Somatic embryogenesis for crop improvement

Review Article Somatic embryogenesis for crop improvement Madhu Kamle 1&2 *, Anju Bajpai 1, Ramesh Chandra 1, Shahina Kalim 2 and Ramesh Kumar 2 GERF Bulletin of Biosciences June 2011, 2(1):54-59 1 Biotechnology Laboratory, Central Institute for Subtropical Horticulture, Rehmankhera, Lucknow, India 2 Department of Biochemistry, Bundelkhand University, Jhansi, India Abstract Somatic embryogenesis (SE) is a process of advancement in vegetative propagation technology for plants, which has a major impact on tree breeding and high value horticultural crops. The process describes the mass multiplication of new and elite varieties within a short span of time within limited resources. The production of true to type plants with new and desired traits against various biotic and abiotic stresses through in vitro selection and genetic enhancement via. transgenic is the needful. The rate of multiplication by conventional breeding methods is slow and nature dependant while, somatic embryogenesis could assist in rapid and mass multiplication of newly released and improved cultivars. A brief overview on somatic embryogenesis for crop improvement process is reviewed in this review article. Keywords: Somatic embryogenesis, propagation, regeneration, molecular markers Introduction Globalization of agriculture is increasingly calling for improved efficiency and competitiveness of the existing production systems. The improvement of fruit trees through conventional breeding techniques has been limited due to inherent problems such as long life cycle with extended juvenile period, floral morphology, existing hybridization barrier, sterility, apomixes and long term inbreeding depression. Conventional propagation methods such as grafting, air layering, stooling etc. for improving many fruit trees already exist but extended juvenility has made these techniques time consuming and cumbersome. The attempt by Haberlandt to establish plant tissue culture systems provided support for a better understanding of the totipotency of plant cells. Plant tissue culture offers an effective solution of such problems of propagation of fruit crops. Improvement of fruit crops through several biotechnological approaches, highly efficient regeneration is a prerequisite (Litz and Gray, 1995). Somatic embryogenesis *Corresponding author: kamle_madhu@yahoo.co.in in fruit crops is emerging as an attractive and at times indispensable adjunct to conventional plant breeding. It is an ideal system for investigation of the whole process of differentiation of plants, as well as the mechanisms of expression of totipotency in plant cells. It has several distinct advantages over the conventional micropropagation (Litz and Gray, 1992). The rapid improvement in the somatic embryogenesis methods allows extensive practical and commercial applications, particularly for in vitro clonal micropropagation. Application of somatic embryogenesis in fruit crop improvement has been limited because of poor germination of somatic embryos and low frequency of recovery of somatic seedlings. However, as a system for large-scale production in bioreactors through repetitive induction, somatic embryogenesis has attracted the attention of fruit breeders and pomologists for the improvement of fruit trees. The desirable traits for fruit tree improvement include delayed ripening, pest resistance, disease resistance, drought and cold tolerance, fruit quality, root ability of mature tissues, reduced juvenility, regular bearing habit and alteration of tree forms and architecture e.g. dwarfing and semi dwarfing capacity. Strategies that have been developed Copyright 2011 Green Earth Research Foundation

GERF Bulletin of Biosciences 2011, 2(1):54-59 55 for improving existing fruit tree cultivars have been based upon the assumption that it is possible to target cells having embryogenic competence and to subtly alter the genotype to affect one or more specific horticultural traits (Litz et al., 1992). Regeneration of fruit trees through somatic embryogenesis particularly in banana, guava, mango, papaya etc. are of great commercial importance for industry. Somatic embryogenesis: A developmental process In plant tissue culture, totipotency of a living plant cell is well known that the nucleus of every living somatic cell contains genetic information necessary to direct the development of the complete plant. Since the first observation of somatic embryo formation in Daucus carota cell suspensions by Steward et al. (1958) and Reinert (1958), the potential for somatic embryogenesis has been shown in a wide range of plant species. Somatic embryogenesis (SE) is a process in which bipolar structures resembling a zygotic embryo, develops from a non zygotic cell without vascular connections with the original tissue. Somatic embryo are used for studying regulation of embryo development but, embryogenesis is a multistep regeneration process starting with the formation of proembryogenic masses followed by somatic embryo formation maturation, desiccation and plant regeneration (Arnold et al., 2002). It is an important system where multiplication can be done at enormous rates. Somatic embryogenesis involves the production of embryo-like