon species, cultivar, explant type, plant growth regulator combinations and culture conditions. Standardi & Catalano (1984) achieved a multiplication rate of 5.3 shoots per bud explant using a 30-day subculture period, and 90% of shoots rooted after three weeks, developing into 150–200 mm high plantlets, with 6–10 leaves within 60 days. A multiplication rate of 2.61 at seven weeks was achieved using 800 µm or 1200 µm transversal micro-cross section (MCS) of *A. deliciosa* 'Hayward' explants, cultured on ½ MS medium supplemented with 3% (w/v) sucrose, 4.5 x 10-3 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.6 x 10-1 µM zeatin in 0.8% agar (w/v), pH 5.8 (Kim et al., 2007).

#### **3.2 Protoplast culture and somatic hybridization**

As dioecy and polyploidy of *Actinidia* can often restrict breeding possibilities, somatic hybridization provides an approach to combine different genetic backgrounds of the same gender or to overcome inter-specific incompatibility, to produce valuable material with desirable traits from two species. Somatic hybridization is generally achieved through protoplast fusion, and methods of protoplast isolation from callus, suspension cultures, leaf mesophyll and cotyledons of various *Actinidia* genotypes and species have been developed. Tsai (1988) isolated protoplasts from calli derived from *A. deliciosa* leaves and stems and used TCCM medium with 0.23 µM 2,4-D, 0.44 µM 6-benzylaminopurine (BAP), 2% coconut milk, 10 g/l sucrose, 1 g/l glucose, 0.3 M mannitol and 0.1 M sorbitol, for preconditioning. Enzymatic degradation of cell walls was achieved in 2% Cellulase Onozuka R-10, 0.5% Macerozyme R-10, 0.5 M mannitol and 3 mM MES. *A. eriantha* protoplasts were isolated from newly growing leaves of *in vitro* culture seedlings, by preconditioning in MS liquid (without NH4NO3), supplemented with 1.0 mg/l 2,4-D and 0.4 M glucose and isolated using 1% Cellulase R-10, 0.5% Macerozyme R-10, 0.05% Pectolyase Y-23 and 3 mM MES (Y.J. Zhang et al., 1998). Plating efficiency after 3 weeks of culture was 19.4%, and calli subsequently recovered and regenerated shoots when cultured on MS media containing 2.28 µM zeatin and 0.57 µM indole-3-acetic acid (IAA).

Xiao & Han (1997) reported successful protoplast fusion of *A. chinensis* and *A. deliciosa*, demonstrating the potential of using this technique to aid breeding programmes. Isolated protoplasts from cotyledon-derived calli for *A. chinensis* (2*n* = 2*x* = 58) and *A. deliciosa* (2*n* = 6*x* = 174) were fused, using a PEG (polyethylene glycol) method and plantlets were regenerated from the fused calli. Xiao et al. (2004), in an attempt to introduce the chilling tolerance characteristics of *A. kolomikta* into *A. chinensis*, fused protoplasts isolated from cotyledon-derived calli of *A. chinensis* (2*n* = 2*x* = 58) and the mesophyll cells of *A. kolomikta*  (2*n* = 2*x* = 58). A number of techniques were employed to confirm that the regenerated plantlets were an inter-specific somatic hybrid (2*n* = 4*x* = 116) and assessment of the chilling tolerance of *in vitro* leaves suggested that the somatic hybrid was more similar to *A. kolomikta*, with a higher capacity of cold resistance than *A. chinensis*.

#### **3.3 Other culture techniques**

Embryo culture techniques, for embryo rescue were developed to recover hybrids from inter-specific crosses in *Actinidia*. From an *A. chinensis* (2*x*) × *A. melanandra* (4*x*) cross, embryo rescue was used successfully to transfer hybrid embryos to *in vitro* culture at an early stage of their development (Mu et al., 1990). Nutrient and hormone requirements were dependent on the stage of embryo development and the endosperm, and nursing tissue was beneficial when globular embryos were cultured. Embryo size and their genetic background are major factors in determining the success of the procedure (Harvey et al., 1995; Kin et al.,

into 150–200 mm high plantlets, with 6–10 leaves within 60 days. A multiplication rate of 2.61 at seven weeks was achieved using 800 µm or 1200 µm transversal micro-cross section (MCS) of *A. deliciosa* 'Hayward' explants, cultured on ½ MS medium supplemented with 3% (w/v) sucrose, 4.5 x 10-3 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.6 x 10-1 µM zeatin

As dioecy and polyploidy of *Actinidia* can often restrict breeding possibilities, somatic hybridization provides an approach to combine different genetic backgrounds of the same gender or to overcome inter-specific incompatibility, to produce valuable material with desirable traits from two species. Somatic hybridization is generally achieved through protoplast fusion, and methods of protoplast isolation from callus, suspension cultures, leaf mesophyll and cotyledons of various *Actinidia* genotypes and species have been developed. Tsai (1988) isolated protoplasts from calli derived from *A. deliciosa* leaves and stems and used TCCM medium with 0.23 µM 2,4-D, 0.44 µM 6-benzylaminopurine (BAP), 2% coconut milk, 10 g/l sucrose, 1 g/l glucose, 0.3 M mannitol and 0.1 M sorbitol, for preconditioning. Enzymatic degradation of cell walls was achieved in 2% Cellulase Onozuka R-10, 0.5% Macerozyme R-10, 0.5 M mannitol and 3 mM MES. *A. eriantha* protoplasts were isolated from newly growing leaves of *in vitro* culture seedlings, by preconditioning in MS liquid (without NH4NO3), supplemented with 1.0 mg/l 2,4-D and 0.4 M glucose and isolated using 1% Cellulase R-10, 0.5% Macerozyme R-10, 0.05% Pectolyase Y-23 and 3 mM MES (Y.J. Zhang et al., 1998). Plating efficiency after 3 weeks of culture was 19.4%, and calli subsequently recovered and regenerated shoots when cultured on MS media containing 2.28

Xiao & Han (1997) reported successful protoplast fusion of *A. chinensis* and *A. deliciosa*, demonstrating the potential of using this technique to aid breeding programmes. Isolated protoplasts from cotyledon-derived calli for *A. chinensis* (2*n* = 2*x* = 58) and *A. deliciosa* (2*n* = 6*x* = 174) were fused, using a PEG (polyethylene glycol) method and plantlets were regenerated from the fused calli. Xiao et al. (2004), in an attempt to introduce the chilling tolerance characteristics of *A. kolomikta* into *A. chinensis*, fused protoplasts isolated from cotyledon-derived calli of *A. chinensis* (2*n* = 2*x* = 58) and the mesophyll cells of *A. kolomikta*  (2*n* = 2*x* = 58). A number of techniques were employed to confirm that the regenerated plantlets were an inter-specific somatic hybrid (2*n* = 4*x* = 116) and assessment of the chilling tolerance of *in vitro* leaves suggested that the somatic hybrid was more similar to *A.* 

Embryo culture techniques, for embryo rescue were developed to recover hybrids from inter-specific crosses in *Actinidia*. From an *A. chinensis* (2*x*) × *A. melanandra* (4*x*) cross, embryo rescue was used successfully to transfer hybrid embryos to *in vitro* culture at an early stage of their development (Mu et al., 1990). Nutrient and hormone requirements were dependent on the stage of embryo development and the endosperm, and nursing tissue was beneficial when globular embryos were cultured. Embryo size and their genetic background are major factors in determining the success of the procedure (Harvey et al., 1995; Kin et al.,

in 0.8% agar (w/v), pH 5.8 (Kim et al., 2007).

**3.2 Protoplast culture and somatic hybridization** 

µM zeatin and 0.57 µM indole-3-acetic acid (IAA).

**3.3 Other culture techniques** 

*kolomikta*, with a higher capacity of cold resistance than *A. chinensis*.

1990). Hirsch et al. (2001) carried out inter-specific hybridizations of different *Actinidia* species and ploidy races, using embryo rescue to obtain hybrid plantlets of *A. kolomikta X A. chinensis, A. polygama X A. valvata, A. arguta X A. polygama* and *A. kolomikta X A. deliciosa*. When optimal media were used, the immature embryos that reached the torpedo stage could be rescued. A series of culture media were developed, which performed as the hybrid embryo's deficient endosperm to ensure embryo survival at the globular and heart stages. Ovule culture has been used also to obtain hybrid plantlets from the inter-specific cross of *A. chinensis X A. kolomikta* (X. Chen et al., 2006).

Endosperm culture is another approach to generating *Actinidia* inter-specific hybrids. Endosperms from F1 and F2 seeds from three inter-specific hybrids (*A. chinensis X A. melanandra; A. arguta X A. melanandra*; and an open pollinated *A. arguta X A. deliciosa*) were induced to form calli, from which plants were recovered by induction of organogenesis or embryogenesis. Media for callus induction and differentiation varied with genotype, and chromosome counts showed evidence of extensive mixoploidy in all hybrids (Mu et al., 1990).

Recently, *in vitro* chromosome doubling using colchicine treatment was reported (J. Wu et al., 2009; 2011). Petiole segments of five diploid *A. chinensis* genotypes, including 'Hort16A', were cultured on half-strength MT basal salt medium, supplemented with 3.0 mg/l BAP, 0.4 mg/l zeatin and 0.5 mg/l IBA for four weeks. Resulting microshoots were treated with 0.05– 0.1% colchicine, and over one-third of the regenerated shoots were confirmed as tetraploid by flow cytometry, with orchard-grown autotetraploid 'Hort16A' plants showing polyploid characteristics such as thicker leaves and flatter flowers, and some plants producing fruit almost double the weight of the original diploid 'Hort16A' fruit (J. Wu et al., 2009).

Cryopreservation is an excellent means of preserving germplasm for long-term storage, and various techniques and methods have been investigated for *Actinidia* germplasm (Bachiri et al., 2001; Hakozaki et al., 1996; Jian & Sun, 1989; Y. Wu et al., 2001; X. Xu et al., 2006; Zhai et al., 2003). Shoot tips from *in vitro* culture of a dwarf *A. chinensis* genotype were pre-cultured in MS medium containing 5% dimethyl sulfoxide (DMSO) and 5% sucrose for four days, followed by dehydration with PVP2 solution (30% glycerol, 15% DMSO, 15% PEG and 13.7% sucrose) for 40 min at 0ºC, and then transferred to liquid nitrogen for storage, with a survival rate of 56.7% upon defrosting shoots (X. Xu et al., 2006). Encapsulation-dehydration protocols used for the preservation of *in vitro* cultured hybrids of *A. arguta X A. deliciosa*, *A. chinensis* and *A. eriantha* gave even higher survival rates, of 85–95% (Bachiri et al., 2001; Y. Wu et al., 2001).

#### **4. Transformation systems**

Since the first report of a transgenic *Actinidia* plant two decades ago (Matsuta et al., 1990), six *Actinidia* species having been transformed, almost exclusively by *Agrobacterium*-mediated transformation. Initially, the development of *Actinidia* transformation focused on the integration into the plant genome of reporter and selectable marker genes (Fraser et al., 1995; Janssen & Gardner, 1993; Uematsu et al., 1991), but transformation of various heterologous genes has followed. These include: *A. rhizogenes rol* genes (Rugini et al., 1991); a soybean β-1,3 endoglucanase cDNA (Nakamura et al., 1999); a rice *OSH1* homeobox gene (Kusaba et al., 1999), and an *Arabidopsis* Na+/H+ antiporter gene (Tian et al., 2011), in attempts to improve kiwifruit disease resistance or drought tolerance; a synthetic gene encoding human epidermal growth factor (Kobayashi et al., 1996); and a grape stilbene synthase (Kobayashi et al., 2000), in attempts to accumulate bioactive compounds; citrus geranylgeranyl diphosphate synthase, phytoene desaturase, β-carotene desaturase, β-carotene hydroxylase and phytoene synthase, to modify the lutein or β-carotene content of kiwifruit (MiSun Kim et al., 2010) and the *A. tumefaciens* isopentyl transferase (*ipt*) gene, to alter vine architecture (Honda et al., 2011).

#### **4.1** *Agrobacterium***-mediated transformation**

*Agrobacterium*-mediated transformation of *Actinidia* is a component of the Plant & Food Research functional genomics platform and has been used to introduce over 100 *Actinidia* genes into various *Actinidia* species. In general, Plant & Food Research *Actinidia* transformation protocols are as follows: Orchard-grown winter mature and dormant canes are maintained at 4°C for 4–6 weeks. To initiate bud break, one-third of a 40 cm cane (with >3 nodes) is immersed in water, and maintained at room temperature under normal light conditions. After four weeks, newly initiated shoots are removed from the canes and shoot sections with a single node (1–2 cm) are soaked in 70% ethanol for 30 s, then surface sterilized with 25% (v/v) commercial bleach (5% active chlorine). After a sterile water rinse, the node sections are cultured on MS media, supplemented with 0.1 mg/l IBA at 24°C ± 2, 16 h photoperiod, with cool white fluorescent light (40 µmol/m2/s). Young leaves harvested from *in vitro* grown shoots are cut into 2 x 5 mm leaf strips. *Agrobacterium tumefaciens* EHA105, harbouring a pART27-derived binary vector (Gleave, 1992), is cultured in 50 ml MGL medium (Tingay et al., 1997) containing 100 mg/l spectomycin dihydrochloride, for 16–20 h at 28°C, with shaking at 250 rpm. At an OD600 nm =1.0-1.5, the bacterial cells are pelleted by centrifugation (5000 g for 10 min) and re-suspended in 10 ml MS media, supplemented 100 μM acetosyringone. Leaf strips are immersed in the *A. tumefaciens* suspension culture for 10 min, blotted dry with sterile filter paper and transferred onto cocultivation media (MS supplemented with 3.0 mg/l zeatin, 0.1 mg/l naphthaleneacetic acid (NAA) and 50 μM of AS). After two days of co-cultivation, the leaf strips are transferred to regeneration and selection medium (MS supplemented with 3.0 mg/l zeatin, 0.1 mg/l NAA, 150 mg/l kanamycin sulphate, 300 mg/l timentin, 30 g/l sucrose and 2.5 g/l Phytagel). The leaf strips produce calli along the cut edges at about four weeks and excised calli are transplanted onto fresh regeneration and selection media. Adventitious buds regenerated from the calli are excised individually and transferred to shoot elongation medium (MS supplemented with 0.1 mg/l IBA, 100 mg/l kanamycin sulphate and 300 mg/l timentin). When shoots reach 1–2 cm in height, they are transplanted onto rooting medium (½ MS basal salts and vitamins supplemented with 1.0 mg/l IBA, 150 mg/l timentin, 50 mg/l kanamycin sulphate, 20 g/l sucrose and 7 g/l agar). Rooted transgenic plants are potted in a ½-litre pot and placed in a containment glasshouse facility. The utility of a plant transformation system is very much dependent upon its efficiency, and several factors that affect *Actinidia* transformation efficiency are discussed below.

#### **4.1.1 Agrobacterium tumefaciens strains**

*A. tumefaciens* strains are defined by their chromosomal background and resident Ti plasmid, and exhibit differences in their capacity to transfer T-DNA to various plant species (Godwin et al., 1991). *A. tumefaciens* LBA4404, A281, C58, EHA101 and EHA105 are the strains commonly used in *Actinidia* transformation. Fraser et al. (1995) reported no marked difference in efficiency of *A. chinensis* transformation between strains A281 (a virulent L,L-

attempts to accumulate bioactive compounds; citrus geranylgeranyl diphosphate synthase, phytoene desaturase, β-carotene desaturase, β-carotene hydroxylase and phytoene synthase, to modify the lutein or β-carotene content of kiwifruit (MiSun Kim et al., 2010) and the *A. tumefaciens* isopentyl transferase (*ipt*) gene, to alter vine architecture (Honda et al., 2011).

*Agrobacterium*-mediated transformation of *Actinidia* is a component of the Plant & Food Research functional genomics platform and has been used to introduce over 100 *Actinidia* genes into various *Actinidia* species. In general, Plant & Food Research *Actinidia* transformation protocols are as follows: Orchard-grown winter mature and dormant canes are maintained at 4°C for 4–6 weeks. To initiate bud break, one-third of a 40 cm cane (with >3 nodes) is immersed in water, and maintained at room temperature under normal light conditions. After four weeks, newly initiated shoots are removed from the canes and shoot sections with a single node (1–2 cm) are soaked in 70% ethanol for 30 s, then surface sterilized with 25% (v/v) commercial bleach (5% active chlorine). After a sterile water rinse, the node sections are cultured on MS media, supplemented with 0.1 mg/l IBA at 24°C ± 2, 16 h photoperiod, with cool white fluorescent light (40 µmol/m2/s). Young leaves harvested from *in vitro* grown shoots are cut into 2 x 5 mm leaf strips. *Agrobacterium tumefaciens* EHA105, harbouring a pART27-derived binary vector (Gleave, 1992), is cultured in 50 ml MGL medium (Tingay et al., 1997) containing 100 mg/l spectomycin dihydrochloride, for 16–20 h at 28°C, with shaking at 250 rpm. At an OD600 nm =1.0-1.5, the bacterial cells are pelleted by centrifugation (5000 g for 10 min) and re-suspended in 10 ml MS media, supplemented 100 μM acetosyringone. Leaf strips are immersed in the *A. tumefaciens* suspension culture for 10 min, blotted dry with sterile filter paper and transferred onto cocultivation media (MS supplemented with 3.0 mg/l zeatin, 0.1 mg/l naphthaleneacetic acid (NAA) and 50 μM of AS). After two days of co-cultivation, the leaf strips are transferred to regeneration and selection medium (MS supplemented with 3.0 mg/l zeatin, 0.1 mg/l NAA, 150 mg/l kanamycin sulphate, 300 mg/l timentin, 30 g/l sucrose and 2.5 g/l Phytagel). The leaf strips produce calli along the cut edges at about four weeks and excised calli are transplanted onto fresh regeneration and selection media. Adventitious buds regenerated from the calli are excised individually and transferred to shoot elongation medium (MS supplemented with 0.1 mg/l IBA, 100 mg/l kanamycin sulphate and 300 mg/l timentin). When shoots reach 1–2 cm in height, they are transplanted onto rooting medium (½ MS basal salts and vitamins supplemented with 1.0 mg/l IBA, 150 mg/l timentin, 50 mg/l kanamycin sulphate, 20 g/l sucrose and 7 g/l agar). Rooted transgenic plants are potted in a ½-litre pot and placed in a containment glasshouse facility. The utility of a plant transformation system is very much dependent upon its efficiency, and several factors that

**4.1** *Agrobacterium***-mediated transformation** 

affect *Actinidia* transformation efficiency are discussed below.

*A. tumefaciens* strains are defined by their chromosomal background and resident Ti plasmid, and exhibit differences in their capacity to transfer T-DNA to various plant species (Godwin et al., 1991). *A. tumefaciens* LBA4404, A281, C58, EHA101 and EHA105 are the strains commonly used in *Actinidia* transformation. Fraser et al. (1995) reported no marked difference in efficiency of *A. chinensis* transformation between strains A281 (a virulent L,L-

**4.1.1 Agrobacterium tumefaciens strains** 

succinamopine strain) and C58 (a virulent strain carrying the nopaline Ti plasmid pTiC58), which both harbour the binary vector pKIWI105. However, Janssen and Gardner (1993) showed A281 produced slightly higher rates of gene transfer than C58 and EHA101 in *A. deliciosa* transformation, and noted that because of source material variability, strain comparisons need to be repeated several times. Strain A281 harbours a tumour-inducing plasmid pTiBo542 (Hood et al., 1986) and an extra copy of the transcription activator of virulence (*vir*) genes, which may account for the higher transformation efficiency.

Comparison of *A. chinensis* callus formation using *A. tumefaciens* A281, GV3101, EHA105 and LBA4404, all harbouring the pART27-10 binary vector, revealed that 27% of leaf strips produced calli using A281, compared with 22.2%, 18.1% or 13.9% when using EHA105, LBA4404 and GV3101, respectively (T. Wang et al., 2007). Both A281 and its nononcogenic derivative, EHA105, have the Ti-plasmid pTiBo542 in a C58 chromosomal background (Hood et al., 1993; 1986), and have been shown to be superior in gene transfer in other plant species, e.g. apple (Bondt et al., 1994). However, high rates of callus formation do not necessarily mean high efficiency of transgenic plant production, and Wang et al. (2007) also found differences in shoot regeneration related to whether cocultivation had been with strains harbouring an oncogenic Ti plasmid (A281) or a nononcogenic Ti plasmid (EHA105). Transformants derived from the use of the disarmed strains EHA105, LBA4404 and GV3101 had callus and regeneration patterns similar to those of control explants, not co-cultivated with *A. tumefaciens*, whereas the use of A281 tended to result in large calli and take about two weeks longer to initiate adventitious buds. Less than 20% of the calli derived from A281 co-cultivation had subsequent shoot and root development, whereas over 70% of calli derived from EHA105, GV3101 and LBA4404 co-cultivation regenerated shoots and roots. Over-proliferation of calli derived from A281 co-cultivation was even more severe in *A. eriantha* and no regenerated shoots were obtained (T. Wang et al., 2006). It is likely that high callus formation and poor adventitious bud and root initiation from the A281 co-cultivated tissue is related to the cointegration into plant genome of the oncogenes.

#### **4.1.2 Species**

Most *Actinidia* transformation systems have been developed for *A. chinensis* and *A. deliciosa,* though transformation of *A. arguta, A. eriantha, A. kolomikta* and *A. latifolia* has been reported. All *Actinidia* genotypes tested have been found to be responsive to a range of tissue culture conditions, and relatively amenable to regeneration protocols (Fraser et al., 1995). Compared with other woody species, e.g. apple (James et al., 1989), relatively high *A. deliciosa* transformation and regeneration rates have been achieved (Uematsu et al., 1991), and *A. chinensis* transformation efficiencies of up to 27.8% have been reported (T. Wang et al., 2007). However, *A. arguta* transformation was less successful when applying the transformation protocols developed for *A. chinensis* or *A. eriantha*, with co-cultivated explants suffering considerable browning and necrosis during callus induction and shoot regeneration stages. Minimizing the extent of explant browning and necrosis was achieved through reducing the basal salt concentration to ½ MS medium, combined with lower light intensity (3.4 µmol/m2/s) during the callus induction and regeneration stages. This resulted in adventitious shoot development and an efficient and reproducible *Agrobacterium*-mediated transformation system for *A. arguta* (Han et al., 2010).

From the production of over 1000 transgenic *Actinidia* plants at Plant & Food Research, the salient features in comparing the transformation of four species are three-fold. *A. arguta*  displays a relatively, low transformation efficiency of 1–10% compared with the 5–20% for *A. deliciosa* and *A. eriantha* and 5–30% for *A. chinensis*; the induction of *A. eriantha* callus is relatively high compared with other species; but the regeneration of *A. eriantha* kanamycinresistant shoots takes much longer than with the other three species.

#### **4.1.3 Co-cultivation conditions**

*Agrobacterium*-mediated DNA delivery to plant cells is initiated through a series of chemical signals exchanged between the host and pathogen, which may activate *vir* genes to signal the bacterium to enter virulence mode. Phenolics, sugars, temperature and pH can affect *Agrobacterium* virulence and presumably its capacity to transform plant cells (Alt-Moerbe et al., 1988). However, the degree to which these factors influence transformation efficiency varies with species and reports. Acetosyringone (AS), one of the phenolic compounds released by wounded plant tissue, and a signal molecule to ensure effective *vir*-induction and T-DNA transfer (Stachel et al., 1985; 1986), has been widely used to increase transformation efficiency in various crops (James et al., 1993; H. Wu et al., 2003). Janssen and Gardner (1993) found the addition of 20 µM AS to the *A. tumefaciens* growth and cocultivation medium increased DNA transfer approximately 2-fold in *A. deliciosa* leaf pieces, whereas highest levels of *A. latifolia* transformation were achieved using 200 µM in the cocultivation medium (Gao et al., 2007). Wang et al. (2006; 2007) used 100 µM AS in bacterial cultures for co-cultivation to improve the efficiency of *A. chinensis* and *A. eriantha* transformation. The inclusion of a suspension cell feeder layer during co-cultivation, separated from the explants by a layer of filter paper, has been used to improve *Actinidia* transformation frequency (Janssen & Gardner, 1993). In addition, as mentioned earlier, light intensity plays a role in the efficiency of *A. arguta* transformation (Han et al., 2010).

#### **4.1.4 Plant regeneration**

Selecting plant cell types or explants that have the ability to differentiate into whole plants is an essential step for the successful production of transgenic plants. Fortunately, *A. deliciosa* and *A. chinensis* callus induction and adventitious bud initiation are relatively straightforward after establishment in tissue culture if appropriate explant material is used. Young leaves, petioles and stem segments have been used successfully for *Actinidia* transformation, and, as with most other crops, the younger the explants, the easier regeneration will be. However, *A. arguta* transformation is one exception to this, as necrosis or browning occurs after *A. tumefaciens* co-cultivation if the explants used are too young (Han et al., 2010).

To maintain *Actinidia* explants in active and amenable condition for co-cultivation with *A. tumefaciens*, it is essential to subculture *in vitro* shoots at 3- to 4-week intervals (Fraser et al., 1995; Wang et al., 2006). MS basal medium has been used successfully for callus induction as well as regeneration in *Actinidia* (Kumar and Sharma 2002). However, optimum application of auxins and cytokinins, and combinations thereof, vary depending on the condition of the explant material used. Fraser et al. (1995) found that for *A. chinensis* regeneration, thidiazuron (TDZ) and kinetin, (0.1 and 10 mg/l) were clearly inferior to other cytokinins.

