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Factors Affecting Somatic Embryogenesis and Transformation in Modern Plant Breeding

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Factors Affecting Somatic Embryogenesis and Transformation in Modern Plant Breeding CSIRO PUBLISHING The South Pacific Journal of Natural and Applied Sciences, 28 , 27-40, 2010 www.publish.csiro.au/journals/spjnas 10.1071/SP10002 Factors affecting somatic embryogenesis and transformation in modern plant breeding Pradeep C. Deo 1, 2 , Anand P. Tyagi 1, Mary Taylor 3, Rob Harding 2 and Doug Becker 2 1Faculty of Science, Technology and Environment (SBCS), The University of the South Pacific, Suva, Fiji. 2Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Brisbane, Australia. 3Centre for Pacific Crops and Trees, Secretariat of the Pacific Community, Suva, Fiji. Abstract Somatic embryogenesis and transformation systems are indispensable modern plant breeding components since they provide an alternative platform to develop control strategies against the plethora of pests and diseases affecting many agronomic crops. This review discusses some of the factors affecting somatic embryogenesis and transformation, highlights the advantages and limitations of these systems and explores these systems as breeding tools for the development of crops with improved agronomic traits. The regeneration of non-chimeric transgenic crops through somatic embryogenesis with introduced disease and pest-resistant genes for instance, would be of significant benefit to growers worldwide. Keywords: Somatic embryogenesis, transformation, plant growth regulators, Agrobacterium, microprojectiles 1. Introduction developed radicals with very fine root hairs (Zimmerman, In plants, embryo-like structures can be generated from 1993). cells other than gametes (i.e. somatic cells) by In monocotyledonous embryogenesis, especially in circumventing the normal fertilization process, hence the graminaceous species, the transition from globular stage term somatic embryos (Parrott, 2000). As somatic follows a series of events all occurring simultaneously. embryos are formed without any fertilization event they This includes the development of scutellum, initiation of are genetically identical to the parent tissue and are the coleoptilar notch, and tissue differentiation with the therefore clones. development of embryogenic vascular system and Somatic embryogenesis may be direct or indirect. accumulation of intracellular storage substances. At the Indirect somatic embryogenesis involves dedifferentiation final stages of maturation, the coleoptile undergoes of organized tissue into callus prior to embryo production enlargement and the embryo axis becomes more whereas direct somatic embryogenesis involves production developed. The embryo axis develops laterally and parallel of embryo from organized tissue without an intervening to the scutellum, while the root apical meristem is callus phase (Slater et al. , 2003). embedded and the shoot apical meristem develops Irrespective of the mode of production, it has been externally and is protected by the coleoptile (Gray, 1996). argued that the anatomical and physiological features of The first leaf primordium appears at the base of the shoot somatic embryos are highly comparable to zygotic apex while development of leaf primordia is preceded by embryos (Cheng and Raghavan, 1985; Boxus, 1989; Gray, the formation of root(s) from the root meristem. As the 1992; Zimmerman, 1993; Bandyopadhyay and Hamill, plantlet develops, numerous root hairs develop on the main 2000). This claim is supported by the work of root (Meinke, 1991). Bandyopadhyay and Hamill (2000) on Eucalyptus nitens, which revealed that both somatic and zygotic embryos 1.1 How is Somatic Embryogenesis Used? have strong similarities in terms of their overall size, Somatic embryogenesis is a valuable tool in plant morphology and internal cellular organization. biotechnology and can be utilized in a number of ways Zimmerman (1993) further argued that the morphological (Zimmerman, 1993; Bandyopadhyay and Hamill, 2000; and temporal development of somatic embryos are very Saiprasad, 2001): similar to that of zygotic embryos and that they both • For large-scale clonal propagation of elite proceed through a series of distinct stages, namely cultivars it provides an alternative approach to globular, heart, torpedo and cotyledon or plantlet stages for conventional micropropagation. dicotyledons (Zimmerman, 1993; Mandal and Gupta, • Synthetic (artificial) seed can be developed from 2002) and globular, elongated, scutellar and coleoptilar somatic embryos potentially facilitating broad- stages for monocotyledons (Gupta and Conger, 1999; acre direct seeding of elite cultivars or providing a Godbole et al. , 2002). These stages typically span a period means of moving germplasm in a less fragile of several days. form than in vitro