structures from somatic cells without gametes fusion. During their development, somatic embryos pass through stages similar to those observed in zygotic embryogenesis. It involves control of 3 consecutive steps: (i) induction of embryogenic lines from explant (ii) maintenance and multiplication of embryogenic lines; (iii) maturation of somatic embryos and conversion into viable plantlets. In somatic embryo, somatic cells develop are induced to form complete embryo similar to that of zygotic embryo (Sharp et al., 1980; Wang et al., 1990) as shown in Fig. 1. Both embryos basically undergo the same stages of development namely globular, heart shaped, torpedo, cotyledonary and mature embryos. They arise naturally in some species in a process known as direct somatic embryogenesis (Williams and Maheswaran, 1986). In contrast, somatic embryos develop from in vitro cultured cells in the process called indirect somatic embryogenesis. Somatic embryos can differentiate either directly from the explant without an intervening callus phase or indirectly after a callus phase (Williams and Maheswaran, 1986). Indirect somatic embryogenesis is the most common method to generate somatic embryos for practical uses has been described in hundreds of species. A special type of indirect somatic embryogenesis is secondary somatic embryogenesis, or repetitive embryogenesis which consists of the production of somatic embryos using somatic embryos as initial explants. Secondary somatic embryogenesis has been described in nearly one hundred species (Raemarkers et al., 1995). Although secondary embryos frequently show low conversion rates to plants, they also can be used in practical applications. Many studies have addressed on problems for control and management of the initial establishment of embryogenic lines and the subsequent conversion step (Sharp et al, 1980; Tisserat et al., 1979). The multiplication step has been comparatively less investigated although it directly contributes to the final plant yield and influences the ability of the resulting embryos to germinate and develop into growing plantlets. Significance of somatic embryogenesis In recent years, development of plant cell, tissue culture technique has a considerable potential for the improvement of several fruit trees. Somatic embryogenesis is a developmental process of somatic cells, which resembles morphologically zygotic embryogenesis. It is an important pathway for regeneration of plants from cell culture system and a method commonly used in large scale production of plants and synthetic seeds. In most of the important fruit crops, tissue culture is well established for plant regeneration via somatic embryogenesis. Many workers have emphasized somatic embryogenesis as a preferred method for genetic improvement and multiplication of valuable germplasm of a number of woody perennials (Gupta and Durzan 1987; Raj Bhansali 1990). Since somatic embryo cultures often originate from a single cell, it is an ideal system for induction of mutations as it helps in preventing chimeras. The rate of somatic embryo germination is very poor, which has become a major hurdle for large-scale plant multiplication of desirable induced mutants. The multiplication of true to type plants through somatic embryogenesis will help in propagating elite and new genotypes in shorter periods of time. As in somatic embryogenesis there is no need for separate root induction, thus the plantlet can be multiplied and acclimatized fast. It has attracted attention in plant biotechnology, because it provides useful systems to produce transgenic plants, as well as material for theproduction of artificial seeds.

56 GERF Bulletin of Biosciences 2011, 2(1):54-59 Fig 1: A pictorial description of somatic embryogenesis in guava. (A) callus induction, (B) somatic embryo induction, (C) regeneration and maturation of embryos, (D) proliferation of plantlets, (E) Rooting (F) acclimatization, (G) potting of plant in soil (H) field establishment. The process of somatic embryogenesis is not only important for the production of plants and secondary products, but also for the transgenic plants and somatic cell genetics. It plays an important role in clonal propagation. When integrated with conventional breeding programs and molecular and cell biological techniques, somatic embryogenesis provides a valuable tool to enhance the pace of genetic improvement of commercial crop species (Stasolla and Yeung, 2003). However, somatic embryogenesis has other practical applications in crop improvement (cell selection, genetic transformation, somatic hybrid and polyploid plant production), germplasm preservation, virus elimination, in vitro metabolite production, and in vitro mycorrhizal initiation. In vitro selection Tissue-cultured cell lines can be selected in vitro for resistance to various stresses. Tissue culture techniques have been widely used for breeding purposes, especially in selection for stress tolerance. Selection is done by placing a stress causing agent in tissue cultures containing dividing cells. An efficient method for obtaining plants with desired characteristics is to add a selective agent that will kill the majority of the cells (except the resistant ones) to a tissue