From the production of over 1000 transgenic *Actinidia* plants at Plant & Food Research, the salient features in comparing the transformation of four species are three-fold. *A. arguta*  displays a relatively, low transformation efficiency of 1–10% compared with the 5–20% for *A. deliciosa* and *A. eriantha* and 5–30% for *A. chinensis*; the induction of *A. eriantha* callus is relatively high compared with other species; but the regeneration of *A. eriantha* kanamycin-

*Agrobacterium*-mediated DNA delivery to plant cells is initiated through a series of chemical signals exchanged between the host and pathogen, which may activate *vir* genes to signal the bacterium to enter virulence mode. Phenolics, sugars, temperature and pH can affect *Agrobacterium* virulence and presumably its capacity to transform plant cells (Alt-Moerbe et al., 1988). However, the degree to which these factors influence transformation efficiency varies with species and reports. Acetosyringone (AS), one of the phenolic compounds released by wounded plant tissue, and a signal molecule to ensure effective *vir*-induction and T-DNA transfer (Stachel et al., 1985; 1986), has been widely used to increase transformation efficiency in various crops (James et al., 1993; H. Wu et al., 2003). Janssen and Gardner (1993) found the addition of 20 µM AS to the *A. tumefaciens* growth and cocultivation medium increased DNA transfer approximately 2-fold in *A. deliciosa* leaf pieces, whereas highest levels of *A. latifolia* transformation were achieved using 200 µM in the cocultivation medium (Gao et al., 2007). Wang et al. (2006; 2007) used 100 µM AS in bacterial cultures for co-cultivation to improve the efficiency of *A. chinensis* and *A. eriantha* transformation. The inclusion of a suspension cell feeder layer during co-cultivation, separated from the explants by a layer of filter paper, has been used to improve *Actinidia* transformation frequency (Janssen & Gardner, 1993). In addition, as mentioned earlier, light

intensity plays a role in the efficiency of *A. arguta* transformation (Han et al., 2010).

Selecting plant cell types or explants that have the ability to differentiate into whole plants is an essential step for the successful production of transgenic plants. Fortunately, *A. deliciosa* and *A. chinensis* callus induction and adventitious bud initiation are relatively straightforward after establishment in tissue culture if appropriate explant material is used. Young leaves, petioles and stem segments have been used successfully for *Actinidia* transformation, and, as with most other crops, the younger the explants, the easier regeneration will be. However, *A. arguta* transformation is one exception to this, as necrosis or browning occurs after *A. tumefaciens* co-cultivation if the explants used are too young

To maintain *Actinidia* explants in active and amenable condition for co-cultivation with *A. tumefaciens*, it is essential to subculture *in vitro* shoots at 3- to 4-week intervals (Fraser et al., 1995; Wang et al., 2006). MS basal medium has been used successfully for callus induction as well as regeneration in *Actinidia* (Kumar and Sharma 2002). However, optimum application of auxins and cytokinins, and combinations thereof, vary depending on the condition of the explant material used. Fraser et al. (1995) found that for *A. chinensis* regeneration, thidiazuron (TDZ) and kinetin, (0.1 and 10 mg/l) were clearly inferior to other cytokinins.

resistant shoots takes much longer than with the other three species.

**4.1.3 Co-cultivation conditions** 

**4.1.4 Plant regeneration** 

(Han et al., 2010).

Differences between NAA and IAA (indole-3-acetic acid) were insignificant. The most satisfactory combination of growth regulator additives was found to be 5 mg/l zeatin combined with 0.1 mg/l of NAA, or 1 mg/l zeatin and 0.5 mg/l BAP combined with 0.1 mg/l of NAA. Zeatin was clearly superior to BAP, when either was used as the sole cytokinin, but a combination of the two cytokinins gave the best overall result, in terms of the numbers of normal-looking shoots produced. Wang et al. (2006) made similar observations with *A. eriantha*  where the highest shoot regeneration rates were obtained using medium containing a combination of 2 mg/l zeatin and 3 mg/l BAP. Uematsu et al. (1991) reported that the regeneration frequency varied with the basal medium used, and B5 basal medium containing zeatin was most suitable for obtaining transformed *A. deliciosa* shoots. Using *A. deliciosa* MCS explants for transformation, Kim et al. (2010) used half-strength MS medium containing 0.001 mg/l 2,4-D and 0.1 gm/l zeatin, for callus induction and shoot regeneration. Calli formed on the surface of MCS segments after two weeks of culturing on selection medium and shoots were regenerated after four weeks. The transformation efficiencies ranged from 2.9 to 22.1% depending on the gene being transformed into the cells. The high degree of callus formation and shoot regeneration of *Actinidia* material from tissue culture makes it possible to obtain transformed shoots at a reasonably high frequency, although it is desirable to minimize callus development and maximize shoot development, to minimize the occurrence of somaclonal variation during these processes.

#### **4.2 Particle bombardment**

As opposed to the biological *Agrobacterium*-mediated transformation process, particle bombardment is a purely physical method for DNA delivery, using DNA-coated microscopic metal particles accelerated towards a target tissue. Qiu et al. (2002) used particle bombardment of *A. deliciosa* suspension cells, with a CaMV 35S transcribed maize *DHN1* gene (induced in response to abiotic stress) fused to the green fluorescent protein (GFP) reporter gene. GFP expression was localized within the cell nucleus after 10 h and was visualized in the cytoplasm (mainly around the plasma membranes) in response to increased osmotic stress (Qiu et al., 2002).

#### **4.3 Other DNA transfer methods**

Although *Agrobacterium-* and particle bombardment-mediated DNA transfer are the most commonly used systems of gene transfer to plants, a polyethylene glycol (PEG)-mediated approach was frequently used in the early 1980s to deliver DNA into protoplasts. Oliveira et al. (1991) used the chloramphenicol acetyl transferase (CAT) gene as a reporter to optimize the conditions for PEG-mediated transfection of *Actinidia* protoplasts, finding that the greatest CAT activity was obtained using 30% PEG 4000 and submitting protoplasts to a 5 min 45°C heat shock, prior to transfection. Using *in vitro* cultured *A. deliciosa* leaves, Raquel & Oliveira (1996) found protoplasts originating from the epidermis and leaf veins had cell division and regeneration ability, and displayed transient expression of a GUS gene introduced by PEG-mediated DNA transfer. Zhu et al. (2003) successfully transferred a GFP gene into *A. arguta* protoplasts by PEG-mediated transfer, with transient GFP expression detected in calli generated from the protoplasts. The physiological conditions of the protoplasts, the PEG concentration, and the time of heat stimulus are factors affecting the efficiency of DNA transfer using this approach. Because of the low yields of transformants and the inability of many species to be regenerated from protoplasts into viable plants, direct DNA uptake methods of transformation are much less frequently adopted than *Agrobacterium*-mediated transformation. However, the successful regeneration of whole plants from *A. chinensis, A. deliciosa* and *A. eriantha* protoplasts has been published (see earlier). Future development of new commercial cultivars produced directly or indirectly via genetic manipulation may see a resurgence in direct DNA uptake methods and protoplast regeneration, as these approaches may be more amenable to some genetic manipulation technologies, such as Zinc finger nuclease targeted site-directed mutagenesis.

#### **5.** *Actinidia* **molecular biology**

Initial molecular studies of *Actinidia* concentrated on fruit tissue, with an emphasis on genes involved in ethylene biosynthesis, cell wall modification, and carbohydrate metabolism (Atkinson & MacRae, 2007 and references therein). The cloning and/or expression of 1 aminocyclopropane-1 carboxylic acid (ACC) oxidase, *S*-adenosyl-*L*-methionine (SAM) synthase and ACC synthase identified some of the key genes involved in ethylene biosynthesis, a control point of fruit ripening. Molecular studies on genes encoding key enzymes in carbohydrate metabolism have included: polygalacturanase; xyloglucan endotransglycosylase/hydrolase; polygalacturonase inhibitor protein; sucrose phosphate synthase; and sucrose synthase. The most widely studied genes in these early forays into *Actinidia* molecular biology were those encoding the cysteine protease, actinidin, which can account for up to 50% of fruit soluble protein. Actinidin genes have been cloned, expressed in transgenic tobacco, the promoter sequenced, and studied in transgenic petunia.

#### **5.1 Expressed sequence tag (EST) databases**

A significant watershed in advancing *Actinidia* molecular biology was the generation of a database of 132,577 expressed sequence tags (EST), from a variety of *Actinidia* species, (Crowhurst et al., 2008). This provided a significant increase in the availability of *Actinidia* transcriptomic data, which prior to this publication were represented by 511 sequences in GenBank (dbEST Jan. 2008). This genetic resource, derived primarily from four species (*A. chinensis*, *A. deliciosa*, *A. arguta* and *A. eriantha*), included a range of tissues and developmental time points (Table 1). The average sequence length of these EST sequences was 503 bases. As expected, a high frequency of redundancy was observed within the *Actinidia* EST dataset and clustering at a 95% threshold, resulting in 23,788 sequences remaining as singletons and 18,070 tentative consensus (TC) sequences, a combined total of 41,858 non-redundant clusters (NRs). Analysis revealed that 28,345 NRs had sufficient homology to *Arabidopsis* sequences (E>1.0e-10) to be assigned a functional classification. Many of the NRs with no *Arabidopsis* homolog did however, have homologs in other crops. Crowhurst et al. (2008) also reported more specific analysis of ESTs of key genes related to distinctive features of *Actinidia* including flavour and aroma, colour, health-beneficial compounds, allergens, and cell wall structure.

Codon usage analysis revealed that *Actinidia* shared many similarities with other dicotyledonous plants, and although codon usage was similar among three *Actinidia* species, it was not identical. A higher GC ratio was seen in coding than in non-coding regions, and this was more marked in *A. deliciosa* and *A. eriantha* than in *A. chinensis*. A modest degree of

and the inability of many species to be regenerated from protoplasts into viable plants, direct DNA uptake methods of transformation are much less frequently adopted than *Agrobacterium*-mediated transformation. However, the successful regeneration of whole plants from *A. chinensis, A. deliciosa* and *A. eriantha* protoplasts has been published (see earlier). Future development of new commercial cultivars produced directly or indirectly via genetic manipulation may see a resurgence in direct DNA uptake methods and protoplast regeneration, as these approaches may be more amenable to some genetic manipulation technologies, such as Zinc finger nuclease targeted site-directed mutagenesis.

Initial molecular studies of *Actinidia* concentrated on fruit tissue, with an emphasis on genes involved in ethylene biosynthesis, cell wall modification, and carbohydrate metabolism (Atkinson & MacRae, 2007 and references therein). The cloning and/or expression of 1 aminocyclopropane-1 carboxylic acid (ACC) oxidase, *S*-adenosyl-*L*-methionine (SAM) synthase and ACC synthase identified some of the key genes involved in ethylene biosynthesis, a control point of fruit ripening. Molecular studies on genes encoding key enzymes in carbohydrate metabolism have included: polygalacturanase; xyloglucan endotransglycosylase/hydrolase; polygalacturonase inhibitor protein; sucrose phosphate synthase; and sucrose synthase. The most widely studied genes in these early forays into *Actinidia* molecular biology were those encoding the cysteine protease, actinidin, which can account for up to 50% of fruit soluble protein. Actinidin genes have been cloned, expressed

in transgenic tobacco, the promoter sequenced, and studied in transgenic petunia.

A significant watershed in advancing *Actinidia* molecular biology was the generation of a database of 132,577 expressed sequence tags (EST), from a variety of *Actinidia* species, (Crowhurst et al., 2008). This provided a significant increase in the availability of *Actinidia* transcriptomic data, which prior to this publication were represented by 511 sequences in GenBank (dbEST Jan. 2008). This genetic resource, derived primarily from four species (*A. chinensis*, *A. deliciosa*, *A. arguta* and *A. eriantha*), included a range of tissues and developmental time points (Table 1). The average sequence length of these EST sequences was 503 bases. As expected, a high frequency of redundancy was observed within the *Actinidia* EST dataset and clustering at a 95% threshold, resulting in 23,788 sequences remaining as singletons and 18,070 tentative consensus (TC) sequences, a combined total of 41,858 non-redundant clusters (NRs). Analysis revealed that 28,345 NRs had sufficient homology to *Arabidopsis* sequences (E>1.0e-10) to be assigned a functional classification. Many of the NRs with no *Arabidopsis* homolog did however, have homologs in other crops. Crowhurst et al. (2008) also reported more specific analysis of ESTs of key genes related to distinctive features of *Actinidia* including flavour and aroma, colour, health-beneficial

Codon usage analysis revealed that *Actinidia* shared many similarities with other dicotyledonous plants, and although codon usage was similar among three *Actinidia* species, it was not identical. A higher GC ratio was seen in coding than in non-coding regions, and this was more marked in *A. deliciosa* and *A. eriantha* than in *A. chinensis*. A modest degree of

**5.** *Actinidia* **molecular biology** 

**5.1 Expressed sequence tag (EST) databases** 

compounds, allergens, and cell wall structure.

CpG suppression was also evident in the three *Actinidia* species, with an XCG/XGG ratio of 0.68–0.71. Analysis of overlapping regions of 3,901 TCs identified 32,764 bi-allelic single nucleotide polymorphisms (SNPs), with one SNP every 417 bp, although some of the SNPs were probably the result of homeologous or paralogous sequences, rather than allelic variation. The allelic SNPs have potential for the development of molecular markers for use in genetic mapping, population genetics and linkage disequilibrium studies or for markerassisted selection. The inter-specific SNPs, identified in orthologous loci from different *Actinidia* species represent species–species variation and have utility in kiwifruit breeding using crosses between different species. Further analysis revealed that over 30% of the *Actinidia* EST NRs had at least one SSR, with dinucleotide repeats, predominantly in the 5' untranslated region, being twice as frequent as trinucleotide repeats, which were more evenly distributed across the gene.


Table 1. Numbers of ESTs derived from various *Actinidia* species and tissues

#### **5.2 An** *Actinidia* **microarray platform**

Characterizing a gene's temporal and spatial expression is critical to understanding its function. Early *Actinidia* molecular studies characterized the expression of a limited number of genes, identified as being differentially expressed during a particular developmental phase (Ledger & Gardner, 1994) or members of a particular gene family (Langenkamper et al., 1998). The *Actinidia* EST database provided a resource for more global gene expression analysis, through the development of a 17,472-feature oligonucleotide microarray of *Actinidia* genes. This microarray represented genes from a variety of species: *A. chinensis*  (51%); *A. deliciosa* (38%); *A. eriantha* (6%); *A. arguta* (3%); and other *Actinidia* species (2%).

Walton et al. (2009) used the *Actinidia* microarray to examine gene expression in *A. deliciosa* meristems and buds in response to the dormancy-breaking hydrogen cyanamide (HC) chemical treatment, over two growing seasons. Although most of the genes that responded early (1–3 days) to HC treatment differed between seasons, there was a high degree of commonality between seasons of genes that showed the greatest change in expression six days post treatment, with 123 genes up-regulated and 35 genes down-regulated at day 6 in both seasons. Quantitative PCR (qPCR) of 35 selected genes validated the microarray data for 97% of up-regulated and 60% of down-regulated genes. Genes that changed in expression upon HC-treatment were classified into distinct profiles, including: i) genes that reached a peak in expression at 3 or 6 days post treatment, then returned to baseline levels by day 15; ii) genes that reached a peak in expression at 3 or 6 days post treatment, followed by a second burst of transcription at 25–40 day post treatment, iii) genes that decreased in expression prior to meristematic activity or external bud growth. Putative function of these HC-responsive *Actinidia* genes, based on homology to other plant genes, indicated that many had been identified in other plant stress-related studies, including a number of genes that had shown similar responses in HC-treated grape, suggesting similar mechanisms in response to HC-treatment in these two crops.

*Actinidia* species are a climacteric fruit, showing a dramatic increase in ethylene production and a high respiration rate during fruit ripening. Generally, kiwifruit are harvested firm, and then enter a period of softening, which is followed by the onset of autocatalytic ethylene production, when fruit soften to "eating ripe" firmness and develop their characteristic flavours and aromas. The final step of the ethylene biosynthetic pathway is the conversion of 1-aminocyclopropane-1 carboxylic acid (ACC) to ethylene by ACC oxidase. Atkinson et al. (2011) examined gene expression changes during the ripening process, using an ACC oxidase-silenced transgenic *Actinidia* line, the fruit of which produce no detectable climacteric ethylene, but could be induced to undergo softening, aroma and flavour development through the application of exogenous ethylene. Using the *Actinidia* microarray, expression of 401 genes changed significantly within 168 h of ethylene treatment, with 25 genes showing a response at 4 h, 81 genes at 12 h, and 183 genes 24 h after application. These ethylene-responsive genes could be grouped into functional categories, including: metabolism; oxidative stress; photosynthesis; regulation; cell wall; hormone; starch; other; and unknown functions. The expression patterns indicated that the majority of photosynthesis- and starch-related genes were down-regulated by ethylene, whereas upand down-regulation of genes in other functional groups were observed in response to ethylene. Validation by qPCR confirmed significant changes in gene expression of a number of genes involved in cell wall modification in response to ethylene, including a polygalacturonase, a pectin lyase, a pectin methylesterase and a xylan-degrading enzyme, as well as genes involved in fruit flavour, ethylene production and perception.

The microarray platform has provided a useful tool for genome-wide gene expression, as is evident from the studies above. However, microarrays have a limited dynamic range, lack the sensitivity required to detect subtle changes in expression, and are essentially a 'closed' platform, limited to examining the expression of only those genes represented on the array. Second-generation sequencing (2ndGS) is becoming the methodology of choice for many genome-wide expression studies (L. Wang et al., 2010), as this is an 'open' platform, capable of detecting any of the genes that are expressed within a particular tissue, organ or cell type at the time of RNA sampling. Analysis of *Actinidia* transcription has been initiated using Illumina 2ndGS, with mRNA-sequence data generated from a range of *A. chinensis* tissues and stages of fruit development (A.P. Gleave & Z. Luo, unpublished).

#### **5.3 Functional genomics in** *Actinidia* **and heterologous hosts**

Prior to the initiation of generating the *Actinidia* EST resource in 2000, reports of functional genomics through expression of *Actinidia* genes in either a heterologous or an *Actinidia* host were somewhat limited (Guo et al., 1999; Lay et al., 1996; Paul et al., 1995; Schroder et al., 1998; Z.C. Xu et al., 1998). The EST resource has facilitated a significant increase in *Actinidia* functional genomics, through expression of genes in various microbial and plant hosts.

by day 15; ii) genes that reached a peak in expression at 3 or 6 days post treatment, followed by a second burst of transcription at 25–40 day post treatment, iii) genes that decreased in expression prior to meristematic activity or external bud growth. Putative function of these HC-responsive *Actinidia* genes, based on homology to other plant genes, indicated that many had been identified in other plant stress-related studies, including a number of genes that had shown similar responses in HC-treated grape, suggesting similar mechanisms in

*Actinidia* species are a climacteric fruit, showing a dramatic increase in ethylene production and a high respiration rate during fruit ripening. Generally, kiwifruit are harvested firm, and then enter a period of softening, which is followed by the onset of autocatalytic ethylene production, when fruit soften to "eating ripe" firmness and develop their characteristic flavours and aromas. The final step of the ethylene biosynthetic pathway is the conversion of 1-aminocyclopropane-1 carboxylic acid (ACC) to ethylene by ACC oxidase. Atkinson et al. (2011) examined gene expression changes during the ripening process, using an ACC oxidase-silenced transgenic *Actinidia* line, the fruit of which produce no detectable climacteric ethylene, but could be induced to undergo softening, aroma and flavour development through the application of exogenous ethylene. Using the *Actinidia* microarray, expression of 401 genes changed significantly within 168 h of ethylene treatment, with 25 genes showing a response at 4 h, 81 genes at 12 h, and 183 genes 24 h after application. These ethylene-responsive genes could be grouped into functional categories, including: metabolism; oxidative stress; photosynthesis; regulation; cell wall; hormone; starch; other; and unknown functions. The expression patterns indicated that the majority of photosynthesis- and starch-related genes were down-regulated by ethylene, whereas upand down-regulation of genes in other functional groups were observed in response to ethylene. Validation by qPCR confirmed significant changes in gene expression of a number of genes involved in cell wall modification in response to ethylene, including a polygalacturonase, a pectin lyase, a pectin methylesterase and a xylan-degrading enzyme, as

well as genes involved in fruit flavour, ethylene production and perception.

and stages of fruit development (A.P. Gleave & Z. Luo, unpublished).

**5.3 Functional genomics in** *Actinidia* **and heterologous hosts** 

The microarray platform has provided a useful tool for genome-wide gene expression, as is evident from the studies above. However, microarrays have a limited dynamic range, lack the sensitivity required to detect subtle changes in expression, and are essentially a 'closed' platform, limited to examining the expression of only those genes represented on the array. Second-generation sequencing (2ndGS) is becoming the methodology of choice for many genome-wide expression studies (L. Wang et al., 2010), as this is an 'open' platform, capable of detecting any of the genes that are expressed within a particular tissue, organ or cell type at the time of RNA sampling. Analysis of *Actinidia* transcription has been initiated using Illumina 2ndGS, with mRNA-sequence data generated from a range of *A. chinensis* tissues

Prior to the initiation of generating the *Actinidia* EST resource in 2000, reports of functional genomics through expression of *Actinidia* genes in either a heterologous or an *Actinidia* host were somewhat limited (Guo et al., 1999; Lay et al., 1996; Paul et al., 1995; Schroder et al., 1998; Z.C. Xu et al., 1998). The EST resource has facilitated a significant increase in *Actinidia* functional genomics, through expression of genes in various microbial and plant hosts.

response to HC-treatment in these two crops.

*Actinidia* genes encoding: a pectin methylesterase inhibitor, with applications in fruit juice production (Hao et al., 2008); Bet v 1 and profilin-homologous allergens (Bublin et al., 2010; Oberhuber et al., 2008); an *L*-galactose-1-phosphate phosphatase and *l*-galactose guanyltransferase, (Laing et al., 2004; 2007) and *L*-galactose dehyrogenase (Shang et al., 2009), involved in vitamin C production; a lycopene beta-cyclase, involved in carotenoid production (Ampomah-Dwamena et al., 2009); three xyloglucan endotransglucosylase/ hydrolases involved in cell wall structure (Atkinson et al., 2009); two terpene synthases, involved in the production of floral sesquiterpenes (Nieuwenhuizen et al., 2009); and three glycosyltransferases of the anthocyanin pathway (Montefiori et al., 2011), have all been successfully expressed in *Escherichia coli,* with the recombinant proteins being used to study protein/enzyme function. The yeast species, *Pichia pastoris* or *Saccharomyces cerevisiae*, have also been used to express recombinant *Actinidia* proteins, a pectin methylesterase inhibitor (Mei et al., 2007), and three alcohol acyltransferases, involved in the production of volatile esters (Gunther et al., 2011) and actinidin, which was found to have a negative effect on *S. cervesiae* growth (Yuwono, 2004).

*In planta* functional genomics of *Actinidia* genes has been used to study genes involved in a variety of processes. Paul et al. (1995) expressed *A. deliciosa* preproactinidin in transgenic *Nicotiana tabacum*, showing that the protein was correctly processed and detrimental to plant growth when it accumulated to high levels. Yin et al. (2010) showed that expression of the *A. deliciosa ETHYLENE INSENSITIVE3*-like *EIL2* and *EIL3* transcription factor cDNAs in *Arabidopsis thaliana* stimulated ethylene production, and up-regulation of host ACC synthase and ACC oxidase gene family members, as well as a number of xyloglucan endotransglycoylase (*XET*) genes. Yin et al. (2010) also used the *N. benthamiana* transient expression system, described by Hellens et al. (2005), to demonstrate transactivation of *A. deliciosa* ripening-related *ACO1* and *XET5* promoters by *EIL2* and *EIL3*, confirming their role in the signal transduction pathway connecting ethylene signalling and ripening processes.

To understand the role of *Actinidia* lipoxygenase (*LOX*) genes, which in other plants are involved in a range of processes, including senescence and fruit ripening, B. Zhang et al. (2006) used transient expression of *A. deliciosa LOX1 and LOX2* genes in *N. benthamiana*. qPCR had shown that *LOX1* increased in expression in ethylene-treated fruit, in contrast to *LOX2 expression,* which was repressed by ethylene. The transient expression studies revealed that *LOX1* significantly accelerated chlorophyll degradation and chlorophyll fluorescence, whereas *LOX2* had no apparent effect on senescence.

Varkonyi-Gasic et al. (2011) expressed cDNAs of nine *Actinidia* MADS-box genes in *A. thaliana,* to determine their role in floral meristem and floral organ fate. Resulting transgenic plants showed a variety of phenotypes. *FUL*-*like* expression promoted floral transition in both long day (LD) and short day (SD) conditions, with a terminal flower phenotype evident in plants showing high levels of transgene expression. Expression of *FUL* promoted flowering, but less efficiently than *FUL*-*like*, and the floral phenotype was as wild-type. *SEP4* expression also promoted floral transition, with many plants showing small and curled leaves, and a reversion to vegetative growth and aerial rosettes during SD conditions. *SEP3* expression had a mild effect on floral transition under LD conditions and *PI* and *AP3-1* expression showed no effect. *A. thaliana* expressing the kiwifruit *AG* flowered earlier than the wild-type under SD conditions, and showed reduced height, curled leaves and a loss of inflorescence indeterminancy. Coupled with information on the patterns of expression of these genes in *Actinidia* vegetative tissue and both normal and aberrant floral organs, these studies gave considerable insights into the role of these MADS-box transcription factors in the specification of *Actinidia* floral organs, phase change and flowering time.