plantlets. In dicotyledonous embryogenesis, small globular • Embryogenesis via callus or secondary embryos initially form which then undergo isodiametric embryogenesis may assist in the application of growth and establish bilateral symmetry. These then gene transfer techniques for further genetic develop into the heart stage embryo in which both improvements. cotyledons and root and shoot meristems are clearly • Somatic embryogenesis systems offer potential established. The development proceeds with the formation models for studying molecular, regulatory and of torpedo and subsequently plantlet stages. The plantlets morphogenetic events in plant embryogenesis. contain green cotyledons, elongated hypocotyls and © The South Pacific Journal of Natural and Applied Sciences, 2010 28 P. Deo et al. : Factors affecting somatic embryogenesis and transformation 1.2 The Advantages of Somatic Embryogenesis for may in fact be linked with increasing mutations associated Mass Propagation with an inability to regenerate. Working with suspension Large-scale production of plants through the cultures of carrot, Evans et al . (1983) showed that multiplication of embryogenic cell lines is the most frequently initiating new cultures and maintaining the commercially attractive application of somatic cultures for less than one year resulted in the regeneration embryogenesis (Jiménez, 2001), and is the most practical of phenotypically normal somatic embryos and plants. application of this technique to benefit agriculture. It has Apparently, somaclonal variation also occurs with many advantages over conventional micropropagation in conventional micropropagation hence new cultures are this respect: also initiated on a regular basis. It should be noted, • It permits the culture of large numbers of somatic however, that somaclonal variation could have tremendous embryos, with up to1.35 million somatic embryos potential for producing novel and useful varieties. capable of being regenerated per litre of medium. • During regeneration, root and shoot formation is 1.4 Role of Plant Growth Regulators (Pgrs) in the simultaneous thus eliminating the need for a root Development of Somatic Embryos induction phase as with conventional The most commonly used protocol for induction of micropropagation. embryogenesis involves the induction of callus in an • The mode of culture permits easy scale-up and auxin-supplemented medium and somatic embryogenesis subculture with low labour inputs. upon transfer of callus to a medium low in growth • Cultures can be manipulated such that embryo regulators (Cheng and Raghavan, 1985; Smith and formation and germination can be synchronized Krikorian, 1989; Smith and Krikorian, 1990; Gray, 1992; maximizing plant output while minimizing labour Zimmerman, 1993). The establishment and maintenance of inputs. embryogenic cultures of nearly all species has relied • As with zygotic embryos, somatic embryos primarily on the manipulation of growth regulators (Smith dormancy can be induced, hence long-term and Krikorian, 1990). In particular, the presence of auxin storage is possible. promotes callus proliferation and inhibits differentiation while the removal or decrease in auxin allows somatic 1.3 Limitations of Somatic Embryogenesis embryo development to progress. Morphogenetic changes Even though somatic embryogenesis offers great can be observed upon transferring callus to an auxin-free potential, it also has some limitations. Firstly, the medium (Cheng and Raghavan, 1985; Zimmerman, 1993). development of somatic embryos tends to be non- It appears that the removal of auxin from the medium synchronous (Zimmerman, 1993; Zegzouti et al. , 2001), provides the signal for the callus cells to embark on an thus embryos of all stages can be present in one culture organized pattern of growth. The fact that embryogenesis system. However, Fujimura and Komamine (1979) can occur upon withdrawal of growth regulators suggests demonstrated that the development of carrot somatic that in the presence of auxin, the proembryonic masses embryos could be synchronized by grouping cell (PEMs) within the culture system may already be aggregates of similar size and density from suspension “primed” to complete the globular stage of embryogenesis cultures using sieving and density gradient centrifugation. and that the PEMs may also contain products inhibitory to Although synchronization of somatic embryos can be the progress of the embryogenesis program (Zimmerman, achieved using these strategies, it appears that the 1993). Consequently, the removal of auxin may result in percentage of somatic embryos regenerated is affected by the inactivation of genes responsible for the presence of the size of cell aggregates. Chee and Cantliff (1989) these inhibitory products, enabling the embryogenesis pointed out that a decrease in the size of cell aggregates
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