culture. This procedure is called in vitro selection (Chawla and Wenzel, 1987). Since the in vitro unit of selection can be a single cell the selection pressure can be uniformly and reproducibly applied. Also, in vitro selection is potentially more efficient than whole plant selection. It is a source of genetic variability that gives rise through genetic modifications during the process of in vitro culture to a phenomenon called somaclonal variation. In vitro selection offers an immense potential for the quick and comprehensive generation of useful somaclones or mutants for resistance against various biotic and abiotic factors. This method has been particularly effective for selecting herbicide tolrant and disease-resistant plants. In practice, involvement of in vitro selection techniques in the crop improvement programme is very limited. Except for few, most of the reports suggest negative correlation between in vitro and in vivo responses for resistance. Genetic transformation Genetic transformation offers the opportunity for genetic manipulation of plants at cellular level and provides the means for modifying single horticultural traits without significantly

GERF Bulletin of Biosciences 2011, 2(1): 54-59 57 altering other aspects of the phenotype (Singh et al., 2004). The main target of gene transfer techniques is to produce improved varieties through the incorporation of important genes into existing cultivars (Singh et al., 2004). However, the plant transformation techniques do not transform all cells, and plants regenerated from transformed tissues via organogenesis are often chimeras. Somatic embryos arise from single cells and for this reason regeneration via somatic embryogenesis reduces the formation of chimeras. Nevertheless, somatic embryogenesis seems to produce fewer rates of somaclonal variations compared with organogenesis. Fruit trees are considered to be recalcitrant material for genetic transformation studies and the main impediment for genetic transformation is the regeneration of transformed plantlets. Choice of explants having competence for transformation and regeneration is a crucial factor. Hence, efficient tissue culture techniques become the base for genetic transformation studies (Giri et al., 2004). The successful regeneration of genetically transformed plants has been achieved in several tropical fruit crop species (Gomez-Lim and Litz, 2004). Applications of genetic transformation technology hold greater potential for fruit crops or perennials than even the herbaceous plants because the gains in genetic improvement can be realized over relatively short periods compared to routinely used techniques of hybridization, selection and mutagenesis which require decades. The present area of research depends on at least three factors that must come together for each species or genotype to be improved: (1) availability of specific genes and promoters for transfer and expression; (2) development of reliable techniques for transfer of the genes to target cells and the selection of transformed cells; and (3) regeneration of transformed cells into whole plants which can be mass-propagated, preferably by asexual means. A variety of potentially useful genes is being characterized and cloned for transfer to agriculturally important plants, many of which also will be useful for the improvement of fruit crops. Likewise, a number of different methods of genetic transformation of plants have been developed, some of which have shown applicability to woody perennials as well. Stable transfer, integration and expression of a model gene (such as NPT or GUS) have been demonstrated only in a few fruit crop species and genes regulating cellular metabolism or physiological aspects of growth and development. Somaclonal variation The purpose of tissue culture can be to preserve the genetic fidelity of the stocks; long-term tissue culture can also be used to increase useful genetic variation. Genetic variability in tissue culture-derived material, called somaclonal variation (Larkin and Scowcroft, 1981) is especially prevalent if the material is kept in a rapidly dividing, non-differentiated state, (callus or cell suspension) for an extended period. In fact, the frequency of somaclonal variation can be 10,000 times higher than spontaneous mutation rates in whole plants (Larkin and Scowcroft, 1981). Genotypic variation among regenerated plants from both somatic and gametic cell cultures (i.e. somaclonal and gametoclonal variations) has been suggested as a useful source of potentially valuable germplasm for plant breeding. In spite of many claims of the potential uses of somaclonal variation, so far there is not a single example of significantly improved new variety of any major crop species developed as a result of somaclonal variation and which is grown commercially. Molecular markers Molecular markers are especially advantageous for improving agronomic traits in perennial fruit crops that is otherwise time consuming and difficult to tag the genes conferring resistance to pathogens, insects, nematodes, tolerance to abiotic stresses, quality parameter and quantitative traits. The search for markers of plant embryogenesis is an important aspect of modern plant breeding (Schell et