Vitamin C is an essential metabolite for plants and animals, and the inability of some animals, including humans, to synthesize vitamin C means that they are dependent upon a dietary source. The *L*-galactose pathway is a significant route for vitamin C production in plants, although the enzyme responsible for the conversion of GDP-*L*-galactose to *L*galactose-1-phosphate remained elusive until Laing et al. (2007) identified homologous genes from *Arabidopsis* and *A. chinensis* encoding a GDP-*L*-galactose guanyltransferase (GGT) capable of carrying out this function. Transient expression of the *A. chinensis GGT* gene in *N. benthamiana* leaves showed a 3-fold increase in vitamin C levels, and coupled with the biochemical studies, confirmed GGT's role in the *L*-galactose pathway. Further studies of *Actinidia* vitamin C production via the *L*-galactose pathway also made use of *in planta* functional genomics. As qPCR results had indicated GGT and GDP-mannose-3',5' epimerase (GME) were key enzymes involved in the high vitamin C content of *A. eriantha*, Bulley et al. (2009) expressed the *A. eriantha GGT* in *Arabidopsis*, identifying plants with over four times the amount of vitamin C in leaves. In *N. benthamiana* leaves, transient *GGT* expression increased vitamin C levels 4.2 fold, a 20% increase resulted from transient *GME* expression, and simultaneous expression of *GGT* and *GME* gave an average increase of 8.6-fold in vitamin C.

Biochemical and gene expression studies on the production β-linalool, an acyclic monoterpene alcohol, were supplemented by transient expression of putative (*S*)-linalool synthase cDNAs from *A. eriantha* and *A. polygama*, to further understand their role in floral aroma (X.Y. Chen et al., 2010). The production of large amounts of linalool in *N. benthamiana* leaves transiently expressing these cDNAs confirmed their function as linalool synthases. A biochemical study of *A. deliciosa* 'Hayward' and its male pollinator 'Chieftain' identified the sesquiterpene (*E,E*)-α-farnesene as the major terpene floral volatile, with germacrene D, (*E*) β-ocimene, (*Z,E*)-α-farnesene, also present (Nieuwenhuizen et al., 2009). Transient expression in *N. benthamiana* leaves of two *A. deliciosa* cDNAs encoding putative terpene synthases (*AFS*1 and *GDS1*), followed by dynamic headspace sampling and GC-MS analyses, showed that expression of *AFS1* resulted in the production of large quantities of (*E,E*)-α-farnesene and smaller quantities of (*Z,E*)-α-farnesene and (E)-β-ocimene. *GDS1*  expression resulted in production of germacrene D.

Glycosyltransferases are responsible for much of the diversity of anthocyanins, a subgroup of the flavonoids that give much of the red, purple and blue pigmentation to plants. Montefiori et al. (2011) characterized two glycosyltransferases (*F3GT1* and *F3GGT1*) from a red-fleshed *A. chinensis*. Recombinant *F3GT1* produced in *E. coli* catalyzed the addition of galactose to the 3-*OH* position in cyanidin, whilst recombinant *F3GGT1* catalyzed the addition of UDP-xylose to cyanadin-3-galactosidase. Confirmation of the roles of these genes in the red pigmentation of fruit flesh was demonstrated firstly through establishing that transient expression of *Arabidopsis PAP1* and *TT8* transcription factors in *A. eriantha* fruit resulted in red pigmentation, localized mainly around the fruit core, and with the major accumulated anthocyanin being cyanidin 3-*O*-xylo-galactoside. Concomitant transient expression of the two *Arabidopsis* genes with an *F3GT1* RNAi construct resulted in little or

these genes in *Actinidia* vegetative tissue and both normal and aberrant floral organs, these studies gave considerable insights into the role of these MADS-box transcription factors in

Vitamin C is an essential metabolite for plants and animals, and the inability of some animals, including humans, to synthesize vitamin C means that they are dependent upon a dietary source. The *L*-galactose pathway is a significant route for vitamin C production in plants, although the enzyme responsible for the conversion of GDP-*L*-galactose to *L*galactose-1-phosphate remained elusive until Laing et al. (2007) identified homologous genes from *Arabidopsis* and *A. chinensis* encoding a GDP-*L*-galactose guanyltransferase (GGT) capable of carrying out this function. Transient expression of the *A. chinensis GGT* gene in *N. benthamiana* leaves showed a 3-fold increase in vitamin C levels, and coupled with the biochemical studies, confirmed GGT's role in the *L*-galactose pathway. Further studies of *Actinidia* vitamin C production via the *L*-galactose pathway also made use of *in planta* functional genomics. As qPCR results had indicated GGT and GDP-mannose-3',5' epimerase (GME) were key enzymes involved in the high vitamin C content of *A. eriantha*, Bulley et al. (2009) expressed the *A. eriantha GGT* in *Arabidopsis*, identifying plants with over four times the amount of vitamin C in leaves. In *N. benthamiana* leaves, transient *GGT* expression increased vitamin C levels 4.2 fold, a 20% increase resulted from transient *GME* expression, and simultaneous expression of *GGT* and *GME* gave an average increase

Biochemical and gene expression studies on the production β-linalool, an acyclic monoterpene alcohol, were supplemented by transient expression of putative (*S*)-linalool synthase cDNAs from *A. eriantha* and *A. polygama*, to further understand their role in floral aroma (X.Y. Chen et al., 2010). The production of large amounts of linalool in *N. benthamiana* leaves transiently expressing these cDNAs confirmed their function as linalool synthases. A biochemical study of *A. deliciosa* 'Hayward' and its male pollinator 'Chieftain' identified the sesquiterpene (*E,E*)-α-farnesene as the major terpene floral volatile, with germacrene D, (*E*) β-ocimene, (*Z,E*)-α-farnesene, also present (Nieuwenhuizen et al., 2009). Transient expression in *N. benthamiana* leaves of two *A. deliciosa* cDNAs encoding putative terpene synthases (*AFS*1 and *GDS1*), followed by dynamic headspace sampling and GC-MS analyses, showed that expression of *AFS1* resulted in the production of large quantities of (*E,E*)-α-farnesene and smaller quantities of (*Z,E*)-α-farnesene and (E)-β-ocimene. *GDS1* 

Glycosyltransferases are responsible for much of the diversity of anthocyanins, a subgroup of the flavonoids that give much of the red, purple and blue pigmentation to plants. Montefiori et al. (2011) characterized two glycosyltransferases (*F3GT1* and *F3GGT1*) from a red-fleshed *A. chinensis*. Recombinant *F3GT1* produced in *E. coli* catalyzed the addition of galactose to the 3-*OH* position in cyanidin, whilst recombinant *F3GGT1* catalyzed the addition of UDP-xylose to cyanadin-3-galactosidase. Confirmation of the roles of these genes in the red pigmentation of fruit flesh was demonstrated firstly through establishing that transient expression of *Arabidopsis PAP1* and *TT8* transcription factors in *A. eriantha* fruit resulted in red pigmentation, localized mainly around the fruit core, and with the major accumulated anthocyanin being cyanidin 3-*O*-xylo-galactoside. Concomitant transient expression of the two *Arabidopsis* genes with an *F3GT1* RNAi construct resulted in little or

the specification of *Actinidia* floral organs, phase change and flowering time.

of 8.6-fold in vitamin C.

expression resulted in production of germacrene D.

no visible red colour in *A. eriantha* fruit, indicating the *F3GT1* gene's critical role in anthocyanin biosynthesis. Concomitant transient expression of *PAP1* and *TT8* with an *F3GGT1* RNAi construct greatly reduced the amount of the major anthocyanin, cyanidin 3- *O*-xylo-galactoside.

Despite the availability of *Actinidia* transformation systems, to date there has been little published research on functional genomics of *Actinidia* genes through over-expression or silencing of genes in *Actinidia.* Such research is ongoing at Plant & Food Research and elsewhere, and the lack of published information is probably because many of these studies are related to fruit characteristics, and the time from initiating transformation to fruiting is at least three years. Of the few reports that have been published, one using gene silencing of ACC oxidase (Atkinson et al., 2011) has been discussed earlier in this chapter. Other studies have involved the over-expression of an *Actinidia Lfy* transcription factor cDNA, in an attempt to enhance early fruit set (Guo et al., 1999) and the silencing of ACC synthetase or ACC oxidase genes in *A. deliciosa* and *A. chinensis* (Li et al., 2003). In both these studies, only the production of transgenic plants was reported, with no analysis of their phenotype. Ledger et al. (2010), however, describe the use of transgenic *A. chinensis* plants to examine the role of *carotenoid cleavage dioxgenase* (*CCD*) genes in branching and vine architecture. The involvement of *CCD* genes, or their orthologs, in branching has been shown through the characterization of branching mutants in a range of annual plant species. Ledger et al. (2010) showed that expression of *A. chinensis CCD7* and *CCD8* cDNAs was able to complement their corresponding *Arabidopsis* branching mutants *max3* and *max4*. In *A. chinensis* plants transformed with a *CCD8* gene silencing construct*,* a number of plants showed greatly reduced *CCD8* expression levels at eight and 13 months of growth in the glasshouse. The *CCD8*-silenced plants showed significantly more primary and higher order branches, and a higher incidence of short branches, compared with control plants, but no difference in internode length on the main stem. Another finding was that leaves on some *CCD8*-silenced plants were slower to senesce and had a greater chlorophyll content than leaves of control plants. This study confirmed the role of *CCD8* in branching, and identified that *CCD8* plays a role in senescence in a deciduous woody perennial plant.

The studies described above give some valuable insights into the enzymatic or structural function of proteins encoded by these *Actinidia* genes and the roles they may play in the plant's phenotypic characteristics. However, in assigning a definitive function to a gene, it is essential also to understand the temporal and spatial regulation of its expression. In many of the studies described above, microarray and/or qPCR analysis were used to determine the transcriptional level of these genes in various tissues and in some cases at different developmental phases of the plant. Another approach to gain insights into the regulation of a gene's expression has been the analysis of promoter-reporter gene fusions in transgenic systems. Lin et al. (1993) fused an upstream region of an actinidin coding region to the β-glucuronidase (GUS) coding region and observed GUS expression during the later stages of transgenic petunia seed pod development, resembling the induction of actinidin in *Actinidia* fruit tissues. Similar promoter-GUS fusions were used to analyse an *A. chinensis* polygalacturonase promoter, and at the breaker stage of transgenic tomato fruit development, GUS expression was observed throughout the inner and outer pericarp, the columella and seeds, and became restricted to the inner pericarp and seeds at the later stages of ripening (Z.Y. Wang et al., 2000).


Table 2. Plant & Food Research's *Actinidia in planta* cDNA over-expression and gene silencing construct collection. Over-expression constructs of full length cDNAs cloned into pART27-derived vectors (Gleave, 1992) and gene silencing constructs of hairpin cDNA structures cloned into pTKO2 (Snowden et al., 2005). cDNAs are under the control of the CaMV35S promoter.

Aromatic amino acid 4 Fruit Ripening Cell wall structure <sup>5</sup> <sup>1</sup>

Anthocyanin biosynthesis 1

Vitamin C Ascorbate biosynthesis 24 6

Allergenicity Allergens 2 1

Transport Transporters 3 1 Unknown Sex-linked 3

miRNA 4

Total 224 59 Table 2. Plant & Food Research's *Actinidia in planta* cDNA over-expression and gene silencing construct collection. Over-expression constructs of full length cDNAs cloned into pART27-derived vectors (Gleave, 1992) and gene silencing constructs of hairpin cDNA structures cloned into pTKO2 (Snowden et al., 2005). cDNAs are under the control of the

Terpenoid Biosynthesis 9 3 Ester Biosynthesis 12 1 Cytochrome P450 22 3

Ethylene biosynthesis and 2 1

Chlorophyll degradation 3 1 Carotenoid biosynthesis 2 2

Phenylpropenoid biosynthesis 1 3

Ubiquitination 2 1 Actinidin 3 2

Branching 1 1 Phase change 2 1 Hormone response 5 4 Cell signalling 1

Cell cycle 1 Nucleotide synthesis & DNA 2 Chromatin remodelling 1 5

Transcriptional machinery 2

*Myb* & *Myb*- 35 3 *bZIP* 19 2 *MADS*-box 15 7 bHLH 13 1 C2-C2 *Dof* 9 C2-C2 *CO*-8 *NAC* Domain 5 *AP2-EREB* 2 1 *WRKY* 1 1 *BZR* 1 1 *WD40* 1

**No. of overexpression** 

**No. of silencing constructs** 

**gene classification** 

Sugars & Acids Sugar metabolism <sup>2</sup>

Defence Antimicrobial peptides 2

Transcription factors

**General Process Metabolic pathway, process or** 

Flavour & Aroma

Colour

Protein Degradation

Plant

Development

DNA and Replication

Transcription

CaMV35S promoter.

Transcription factors play a central role in regulating gene expression, through activation or repression of target promoters, and are able to regulate complex developmental processes or entire metabolic pathways co-ordinately. The dual-luciferase reporter vector system, developed to identify transcription factor activation of promoters (Hellens et al., 2005), has been applied to studying transactivation of promoters of *Actinidia* ripening-related *ACO1* and *XET5* genes, by *EIL2* and *EIL3* (Yin et al., 2010). Exploiting this promoter activation tool is reliant on having a cloned *Actinidia* transcription factors; Plant & Food Research has developed such a resource, which includes *Actinidia* cDNA over-expression and RNAi gene silencing constructs, including 109 *Actinidia* transcription factors for use in many of the *in planta* functional genomics approaches described above (Table 2).

#### **5.4 Molecular markers and mapping**

Molecular markers have been used to carry out genetic characterization of the *Actinidia* genus. They allow germplasm enhancement through systematic crossing of plants, selected on the basis of their intra- and inter-specific phylogenic relationships and patterns of allelic diversity, and the selection of parent plants with desirable alleles for use in breeding programmes. Markers are also used to determine hybridity, pedigree, and for quality control during crossing. The development and application of molecular markers closely linked to desirable traits has the potential to assist kiwifruit breeding greatly through the early selection of those progeny, with a high probability of carrying the genetic information for the desired trait. Various genetic markers have been developed in *Actinidia* using restriction fragment length polymorphisms (RFLPs) (Crowhurst et al., 1990), amplified fragment length polymorphisms (AFLPs) (Novo et al., 2010; Testolin et al., 2001; X.G. Xiao et al., 1999), random amplified polymorphic DNAs (RAPDs) (Gill et al., 1998; H.W. Huang et al., 2002a; Shirkot et al., 2002), SSRs (Fraser et al., 2004; W.G. Huang et al., 1998; Korkovelos et al., 2008) or SNPs (Zhou et al., 2011). Much of the early molecular marker development was primarily to investigate the molecular phylogeny of *Actinidia* species, which in general was consistent with the traditional morphology-based classification. Studies were aimed also at sex determination, and molecular markers confirmed that the dioecious nature in *Actinidia* was a consequence of sex-determining genes localized on a pair of chromosomes that function like an XX/XY system (Gill et al., 1998; Harvey et al., 1997; Testolin et al., 1995). Although the genetic basis for sex determination in kiwifruit remains unknown, RAPD markers linked to this trait led to the development of sequence-characterized amplified regions (SCARs) (Gill et al., 1998). These are now deployed routinely in markerassisted kiwifruit breeding, to eliminate male plants from crosses at the seedling stage, to select males when breeding for pollinizers, or to ensure a desirable male-to-female ratio of progeny are planted when characterizing families.

A framework *Actinidia* linkage map was first constructed using SSRs and the pseudo-test cross mapping strategy, often used for mapping out-crossing species, followed by the integration of AFLP markers (Testolin et al., 2001). Markers were screened over 94 individuals from a population generated from an inter-specific cross of a diploid *A. chinensis*  female and a diploid *A. callosa* male. Linkage maps were produced for each parent, with the female framework map having 160 loci, 38 linkage groups and covering 46% of the estimated genome length, and the male framework map having 116 loci, 30 linkage groups, and covering 34% of the estimated genome length. The maps were produced with LOD scores ≥2 (as an indication of coinheritance of loci). Continued progress in *Actinidia* mapping led to a significant advancement in *Actinidia* genetics, with the generation of a gene-rich linkage map of *A. chinensis,* constructed using 644 SSRs, and defining the 29 chromosomes of the haploid genome (Fraser et al., 2009). Again, SSRs were the marker of choice, owing to their abundance, distribution in coding and non-coding regions, reproducibility, Mendelian mode of inheritance and co-dominant nature. The inherent variability of SSRs, because of the high mutation rate, makes SSRs highly informative genetic markers. The linkage maps were produced using a mapping population of 272 individuals, created through an intraspecific cross of diploid *A. chinensis* parents, selected from two very distinct geographical locations in China, and exhibiting a diversity of fruiting characteristics. Resulting comprehensive genetic linkage maps of the male and female parents were produced and an integrated map of the cross was generated, using co-dominant SSR markers. The female and male linkage maps were composed of 464 and 365 markers, respectively, with markers estimated to be within 10 cM of each other in over 96% of the female genome and 94% of the male genome. The robustness of the maps was reflected by the LOD scores of 4–10. Using sex-linked SCAR markers, linkage group 17 was identified as the putative X and Y chromosomes. The sex-determining locus appeared to be sub-telomeric, occupying only a small portion of the chromosome, with little evidence of recombination in this region. These genetic linkage maps provide a valuable resource for the supply of markers for the breeding of novel cultivars, as tools for comparative and quantitative trait mapping. They will contribute to further investigations on the evolution and function of genetic control mechanisms in kiwifruit. They are an essential part of assembling the genome sequence of *Actinidia*.

#### **5.5 Genome sequencing**

Although the *Actinidia* EST database is a useful resource, it at best represents only 50–60% of the genes within the *Actinidia* genome, and contains no information on elements such as promoters, terminators and introns that play important roles in controlling gene expression. In addition, EST libraries, by the nature of their construction, under-represent genes that are expressed at relatively low levels and yet could play a critical role in a particular trait. Understanding key traits requires detailed information of not only the transcribed regions of a genome but also the intergenic and intron sequences, information that can be gained from the whole genome sequence (WGS) and its subsequent annotation. The advent of secondgeneration sequencing (2ndGS) and advances in data handling and assembly software have now made it feasible to determine the WGS of a plant species at a fraction of the cost of the Sanger technology used to generate the WGS of *Arabidopsis,* for example (The Arabidopsis Genome Initiative, 2000). Plant & Food Research has recently initiated a research effort to determine the WGS of a diploid *A. chinensis* genotype, the haploid genome of which is predicted to be 650 Mbp. With no di-haploid or homozygous *Actinidia* genotypes available, the heterozygosity of the diploid *A. chinensis* may create some problems in WGS assembly. To minimize this, a genotype that has undergone two generations of sib-crossing and has an inbreeding coefficient of 0.375 has been selected. Genome sequencing is being carried out using an Illumina sequencing platform, using a variety of libraries and resulting sequencing data assembled into scaffolds. WGS assembly is being complemented by BAC-end sequencing, using an *A. chinensis* BAC library (Hilario et al., 2007), and use of the geneticlinkage map discussed earlier.

scores ≥2 (as an indication of coinheritance of loci). Continued progress in *Actinidia* mapping led to a significant advancement in *Actinidia* genetics, with the generation of a gene-rich linkage map of *A. chinensis,* constructed using 644 SSRs, and defining the 29 chromosomes of the haploid genome (Fraser et al., 2009). Again, SSRs were the marker of choice, owing to their abundance, distribution in coding and non-coding regions, reproducibility, Mendelian mode of inheritance and co-dominant nature. The inherent variability of SSRs, because of the high mutation rate, makes SSRs highly informative genetic markers. The linkage maps were produced using a mapping population of 272 individuals, created through an intraspecific cross of diploid *A. chinensis* parents, selected from two very distinct geographical locations in China, and exhibiting a diversity of fruiting characteristics. Resulting comprehensive genetic linkage maps of the male and female parents were produced and an integrated map of the cross was generated, using co-dominant SSR markers. The female and male linkage maps were composed of 464 and 365 markers, respectively, with markers estimated to be within 10 cM of each other in over 96% of the female genome and 94% of the male genome. The robustness of the maps was reflected by the LOD scores of 4–10. Using sex-linked SCAR markers, linkage group 17 was identified as the putative X and Y chromosomes. The sex-determining locus appeared to be sub-telomeric, occupying only a small portion of the chromosome, with little evidence of recombination in this region. These genetic linkage maps provide a valuable resource for the supply of markers for the breeding of novel cultivars, as tools for comparative and quantitative trait mapping. They will contribute to further investigations on the evolution and function of genetic control mechanisms in kiwifruit. They are an essential part of assembling the

Although the *Actinidia* EST database is a useful resource, it at best represents only 50–60% of the genes within the *Actinidia* genome, and contains no information on elements such as promoters, terminators and introns that play important roles in controlling gene expression. In addition, EST libraries, by the nature of their construction, under-represent genes that are expressed at relatively low levels and yet could play a critical role in a particular trait. Understanding key traits requires detailed information of not only the transcribed regions of a genome but also the intergenic and intron sequences, information that can be gained from the whole genome sequence (WGS) and its subsequent annotation. The advent of secondgeneration sequencing (2ndGS) and advances in data handling and assembly software have now made it feasible to determine the WGS of a plant species at a fraction of the cost of the Sanger technology used to generate the WGS of *Arabidopsis,* for example (The Arabidopsis Genome Initiative, 2000). Plant & Food Research has recently initiated a research effort to determine the WGS of a diploid *A. chinensis* genotype, the haploid genome of which is predicted to be 650 Mbp. With no di-haploid or homozygous *Actinidia* genotypes available, the heterozygosity of the diploid *A. chinensis* may create some problems in WGS assembly. To minimize this, a genotype that has undergone two generations of sib-crossing and has an inbreeding coefficient of 0.375 has been selected. Genome sequencing is being carried out using an Illumina sequencing platform, using a variety of libraries and resulting sequencing data assembled into scaffolds. WGS assembly is being complemented by BAC-end sequencing, using an *A. chinensis* BAC library (Hilario et al., 2007), and use of the genetic-

genome sequence of *Actinidia*.

linkage map discussed earlier.

**5.5 Genome sequencing** 

#### **6. Future perspectives and challenges**

Completion of the *A. chinensis* WGS will be the first within the Ericales, a large and diverse order that includes persimmon, blueberry, cranberry and tea, and the benefits to be gained in having the *Actinidia* WGS are enormous. Genome annotation is a key to the utility of any WGS, and the advances in transcriptome sequencing will greatly aid the defining and delineating of genes. Building on the availability of the genome sequence, characterization of the interrelationships between the *Actinidia* genome, transcriptome, proteome and metabolome, and functional genomics of alleles, will greatly aid in the understanding of biological processes, phenotypes and traits of kiwifruit. The annotated *A. chinensis* WGS will also provide a reference genome for the sequencing of genomes of other *Actinidia* species, to examine inter-species variability, and to identify SNPs. The knowledge gained from these efforts will open up greater opportunities for molecular breeding in kiwifruit, allowing the use of molecular markers for selective and accelerated introgression of desirable traits from the diverse *Actinidia* germplasm, to create new and novel cultivars.

As detailed in this chapter, much of the molecular research in *Actinidia* has targeted traits such as fruit flavour, aroma, ripening and colour, which could be exploited in the development of new cultivars with novel fruit characteristics. There has been very little molecular research in *Actinidia* targeted towards pathogens and disease. However, the recent devastating effect on commercial kiwifruit orchards in parts of Italy, due to kiwifruit canker, caused by the bacterium *Pseudomonas syringae* pv. *actinidiae* (Psa), may well change the emphasis of the immediate future of kiwifruit research. Although Psa was identified in Italy in 1992 (Scortichini, 1994), the bacterium caused little problem, until severe disease outbreaks in both *A. deliciosa* and *A. chinensis* cultivars in 2009. The presence of Psa has now been reported in most of the major kiwifruit growing regions of the world, although there appear to be a number of haplotypes, differing in their virulence. Minimizing the impact of Psa on the global kiwifruit industry will require a coordinated effort by pathologists, physiologists, breeders and growers. Many of the molecular tools, the knowledge and the *Actinidia* resources described in this chapter will aid in the understanding of the plant– pathogen interactions, the plant's response to infection, the identification and mapping of *Actinidia* genes offering Psa resistance, and ultimately the development of kiwifruit cultivars resistant to Psa, through breeding or genetic manipulation.

#### **7. References**


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Atkinson, R.G. & MacRae, E. (2007). Kiwifruit. In Pua, E.C. & Davey, M.R., eds, *Biotechnology* 

Bachiri, Y.; Song, G.Q.; Plessis, P.; Shoar-Ghaffari, A.; Rekab, T. & Morisset, C. (2001).

Barbieri, C. & Morini, S. (1987). Plant regeneration from Actinidia callus cultures. *Journal of* 

Bondt, A.d.; Eggermont, K.; Druart, P.; Vil, M.d.; Goderis, I.; Vanderleyden, J. & Broekaert,

Bublin, M.; Pfister, M.; Radauer, C.; Oberhuber, C.; Bulley, S.; DeWitt, A.M.; Lidholm, J.;

Bulley, S.M.; Rassam, M.; Hoser, D.; Otto, W.; Schunemann, N.; Wright, M.; MacRae, E.;

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## **Biotechnological Tools for Garlic Propagation and Improvement**

Alejandrina Robledo-Paz1 and Héctor Manuel Tovar-Soto2 *1Postgrado en Recursos Genéticos y Productividad-Semillas Colegio de Postgraduados Km. 36.5 Carretera México-Texcoco, Montecillo, Edo. Méx. 2Instituto Tecnológico de Cd. Altamirano Pungarabato Pte. S/N. Ciudad Altamirano, Guerrero* 

*México* 

#### **1. Introduction**

30 Innovations in Biotechnology

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kiwifruit shoot-tips by vitrification and their regeneration. *Acta Horticulturae Sinica*,

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in regulating fruit ripening. *Plant Physiology*, Vol. 153, Issue 3, pp. 1280-1292, ISSN

carrying the actinidin gene influence the growth of Saccharomyces cerevisiae. *World Journal of Microbiology & Biotechnology*, Vol. 20, Issue 5, pp. 441-447, ISSN

plantlets regenerated from cryopreserved in vitro cultured grape and kiwi shoot-

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from in vitro-cultured seedling leaf protoplasts of Actinidia eriantha Benth. *Plant* 

Polymorphisms (Snps) in the Actinidia Chinensis Species Complex (Actinidiaceae).

expression of GFP gene in callus protoplasts of Actinidia arguta. *Journal of Henan* 

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Garlic (*Allium sativum* L.) is a monocotyledonous herb belonging to the genus *Allium* and the family Alliaceae and it is the second most widely distributed species of this genus throughout the world, (Kamenetsky, 2007). Garlic is used as food flavoring or as a medicinal plant. It can be preserved in oil or vinegar or processed into products such as garlic salt, garlic juice, concentrated garlic or most commonly, dehydrated garlic (Brewster, 1994). Although there have been different hypothesis as to the origin of garlic and it was even thought that it was a Mediterranean plant, Vavilov (1926) and Kazakova (1971) suggested Central Asia as its primary center. Years later, this hypothesis was confirmed by the discovery of fertile clones of a primitive garlic type in the Tien-Shan mountains in Kyrgyzstan (Etoh, 1986; Kotlinska et al., 1991) and by studies using biochemical and molecular markers (Pooler & Simon, 1993).

Fritsch & Friesen (2002) put forward the idea that *Allium sativum* is a complex made up by three main groups: (a) Sativum, (b) Longicuspis and (c) Ophioscorodon, and two subgroups: Subtropical and Pekinense. The commercial types of garlic can be divided into: (1) violet or Asian, which is cultivated in subtropical regions, (2) pink, which needs long photoperiods and has low requirements for cold, (3) white, which needs long photoperiods, has medium to high requirements for cold, and (4) purple, which needs long photoperiods and periods of cold (Heredia-García, 2000). They can also be classified into hard-neck and soft-neck garlic. Hard-neck garlic forms a floral scape whose flowers normally abort and whose end produces topsets, while soft-necked garlic does not form a scape. The majority of garlic cultivated for commercial purposes is soft-neck type because it is easier to cultivate and it has a longer shelf life (Kamenestsky, 2007).

Garlic is grown all over the world from temperate to subtropical climates (Fritsch & Friesen, 2002). Production and world cultivated area have increased over years. In 2004 production was of 14'071,335 t obtained from an area of 1'129,714 ha; while in 2007 15'799,909 t were produced on 1'220,314 ha. The main producer of garlic is China, with 17'967,857 t, accounting for 80.6% of the world production, followed by India (1'070,000 t) and the Republic of Korea (380,000 t) (FAOSTAT, 2009) (Table 1). Garlic bulbs are composed of aggregate of cloves which have their origin in the axillary buds. The cloves are made up of a protective sheath (which is dry and thin at maturity), a thickened storage sheath leaf (which represents the major part of the clove and it is also the usable part), and sprouting and foliage leaves which protect the apical meristem (Mann, 1952; Purseglove, 1988). The number of cloves per bulb varies with the cultivar, but bulbs with a maximum of 16 cloves are preferred.


Table 1. Main garlic-producing countries in the world (FAOSTAT, 2009).

Currently, garlic propagates vegetatively through cloves or through topsets that develop in the plant´s inflorescences (which can prevent the plant from producing flowers and seeds). Kamenetsky & Rabinowitch (2001) explain that lack of fertility could be due to the fact that in past the floral scapes were removed and plants with low flowering ability were selected in order to obtain bigger bulbs (Kamenetsky & Rabinowitch, 2001; Etoh & Simon, 2002). Nowadays, in some places the bulbs are harvested before the flowering time to avoid their rotting or to use the scapes as vegetable (Etoh & Simon, 2002). In addition, the sterility of the garlic has been mainly attributed to chromosomal deletions, and also to differences in the length of homologous chromosome, to loss of genes involved in gametogenesis, to hypertrophy of the tapetal layer of the anthers at the post-meiotic stage, to microspore degeneration before or after the tetrade stage, to nutritional competition between the topsets and flowers, and to infestation with microorganisms (rickettsias) (Novak, 1972; Konvicka et al., 1978; Etoh, 1985; Pooler & Simon, 1994).

#### **2. Chemical composition and medicinal traits**

The main components of the garlic bulb are water (65%) and carbohydrates (26-30%), especially fructose polymers (Table 2). Other components are lipids, proteins, fiber, minerals and saponins (Lawson, 1996). Elements such as selenium (700µg per 100g of fresh weight), sulphur, zinc, magnesium, iron, sodium, calcium, as well as vitamins A, C, E and B-complex vitamins (thiamin, riboflavin, niacin) and phenols are also present in the garlic bulb (Koch & Lawson, 1996; Vinson et al., 1998). Garlic produces organosulphur compounds such as the γglutamylcysteines and alliin ((+)-S-(2-propenyl)-L-cysteine sulfoxide) which confers its flavor, odor and biological activity (Block, 1985). The alliin can account for 1.4% of the fresh weight of bulb (Keusgen, 2002). It has been found that the activity of alliinase, the enzyme

have their origin in the axillary buds. The cloves are made up of a protective sheath (which is dry and thin at maturity), a thickened storage sheath leaf (which represents the major part of the clove and it is also the usable part), and sprouting and foliage leaves which protect the apical meristem (Mann, 1952; Purseglove, 1988). The number of cloves per bulb varies with the

1 China 17'967,857 80.64 2 India 1'070,000 4.80 3 Republic of Korea 380,000 1.71 4 Russian Federation 227,270 1.02 5 Myanmar (before Burma) 200,000 0.90 6 United States of America 178,760 0.80 7 Egypt 174,659 0.78 8 Bangladesh 154,831 0.69 9 Spain 154,000 0.69 10 Ukraine 150,100 0.67

Currently, garlic propagates vegetatively through cloves or through topsets that develop in the plant´s inflorescences (which can prevent the plant from producing flowers and seeds). Kamenetsky & Rabinowitch (2001) explain that lack of fertility could be due to the fact that in past the floral scapes were removed and plants with low flowering ability were selected in order to obtain bigger bulbs (Kamenetsky & Rabinowitch, 2001; Etoh & Simon, 2002). Nowadays, in some places the bulbs are harvested before the flowering time to avoid their rotting or to use the scapes as vegetable (Etoh & Simon, 2002). In addition, the sterility of the garlic has been mainly attributed to chromosomal deletions, and also to differences in the length of homologous chromosome, to loss of genes involved in gametogenesis, to hypertrophy of the tapetal layer of the anthers at the post-meiotic stage, to microspore degeneration before or after the tetrade stage, to nutritional competition between the topsets and flowers, and to infestation with microorganisms (rickettsias) (Novak, 1972; Konvicka et

The main components of the garlic bulb are water (65%) and carbohydrates (26-30%), especially fructose polymers (Table 2). Other components are lipids, proteins, fiber, minerals and saponins (Lawson, 1996). Elements such as selenium (700µg per 100g of fresh weight), sulphur, zinc, magnesium, iron, sodium, calcium, as well as vitamins A, C, E and B-complex vitamins (thiamin, riboflavin, niacin) and phenols are also present in the garlic bulb (Koch & Lawson, 1996; Vinson et al., 1998). Garlic produces organosulphur compounds such as the γglutamylcysteines and alliin ((+)-S-(2-propenyl)-L-cysteine sulfoxide) which confers its flavor, odor and biological activity (Block, 1985). The alliin can account for 1.4% of the fresh weight of bulb (Keusgen, 2002). It has been found that the activity of alliinase, the enzyme

**(t)** 

**Production (%)** 

cultivar, but bulbs with a maximum of 16 cloves are preferred.

**Rank Country Production** 

 World 22'282,060 Table 1. Main garlic-producing countries in the world (FAOSTAT, 2009).

al., 1978; Etoh, 1985; Pooler & Simon, 1994).

**2. Chemical composition and medicinal traits** 

that hydrolyzes the sulphur compounds in garlic, is 10 times higher in bulbs than in leaves (Rabinkov et al., 1994) (Table 2).

Apart from its use for food flavoring, garlic also has medicinal uses for the relief of various ailments such as those caused by aging, arthritis, cancer, artheroesclerosis, immune deficiencies, blood glucose level, respiratory diseases, etc. (Keusgen, 2002; Raham, 2001). Likewise, it has been observed that garlic has antioxidant properties, it reduces blood cholesterol and triglycerides levels, lowers blood pressure and the possibility of blood clot formation and improves arterial oxygenation (Augusti, 1990; Abrams & Fallon, 1998; Bordia et al., 1998). Garlic's effect on reduction of lipids has been most extensively studied. The properties mentioned above are directly related to the sulphur compounds found in the garlic bulb. Alliin is also attributed the antibiotic effect on microorganisms such as *Helicobacter pylori* (bacterium which is associated with stomach cancer), *Salmonella typhi*, yeasts, *Trypanosoma* and *Staphylococcus epidermis.* Its inhibitory effect has also been observed on pathogenic fungi (*Aspergillus*, *Cryptoccocus neoformis*, dermatophytes) (Keusgen, 2002).


\*Excluding protein and inorganic sulphate (0.5%)

Table 2. Chemical composition of garlic bulb (Lawson, 1996).

#### **3. Pests and diseases during garlic cultivation and storage**

Garlic plant can be affected by various diseases caused by viruses, fungi and bacteria. The viruses that tend to cause it severe damages are potyviruses, such as Leek Yellow Stripe Virus (LYSV), Garlic Yellow Streak Virus (GYSV) and Onion Yellow Dwarf Virus (OYDV) (Bos, 1982; Walkey, 1987). Some carlaviruses, like Common Latent Virus (GCLV) and Shallot Latent Virus (SLV) can also infect the garlic plant (Messiaen et al., 1994). One of the most widely spread diseases in garlic producing countries is white rot, caused by the fungus *Sclerotium cepivorum,* which provokes wilting of the plant and rotting of the bulb. Its sclerotia can survive in soil for up to 20 years, therefore limiting garlic production (Delgadillo-Sánchez, 2000). As far as the fungus *Penicillium corymbiferum* is concerned, this attacks plants weakened by other pathogens, and although infested plants survive the infection, bulbs present symptoms during the storage period. Various bacteria (*Bacillus*  spp., *Erwinia* spp., *Pseudomonas* spp.) can also cause damages on bulbs upon storage. Garlic can also be affected by pests like thrips (*Thrips tabaci*), which are insects that infest plants from early developmental stages and cause severe foliage damages. For this reason, thrips are considered the most noxious pest affecting this crop plant. Mites (*Rhizoglyphus* spp.) are another garlic pest that invade the bulbs and limit their sprouting ability (Bujanos-Muñiz & Marín-Jarillo, 2000). On the other hand, bulb nematode (*Ditylenchus dipsaci*) causes the root knot disease, characterized by yellowing and rolling of leaves, as well as rotting of the bulb's base.

#### **4. Breeding**

Commercial garlic cultivars only propagate themselves vegetatively, the increase of genetic variation through conventional crossing is very low, or even absent. For this reason, clonal selection, induced mutations, somaclonal variation or genetic engineering are the only options for breeding improved cultivars (Robinson, 2007). Clonal selection has been the most widely used method for generating new garlic material. It is based on the variability existing in populations as a result of cross pollination between various garlic types and its ancestors when this plant still had the ability of sexual reproduction (Etoh & Simon, 2002; Koul et al., 1979). On the other hand, although mutations may be a source of variability, they are rather limited; therefore, breeding using this strategy has not resulted in significant progress (Etoh & Simon, 2002). The lack of sexuality in garlic limits the increase of variability that is useful for breeding for economically important traits, such as tolerance to biotic and abiotic stress, earliness, yield and quality (Kamenetsky, 2007). Moreover, vegetative propagation has various disadvantages for the crop: (a) a low multiplication rate (5 to 10 per year), (b) expensive and short-term storage that requires wide spaces, (c) transmission of phytopathogens (fungi, viruses, nematodes) through generations and from one production area to another, which can cause a yield decrease of up to 70%, and (d) loss of product quality (Kamenetsky, 2007; Walkey, 1990; Nagakubo et al., 1993).

#### **5. Biotechnology for garlic propagation, preservation and breeding**

Biotechnological tools such as micropropagation, meristems culture (in order to obtain virus-free plants), somaclonal variation, and genetic transformation, have contributed to propagation, preservation and breeding of garlic.

#### **5.1 Micropropagation**

Studies related to the application of tissue culture techniques such as micropropagation for garlic production started in 1970. This technique proved to be advantageous over clove reproduction, as it only requires cells or small tissue fragments to generate high number of plants. Micropropagation can be carried out via two morphogenetic ways: (1) organogenesis, which results in the formation of organs (shoots or roots), and (2) somatic embryogenesis, which leads to the formation of structures having a similar or equal morphology to that of a zygotic embryo. Both processes can involve (indirect) or not (direct) a previous callus phase. Morphogenetic ability in garlic decreases as the callus grows older and the emergence of abnormal plants increases (Novak, 1990). For this reason, regeneration that does not involve a previous callus phase is preferred. Embryogenesis possesses a series of advantages over organogenesis, such as higher potential for high plant output, lower labour requirement and lower cost (Sata et al., 2001). Several micropropagation protocols have been established using both ways of morphogenesis and different explant types; however, most protocols have been developed following the organogenetic way.

#### **5.1.1 Organogenesis**

34 Innovations in Biotechnology

Its sclerotia can survive in soil for up to 20 years, therefore limiting garlic production (Delgadillo-Sánchez, 2000). As far as the fungus *Penicillium corymbiferum* is concerned, this attacks plants weakened by other pathogens, and although infested plants survive the infection, bulbs present symptoms during the storage period. Various bacteria (*Bacillus*  spp., *Erwinia* spp., *Pseudomonas* spp.) can also cause damages on bulbs upon storage. Garlic can also be affected by pests like thrips (*Thrips tabaci*), which are insects that infest plants from early developmental stages and cause severe foliage damages. For this reason, thrips are considered the most noxious pest affecting this crop plant. Mites (*Rhizoglyphus* spp.) are another garlic pest that invade the bulbs and limit their sprouting ability (Bujanos-Muñiz & Marín-Jarillo, 2000). On the other hand, bulb nematode (*Ditylenchus dipsaci*) causes the root knot disease, characterized by yellowing and rolling of leaves, as

Commercial garlic cultivars only propagate themselves vegetatively, the increase of genetic variation through conventional crossing is very low, or even absent. For this reason, clonal selection, induced mutations, somaclonal variation or genetic engineering are the only options for breeding improved cultivars (Robinson, 2007). Clonal selection has been the most widely used method for generating new garlic material. It is based on the variability existing in populations as a result of cross pollination between various garlic types and its ancestors when this plant still had the ability of sexual reproduction (Etoh & Simon, 2002; Koul et al., 1979). On the other hand, although mutations may be a source of variability, they are rather limited; therefore, breeding using this strategy has not resulted in significant progress (Etoh & Simon, 2002). The lack of sexuality in garlic limits the increase of variability that is useful for breeding for economically important traits, such as tolerance to biotic and abiotic stress, earliness, yield and quality (Kamenetsky, 2007). Moreover, vegetative propagation has various disadvantages for the crop: (a) a low multiplication rate (5 to 10 per year), (b) expensive and short-term storage that requires wide spaces, (c) transmission of phytopathogens (fungi, viruses, nematodes) through generations and from one production area to another, which can cause a yield decrease of up to 70%, and (d) loss

of product quality (Kamenetsky, 2007; Walkey, 1990; Nagakubo et al., 1993).

propagation, preservation and breeding of garlic.

**5.1 Micropropagation** 

**5. Biotechnology for garlic propagation, preservation and breeding** 

Biotechnological tools such as micropropagation, meristems culture (in order to obtain virus-free plants), somaclonal variation, and genetic transformation, have contributed to

Studies related to the application of tissue culture techniques such as micropropagation for garlic production started in 1970. This technique proved to be advantageous over clove reproduction, as it only requires cells or small tissue fragments to generate high number of plants. Micropropagation can be carried out via two morphogenetic ways: (1) organogenesis, which results in the formation of organs (shoots or roots), and (2) somatic embryogenesis, which leads to the formation of structures having a similar or equal

well as rotting of the bulb's base.

**4. Breeding** 

Meristem culture is a technique used for obtaining virus-free plants, and also for micropropagation. Messiaen et al. (1970) were the first in regenerating garlic plants from meristems. Shoot or bud formation from callus was achieved using a combination of 6 furfurylaminopurine (kinetin), indol-3-acetic acid (IAA) and 2,4-diclorophenoxiacetic acid (2,4-D) (9.28µM, 11.4µM and 4.5µM, respectively). Likewise, Havránek & Novak (1973) obtained numerous growth areas on calli produced from meristems on a culture medium with 2,4-D. The subculture of calli to a medium containing IAA (11.4µM) and kinetin (46.5µM) induced formation of adventitious shoots.

In a different work, calli obtained from meristems of three garlic varieties (*Rose de Lautrec*, *California Early* and *California Late*) cultivated in a medium with 2,4-D (4.5μM) and IAA (5.7 μM) produced adventitious shoots when transferred to a medium with IAA (5.7μM) and kinetin (4.6μM) (Kehr & Shaeffer, 1976). For his part, Abo-El-Nil (1977) started cultures from meristems, stems and leaf discs of the variety *Extra Early White*, on a medium with pchlorophenoxyacetic acid (p-CPA) (10μM), 2,4-D (2μM) and kinetin (0.5μM), from which callus formation was achieved, which in turn resulted in the formation of adventitious shoots in the presence of kinetin (10μM) and IAA (10μM). In other works, meristems were cultivated on B5 medium (Gamborg et al., 1968) with 2.5μM 2-isopentenyladenine (2ip) and 0.55μM α-naphtalenacetic acid (NAA) (Bhojwani, 1980), or in Linsmaier and Skoog (LS) (Linsmaier & Skoog, 1965) medium with 9μM N6-benzyladenine (BA) alone or with 11.1μM NAA, and multiple shoots were obtained (Osawa et al., 1981).

There are only a few reports on suspension cell culture in garlic. For instance, Nagasawa & Finer (1988) were the first to establish suspension cell cultures obtained from calli derived from meristems of the cultivar *Howaito-Roppen* grown in presence of NAA (5.5μM) and BA (9μM). Adventitious shoots with leaf primordia started differentiating only after transferring the calli to agar-solidified medium. Likewise, Kim et al. (2003) obtained cells in suspension after cultivating shoots of the cultivar *Danyang* differentiated *in vitro* in Murashige and Skoog (MS) (1962) medium with 2.5µM 2iP. These cultures regenerated an average of 21.5 shoots per explant when they were exposed to a light intensity of 50μmol m-2 s-1. Thirty bulbs developed per explant in a medium containing 11% sucrose and 135 bulbs in the presence of 10μM jasmonic acid.

Subsequently, Nagakubo et al. (1993) developed a micropropagation protocol for six varieties (*Isshuwase*, *Isshu-gokuwase*, *Shanhai*, *Santo*, *Furano* and *Howaito-roppen*) starting from shoot-tips which were cultivated in a medium supplemented with NAA (1μM) and BA (1µM). Regenerated adventitious shoots were subcultured for four generations in presence of NAA (5µM) and BA (10µM) for their multiplication. The application of this protocol enables the production of 256 plants from one shoot-tip in 10 months. A novel regeneration protocol was developed by dissecting and sectioning longitudinally the shoots developed from cloves of the cultivar *Extra Select Sets*. These shoots were cultivated on a medium with BA (8µM) and NAA (0.1µM), and after five weeks they produced eight more shoots compared to the ones that had been kept intact (Mohamed-Yassen et al., 1994).

The roots produced by cloves have proved to be a good explant for plant regeneration. When the root tips are cultivated in a medium with NAA (1μM) and BA (10µM) the shoot formation is achieved for 75% of the explants, without an intermediate callus phase. It is estimated that by using this method up to 380 shoots could be produced starting from a single clove (Haque et al., 1997). Other protocols have been developed, which involve callus formation from this type of explants upon their cultivation in MS or N6 culture media (Chu et al., 1975) supplemented with 2,4-D (4.5µM) alone or combined with kinetin (4.7µM). Transferring the calli to a medium with 4.4µM BA allows the regeneration of 169 plants per gram of callus, which have the ability of forming microbulbs (Robledo-Paz et al., 2000). Khan et al. (2004) also regenerated adventitious shoots from calli developed from root tips of two garlic varieties. The highest callus formation frequency was observed when a combination of 2,4-D (6.8µM) and kinetin (23.8µM) was used. Shoot differentiation rose exponentially with increasing BA concentration, reaching the highest value at 45µM BA, while shoot rooting occurred in the absence of growth regulators. A variation in the number of shoots and their regeneration time was observed depending on the genotype used. Approximately 75% of the regenerated plants established successfully when transferred to greenhouse.

The roots developed from adventitious shoots obtained *in vitro* also allowed garlic micropropagation when cultivated on a medium that induced callus formation and then transferred to a medium with BA (13.3μM) and 4-amino-3,5,6-trichloro-picolinic acid (picloram) (1.4μM). This method enables the regeneration of 5.4 shoots per explant (Myers & Simon, 1998a). A protocol named one-step was developed when the same type of explants was cultivated on a modified B5 medium supplemented with 0.1μM 2,4-D, 11.1µM NAA and 13.6µM BA, under two light conditions (16 hours photoperiod and complete darkness). In general, the root tips cultivated under low light conditions displayed the highest percentage of organogenic calli. The application of this protocol allowed the formation of callus and the regeneration of 250 shoots per gram of callus in the same culture medium and under the same light conditions (Martín-Urdíroz et al., 2004). Zheng et al. (2003) also obtained adventitious shoots by using apical and non-apical root fragments, originating from plants generated *in vitro* of four cultivars grown in Europe (*Messidrome*, *Morado de Cuenca*, *Morasol* and *Printanor*). The explants were cultivated on MS medium with 4.5µM 2,4- D and 0.5µM 2iP in order to induce calli formation, which were then transferred to a medium containing 4.7µM kinetin to promote shoot differentiation. The highest regeneration rate was obtained when non-apical fragments were used, although the difference was not significant.