al., 1994). By using marker assisted selection, the breeder could select resistant genotypes molecular markers that are tightly linked to resistant genes. For the commercial utilization of process of in vitro propagation of fruit trees, it is imperative to assess genetic fidelity of the regenerants especially before their transplantation to field. Till now, several strategies have been used to assess the genetic fidelity of in vitro raised plants but, these have their own limitations. On the contrary, molecular markers facilitate the screening of in vitro raised plants with greater precision. Since, these markers are remains unaffected by the environmental factors and produce more reliable and reproducible results. Several physiological, biochemical and molecular markers associated with embryogenic competence of cells have been reported, including isozymes and molecular markers. Isozymes patterns are helpful tools for a better understanding of the basic mechanisms of cellular differentiation and further plant development. Apart from the classical studies in which

58 GERF Bulletin of Biosciences 2011, 2(1):54-59 isozymes were used as markers for events at the later stages of the growing plant (e.g. organogenesis or seed germination), during the last twenty years their usefulness as markers during early development of the plant ( i.e. early embryogenesis) was also demonstrated. Tissue culture techniques and, more specifically, somatic embryogenesis allowed the application of isozymes analysis to the embryogenic process, because they permitted the availability of relatively high amounts of plant material in the desired developmental stage. An extensive review on the use of isozymes as biochemical markers in somatic embryogenesis has been already published (Schell et al., 1994). There are several candidate genes that could be used as molecular markers of single competent cells. One of these genes, the Somatic Embryogenesis Receptor like Kinase (SERK) gene, was found to mark single Daucus and Dactylis suspension cells that are competent to form somatic embryos (Somleva et al., 2000). Potential applications of somatic embryogenesis 1) Mass scale production: Somatic embryos have a great advantage over conventional breeding of propagation. Tremendous amount of embryos can be produced from single explant. 2) Rapid Multiplication: Rapid multiplication through production of somatic embryogenesis in cell cultures, and use of bioreactors for scale-up technology. 3) Shortening of breeding cycle: The process shortens the breeding cycle of tree species and thus increases the germination of hybrid embryos. 4) Transgenics:Development of somatic embryogenesis pathway could lead to exploitation of transgenic against various biotic and abiotic stresses. 5) Molecular and biochemical: Provides important source for the analysis of molecular and biochemical events that occur during induction and maturation of embryos. Conclusion Somatic embryogenesis is a promising method for the establishment of protocols for rapid multiplication of new and elite genotypes, synthetic seed production, in vitro selection approaches for various biotic and abiotic stresses and for studies of genetic manipulation. Gene transfer into embryogenic plant cells is already challenging conventional plant breeding, and has become an indispensable tool for crop improvement. One of the most important prerequisites for genetic manipulation of plants in vitro is the ability to grow somatic cells in sterile plant growth medium and to regenerate viable plants from these cultures. Somatic embryogenesis, therefore, is a more efficient pathway for studies involving production of genetically transformed plants. References 1. Arnold SV, Sabala I, Bozhkov P, Dyachok J and Filonova L (2002). Developmental pathways of somatic embryogenesis. Plant Cell Tiss. Org. Cult. 69: 233-249. 2. Chawla HS and Wenzel G (1987). In vitro selection for fusaric acid resistant barley plants. Plant Breeding. 99: 159-163. 3. Giri C, Shyamkumar B and Anjaneyulu C (2004). Progress in tissue culture, genetic transformation and applications of biotechnology to trees: An overview. Trees-Struct. Funct. 18: 115-135. 4. Gomez-Lim MA and Litz RE (2004). Genetic transformation of perennial tropical fruits. In Vitro Cell. Devel. Biol. 40: 442-449. 5. Gupta PK and Durzan DJ (1987). Biotechnology of somatic polyembryogenesis and plantlet regeneration in loblolly pine. Biotechnology. 5: 147-151. 6. Larkin PJ and Scowcroft WR (1981). Somaclonal variation - a novel source of variability from cell cultures for plant improvement. Theor. Appl. Genet., 60: 197-214. 7. Litz RE and Gray DJ (1992). Organogenesis and somatic embryogenesis. In: Biotechnology of perennial fruit crops. (Eds. Hammerschlag FA and Litz RE) p. 3-34, CAB International, Wallingford. U.K. 8. Litz RE and Gray DJ (1995). Somatic embryogenesis for agricultural improvement. World J. Microbio. Biotech. 11: 416-425. 9. Raemakers CJJM, Jacobsen E and Visser RGF (1995). Secondary somatic embryogenesis and applications in plant breeding. Euphytica. 81: 93-107. 10. Raj-Bhansali R (1990). Somatic embryogenesis and regeneration of plantlets in pomegranate. Ann. Bot.

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