In a different work, Ayabe & Sumi (1998) used the stem disc (consisting in the apical meristem and the lateral buds of the clove) to regenerate plants of the cultivar *Fukuchihowaito*. When this was cut into various fragments and then cultivated on a medium with BA (0.4µM), 20-25 adventitious shoots were obtained. The same result was observed when

of NAA (5µM) and BA (10µM) for their multiplication. The application of this protocol enables the production of 256 plants from one shoot-tip in 10 months. A novel regeneration protocol was developed by dissecting and sectioning longitudinally the shoots developed from cloves of the cultivar *Extra Select Sets*. These shoots were cultivated on a medium with BA (8µM) and NAA (0.1µM), and after five weeks they produced eight more shoots

The roots produced by cloves have proved to be a good explant for plant regeneration. When the root tips are cultivated in a medium with NAA (1μM) and BA (10µM) the shoot formation is achieved for 75% of the explants, without an intermediate callus phase. It is estimated that by using this method up to 380 shoots could be produced starting from a single clove (Haque et al., 1997). Other protocols have been developed, which involve callus formation from this type of explants upon their cultivation in MS or N6 culture media (Chu et al., 1975) supplemented with 2,4-D (4.5µM) alone or combined with kinetin (4.7µM). Transferring the calli to a medium with 4.4µM BA allows the regeneration of 169 plants per gram of callus, which have the ability of forming microbulbs (Robledo-Paz et al., 2000). Khan et al. (2004) also regenerated adventitious shoots from calli developed from root tips of two garlic varieties. The highest callus formation frequency was observed when a combination of 2,4-D (6.8µM) and kinetin (23.8µM) was used. Shoot differentiation rose exponentially with increasing BA concentration, reaching the highest value at 45µM BA, while shoot rooting occurred in the absence of growth regulators. A variation in the number of shoots and their regeneration time was observed depending on the genotype used. Approximately 75% of the regenerated plants established successfully

The roots developed from adventitious shoots obtained *in vitro* also allowed garlic micropropagation when cultivated on a medium that induced callus formation and then transferred to a medium with BA (13.3μM) and 4-amino-3,5,6-trichloro-picolinic acid (picloram) (1.4μM). This method enables the regeneration of 5.4 shoots per explant (Myers & Simon, 1998a). A protocol named one-step was developed when the same type of explants was cultivated on a modified B5 medium supplemented with 0.1μM 2,4-D, 11.1µM NAA and 13.6µM BA, under two light conditions (16 hours photoperiod and complete darkness). In general, the root tips cultivated under low light conditions displayed the highest percentage of organogenic calli. The application of this protocol allowed the formation of callus and the regeneration of 250 shoots per gram of callus in the same culture medium and under the same light conditions (Martín-Urdíroz et al., 2004). Zheng et al. (2003) also obtained adventitious shoots by using apical and non-apical root fragments, originating from plants generated *in vitro* of four cultivars grown in Europe (*Messidrome*, *Morado de Cuenca*, *Morasol* and *Printanor*). The explants were cultivated on MS medium with 4.5µM 2,4- D and 0.5µM 2iP in order to induce calli formation, which were then transferred to a medium containing 4.7µM kinetin to promote shoot differentiation. The highest regeneration rate was obtained when non-apical fragments were used, although the

In a different work, Ayabe & Sumi (1998) used the stem disc (consisting in the apical meristem and the lateral buds of the clove) to regenerate plants of the cultivar *Fukuchihowaito*. When this was cut into various fragments and then cultivated on a medium with BA (0.4µM), 20-25 adventitious shoots were obtained. The same result was observed when

compared to the ones that had been kept intact (Mohamed-Yassen et al., 1994).

when transferred to greenhouse.

difference was not significant.

protoplasts isolated from shoot primordia were cultured in the presence of NAA (0.5µM) and 2iP (2.4µM), adenine and coconut milk (Ayabe et al., 1995). Barandiaran et al. (1999) used immature bulbs of 23 accessions as a source of axillary buds, which were cultivated during six weeks on B5 medium with 2.5µM 2iP and 0.55µM NAA (establishment phase). Multiplication of regenerated shoots was done on the same culture medium and 20 weeks later shoot clusters were separated in order to cultivate them individually and to induce bulb formation at a low temperature (4°C). Although plants and bulbs were obtained for all accessions under tested conditions, response depended on genotype (accession). Three months later, 60% of bulbs that were transferred to soil survived and produced shoots. This protocol allowed the use of the same culture medium for all phases of micropropagation (establishment, multiplication and bulb formation) and for all accessions, which enabled the handling of all materials tested at the same time, as only three subcultures were required over a period of seven months. Primordial leaf obtained from cloves are also able to produce adventitious shoots when cultivated on a medium with 2,4-D (4.5µM), and develop into plants when transferred onto a medium containing picloram (1.4µM ) and BA (13.3µM) (Myers & Simon, 1999). Haque et al. (2003) developed a protocol for plant regeneration and bulb formation from shoot and root meristems of the cultivar *Bangladesh Local*. Meristems were cultivated on MS medium without growth regulators or containing various concentrations of BA (1-10µM) and NAA (1-5µM). None of the combinations of regulators tested produced a higher response than the one observed in their absence (95.5%). In fact, the presence of these compounds suppressed shoot formation in a directly proportional manner to concentration; 45% of root explants formed adventitious shoots, 60% of which produced bulbs. Although a higher number of buds resulted in shoot formation, the root meristems produced more shoots per explant (20). Bulbs derived from root meristems were smaller than the ones derived from bud meristems.

On the other hand, Luciani et al. (2006) tested different explants for micropropagation of variety *069*, which were cultivated on BDS medium (Dustan & Short, 1977), supplemented with picloram, 2,4-D and BA. The basal plates and meristems resulted in the highest values of shoot regeneration, and 2,4-D proved to be better than picloram for inducing callus and shoot formation. By using a combination of 0.25µM 2,4-D and 4.43µM BA, 100% of explants were able to produce calli, which differentiated into both embryos and shoots. It is worth mentioning that *in vitro* propagation is frequently associated with a process known as hyperhydricity or vitrification, which is a physiological disorder caused by the *in vitro* culture conditions that affects the behavior of regenerated plants. This disorder promotes abnormalities at physiological, anatomical and morphological level, which limit the successful establishment of differentiated plants upon their transfer to greenhouse. Hyperhydric plants have a slow growth rate, thick and deformed stems. Their leaves are translucent, thick and wet (Olmos & Hellin, 1998; Kevers et al., 2004).

A study of biochemical and ultrastructural traits of hyperhydric garlic shoots regenerated *in vitro* was carried out by Wu et al. (2009), who observed that organelles such as mitochondria and chloroplasts were compressed against cell wall, in these shoots. In addition, protein content decreased significantly and O2 and H2O2 generation rate increased 45.3% and 63.9%, respectively. Activity of oxidative stress-related enzymes (lipoxygenase, superoxid dismutase, peroxidase, catalase, ascorbate peroxidase) also increased. Moreover, a rise in the level of electrolytes lixiviation was observed, indicating a damage of membrane lipids. Authors concluded that hyperhydric condition of tissues is closely linked to oxidative stress.

#### **5.1.2 Somatic embryogenesis**

Formation of structures called embryoids was reported for the first time in 1977. They differentiated from calli obtained from stem tips, bulb leaf discs cultivated in the presence of kinetin (20µM) and IAA (10µM) (Abo-El-Nil, 1977). This response was observed again after a long time when basal plates and floral receptacles were cultivated on a medium containing NAA (1µM) and BA (10µM) (Xue et al., 1991; Al-Zahim et al., 1999). Likewise, Ali & Metwally (1992) induced embryo formation from calli generated from root segments; however, regeneration rate was low. In a different work, Barrueto-Cid et al. (1994) established cultures in suspension of the variety *Chonan* starting from calli initiated on MS medium with 5µM 2,4-D, 5µM picloram and 10µM kinetin. Calli were subcultured onto a medium with 4.5µM 2,4-D and hydrolyzed casein before using them for cell suspension cultures. Plant regeneration occurred after transferring cells to a medium containing 77-153µM adenine.

Later, Sata et al. (2001) obtained somatic embryos directly from basal sections of cloves of the cultivar *Malepur* grown on White medium (White, 1963) supplemented with 4.5µM 2,4-D and 2.3µM kinetin. Under these conditions, each explant formed 20 to 25 embryos, which in the presence of higher concentrations of 2,4-D and kinetin turned into masses of hyperhydric tissue. In the same way, Fereol et al. (2002) produced somatic embryos and plants of the variety *Rouge de la Réunion* after cultivating calli obtained from root tips on a modified B5 medium supplemented with 2,4-D (0.4µM) and kinetin (2.3µM). Thirty percent of the somatic embryos developed into plants which acclimated successfully to greenhouse conditions. Later, Fereol et al. (2005b) established a protocol for embryo regeneration through suspension cultures by using young leaf sections from cloves of the variety *Morasol*. Embryogenic calli were obtained when explants were grown on B5 medium with 4.5µM 2,4- D and 0.47µM kinetin, then transferred to a modified B5 medium with 2.2, 1.1, 1.1, 0.4µM 2,4-D, IAA, NAA and kinetin, respectively, plus 175mM sucrose and 2mM proline. Calli were maintained on this medium for five months and were later used to initiate suspension cultures in a modified N6 medium supplemented with 1.3µM 2,4-D, 0.4µM BA and 131mM sucrose. Embryo production was induced on a medium with 2.3µM kinetin and 0.4µM 2,4- D. Embryos developed into plants, which could produce microbulbs *in vitro*. By using the same explants type and the same culture conditions described above, induction of embryogenic suspension cultures of four garlic cultivars (*Rouge de la Réunion*, *Morasol*, *Messidrome* and *Printanor*) was achieved. Ninety percent of calli differentiated into embryos at globular stage after two months of culture. Out of the regenerated embryos, 50% developed into plants that were successfully established in greenhouse. The histological analysis of the culture revealed that regenerated somatic embryos had a unicellular origin (Fereol et al., 2005a).

#### **5.2 Meristem culture**

Meristem culture technique has been widely used for the production of virus-free clones. Virus elimination through meristem culture is based on the fact that these meristematic cells are free or almost free of virus and therefore plants regenerated from them will also be virus-free (Salomon, 2002). For this purpose, it is recommended to isolate explants of maximum 5mm, although sometimes their size may limit their establishment *in vitro*. Meristem culture enabled virus-free plants to be produced in various regions in the world

Formation of structures called embryoids was reported for the first time in 1977. They differentiated from calli obtained from stem tips, bulb leaf discs cultivated in the presence of kinetin (20µM) and IAA (10µM) (Abo-El-Nil, 1977). This response was observed again after a long time when basal plates and floral receptacles were cultivated on a medium containing NAA (1µM) and BA (10µM) (Xue et al., 1991; Al-Zahim et al., 1999). Likewise, Ali & Metwally (1992) induced embryo formation from calli generated from root segments; however, regeneration rate was low. In a different work, Barrueto-Cid et al. (1994) established cultures in suspension of the variety *Chonan* starting from calli initiated on MS medium with 5µM 2,4-D, 5µM picloram and 10µM kinetin. Calli were subcultured onto a medium with 4.5µM 2,4-D and hydrolyzed casein before using them for cell suspension cultures. Plant regeneration occurred after transferring cells to a medium

Later, Sata et al. (2001) obtained somatic embryos directly from basal sections of cloves of the cultivar *Malepur* grown on White medium (White, 1963) supplemented with 4.5µM 2,4-D and 2.3µM kinetin. Under these conditions, each explant formed 20 to 25 embryos, which in the presence of higher concentrations of 2,4-D and kinetin turned into masses of hyperhydric tissue. In the same way, Fereol et al. (2002) produced somatic embryos and plants of the variety *Rouge de la Réunion* after cultivating calli obtained from root tips on a modified B5 medium supplemented with 2,4-D (0.4µM) and kinetin (2.3µM). Thirty percent of the somatic embryos developed into plants which acclimated successfully to greenhouse conditions. Later, Fereol et al. (2005b) established a protocol for embryo regeneration through suspension cultures by using young leaf sections from cloves of the variety *Morasol*. Embryogenic calli were obtained when explants were grown on B5 medium with 4.5µM 2,4- D and 0.47µM kinetin, then transferred to a modified B5 medium with 2.2, 1.1, 1.1, 0.4µM 2,4-D, IAA, NAA and kinetin, respectively, plus 175mM sucrose and 2mM proline. Calli were maintained on this medium for five months and were later used to initiate suspension cultures in a modified N6 medium supplemented with 1.3µM 2,4-D, 0.4µM BA and 131mM sucrose. Embryo production was induced on a medium with 2.3µM kinetin and 0.4µM 2,4- D. Embryos developed into plants, which could produce microbulbs *in vitro*. By using the same explants type and the same culture conditions described above, induction of embryogenic suspension cultures of four garlic cultivars (*Rouge de la Réunion*, *Morasol*, *Messidrome* and *Printanor*) was achieved. Ninety percent of calli differentiated into embryos at globular stage after two months of culture. Out of the regenerated embryos, 50% developed into plants that were successfully established in greenhouse. The histological analysis of the culture revealed that regenerated somatic embryos had a unicellular origin

Meristem culture technique has been widely used for the production of virus-free clones. Virus elimination through meristem culture is based on the fact that these meristematic cells are free or almost free of virus and therefore plants regenerated from them will also be virus-free (Salomon, 2002). For this purpose, it is recommended to isolate explants of maximum 5mm, although sometimes their size may limit their establishment *in vitro*. Meristem culture enabled virus-free plants to be produced in various regions in the world

**5.1.2 Somatic embryogenesis** 

containing 77-153µM adenine.

(Fereol et al., 2005a).

**5.2 Meristem culture** 

(Walkey, 1987; Bhojwani et al., 1982; Peña-Iglesias & Ayuso, 1982; Bertacinni et al., 1986; Conci & Nome, 1991). In Slovenia, Eastern Europe, a trial was conducted to eliminate the OYDV in plants of the cultivar *Ptujksi-spomladanski* through thermotherapy and meristem culture. Meristems of 0.3-0.6mm were first cultivated on B5 medium with 1µM IAA and 1µM BA, then transferred to a multiplication medium containing 5µM jasmonic acid and 5µM 2iP. Meristems obtained from plants that had undergone thermotherapy regenerated a lower number of shoots (1.0-2.2) than the non-treated plants (9.3); 90 to 100% plants were found to be free of the OYDV (Ucman et al., 1998).

Sidaros et al. (2004) attempted to produce plants of three garlic cultivars (*Chinese*, *Italian* and *Balady*) through meristem culture and chemotherapy. Chemotherapy was carried out by using virazole [or ribavirin (1-β-D-ribofuranosil-1,2,4-triazole-3-carboxamide)] in culture medium. The highest percentage (100%) of virus-free plants was obtained when meristems of 3mm were cultivated on MS medium containing 50mg L-1 virazole. In a different study, thermotherapy, chemotherapy and meristem culture were combined in order to obtain plants of the varieties *Taiwan* and *Chileno* free of potyvirus. Thermotherapy consisted in maintaining regenerated plants from embryos dissected from cloves that showed negative results on an ELISA (Enzyme Linked Immuno Absorbent Assay) for potyvirus during one week at 32°C, followed by two weeks at 36°C, and three weeks at 38°C. Embryos were removed from cloves of these plants and cultivated in presence of 205µM ribavirin. Meristems (0.1-0.5mm) of regenerated plants that showed negative results by ELISA were used to generate new plants. Thermotherapy had a more negative effect on plant survival than meristem culture and chemotherapy. However, thermotherapy proved to be more efficient for virus elimination (60.0 to 70.9%) than meristem culture (64.0%), while chemotherapy was not efficient for potyvirus elimination. On the other hand, 10.7% of plants of the cultivar Taiwan grown in field became reinfected, while the Chileno cultivar showed an 8.9% of reinfection after three consecutive cycles of the crop (Ramírez-Malagón et al., 2006).

The use of stems and scape tips of the variety *Red Six Cloves* allows formation of adventitious shoots when cultivated on a medium with NAA (2.6µM) and kinetin (2.3µM). These shoots developed into plants free of the garlic mosaic virus (GMV) 65 days after starting the culture (Ma et al., 1994). Alternative protocols have been developed for generating virus-free plants starting from inflorescence meristems, bulbils or roots, as apart from being virus free they are available in higher numbers than the apical meristems (Appiano & D'Agostino, 1983). In this way, Verbeek et al. (1995) cultivated meristems obtained from cloves and bulbils (0.15-1.00mm), 71-71% of which regenerated plants; 38% of explants obtained from cloves and 25% of the explants obtained from bulbils were found to be virus-free. In addition, it was observed that meristems smaller than 0.4mm failed to produce shoots. Similarly, Ebi et al. (2000) established a system for elimination of mite-borne mosaic virus using meristems (0.2-0.4mm) obtained from bulbils. These meristems produced plants after being cultivated on MS medium with 5.4µM NAA. The immunoblot assay indicated that several of regenerated plants were virus-free. Senula et al. (2000) obtained plants of 87 accessions free of the viruses OYDV, LYSV, GCLV, SLV and MbFV by cultivating meristems of 0.3-0.8mm originated from bulbils. OYDV and LYSV were eliminated in 85-95% of the regenerated plants. Addition of ribavirin to culture medium reduced regeneration potential, but increased virus elimination. Later, Xu et al. (2001) regenerated virus-free plants from meristems obtained from inflorescences of nine lines. Explants were cultivated on B5 medium containing 0.22µM BA and 0.3mM adenine. By using this protocol it was possible to obtain 50-90% plants free of the OYDV, 70-100% plants free of the LYSV and 60-80% free of the SLV. Production of bulbs from virus-free plants was higher than from infected plants. Four to five years would be necessary to obtain virus-free elite seeds that can be established in the field. The economical analysis indicated a net profit of 50.3 to 244.5% (depending on the genotype) for garlic seed producers.

#### **5.3 Somaclonal variants**

Tissue culture tools such as *in vitro* selection, embryo rescue, somatic hybridization, genetic transformation and somaclonal variation can be used to generate crop variation. Larkin and Scrowcroft (1981) defined somaclonal variation as the phenotypic variation seen in plants regenerated *in vitro* with respect to the original plant. At genetic level, somaclonal variation can be brought about by various DNA changes that include: (a) chromosomal rearrangements, (b) aneuploidy, (c) poliploidy, (d) modification of gene expression by methylation, amplification, inactivation or reactivation, (e) genetic conversion, (f) somatic recombination, (g) transposons movement, (h) genes mutations, etc. (Scowcroft, 1984; Peschke & Phillips, 1991).

Various experiments have been undertaken in garlic in order to generate somaclonal variants that could be used in its improvement. For instance, Novak (1983) treated meristems 0.5-0.7mm with a solution of colchicine (3g L-1) to induce polyploidy. Meristems were treated in two different ways: (1) cultured for 7 days on a solid medium with colchicine, and (2) cultured for 2 days in a liquid medium with colchicine. The latter treatment proved to be more effective. By using this experimental strategy, 35% of regenerated plants were found to be tetraploid, and 14% chimaeras with diploid and tetraploid cells. Dolezel et al. (1986) pointed out that in garlic probability of generating somaclonal variation is higher when disorganized growth occurs, specially for longer periods of time. Similarly, plants generated from old explants or with a high level of differentiation have an increased possibility of suffering this type of variation. The cultivar *Frankon* is resistant to populations of the nematode *Ditylenchus dispsaci* found in Israel, but produces bulbs and cloves of small size and has low yield. A protocol for *in vitro*  regeneration was developed to generate somaclonal variants that can produce bulbs with commercial traits. This protocol consisted in cultivating basal plates on BDS medium supplemented with 2.2µM 2,4-D and 2.3µM kinetin to induce callus formation, which were then transferred to a medium containing 9.5µM kinetin and 11.7µM IAA to develop adventitious shoots. Once shoots produced roots, they were transplanted to soil in greenhouse. Assessment of plant characteristics and bulb development revealed that there was variation in bulb size and color, and also in number and size of cloves per bulb compared to the original plant type, which indicates that this could represent a promising material for generation of improved somaclonal variants (Koch & Salomon, 1994).

In a different study, Madhavi et al. (1991) compared ability of calli (organized and disorganized) and bulbs to produce sulphur compounds like alliin. Apart from finding differences in sulphur level compounds, they also observed changes related to proteins, aminoacids, carbohydrates and enzymes. For instance, specific activity of the enzyme alliin lyase in callus was 50% lower than in bulb, while enzymes amino transferase, malate dehydrogenase, polyphenol oxidase, peroxidase and alkaline phosphatase displayed a higher activity than in bulb. Incorporation of precursors to volatile fraction was also higher

using this protocol it was possible to obtain 50-90% plants free of the OYDV, 70-100% plants free of the LYSV and 60-80% free of the SLV. Production of bulbs from virus-free plants was higher than from infected plants. Four to five years would be necessary to obtain virus-free elite seeds that can be established in the field. The economical analysis indicated a net profit

Tissue culture tools such as *in vitro* selection, embryo rescue, somatic hybridization, genetic transformation and somaclonal variation can be used to generate crop variation. Larkin and Scrowcroft (1981) defined somaclonal variation as the phenotypic variation seen in plants regenerated *in vitro* with respect to the original plant. At genetic level, somaclonal variation can be brought about by various DNA changes that include: (a) chromosomal rearrangements, (b) aneuploidy, (c) poliploidy, (d) modification of gene expression by methylation, amplification, inactivation or reactivation, (e) genetic conversion, (f) somatic recombination, (g) transposons movement, (h) genes mutations, etc. (Scowcroft, 1984;

Various experiments have been undertaken in garlic in order to generate somaclonal variants that could be used in its improvement. For instance, Novak (1983) treated meristems 0.5-0.7mm with a solution of colchicine (3g L-1) to induce polyploidy. Meristems were treated in two different ways: (1) cultured for 7 days on a solid medium with colchicine, and (2) cultured for 2 days in a liquid medium with colchicine. The latter treatment proved to be more effective. By using this experimental strategy, 35% of regenerated plants were found to be tetraploid, and 14% chimaeras with diploid and tetraploid cells. Dolezel et al. (1986) pointed out that in garlic probability of generating somaclonal variation is higher when disorganized growth occurs, specially for longer periods of time. Similarly, plants generated from old explants or with a high level of differentiation have an increased possibility of suffering this type of variation. The cultivar *Frankon* is resistant to populations of the nematode *Ditylenchus dispsaci* found in Israel, but produces bulbs and cloves of small size and has low yield. A protocol for *in vitro*  regeneration was developed to generate somaclonal variants that can produce bulbs with commercial traits. This protocol consisted in cultivating basal plates on BDS medium supplemented with 2.2µM 2,4-D and 2.3µM kinetin to induce callus formation, which were then transferred to a medium containing 9.5µM kinetin and 11.7µM IAA to develop adventitious shoots. Once shoots produced roots, they were transplanted to soil in greenhouse. Assessment of plant characteristics and bulb development revealed that there was variation in bulb size and color, and also in number and size of cloves per bulb compared to the original plant type, which indicates that this could represent a promising

material for generation of improved somaclonal variants (Koch & Salomon, 1994).

In a different study, Madhavi et al. (1991) compared ability of calli (organized and disorganized) and bulbs to produce sulphur compounds like alliin. Apart from finding differences in sulphur level compounds, they also observed changes related to proteins, aminoacids, carbohydrates and enzymes. For instance, specific activity of the enzyme alliin lyase in callus was 50% lower than in bulb, while enzymes amino transferase, malate dehydrogenase, polyphenol oxidase, peroxidase and alkaline phosphatase displayed a higher activity than in bulb. Incorporation of precursors to volatile fraction was also higher

of 50.3 to 244.5% (depending on the genotype) for garlic seed producers.

**5.3 Somaclonal variants** 

Peschke & Phillips, 1991).

in organized calli. The somaclone *118.15*, derived from variety *Rosado,* which is grown commercially in Argentina, possesses agronomically desirable traits. A cytological and phenotypical study revealed that both somaclone and original type had the same chromosome number (2n = 16). In addition, some individuals of this clone contained polyploid, aneuploid and haploid cells, probably derived from processes such as endomitosis, nuclear fusion or homologue chromosome pairing in somatic cells. Binucleate cells and differences in length of chromosome pairs were also observed. Plants of clone *118.15* were taller, had a higher diameter of pseudostem and produced bulbs with less, but bigger cloves than the original type (Ordoñez et al., 2002).

El-Aref (2002) regenerated plants of cultivar *Balady* from leaves and roots cultivated first on BDS medium with 2,4-D (4.5µM) and kinetin (9.5µM) for callus initiation, then in presence of 9µM BA and 5.5µM NAA for plant regeneration. These plants formed bulbs which were planted in soil to generate new plants. Isoenzyme analysis showed that 9 out of 29 regenerated plants were different from original plant with regard to some of the enzymes analyzed (phosphatase acid, alcohol dehydrogenase, malate dehydrogenase, esterase). Parental bands were found in all plants, and 5 new bands were observed in 31% of them. Esterase and acid phosphatase displayed a higher polymorphism than alcohol dehydrogenase and malate dehydrogenase. Later, Mukhopadhyay et al. (2005) studied chromosome stability in plants of cultivar *Rossete* generated from callus. It was observed that plants generated from calli iniciated on solid MS medium with 2,4-D (9µM) and kinetin (0.93µM) and sub-cultured in liquid medium with NAA and kinetin exhibited chromosome stability, while the ones grown solely on the initiation solid medium contained hypo- or hyperdiploid cells along with the diploid ones. Frequency of aneuploid cells (2.2-48.9%) increased with callus age.

Recently, Badria & Ali (1999) identified somaclonal variants regenerated from calli obtained from root meristems cultivated on MS medium with kinetin, 2,4-D and IAA. The somaclonal variants formed bulbs without division in the first generation, and displayed normal phenotype in the following generation. After four cycles in field, somaclonal variants that exhibited significant differences of bulb characteristics were found. Cytogenetical analysis revealed that these somaclonal variants had the same chromosome number as original plants. Quantification of alliicin production showed that some somaclones contained three times more of this compound (14.5mg g-1) than control plants (3.8mg g-1). Authors suggest that this technique could be useful for improving alliicin content in garlic.

#### **5.4 Germplasm conservation**

Conservation of valuable garlic accessions involves their yearly cultivation, as bulbs cannot be stored for long periods of time (6 months at -3°C). This practice is expensive as it requires land use and manpower. Moreover, germplasm grown in field is exposed to environmental changes, pests and diseases that reduces its quality (Panis & Lambardi, 2006). The majority of plant germplasm is stored in seed repositories at temperatures between -15 and -20°C. However, for species whose seeds are recalcitrant (they cannot be dried to humidity levels low enough for storage) or for species that do not produce seeds, like garlic, slow growth storage and cryopreservation are the only tools for conserving them. Slow growth storage involves a condition that maintains tissue growth at a minimum and it allows the medium term storage of material (Botau et al., 2005). It is based on using organs cultured *in vitro* at 4°C and 10 to 15°C for plants growing in temperate and tropical areas, respectively (Keller et al., 2006).

Cryopreservation is one of the most commonly used tools for germplasm conservation because it requires minimum amount of space and maintenance. In addition, it reduces loss of accessions by contamination, human errors and somaclonal variation which may occur during slow growth storage (Panis & Lambardi, 2006; Sakai & Engelman, 2007). This technique involves the use of liquid nitrogen (which has a freezing temperature of -196°C) for long-term storage of plant material. At this temperature, the majority of biochemical and physical processes are effectively stopped. Cryopreservation is only useful if formation of intracellular ice crystals does not take place, as they may cause irreversible damage to the cell membrane (Panis & Lambardi, 2006). Ice formation without an extreme reduction of the cell water content can only be avoided by a process known as vitrification, in which an aqueous solution turns into an amorphus and glassy state (Sakai, 2000). This procedure substitutes cellular dehydration that occurs during freezing by a reduction of cell water content that is achieved by treating tissues prior to the cooling process with highly concentrated solutions (PVS2, PVS3) containing glycerol, ethylen glycol, dimethylsulfoxid and sucrose, or by air drying (Sakai & Engelman, 2007).

Some countries are already making use of the previously mentioned techniques to conserve their valuable germplasm. For example, China possesses *in vitro* virus-free germplasm banks and their respective databases. These banks have been established taking into account factors such as genotype, culture medium components, light conditions, temperature of incubation rooms, etc. Moreover, studies have been carried out in order to optimize conservation conditions. In this respect, Xu et al. (2005) studied the behavior of six genotypes during their *in vitro* storage. They found out differences in conservation period, depending on genotype. Two genotypes, namely *Cangshan Zaotai* and *Tianjin Baodi* could be stored for 25 months and had a survival percentage of 100%. They also observed that shoots grown at low temperatures on B5 medium with 1.3-2.2µM BA, 0.5-1.6µM NAA and 38-115µM of abscisic acid could be conserved for a longer time. Evaluation of stored material indicated that it was genetically stable and 0.1-0.2% of it became infected with virus. Similarly, the Institute of Plant Genetics and Crop Plant Research at Gatersleben, Germany, one of the biggest gene banks in Europe, possesses a collection of 3039 accessions of species of the genus *Allium*, including the European garlic core collection. Before storage of germplasm, virus elimination is undertaken through meristem culture, then either slow growth storage is carried out for 12 months at 2 and 19°C or cryopreservation-vitrification using a mix of glycerol and sucrose 1:1 as cryoprotectant inside the aluminum foil (Keller et al., 2006).

In the United States, investigations have been carried out for cryopreservation of garlic accessions at the Western Regional Plant Introduction Station, Pullman, Washington. In this respect, Ellis et al. (2005) tested two vitrification solutions to cryopreserve 12 accessions. Shoot tips excised from cloves were treated with the vitrification solutions 2 (PVS2; 15% DMSO, 15% ethylene glycol, 30% glycerol, 0.4M sucrose) and 3 (PVS3; 50% sucrose, 50% glycerol). Eleven out of the 12 accessions could be successfully cryopreserved by using vitrification solutions 2 and 3 as cryoprotectants. Cryopreservation resulted in better regrowth of 7 and 3 accessions when PVS2 and PVS3, respectively, was used. Only one

based on using organs cultured *in vitro* at 4°C and 10 to 15°C for plants growing in

Cryopreservation is one of the most commonly used tools for germplasm conservation because it requires minimum amount of space and maintenance. In addition, it reduces loss of accessions by contamination, human errors and somaclonal variation which may occur during slow growth storage (Panis & Lambardi, 2006; Sakai & Engelman, 2007). This technique involves the use of liquid nitrogen (which has a freezing temperature of -196°C) for long-term storage of plant material. At this temperature, the majority of biochemical and physical processes are effectively stopped. Cryopreservation is only useful if formation of intracellular ice crystals does not take place, as they may cause irreversible damage to the cell membrane (Panis & Lambardi, 2006). Ice formation without an extreme reduction of the cell water content can only be avoided by a process known as vitrification, in which an aqueous solution turns into an amorphus and glassy state (Sakai, 2000). This procedure substitutes cellular dehydration that occurs during freezing by a reduction of cell water content that is achieved by treating tissues prior to the cooling process with highly concentrated solutions (PVS2, PVS3) containing glycerol, ethylen glycol, dimethylsulfoxid

Some countries are already making use of the previously mentioned techniques to conserve their valuable germplasm. For example, China possesses *in vitro* virus-free germplasm banks and their respective databases. These banks have been established taking into account factors such as genotype, culture medium components, light conditions, temperature of incubation rooms, etc. Moreover, studies have been carried out in order to optimize conservation conditions. In this respect, Xu et al. (2005) studied the behavior of six genotypes during their *in vitro* storage. They found out differences in conservation period, depending on genotype. Two genotypes, namely *Cangshan Zaotai* and *Tianjin Baodi* could be stored for 25 months and had a survival percentage of 100%. They also observed that shoots grown at low temperatures on B5 medium with 1.3-2.2µM BA, 0.5-1.6µM NAA and 38-115µM of abscisic acid could be conserved for a longer time. Evaluation of stored material indicated that it was genetically stable and 0.1-0.2% of it became infected with virus. Similarly, the Institute of Plant Genetics and Crop Plant Research at Gatersleben, Germany, one of the biggest gene banks in Europe, possesses a collection of 3039 accessions of species of the genus *Allium*, including the European garlic core collection. Before storage of germplasm, virus elimination is undertaken through meristem culture, then either slow growth storage is carried out for 12 months at 2 and 19°C or cryopreservation-vitrification using a mix of glycerol and sucrose 1:1 as

In the United States, investigations have been carried out for cryopreservation of garlic accessions at the Western Regional Plant Introduction Station, Pullman, Washington. In this respect, Ellis et al. (2005) tested two vitrification solutions to cryopreserve 12 accessions. Shoot tips excised from cloves were treated with the vitrification solutions 2 (PVS2; 15% DMSO, 15% ethylene glycol, 30% glycerol, 0.4M sucrose) and 3 (PVS3; 50% sucrose, 50% glycerol). Eleven out of the 12 accessions could be successfully cryopreserved by using vitrification solutions 2 and 3 as cryoprotectants. Cryopreservation resulted in better regrowth of 7 and 3 accessions when PVS2 and PVS3, respectively, was used. Only one

temperate and tropical areas, respectively (Keller et al., 2006).

and sucrose, or by air drying (Sakai & Engelman, 2007).

cryoprotectant inside the aluminum foil (Keller et al., 2006).

genotype displayed good recovery in both solutions, which indicated that response depended on genotype.

In Romania, investigations were carried out to establish conditions for slow growth of Romanian garlic landraces. Shoots regenerated from leaf discs were cultured under a slow growth condition that consisted in a medium lacking sucrose at 16°C and under normal conditions on a medium with 3% of sucrose at 24°C. After four weeks, shoots cultivated under slow growth conditions reduced their growth rate by 42%. Shoots grown on sucrosefree medium displayed shorter internodes, but higher foliar surface, and shorter roots. Some landraces proved to be more sensitive to lack of sucrose than to low temperature. Differences in response to growth conditions tested were also observed among the different genotypes under study. The developed protocol allowed medium-term preservation of landraces under study (Botau et al., 2005).

Other investigation groups have focused their efforts on defining the best conditions for slow growth storage or cryopreservation. For example, Makowska et al. (1999) studied the response of apexes from different sources (cloves and bulbils) to cryopreservation after being treated with vitrification solutions. They found out that after freezing, apexes treated with PVS2 solution (30% glycerol, 15% ethylene glycol, 15% DMSO, 0.4M sucrose) restored their growth in a higher proportion compared to the ones that had been submerged in solution PVS3 (40% glycerol and 40% sucrose). A higher number of apexes excised from big bulbils restored their growth, while the ones dissected from small bulbils failed to regrow. On the other hand, apexes obtained from cloves had higher survival rate than the ones obtained from bulbils.

Later, Sudarmonowati (2001) tested different vitrification solutions in an attempt to define the most suitable method for cryopreservation of embryogenic calli of cultivar *Lumbu Hijau*. Calli with embryos at globular stage were precultured on MS medium with 9µM BA and 0.4M sucrose for 1 to 7 days. Next, they were submerged in MS medium with 2M glycerol and 0.4M sucrose (loading solution) in order to be later exposed to different vitrification solutions for various periods of time (5 to 60 min.). Calli were placed in cryotubes with one drop of vitrification solution, then plunged into liquid nitrogen for 30 minutes. After freezing, calli were first plated on MS medium with 5µM 2iP, 2.3µM kinetin and 0.4M sucrose, and later on the same medium containing a lower amount of sucrose. Of the three vitrification solutions tested, the one that contained a mix of glycerol (22%), ethylene glycol (17%), propylene glycol (17%) and DMSO (7%) proved to be better for calli conservation. The highest calli survival percentage was 30%, which indicated that it was necessary to improve the tested methodology.

Recently, Hassan et al. (2007) established slow growth cultures of two garlic varieties grown in Egypt (*Balady* and *Seds 10*). They cultivated bulblets on MS medium with 0.35M sucrose, 5g L-1 charcoal and 0.04µM BA, then on a medium with different concentrations of sorbitol and sucrose (0.1, 0.2 and 0.4M). Cultures were incubated in darkness at 4°C for their conservation. Bulblets did not develop neither shoots nor roots under these conditions during the first three months. Addition of 0.1M sorbitol to culture medium delayed growth of shoots and roots of cultivar *Balady* to 6, 12 and 18 months, while sucrose (0.1 or 0.2M) had the same effect on bulblets of cultivar *Seds* 40. The survival rate was of 100% after 18 months of maintaining cultures under these conditions.

#### **5.5 Genetic transformation**

Although several methods are available to introduce DNA into plant cells, most of them have been developed using the bacterium *Agrobacterium tumefaciens* (direct method) or biolistic (indirect method) as a vehicle. *A. tumefaciens* is a soil bacterium having natural ability to transfer part of its DNA to plant cells of various species causing formation of crown gall tumors (Hooykaas & Schilperoot, 1992). This ability is conferred by the Ti plasmid (tumorinducing), which contains a region called T-DNA, that is transferred to the host cell with helping of virulence genes, also present in Ti plasmid. The T-DNA contains genes that are involved in production of cytokinins (2-isopentyl-AMP) and auxins (IAA), which are responsible for tumor formation (Leemans et al., 1982; Barry et al., 1984). Genetic manipulation of this plasmid has resulted in the replacement of genes contained in the T-DNA of the wild strain by genes that confer desirable traits to transformed plants (Christou, 1996).

A series of physical, electrical and chemical methods (e.g. electroporation and biolistic) have been generated to introduce DNA directly into plant cells (Songstad et al., 1995). Starting from the successful transformation of monocotyledonous plants, such as maize, and soybean by using the biolistic method (McCabe et al., 1988; Fromm et al., 1990), this has become one of the most used systems for gene transfer. This method consists in bombarding target cells with DNA-coated gold or tungsten microparticles accelerated to very high speeds by a gene gun, which allows them to cross the cell walls. Although there are various types of gene guns, the PDS1000 helium designed by Dupont has been the most widely used, specially for transformation of monocotyledonous plants (Vain et al., 1993; Christou, 1995). A great variety of genetic transformation protocols have been developed, but this technology has not been applied with the same efficiency in every species. The species of the genus *Allium* represent an example in this respect, particularly garlic, for which only a small number of publications are available.

#### **5.5.1 Via biolistic**

It was not until 1998 that a protocol for garlic transformation was reported for the first time. Barandiaran et al*.* (1998) bombarded leaf tissue, immature bulbs, cloves and callus of the cultivar *Morado de Cuenca* with four constructs (pDE4, pCW101, pActl-D and pAHC25). Out of these vectors, the one carrying the reporter *uidA* gene (*gusA*) (coding for β-glucuronidase) under control of the promoter 35S from cauliflower mosaic virus (CaMV35S) and the terminator of the nopaline synthase gene (NOS), allowed expression of the *uidA* gene in 43.3% of leaf explants, 76.7% of bulbs, 23.3% of clove tissue and 13% of calli. Transitory expression of the *uidA* gene could only be detected after treating tissues with a nuclease inhibitor (aurintricarboxylic acid). However, regeneration of transgenic plants could not be achieved by using this protocol. Similarly, Myers & Simon (1998) bombarded cell suspensions of *RAL27* clone with *gusA* and *nptII* (conferring resistance to kanamycin) genes which were under control of CaMV35S and NOS promoters, respectively. After 14-16 weeks on selection medium, shoots were regenerated on calli. Incorporation of *gus* and *nptII* genes into garlic transgenic plants was confirmed by PCR assays.

Later, Ferrer et al. (2000) used biolistics to introduce the reporting gene *uidA* and the selection gene *bar*, which codes for N-acetyl-transpherase, into leaf tissue, basal plate discs and embryogenic calli of cultivar *Moraluz*. The *uidA* and *bar* genes were under control of CaMV35S and maize ubiquitin (Ubi) promoters, respectively. Maximum expression of *uidA* 

Although several methods are available to introduce DNA into plant cells, most of them have been developed using the bacterium *Agrobacterium tumefaciens* (direct method) or biolistic (indirect method) as a vehicle. *A. tumefaciens* is a soil bacterium having natural ability to transfer part of its DNA to plant cells of various species causing formation of crown gall tumors (Hooykaas & Schilperoot, 1992). This ability is conferred by the Ti plasmid (tumorinducing), which contains a region called T-DNA, that is transferred to the host cell with helping of virulence genes, also present in Ti plasmid. The T-DNA contains genes that are involved in production of cytokinins (2-isopentyl-AMP) and auxins (IAA), which are responsible for tumor formation (Leemans et al., 1982; Barry et al., 1984). Genetic manipulation of this plasmid has resulted in the replacement of genes contained in the T-DNA of the wild

A series of physical, electrical and chemical methods (e.g. electroporation and biolistic) have been generated to introduce DNA directly into plant cells (Songstad et al., 1995). Starting from the successful transformation of monocotyledonous plants, such as maize, and soybean by using the biolistic method (McCabe et al., 1988; Fromm et al., 1990), this has become one of the most used systems for gene transfer. This method consists in bombarding target cells with DNA-coated gold or tungsten microparticles accelerated to very high speeds by a gene gun, which allows them to cross the cell walls. Although there are various types of gene guns, the PDS1000 helium designed by Dupont has been the most widely used, specially for transformation of monocotyledonous plants (Vain et al., 1993; Christou, 1995). A great variety of genetic transformation protocols have been developed, but this technology has not been applied with the same efficiency in every species. The species of the genus *Allium* represent an example in this respect, particularly garlic, for which only a small

It was not until 1998 that a protocol for garlic transformation was reported for the first time. Barandiaran et al*.* (1998) bombarded leaf tissue, immature bulbs, cloves and callus of the cultivar *Morado de Cuenca* with four constructs (pDE4, pCW101, pActl-D and pAHC25). Out of these vectors, the one carrying the reporter *uidA* gene (*gusA*) (coding for β-glucuronidase) under control of the promoter 35S from cauliflower mosaic virus (CaMV35S) and the terminator of the nopaline synthase gene (NOS), allowed expression of the *uidA* gene in 43.3% of leaf explants, 76.7% of bulbs, 23.3% of clove tissue and 13% of calli. Transitory expression of the *uidA* gene could only be detected after treating tissues with a nuclease inhibitor (aurintricarboxylic acid). However, regeneration of transgenic plants could not be achieved by using this protocol. Similarly, Myers & Simon (1998) bombarded cell suspensions of *RAL27* clone with *gusA* and *nptII* (conferring resistance to kanamycin) genes which were under control of CaMV35S and NOS promoters, respectively. After 14-16 weeks on selection medium, shoots were regenerated on calli. Incorporation of *gus* and *nptII* genes

Later, Ferrer et al. (2000) used biolistics to introduce the reporting gene *uidA* and the selection gene *bar*, which codes for N-acetyl-transpherase, into leaf tissue, basal plate discs and embryogenic calli of cultivar *Moraluz*. The *uidA* and *bar* genes were under control of CaMV35S and maize ubiquitin (Ubi) promoters, respectively. Maximum expression of *uidA* 

strain by genes that confer desirable traits to transformed plants (Christou, 1996).

**5.5 Genetic transformation** 

number of publications are available.

into garlic transgenic plants was confirmed by PCR assays.

**5.5.1 Via biolistic** 

was observed in calli and leaves. In a different investigation, Sawehel (2002) developed a transformation system using calli derived from immature cloves of cultivar *Giza 3*. Calli were bombarded with the plasmid pBI22.23 containing the *hpt* gene (coding for the enzyme hygromycin phosphotranspherase that confers resistance to antibiotic hygromycin), and the reporting gene *gusA.* Calli had been previously treated with aurintricarboxilic acid to inhibit activity of endogenous nucleases. Southern blot assays and histochemical analysis proved that this system allowed the transfer, expression and stable integration of transgenes into the garlic genomic DNA.

At the same time, Park et al. (2002) obtained transgenic plants resistant to herbicide chlorsulfuron after bombarding calli of cultivar *Danyang* with the plasmid pC1301-ALS, which contains *gus, hpt* and *als* (coding for acetolactate synthase) genes, under control of the promoter CaMV35S. Out of 1900 calli, 12 grew and regenerated plants resistant to chlorsulphuron (3mg L-1), which formed bulbs and reached maturity. PCR, Southern blot and Northern blot assays confirmed the expression and integration of transgenes into the genome.

In a different work, Robledo-Paz et al. (2004) established a transformation protocol using embryogenic calli derived from root tips of cultivar *GT96-1*. Calli were bombarded with the plasmid pWRG1515 containing *hpt* and *gusA* genes*,* both under the control of the promoter CaMV35S, and the 3' region of the *nos* gene*.* Putative transgenic calli were identified after four months of culturing them on a medium containing hygromycin (20mg L-1), and later developed into plants. Molecular (Southern blot) and histochemical (GUS) analysis confirmed transgenic nature of regenerated plants. Transformation efficiency was of 2.2 clones per fresh weight gram of bombarded callus.

#### **5.5.2 Via** *Agrobacterium tumefaciens*

Kondo et al. (2000) were the first to achieve the establishment of a transformation protocol in garlic using *A. tumefaciens* as a vehicle. They infected morphogenetic calli with the strain EHA101 carrying the plasmid pIG121, which in turn contained *nptII, hph* and *uidA* genes under control of the promoter CaMV35S. By using this protocol it was possible to regenerate 15 transgenic plants from 1000 inoculated calli grown on a selective culture medium for five months. Zheng et al. (2004) presented a transformation system that apart from producing plants resistant to antibiotics or herbicides, also enabled introduction of genes for resistance to insects. Inoculation of calli of three European cultivars was undertaken using the strain AGLO carrying four different plasmids containing *gusA* and *hpt* genes*,* and also *cry1Ca* and *HO4* genes from *Bacillus thuringiensis,* which confers resistance to the insect *Spodoptera exigua*. The highest transformation frequency (1.47%) was achieved with the cultivar *Printanor* and the plasmid pPB34. Of regenerated plants, only the ones that integrated the *cry1Ca* gene had a good growth under greenhouse conditions and had the ability to form bulbs. These plants were totally resistant to *Spodoptera exigua* in bioassays carried out *in vitro.* Later, Eady et al. (2005) inoculated immature embryos with the strain LBA4404 carrying the vector pBIN *m-gfp-ER* containing the gen *gfp* (encoding for the green flourescent protein) and the gene *nptII.* Out of the 3200 infected embryos, only two transgenic plants (0.06%) were regenerated. Khar et al. (2005) studied the transitory expression of the reporter gene *gusA* in two garlic cultivars after infecting them with a *A. tumefaciens* strain carrying two plasmids. Plasmid pCAMBIA 1301 induced a higher transformation frequency (7.4%) than plasmid pTOK233 (4.1%). Genes conferring resistance to fungi are still not being commercially used for fighting diseases caused by these phytopathogens. In garlic, Robledo-Paz (2010, personal communication) incorporated chitinase and glucanase genes in an attempt to confer resistance to the fungus *Sclerotium cepivorum.* These experiments revealed that regenerated transgenic plants were not totally resistant to the fungus, but displayed a delay in the infection speed.

#### **5.6 Molecular markers**

In order to make a more efficient use of garlic germplasm cultivated in various regions of the world, it is necessary to evaluate and characterize the available genetic diversity (Ordás et al., 1994). As the descriptions based on anatomical and morphological characteristics are incomplete and they can be affected by environmental factors, other methods are required to perform this characterization (García-Lampasona et al., 2003). Polymorphism of molecules such as isozymes and DNA can be used to characterize plant germplasm, specially in cases where morphological and biochemical differences are not conspicuous. Although isozyme analysis represented the first application of molecular markers in the genus *Allium,* its main drawback is the low number of enzymatic systems available in garlic. In addition, these markers may suffer changes induced by the developmental stage of plant material analyzed and by environment (Pooler & Simon, 1993; Klaas & Friesen, 2002).

DNA-based markers are less affected by age, physiological condition of the sample and environmental factors. They are not tissue specific and can be detected in any developmental stage of an organism. DNA markers such as RAPDs (Random Amplified Polymorphic DNA), AFLPs (Amplified Fragment Length Polymorphism), SSR (Simple Sequence Repeats) and DNA fingerprinting have been of great use for various studies in garlic. Isozyme analysis, RAPDs and AFLPs have enabled the study of phylogenetic relationships between different garlic clones and determination of their place of origin (Pooler & Simon, 1993; Maaβ & Klaas, 1995; Bradley et al., 1996; Al-Zahim et al., 1997; Lallemand et al., 1997; Ipek & Simon, 1998; García-Lampasona et al., 2003; Buso et al., 2008; Ipek et al., 2008; Abdoli et al., 2009). The use of molecular markers is indispensable for the establishment of core collections that should contain unique, varied and completely identified accessions in order to reduce costs and labour required for maintenance of collections *in situ* (Ipek et al., 2008). On the other hand, germplasm exchange between garlic producing countries can give rise to that a clone be called in different ways in various countries. If this occurred, the germplasm banks could be constituted by duplicated accessions. In this respect, molecular markers such as DNA fingerprinting and AFLPs have been used to detect duplicated accessions in collections (Bradley et al., 1996; Ipek et al., 2003). The use of AFLPs revealed that 64% of the U.S. National Plant Germplasm System's garlic collection was duplicated (Volk et al., 2004). Moreover, molecular markers can be used for detection of somaclonal variants generated by *in vitro* culture (Al-Zahim et al., 1999; Saker & Sawahel, 1998; Sánchez-Chiang & Jiménez, 2009), for determination of fertile clones (Hong et al., 2000; Etoh & Hong, 2001), disease resistance (Nabulsi et al., 2001) and clones producing S-amino acids (Ovesná et al., 2007).

#### **6. Conclusion**

Biotechnological tools such as plant tissue culture can help overcome problems associated with vegetative propagation of garlic, specially the low multiplication rate and disease dispersion. Although plant regeneration has been achieved from different explants types, use of root tips has advantages over other explants due to their virus-free condition and to their availability in a relatively high number (30 or more per clove). Moreover, roots developed from bulbs obtained *in vitro* can also be used for tissue culture (Robledo-Paz et al., 2000). On the other hand, production of virus-free plants via meristem culture combined with thermotherapy and chemotherapy can reduce losses caused by phytopathogens, even when propagation of virusfree material is relatively expensive (Salomon, 2002). Tissue culture has also been applied to the establishment of germplasm banks in various parts of the world where valuable garlic collections are maintained for medium (slow growth) and long term (cryopreservation).

Techniques such as somaclonal variation and genetic engineering could play an important role in the genetic improvement of garlic because they generate genetic variability. However, the somaclonal variants with commercial potential are scarce, and further experiments are necessary to identify the optimal explant type and the culture conditions that enable formation of somaclones (Novak, 1990). In addition, although there have been advances in the field of genetic transformation in garlic, more investigations are required to establish reproducible and efficient protocols. This task will require selection of suitable target cells for inoculation with *Agrobacterium* or biolistics (e.g. embryogenic calli) (Myers & Simon, 1998b; Robledo-Paz et al., 2004), strategies for transgene expression, a suitable selection method and efficient protocols for plant regeneration (McEloy & Brettell, 1994; Hansen & Wright, 1999). Furthermore, molecular markers will be key pieces in phylogenetic and taxonomic studies (Maaβ & Klaas, 1995) and germplasm conservation (Ipek et al., 2008). Moreover, they will be used for detection of somaclonal variants (Al-Zahim et al., 1999), fertile genotypes (Etoh & Hong, 2001), disease resistant genotypes (Nabulsi et al., 2001) and clones producing compounds of economical importance (Ovesná et al., 2007) which can be used for improving this important crop plant.

#### **7. References**

46 Innovations in Biotechnology

to fungi are still not being commercially used for fighting diseases caused by these phytopathogens. In garlic, Robledo-Paz (2010, personal communication) incorporated chitinase and glucanase genes in an attempt to confer resistance to the fungus *Sclerotium cepivorum.* These experiments revealed that regenerated transgenic plants were not totally

In order to make a more efficient use of garlic germplasm cultivated in various regions of the world, it is necessary to evaluate and characterize the available genetic diversity (Ordás et al., 1994). As the descriptions based on anatomical and morphological characteristics are incomplete and they can be affected by environmental factors, other methods are required to perform this characterization (García-Lampasona et al., 2003). Polymorphism of molecules such as isozymes and DNA can be used to characterize plant germplasm, specially in cases where morphological and biochemical differences are not conspicuous. Although isozyme analysis represented the first application of molecular markers in the genus *Allium,* its main drawback is the low number of enzymatic systems available in garlic. In addition, these markers may suffer changes induced by the developmental stage of plant material analyzed

DNA-based markers are less affected by age, physiological condition of the sample and environmental factors. They are not tissue specific and can be detected in any developmental stage of an organism. DNA markers such as RAPDs (Random Amplified Polymorphic DNA), AFLPs (Amplified Fragment Length Polymorphism), SSR (Simple Sequence Repeats) and DNA fingerprinting have been of great use for various studies in garlic. Isozyme analysis, RAPDs and AFLPs have enabled the study of phylogenetic relationships between different garlic clones and determination of their place of origin (Pooler & Simon, 1993; Maaβ & Klaas, 1995; Bradley et al., 1996; Al-Zahim et al., 1997; Lallemand et al., 1997; Ipek & Simon, 1998; García-Lampasona et al., 2003; Buso et al., 2008; Ipek et al., 2008; Abdoli et al., 2009). The use of molecular markers is indispensable for the establishment of core collections that should contain unique, varied and completely identified accessions in order to reduce costs and labour required for maintenance of collections *in situ* (Ipek et al., 2008). On the other hand, germplasm exchange between garlic producing countries can give rise to that a clone be called in different ways in various countries. If this occurred, the germplasm banks could be constituted by duplicated accessions. In this respect, molecular markers such as DNA fingerprinting and AFLPs have been used to detect duplicated accessions in collections (Bradley et al., 1996; Ipek et al., 2003). The use of AFLPs revealed that 64% of the U.S. National Plant Germplasm System's garlic collection was duplicated (Volk et al., 2004). Moreover, molecular markers can be used for detection of somaclonal variants generated by *in vitro* culture (Al-Zahim et al., 1999; Saker & Sawahel, 1998; Sánchez-Chiang & Jiménez, 2009), for determination of fertile clones (Hong et al., 2000; Etoh & Hong, 2001), disease resistance (Nabulsi et al., 2001) and clones

Biotechnological tools such as plant tissue culture can help overcome problems associated with vegetative propagation of garlic, specially the low multiplication rate and disease dispersion.

resistant to the fungus, but displayed a delay in the infection speed.

and by environment (Pooler & Simon, 1993; Klaas & Friesen, 2002).

producing S-amino acids (Ovesná et al., 2007).

**6. Conclusion** 

**5.6 Molecular markers** 


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### **Plant Beneficial Microbes and Their Application in Plant Biotechnology**

#### Anna Russo1, Gian Pietro Carrozza4, Lorenzo Vettori2, Cristiana Felici4, Fabrizio Cinelli3 and Annita Toffanin4 *1Department of Biological and Environmental Sciences and Technologies, University of Salento 2Department of Agriculture Biotechnology, University of Florence 3Department of Fruit Science and Plant Protection of Woody Species 'G. Scaramuzzi', University of Pisa 4Department of Crop Plant Biology, University of Pisa Italy*

#### **1. Introduction**

56 Innovations in Biotechnology

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Regeneration in Basal Plate and Receptacle Derived-Callus Culture of Garlic (*Allium sativum* L.). *J. Japan Soc. Hort. Sci.*, Vol. 60, pp. 627-634, ISSN 1882-3351. Zheng, S. J., Henken, B., Ahn, Y. K., Krens, F. A., & Kik, C. (2004). The Development of a

Reproducible *Agrobacterium tumefaciens* Transformation System for Garlic (*Allium sativum* L.) and the Production of Transgenic Resistant to Beet Armyworm

Cultivar Independent Plant Regeneration System From Callus Derived From Both Apical and Non-Apical Root Segments of Garlic (*Allium sativum* L.). *In Vitro Cell*  Plants are involved in a complex network of interactions with microorganisms; some of those are beneficial, others are detrimental, but the former are by far the largest and still widely unexplored part. This chapter reviews the status of development and application of beneficial microbes that provide an option for future prospects.

There is a growing worldwide demand for sound and ecologically compatible environmentally friendly techniques in agriculture, capable of providing adequate nourishment for the increasing human population and of improving the quality and quantity of certain agricultural products. For these reasons, the application of beneficial microorganisms is an important alternative to some of the traditional agricultural techniques which, as it has been well documented, very often severely alter the agroecosystem balance and cause serious damage to health. For example, contamination of groundwater by leaching of nitrogen fertilizers, accumulation of nitrates and persistence of chemicals used in crop protection in edible portion of foods are cause of grave concern.

The use of beneficial microorganisms in the replacement or the reduction of chemicals has been so far attested (Dobbelaere et al., 2003; Burdman et al., 2000). Beneficial microorganisms such as diazotrophs bacteria, biological control agents (BCAs), plant growth promoting rhizobacteria (PGPRs) and fungi (PGPFs), can play a key role in this major challenge, as they fulfil important ecosystem functions for plants and soil (Whipps, 1997; Raaijmakers et al., 2009; Hermosa et al., 2011). Moreover, modern agriculture, based on the cultivation of a very limited number of crop species and cultivars, is susceptible to epidemic diseases traditionally contrasted through the use of chemicals. With most crops, no effective fungicides are available against a lot of fungal diseases. Plant growth stimulation and crop protection may be improved by the direct application of a number of microorganisms known to act as bio-fertilizers and/or bio-protectors. How beneficial microorganisms really do act to improve plant rooting is only partially known, as several aspects have to be considered, including (i) the production of metabolites related to root development growth and pathogen control (phytohormones, antimicrobials, antibiotics), and (ii) the difficulty to discriminate the direct effects on the specific/total activities and the indirect effects due to the enhanced availability of nutrients and growth regulators.

Though over the past 150 years bacteria and fungi have been repeatedly demonstrated to promote plant growth and suppress plant pathogens, this knowledge has yet to be extensively exploited in agricultural biotechnology (Berg, 2009).

#### **2. Plant-microorganism interactions: Ecological implications**

Soil-borne microorganisms interact with plant roots and soil constituents at the root-soil interface, where root exudates and decaying plant material provide sources of carbon compounds for the heterotrophic biota (Barea et al., 2005; Bisseling et al., 2009). The number of bacteria in the rhizosphere (the narrow region of soil that is directly influenced by root secretions and associated soil microorganisms) and rhizoplane (the external surface of roots together with closely adhering soil particles and debris) is higher than in the soil devoid of plants; this happens because soils devoid of plants are poor in many attractive substances secreted from the roots. As soon as a seed starts to germinate, a relatively large amount of carbon and nitrogen compounds i.e., sugars, organic acid, aminoacids, and vitamins are excreted into the surrounding environment. This attracts a large population of microorganisms inducing vigorous competition between the different species (Okon, 1994). Moreover, rhizosphere microbiomes typically differ between plant species (Bisseling et al., 2009).

Beneficial microorganisms are known to be biocontrol agents and/or growth promoters. There are several modes of action by which they can be beneficial to plant health, which can be related to an indirect or a direct positive effect. Microorganisms have indirect positive effects on plants, affecting adversely the population density, dynamics and metabolic activities of soil-borne pathogens, mainly through competition, antibiosis, lysis, and hyperparasitism. Competition takes place for space and nutrients at the root surface; competitive colonization of the rhizosphere and successful establishment in the root zone is a prerequisite for effective biocontrol. Antagonistic microorganisms can often produce a range of different antimicrobial secondary metabolites, and/or extracellular lytic enzymes. Hyperparasitism is well documented for *Trichoderma*; it involves secretion of chitinases and cellulases, contact with the pathogen, coiling of hyphae around the hyphae of the pathogen, enzymatic digestion of its cell wall, and penetration. Direct positive effects on plants are exerted by rhizosphere microorganisms through a phytostimulation and a biofertilization of plants; these processes involve production of phytohormones, non-symbiotic nitrogen fixation, and the increase of availability of phosphate and other nutrients in the soil (Burdman et al., 2000). Numerous compounds that are toxic to pathogens, such as HCN, phenazines, pyrrolnitrin, and pyoluteorin as well as, enzymes, antibiotics, metabolites and phytohormones are the means by which PGPRs act; similarly other phenomena such as quorum sensing and chemotaxis, are vital for rhizosphere colonization (Castro-Sowinski et al., 2007; Ramette et al., 2011; Jousset et al., 2011).

Under iron-limiting conditions of soil habitats and plant surfaces, PGPRs can produce lowmolecular weight compounds called siderophores, that sequester iron in a competitive way,

microorganisms really do act to improve plant rooting is only partially known, as several aspects have to be considered, including (i) the production of metabolites related to root development growth and pathogen control (phytohormones, antimicrobials, antibiotics), and (ii) the difficulty to discriminate the direct effects on the specific/total activities and the

Though over the past 150 years bacteria and fungi have been repeatedly demonstrated to promote plant growth and suppress plant pathogens, this knowledge has yet to be

Soil-borne microorganisms interact with plant roots and soil constituents at the root-soil interface, where root exudates and decaying plant material provide sources of carbon compounds for the heterotrophic biota (Barea et al., 2005; Bisseling et al., 2009). The number of bacteria in the rhizosphere (the narrow region of soil that is directly influenced by root secretions and associated soil microorganisms) and rhizoplane (the external surface of roots together with closely adhering soil particles and debris) is higher than in the soil devoid of plants; this happens because soils devoid of plants are poor in many attractive substances secreted from the roots. As soon as a seed starts to germinate, a relatively large amount of carbon and nitrogen compounds i.e., sugars, organic acid, aminoacids, and vitamins are excreted into the surrounding environment. This attracts a large population of microorganisms inducing vigorous competition between the different species (Okon, 1994). Moreover,

indirect effects due to the enhanced availability of nutrients and growth regulators.

rhizosphere microbiomes typically differ between plant species (Bisseling et al., 2009).

Beneficial microorganisms are known to be biocontrol agents and/or growth promoters. There are several modes of action by which they can be beneficial to plant health, which can be related to an indirect or a direct positive effect. Microorganisms have indirect positive effects on plants, affecting adversely the population density, dynamics and metabolic activities of soil-borne pathogens, mainly through competition, antibiosis, lysis, and hyperparasitism. Competition takes place for space and nutrients at the root surface; competitive colonization of the rhizosphere and successful establishment in the root zone is a prerequisite for effective biocontrol. Antagonistic microorganisms can often produce a range of different antimicrobial secondary metabolites, and/or extracellular lytic enzymes. Hyperparasitism is well documented for *Trichoderma*; it involves secretion of chitinases and cellulases, contact with the pathogen, coiling of hyphae around the hyphae of the pathogen, enzymatic digestion of its cell wall, and penetration. Direct positive effects on plants are exerted by rhizosphere microorganisms through a phytostimulation and a biofertilization of plants; these processes involve production of phytohormones, non-symbiotic nitrogen fixation, and the increase of availability of phosphate and other nutrients in the soil (Burdman et al., 2000). Numerous compounds that are toxic to pathogens, such as HCN, phenazines, pyrrolnitrin, and pyoluteorin as well as, enzymes, antibiotics, metabolites and phytohormones are the means by which PGPRs act; similarly other phenomena such as quorum sensing and chemotaxis, are vital for rhizosphere colonization (Castro-Sowinski et

Under iron-limiting conditions of soil habitats and plant surfaces, PGPRs can produce lowmolecular weight compounds called siderophores, that sequester iron in a competitive way,

extensively exploited in agricultural biotechnology (Berg, 2009).

al., 2007; Ramette et al., 2011; Jousset et al., 2011).

**2. Plant-microorganism interactions: Ecological implications** 

thus depriving pathogenic fungi of this essential and often scarcely bioavailable element (Pedraza et al., 2007).

Many rhizosphere microorganisms can induce a systemic response in plants, activating plant defence mechanisms. Inoculation with non-pathogenic root zone bacteria can trigger signalling pathways that lead to higher pathogen resistance of the host, the so-called induced systemic resistance (ISR). Several of the bacteria that have been used to study beneficial effects under abiotic stress conditions, such as *Bacillus* sp., have been shown to induce ISR (Chakraborty et al., 2006). Some PGPRs elicit physical or chemical changes related to plant defense, a process often referred to as ISR, and/or tolerance to abiotic stress, such as drought, salt and nutrient excess or deficiency. For the latter PGPR-induced changes in plants, it has been proposed the term "induced systemic tolerance" (IST). IST relates to an enhanced tolerance to abiotic stresses (Yang et al., 2009). The metabolic pathways for signal transduction in plant defense responses can intercommunicate with other plant stress responses. In addition, the genes that are involved in plant responses to biotic and abiotic stresses can be co-regulated (Dimkpa et al*.*, 2009).

The effect of the growth promotion exerted by PGPRs is mainly related to the release of metabolites and nitrogen fixation processes, the provision of bioavailable phosphorus for plant uptake, sequestration of iron by siderophores, production of plant hormones like auxins, cytochinins and gibberellins, and lowering of plant ethylene levels (Glick, 1995; Glick et al., 1999; Tortora et al., 2011). On the contrary, biocontrol occurs through an indirect action of the BCAs that interact with soil pathogens through several mechanisms such as antibiosis (production of antimicrobial compounds), competition for iron and nutrients or for colonization sites, predation and parasitism, induction of resistance factors (for example the plant is strongly stimulated to synthesize substance called phytoalexins, small molecules with antibiotic activity, which can inhibit the growth of many pathogenic microorganisms), production of enzymes such as chitinase, glucanase, protease and lipase (Whipps, 2001). Growth promotion and biocontrol can be due to the same microorganism that positively influences the development of the plant through different mechanisms, for instance the increased availability and assimilation of the mineral nutritional components, the release of growth factors and the suppression of pathogenic microorganisms. This is translated in more resistant and healthy plants. In addition, PGPR species are able to metabolize numerous and varying carbon sources, to multiply quickly and above all to show a greater competence in colonizing the rhizosphere in comparison to deleterious microorganisms.

The beneficial bacteria are widely studied by microbiologists and agronomists because of their potential in increasing plant production (Somers et al., 2004). The research involving the use of PGPRs were made mainly on herbaceous plants in open field environments and in horticultural crops. Moreover, their application has recently expanded both in forestry and in phytoremediation of contaminated soils. Strains belonging to the genera *Azospirillum* (Okon & Labandera-Gonzalez, 1994; Okon & Itzigshon, 1995; Dobbelaere et al., 2001), *Bacillus* (Reddy & Rahe, 1989; Kokalis-Bourelle et al., 2002; Kokalis-Burelle et al., 2006) and *Pseudomonas* (McCullaugh et al., 1996; Meyer et al., 2010) have been used in experimental tests on a wide range of economically important crops.

Endophytic bacteria, those bacteria that dwell intercellularly in association with plants for most, if not all, of their life cycles (Bacon & Hinton, 2007), have been used for biological control of various plant diseases, as well as for enhanced plant agronomic characteristics, such as increased drought tolerance and nitrogen efficiency.

These bacteria, that include anaerobic, aerobic, and microaerobic species, live within the intercellular spaces of plant, where they feed on apoplastic nutrients, as non-pathogens. They can be found within a wide variety of plant tissue, including seeds, fruit, stems, roots and tubers (Surette et al., 2003). Among them are comprised bacterial diazotrophs that do not form nodules on hosts, such as *Azospirillum* species, and some *Rhizobium* species. Isolated from a large diversity of plants (Rosenblueth & Martínez-Romero, 2006), in general they occur at lower population density than rizospheric bacteria or bacterial pathogens and can positively affect host plant growth (Long et al., 2008). What makes bacterial endophytes suitable as biocontrol agents is their colonization of an ecological niche similar to that of phytopathogens (Ryan et al., 2008).

Endophytes can be strictly dependent on the host plant for their growth and survival ("obligate endophytes"); alternatively, "facultative endophytes" have a stage in their life cycle in which they exist outside host plants (Hardoim et al., 2008). The latter group probably comprises the vast majority of the microorganisms that can thrive inside plants. These endophytes often originate from the soil, initially infecting the host plant by colonizing, for instance, the cracks formed in lateral root junctions and then quickly spreading to the intercellular spaces in the root. Hence, to be ecologically successful, endophytes that infect plants from soil must be competent root colonizers. Endophytic colonization of the plant interior is presumably similar, at least in the initial phases, to colonization of plant roots by rhizobacteria. Competitive rhizosphere bacteria, for example members of the genera *Pseudomonas* (e.g. *P. fluorescens*), *Azospirillum* (e.g. *A. brasilense*) and *Bacillus* (Pedraza et al., 2007; Mano & Morisaki, 2008), are often also found as colonizers of the internal tissue of plants. A suite of environmental and genetic factors is presumed to have a role in enabling a specific bacterium to become endophytic. Inside the plant tissues, modulation of plant physiology by tinkering with the plant ethylene levels has emerged as a major strategy, because any effect on this plant stress signal has major impacts on the bacterial niche. How bacteria modulate plant ethylene concentrations is the key to their ecological success or competence as endophytes. The concept of "competent endophytes" has been proposed as a way to characterize those bacteria that possess key genetic machinery required to colonize the endosphere and to persist in it. This is in contrast to "opportunistic endophytes", which are competent rhizosphere colonizers that might become endophytic by coincidentally entering root tissue, but lack genes that are a key to their ecological success inside the plant. Moreover, it is possible to distinguish "passenger endophytes" that, in the absence of any machinery for efficient root colonization or entry, might enter plants purely as a result of chance events (Rosenblueth & Martínez-Romero, 2006; Mercado-Blanco & Bakker, 2007).

Bacterial endophytes, used for biological control of various plant deseases and for improved plant agronomic characteristics, may be of particular interest as they have the advantage of being relatively protected from the competitive soil environment; moreover, they usually grow in the same plant tissue where bacterial plant pathogens are detected (Bulgari et al., 2009). Their importance to crop production systems is only just beginning to be appreciated: so far, they have been shown to promote growth in potatoes, tomatoes, and rice, and they have been shown to be capable of inducing both biotic and abiotic stress resistance (Surette et al., 2003). A large number of mechanisms are being proposed to explain this effect: production of antimicrobial compounds, macronutrient competition, siderophore production, induced systemic resistance. This array of proposed mechanisms reflects the high diversity of endophytic bacteria.

#### **2.1 Tolerance to salinity**

60 Innovations in Biotechnology

control of various plant diseases, as well as for enhanced plant agronomic characteristics,

These bacteria, that include anaerobic, aerobic, and microaerobic species, live within the intercellular spaces of plant, where they feed on apoplastic nutrients, as non-pathogens. They can be found within a wide variety of plant tissue, including seeds, fruit, stems, roots and tubers (Surette et al., 2003). Among them are comprised bacterial diazotrophs that do not form nodules on hosts, such as *Azospirillum* species, and some *Rhizobium* species. Isolated from a large diversity of plants (Rosenblueth & Martínez-Romero, 2006), in general they occur at lower population density than rizospheric bacteria or bacterial pathogens and can positively affect host plant growth (Long et al., 2008). What makes bacterial endophytes suitable as biocontrol agents is their colonization of an ecological niche similar to that of

Endophytes can be strictly dependent on the host plant for their growth and survival ("obligate endophytes"); alternatively, "facultative endophytes" have a stage in their life cycle in which they exist outside host plants (Hardoim et al., 2008). The latter group probably comprises the vast majority of the microorganisms that can thrive inside plants. These endophytes often originate from the soil, initially infecting the host plant by colonizing, for instance, the cracks formed in lateral root junctions and then quickly spreading to the intercellular spaces in the root. Hence, to be ecologically successful, endophytes that infect plants from soil must be competent root colonizers. Endophytic colonization of the plant interior is presumably similar, at least in the initial phases, to colonization of plant roots by rhizobacteria. Competitive rhizosphere bacteria, for example members of the genera *Pseudomonas* (e.g. *P. fluorescens*), *Azospirillum* (e.g. *A. brasilense*) and *Bacillus* (Pedraza et al., 2007; Mano & Morisaki, 2008), are often also found as colonizers of the internal tissue of plants. A suite of environmental and genetic factors is presumed to have a role in enabling a specific bacterium to become endophytic. Inside the plant tissues, modulation of plant physiology by tinkering with the plant ethylene levels has emerged as a major strategy, because any effect on this plant stress signal has major impacts on the bacterial niche. How bacteria modulate plant ethylene concentrations is the key to their ecological success or competence as endophytes. The concept of "competent endophytes" has been proposed as a way to characterize those bacteria that possess key genetic machinery required to colonize the endosphere and to persist in it. This is in contrast to "opportunistic endophytes", which are competent rhizosphere colonizers that might become endophytic by coincidentally entering root tissue, but lack genes that are a key to their ecological success inside the plant. Moreover, it is possible to distinguish "passenger endophytes" that, in the absence of any machinery for efficient root colonization or entry, might enter plants purely as a result of chance events (Rosenblueth & Martínez-Romero,

Bacterial endophytes, used for biological control of various plant deseases and for improved plant agronomic characteristics, may be of particular interest as they have the advantage of being relatively protected from the competitive soil environment; moreover, they usually grow in the same plant tissue where bacterial plant pathogens are detected (Bulgari et al., 2009). Their importance to crop production systems is only just beginning to be appreciated: so far, they have been shown to promote growth in potatoes, tomatoes, and rice, and they have been shown to be capable of inducing both biotic and abiotic stress resistance (Surette et al., 2003). A large number of mechanisms are being proposed to explain this effect:

such as increased drought tolerance and nitrogen efficiency.

phytopathogens (Ryan et al., 2008).

2006; Mercado-Blanco & Bakker, 2007).

Soil salinity in arid regions is frequently an important limiting factor for cultivating agricultural crops. PGPR-elicited plant tolerance against salt stress has been intensively studied, showing that inoculation with endophytic bacteria can mitigate the effects of salt stress in different plant species.

High K+/Na+ ratios were found in salt-stressed maize in which selectivity for Na+, K+ and Ca2+ was altered upon inoculation with *Azospirillum* (Hamdia et al., 2004).

Similarly, inoculation of pepper with *Bacillus* sp.TW4 led to relief from osmotic stress, which is often manifested as salinity (and/or drought) stress. In these plants, genes linked with ethylene metabolism under abiotic stress were down-regulated (Sziderics et al., 2007). Because *Bacillus* sp. TW4 showed ACC deaminase activity, the authors speculated that the enzyme may be involved in the lower expression of these genes. Salt stress has also been shown to affect nodulation during *Phaseolus*–*Rhizobium* interaction. However, secondary inoculation of the salt-stressed plants with *Azospirillum* caused an extended exudation of plant flavonoids compared to *Rhizobium* alone, implying an induction of flavonoid genes in the presence of *Azospirillum* (Dardanelli et al*.*, 2008). Thus, the co-inoculation of plants with different bacterial species may contribute to relieving abiotic stress.

IST to salt stress was also noted with *Arabidopsis* (Zhang et al*.*, 2008) using *Bacillus subtilis* GB03, a species that has previously been used as a commercial biological control agent. Interestingly, some of the volatile organic compounds (VOCs) that are emitted from *B. subtilis* GB03 (Ryu et al., 2004) are bacterial determinants involved in IST. The response to saline stress has also been evidenced in barley seedlings where inoculation with *Azospirillum* seemed to mitigate NaCl stress (Zawoznik et al., 2011).

#### **2.2 Drought tolerance**

Land surface becoming arid or semi-arid has been rising progressively in these last decades; water use efficiency is a current priority for the United Nations policy and a key issue for plant research. Under water stress conditions, leaf transpiration and leaf conductance decrease, and the water use efficiency rises; this mechanism keeps plant growth under water-limited environments (Aroca & Ruiz-Lozano, 2009).

Plant responses to drought include an increase in abscisic acid (ABA) levels, that cause stomatal closure to minimize water loss; these events involve production of activated oxygen species (Cho et al., 2008). Other plant-signalling compounds are involved in regulating stomatal closure, such as methyl jasmonate, salicylic acid and ethylene.

The mechanisms that allow plants to cope with drought stress are regulated by changes in gene expression; drought regulated genes can be divided in two groups: functional genes (encoding for transporters, detoxification enzymes, osmolyte biosynthesis enzymes etc.) and regulatory genes, that encode for transcription factors (Aroca& Ruiz-Lozano, 2009). On the whole, the beneficial effects of PGPR on plant drought tolerance is caused by changes in hormonal contents, mainly that of ABA, ethylene and cytokinins.

ABA is involved in the enhancement of plant drought tolerance by PGPR; *Arabidopsis* plants inoculated with *A. brasilense* Sp245 showed more ABA content than non-inoculated ones (Cohen et al*.*, 2008).

Different strains of *A. lipoferum* were used to inoculate wheat seedlings under drought stress. Inoculation alleviated the plant drought stress, increasing wheat growth and yield; different strains performed differently (Arzanesh et al., 2011).

Exactly how a beneficial bacterium induces changes in plant root morphology is not yet clear. Bacterial production of hormone-like substances and their ability to stimulate endogenous hormone levels were believed to play the key role in this process (Dobbelaere et al., 1999). However, more recently, it has been found that, under aerobic conditions, *A. brasilense* produces significant amounts of the small diffusible gas, nitric oxide, which has been shown to act as a signalling molecule in an IAA-induced pathway involved in adventitious root development (Creus et al., 2005; Molina-Favero et al., 2008).

At the transcriptional level, the bacterium *P. polymyxa* caused the induction of a droughtresponsive gene, *ERD15*, isolated from drought-stressed *A. thaliana* (Timmusk & Wagner, 1999). The inoculated plants were more tolerant to drought stress than non-inoculated ones; that could be caused by a mild biotic stress that could help plants cope with subsequent drought stress.

#### **3. Experimental considerations about plant-beneficial bacteria**

In the Mediterranean area the use of microorganisms became indeed widespread in the '80s, in coincidence with the sudden spread of soybean crop, that required the inoculation of the nitrogen fixing *Bradyrhizobium japonicum*, mainly applied to seeds as peat based or liquid inocula at sowing. Operators became familiar with the use microorganisms in agriculture. After that, despite the huge potentiality of beneficial microorganisms, a relative low diffusion must be highlighted, owing to "inconsistent" results in field experiments, but also owing to prejudices derived from the easy and large availability of chemicals. At the moment, as a consequence of (i) a growing interest towards low input agriculture systems (organic farming, biodinamics, natural farming), (ii) a favourable opinion of consumers for food with no chemicals, and (iii) the increased difficulties in the employment of chemicals according to the most recent laws, we are assisting at a "microbiological revolution", and the use of microorganisms is increasing.

The use of beneficial microorganims is mostly oriented to improve plant growth and protection in an agricultural context, nevertheless several applications in a wider environmental sense could be prospected, as reported by our group in scientific literature. *Pseudomonas fluorescent* (Russo et al., 1996; 2001; 2005), *Bacillus subtilis* (Felici et al., 2008), *Rhizobium* spp (Toffanin et al., 2000; Casella et al., 2006), are some of beneficial bacteria applied in our experimental/scientific work as biofertilizers and/or biocontrol agents in agriculture. Other potential applications currently include micropropagation, bioremediation and phytoremediation, phosphate solubilization, soil aggregation, sewage treatment, bioleaching, oil recovery, coal scrubbing and biogas production.

whole, the beneficial effects of PGPR on plant drought tolerance is caused by changes in

ABA is involved in the enhancement of plant drought tolerance by PGPR; *Arabidopsis* plants inoculated with *A. brasilense* Sp245 showed more ABA content than non-inoculated ones

Different strains of *A. lipoferum* were used to inoculate wheat seedlings under drought stress. Inoculation alleviated the plant drought stress, increasing wheat growth and yield;

Exactly how a beneficial bacterium induces changes in plant root morphology is not yet clear. Bacterial production of hormone-like substances and their ability to stimulate endogenous hormone levels were believed to play the key role in this process (Dobbelaere et al., 1999). However, more recently, it has been found that, under aerobic conditions, *A. brasilense* produces significant amounts of the small diffusible gas, nitric oxide, which has been shown to act as a signalling molecule in an IAA-induced pathway involved in

At the transcriptional level, the bacterium *P. polymyxa* caused the induction of a droughtresponsive gene, *ERD15*, isolated from drought-stressed *A. thaliana* (Timmusk & Wagner, 1999). The inoculated plants were more tolerant to drought stress than non-inoculated ones; that could be caused by a mild biotic stress that could help plants cope with subsequent

In the Mediterranean area the use of microorganisms became indeed widespread in the '80s, in coincidence with the sudden spread of soybean crop, that required the inoculation of the nitrogen fixing *Bradyrhizobium japonicum*, mainly applied to seeds as peat based or liquid inocula at sowing. Operators became familiar with the use microorganisms in agriculture. After that, despite the huge potentiality of beneficial microorganisms, a relative low diffusion must be highlighted, owing to "inconsistent" results in field experiments, but also owing to prejudices derived from the easy and large availability of chemicals. At the moment, as a consequence of (i) a growing interest towards low input agriculture systems (organic farming, biodinamics, natural farming), (ii) a favourable opinion of consumers for food with no chemicals, and (iii) the increased difficulties in the employment of chemicals according to the most recent laws, we are assisting at a "microbiological revolution", and

The use of beneficial microorganims is mostly oriented to improve plant growth and protection in an agricultural context, nevertheless several applications in a wider environmental sense could be prospected, as reported by our group in scientific literature. *Pseudomonas fluorescent* (Russo et al., 1996; 2001; 2005), *Bacillus subtilis* (Felici et al., 2008), *Rhizobium* spp (Toffanin et al., 2000; Casella et al., 2006), are some of beneficial bacteria applied in our experimental/scientific work as biofertilizers and/or biocontrol agents in agriculture. Other potential applications currently include micropropagation, bioremediation and phytoremediation, phosphate solubilization, soil aggregation, sewage treatment, bioleaching,

adventitious root development (Creus et al., 2005; Molina-Favero et al., 2008).

**3. Experimental considerations about plant-beneficial bacteria** 

hormonal contents, mainly that of ABA, ethylene and cytokinins.

different strains performed differently (Arzanesh et al., 2011).

(Cohen et al*.*, 2008).

drought stress.

the use of microorganisms is increasing.

oil recovery, coal scrubbing and biogas production.

*Azospirillum brasilense* is a free-living, aerobic Gram-negative bacterium, that fixes N2 under microaerobic conditions, highly motile, displaying a mixed pattern of flagellation, which offers these microrganisms the advantage of moving towards favorable nutrient conditions. These bacteria have been isolated in particular from the rizosphere of cereals and grasses, in soils with low organic content and low doses of nitrogen fertilization (Dobbelaere et al., 2001; 2003). They are able to penetrate the roots and grow endophytically in the intercellular spaces; they have been isolated from a large variety of soils and locations worldwide, in tropical and temperate regions (Steenhoudt & Vanderleyden, 2000). The *Azospirillum* species are plant growth promoting rhizobacteria, which positively affect the growth and the yield of many plants of agricultural and ecological importance (Bashan et al., 2004). Since the '80s, *Azospirillum* species have been extensively studied for their potential in improving the growth and yield of cereal crops, particularly in sub-tropical regions, firstly speculating that their ability in freely fixing nitrogen could improve soil fertility and increase nutrient uptake of plants. In the last years, much evidence has arisen that the beneficial effects of *Azospirillum* species depend on an array of contributions, such as production of phytohormones and other bioactive substances, rather than the bacterial nitrogen fixation. Multiple mechanisms are currently suggested to explain the beneficial effects on plant growth (Bashan & de-Bashan, 2010). *Azospirillum* is used in many Countries as bacterial inoculant, alone or together with other bacteria and vesicular arbuscular mycorrhizal (VAM) fungi, for many crops (Bashan et al., 2004). The effects on the yield have not been reported to be always positive; they depend on the bacterial strain, the inoculated plant cultivar, and the environmental conditions (Pandey et al., 1998). In particular, temperature has turned out to be of crucial importance when this bacterium is inoculated in winter crops, where responses can be low or non-significant (Kaushik et al., 2001). Hence, the growth response of inoculated crops is not completely predictable (Hartmann & Bashan, 2009); nevertheless, much progress has been made in this field, and the practical field application of *Azospirillum* is expanding worldwide, especially in Central and South America. It has been estimated that there were 300,000 ha inoculated fields in Mexico in 2007, while in Argentina over 220,000 ha of wheat and corn were inoculated in 2008.

*Azospirillum brasilense* has been proposed in our studies in different fields, ranging from agriculture (crops, micropropagation, grape and olive propagation, ornamental plants nursery) to environmental sciences (bioremediation, environmental engineering), as reported below.

#### **3.1 Micropropagation**

Micropropagation is an efficient method of propagating large numbers of genetically uniform plants (Honda & Kobayashi, 2004), although serious problems concerning specific steps including explant sterilization, media manipulation, and acclimatization phase, can often invalidate its success, making the plantlets production a cost-intensive process.

*In vitro* bacterization of potato plantlets has been shown to enhance their transplant stress tolerance thereby eliminating the need of an expensive greenhouse hardening step, which even now is commonly used by pre-elite seed potato producers. Plants bacterized *in vitro* with *Pseudomonas fluorescens* strains CHA0 and IP10 were found to have a significantly higher fresh shoot weight compared to non-bacterized plants in the same system. Arbuscular mycorrhizal (AM) fungi have also been shown to reduce drought stress and increase disease resistance.

We have investigated the possibility of using the PGPR *Azospirillum brasilense* Sp245 to improve the micropropagation of cherry plum (*Prunus cerasifera*) trees (Russo et al., 2008). We have examined the ability of *A. brasilense* to promote rooting of explants during *in vitro* culture in growth-chamber tests and to promote plant growth and plant health during *ex vitro* acclimatization in greenhouse. In the presence of indolebutyric acid (IBA), both rooting and growth of *P. cerasifera* cuttings were significantly improved by bacteria inoculation. During the acclimatation phase, the main positive effect of inoculation was an increased biomass production, as compared with uninoculated control, suggesting that during acclimatation the rhizobacterium produced phytohormones, increased the nutrient uptake of the roots, and caused an improvement of overall plant performance. An ability to protect plants against pathogen attack was also observed, with a plant survival rate of nearly 100% in inoculated plants as compared to 0% in the negative control. This protective effect was demonstrated both *in vitro* and *in vivo* against the pathogenic fungus *Rhizoctonia* spp.

The effectiveness of *A. brasilense* cells application to micropropagated plantlets at the time of transplanting from *in vitro* culture to acclimatization conditions, has been furthermore assessed on three different fruit tree rootstocks: Mr.S2/5 (*Prunus cerasifera* X *P. spinosa*), GF 677 (*Prunus persica* X *P. amygdalus*), MM 106 (Northen spy X M1). This is a critical phase, in which plantlets are subjected to numerous environmental stresses that may lead to significant plant loss. Plant growth and plant health at the end of *post-vitrum* acclimatization, both in growth-chamber and greenhouse trials, respectively, were evaluated.

After 60 days, growth parameters were positively affected by Sp245 inoculum. In the case of Mr.S 2/5, an increase in rootstock stem length and node number by 37% and 42%, respectively, compared to the control was noted. In the case of GF 677, the bacterial inoculum increased stem length and node number by up to the 75% and 65%, respectively, compared to the control. The inoculum did not exert on MM 106 for both parameters suggesting that the effects of Sp245 could depend on a specific clone-microbe association. In all the cases, however, a higher vigor, consistent with a wider leaf area, was present in the inoculated plantlets demonstrating that the use of *Azospirillum* can significantly contribute to optimize plant performance during the phase of adaptation of plants to *post-vitrum* conditions (Vettori et al., 2010).

Considering that the main obstacles to an intensive and widespread use of beneficial microorganisms, at the commercial level, have been so far the so called "inconsistent" results in field application, mainly related to biotic and abiotic adverse environmental factors, the *in vitro* and *post vitro* inoculation with bacteria may be a way of overcoming a part of these difficulties.

#### **3.2 Co-inoculation strategies**

In recent years, a number of studies on co-inoculation of two or more beneficial microorganisms for better crop productivity have been reported. Positive effects, such as increase in biomass parameters, nitrogen-content and yield have been found in legumes inoculated with *Rhizobium* and *Azospirillum*. These positive effects may be attributed to early and increased nodulation, enhanced N2 fixation rates, and a general improvement of root development. Stimulation of nodulation following the inoculation with *Azospirillum* may be derived from an increase in production of lateral roots, root hair density and branching, but

We have investigated the possibility of using the PGPR *Azospirillum brasilense* Sp245 to improve the micropropagation of cherry plum (*Prunus cerasifera*) trees (Russo et al., 2008). We have examined the ability of *A. brasilense* to promote rooting of explants during *in vitro* culture in growth-chamber tests and to promote plant growth and plant health during *ex vitro* acclimatization in greenhouse. In the presence of indolebutyric acid (IBA), both rooting and growth of *P. cerasifera* cuttings were significantly improved by bacteria inoculation. During the acclimatation phase, the main positive effect of inoculation was an increased biomass production, as compared with uninoculated control, suggesting that during acclimatation the rhizobacterium produced phytohormones, increased the nutrient uptake of the roots, and caused an improvement of overall plant performance. An ability to protect plants against pathogen attack was also observed, with a plant survival rate of nearly 100% in inoculated plants as compared to 0% in the negative control. This protective effect was

demonstrated both *in vitro* and *in vivo* against the pathogenic fungus *Rhizoctonia* spp.

growth-chamber and greenhouse trials, respectively, were evaluated.

conditions (Vettori et al., 2010).

**3.2 Co-inoculation strategies** 

part of these difficulties.

The effectiveness of *A. brasilense* cells application to micropropagated plantlets at the time of transplanting from *in vitro* culture to acclimatization conditions, has been furthermore assessed on three different fruit tree rootstocks: Mr.S2/5 (*Prunus cerasifera* X *P. spinosa*), GF 677 (*Prunus persica* X *P. amygdalus*), MM 106 (Northen spy X M1). This is a critical phase, in which plantlets are subjected to numerous environmental stresses that may lead to significant plant loss. Plant growth and plant health at the end of *post-vitrum* acclimatization, both in

After 60 days, growth parameters were positively affected by Sp245 inoculum. In the case of Mr.S 2/5, an increase in rootstock stem length and node number by 37% and 42%, respectively, compared to the control was noted. In the case of GF 677, the bacterial inoculum increased stem length and node number by up to the 75% and 65%, respectively, compared to the control. The inoculum did not exert on MM 106 for both parameters suggesting that the effects of Sp245 could depend on a specific clone-microbe association. In all the cases, however, a higher vigor, consistent with a wider leaf area, was present in the inoculated plantlets demonstrating that the use of *Azospirillum* can significantly contribute to optimize plant performance during the phase of adaptation of plants to *post-vitrum*

Considering that the main obstacles to an intensive and widespread use of beneficial microorganisms, at the commercial level, have been so far the so called "inconsistent" results in field application, mainly related to biotic and abiotic adverse environmental factors, the *in vitro* and *post vitro* inoculation with bacteria may be a way of overcoming a

In recent years, a number of studies on co-inoculation of two or more beneficial microorganisms for better crop productivity have been reported. Positive effects, such as increase in biomass parameters, nitrogen-content and yield have been found in legumes inoculated with *Rhizobium* and *Azospirillum*. These positive effects may be attributed to early and increased nodulation, enhanced N2 fixation rates, and a general improvement of root development. Stimulation of nodulation following the inoculation with *Azospirillum* may be derived from an increase in production of lateral roots, root hair density and branching, but also from the differentiation of a greater number of epidermal cells into root hairs susceptible for infection by rhizobia. Nodulation by rhizobia co-inoculated with *Azospirillum* may also be enhanced by an increased secretion of root flavonoid substances that are involved in the activation of the nodulation genes in *Rhizobium* (Dobbelaere et al., 2001). Considerable results have also been obtained on grain yield, N, P, K content in wheat coinoculated with *Azospirillum brasilense* and *Rhizobium meliloti* (Askary et al., 2009).

The effects of co-inoculation of bacteria and fungi has also been reported. *Azospirillum*–AM fungus combination seems suitable for sustainable agriculture practices, since both types of microorganisms are compatible with each other. The stimulatory effect of the *Azospirillum* inocula on root growth did not significantly influence the mycorrhization, regardless of the AM fungus involved, either in wheat or in maize plants, grown in the greenhouse and/or under field conditions. The effect of *Azospirillum brasilense* Sp245 was assessed at greenhouse level in three different cultivars of durum wheat, in the presence of indigenous AM fungi, and in maize plants artificially inoculated with *Glomus mosseae* and *Glomus macrocarpum*. At field level, the establishment of natural AM fungal symbiosis was evaluated with the commercial strain *Azospirillum lipoferum* CRT1 in maize plants (Russo et al., 2005). Positive effects of *Azospirillum brasilense* and arbuscular mycorrhizal colonization on rice growth and drought resistance have also been attested (Ruíz-Sánchez et al., 2011).

On the other hand, the combination of two rhizobacteria had no synergistic or comparable effects on plant biomass, with respect to their single applications. Indeed, individual inoculation of *B. subtilis* and *A. brasilense* Sp245 positively affected the growth in dry weight of both shoots and roots of tomato plants, but the combination of the two rhizobacteria had no synergistic or comparable effects on plant biomass. *In vitro* tests and cellular analysis of root tips revealed a growth inhibition of the primary root, which is not related to a reduced persistence in the rhizosphere of one or both bacteria (Felici et al., 2008). Moreover coinoculation with mycorrhiza and rhizobia of different bean genotypes resulted in the reduction in the trehalose content and the authors concluded supporting the idea of using rhizobial or mycorrhizal inoculation separately (Ballesteros-Almanza et al., 2010).

These results suggest that mixing different microorganisms in the same inocula/treatment can cause interferences and consequent bad or lower than expected performances. Interactions and antagonist phenomena in contaminant species against *Bradyrhizobium japonicum*, obtained from the same soybean inocula preparation, had already been evidenced in the '80s.

#### **3.3 Bioremediation and phytoremediation**

Phytoextraction, actuated by hyperaccumulating or non-hyperaccumulating species, could be improved by using a plant-microbe system (Zhuang et al. 2007), thus contributing to novel promising methods for the cleaning-up of soils contaminated by heavy metals. Rhizobacteria of the genus *Azospirillum* have been extensively used for crop phytostimulation as above stated, thanks to the positive interaction between bacteria and plants at root level (Dobbeleare et al., 2001; Dobbeleare et al., 2003; Russo et al., 2005; Russo et al., 2008).

The implementation of lead phytoextraction in contaminated industrial soils by applying *A. brasilense* Sp245 to plants of indigenous species belonging to Mediterranean forestry was investigated. The possible phytoextraction ability was evaluated in *Myrtus communis*  L*.* and *Laurus nobilis* L., previously selected among other plant species that were found able to grow in the contaminated areas, on the basis of the Pb content (Emission Spectrophotometer Atomic Plasma, ICP-AES), the growing speed and the vegetative *habitus*. By trials carried out in greenhouse, it was shown that *A. brasilense* Sp245 can enhance the plant growth in Pb contaminated soil and affect the plant total lead content. Greenhouse trials were performed for 2 and 9 months, and plants were grown in pot in the presence of two level of Pb (312 and 4345 ppm).

The presence of Sp245 positively affected the total amount of Pb that was removed by plants, either as total biomass produced (Figure 1) or as specific Pb concentration, as a consequence of the incremented root growth, attesting the synergic effect of plants and microorganisms in a bioremediation system, and as higher specific Pb concentration (Table 1 and Table 2). Moreover the bioconcentration factor (Pb in plant tissues/Pb in soil) and translocation factor (Pb in leaves and shoots/Pb in roots) were significantly affected by the presence of *A*. *brasilense* Sp245, attesting the synergetic effect of plants and microorganisms in a rhizoremediation system.

Fig. 1. Effect of *Azospirillum brasilense* Sp245 inoculation on root system in *Laurus nobilis* and *Myrtus communis* after nine months of pot cultivation with Pb polluted soil.


Table 1. Effect of *A. brasilense* Sp245 inoculation on Pb uptake (mg/Kg d.m.) by each plant of *Laurus nobilis* and *Myrtus communis* after two and nine months of pot cultivation with soils at different Pb concentration (Pb- : 312 mg/Kg and Pb+ : 4345 mg/Kg). Within each shrub species and each period means with the same letter were not significantly different according to the SNK'test (P ≤0.05).


Table 2. Effect of *A. brasilense* Sp245 inoculation on biomass produced (g/plant d.m.) by *Laurus nobilis* and *Myrtus communis* after two and nine months of pot cultivation with Pb polluted soil. Within shrub species means with the same letter were not significantly different according to the SNK'test (P ≤0.05).

#### **3.4 Naturalistic engineering and endotherapy**

The increased root density and branching, the improving in rooting of cuttings and the better adaptation to biotic and abiotic stresses derived from beneficial microorganisms, may represent an advantage in soil bioengineering and landscape construction. The role of vegetation in slope stability and restoration of steep rock faces with shrubs and trees is difficult due to extreme microclimatic and edaphic conditions (Beikircher et al., 2010). Inoculation with *A. brasilense* Sp245 of plant material used for restoration of drought-prone sites during preconditioning, can increase the drought tolerance and can play a synergetic and pivotal role in that phase. Work in progress with some angiosperm species, known for their vulnerability, gives us good expectation.

Another promising field could be the use of selected beneficial microorganisms in endotherapy, or trunk injection. This is an alternative method of treatment of urban woodland plant, with many advantages compared to traditional air treatments, including the absence of spraying of chemicals, and hence the complete harmlessness for the health of citizens, birds and other animals (Sánchez-Zamora & Fernández-Escobar, 2004; Hubbard & Potter, 2006).

#### **4. Conclusions**

66 Innovations in Biotechnology

Spectrophotometer Atomic Plasma, ICP-AES), the growing speed and the vegetative *habitus*. By trials carried out in greenhouse, it was shown that *A. brasilense* Sp245 can enhance the plant growth in Pb contaminated soil and affect the plant total lead content. Greenhouse trials were performed for 2 and 9 months, and plants were grown in pot in

The presence of Sp245 positively affected the total amount of Pb that was removed by plants, either as total biomass produced (Figure 1) or as specific Pb concentration, as a consequence of the incremented root growth, attesting the synergic effect of plants and microorganisms in a bioremediation system, and as higher specific Pb concentration (Table 1 and Table 2). Moreover the bioconcentration factor (Pb in plant tissues/Pb in soil) and translocation factor (Pb in leaves and shoots/Pb in roots) were significantly affected by the presence of *A*. *brasilense* Sp245, attesting the synergetic effect of plants and microorganisms

Fig. 1. Effect of *Azospirillum brasilense* Sp245 inoculation on root system in *Laurus nobilis* and

Pb uptake Effect of *A. brasilense* Sp 245

(mg/Kg dry matter)

*Laurus nobilis* Pb- Pb+ Pb- Pb+ Two months 63b 430a 77b 438a Nine months 79c 466b 94c 534a *Myrtus communis* Pb- Pb+ Pb- Pb+ Two months 103c 806b 116c 954a Nine months 191d 1176b 305c 1324a Table 1. Effect of *A. brasilense* Sp245 inoculation on Pb uptake (mg/Kg d.m.) by each plant of *Laurus nobilis* and *Myrtus communis* after two and nine months of pot cultivation with soils at different Pb concentration (Pb- : 312 mg/Kg and Pb+ : 4345 mg/Kg). Within each shrub species and each period means with the same letter were not significantly different

*Myrtus communis* after nine months of pot cultivation with Pb polluted soil.

Shrub species Pb uptake

the presence of two level of Pb (312 and 4345 ppm).

in a rhizoremediation system.

according to the SNK'test (P ≤0.05).

Agriculture is the oldest economic sector in the world, and is more dependent on fertile soils and a stable climate than any other trade. At the same time, it has a huge influence on the ecological balance, water and soil quality, and on the preservation of biological diversity. Since the middle of the last century, agricultural techniques and economic framework conditions worldwide have undergone such a radical transformation that agriculture has became a major source of environmental pollution.

The investigation about ecologically compatible techniques in agriculture and environmental sciences can take essential advantage from the use of beneficial microorganisms as plantmicrobe interactions fulfil important ecosystem functions.

Plant diseases are a major cause of yield losses and ecosystem instability worldwide. Use of agrochemicals to protect crop against plant pathogens has been increasing along with the intensification of agricultural production over the last few decades.

New biotechnological methods for crop protection are based on the use of beneficial microorganisms applied as biofertilizers and/or biocontrol agents; this approach represents an important tool for plant disease control, and could lead to a substantial reduction of chemical fertilizer use, which is an important source of environmental pollution. Nevertheless, despite dedicated efforts to study beneficial microorganisms, relatively few products have been registered for agricultural use and they count for a very small fraction of the potential market. This is a consequence of several limitations affecting their commercial expansion, which are mainly related to the survival capability of microorganisms under various environmental conditions.

Moreover, nowadays, microbial inoculants, some of which have a historical record for safe use since 1896 (the well-known rhizobia, for the inoculation of legumes) or since the 1930s (e.g. *Bacillus thuringiensis*, for the biological control of invertebrate pests) are being widely applied in modern agriculture as biofertilizers and biocontrol agents. Other interesting applications include micropropagation, bioremediation and phytoremediation, phosphate solubilization, soil aggregation, sewage treatment, bioleaching, oil recovery, coal scrubbing and biogas production, and represent incoming fields of application.

In short, from the examples and references cited above, it is manifest that useful microorganisms of agricultural importance represent an alternative and ecological strategy for disease management, in order to reduce the use of chemicals in agriculture and to improve cultivar performance. At the same time, their application is a highly efficient way to resolve environmental problems, for example through bioremediation and bioengineering. However, although beneficial microorganisms hold a great promise for dealing with different environmental problems, it is important to aknowledge that much of this promise has yet to be realized. Indeed, much needs to be learned about how microorganisms interact with each other and with the environments. For the future development of biotechnology in this field, the contribution of a combination of scientific disciplines is of primary importance to promote sustainable practices in plant production system, as well as in conservation and ecosystem restoration.

#### **5. References**


New biotechnological methods for crop protection are based on the use of beneficial microorganisms applied as biofertilizers and/or biocontrol agents; this approach represents an important tool for plant disease control, and could lead to a substantial reduction of chemical fertilizer use, which is an important source of environmental pollution. Nevertheless, despite dedicated efforts to study beneficial microorganisms, relatively few products have been registered for agricultural use and they count for a very small fraction of the potential market. This is a consequence of several limitations affecting their commercial expansion, which are mainly related to the survival capability of microorganisms under

Moreover, nowadays, microbial inoculants, some of which have a historical record for safe use since 1896 (the well-known rhizobia, for the inoculation of legumes) or since the 1930s (e.g. *Bacillus thuringiensis*, for the biological control of invertebrate pests) are being widely applied in modern agriculture as biofertilizers and biocontrol agents. Other interesting applications include micropropagation, bioremediation and phytoremediation, phosphate solubilization, soil aggregation, sewage treatment, bioleaching, oil recovery, coal scrubbing

In short, from the examples and references cited above, it is manifest that useful microorganisms of agricultural importance represent an alternative and ecological strategy for disease management, in order to reduce the use of chemicals in agriculture and to improve cultivar performance. At the same time, their application is a highly efficient way to resolve environmental problems, for example through bioremediation and bioengineering. However, although beneficial microorganisms hold a great promise for dealing with different environmental problems, it is important to aknowledge that much of this promise has yet to be realized. Indeed, much needs to be learned about how microorganisms interact with each other and with the environments. For the future development of biotechnology in this field, the contribution of a combination of scientific disciplines is of primary importance to promote sustainable practices in plant production

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