Introduction

technique to overcome some of the limitations of conventional clonal propagation. 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. Starting with the first accounts of somatic embryogenesis in carrot, there have been a steadily increasing number of reports of embryogenic induction from somatic cells in a variety of plants, initially confined to members of the carrot family (Umbelliferae) and later extending to members of angiosperm and gymnosperm families. No consolidated listing of these plants is currently available, but separate listings of herbaceous dicots (Brown et al., 1995), herbaceous monocots (Krishnaraj and Vasil, 1995), woody angiosperms and gymnosperms (Dunstan et al., 1995), and angiosperms in general (Thorpe and Stasolla, 2001) have been published in recent years.

The most promising application of somatic embryo is in the field of genetic engineering where by means of somatic embryos specific and directed changes are introduced into elite individuals. As an embryo originates from a single cell or a group of cells, plants derived from somatic embryos tend to be genetically alike (Yasuda et al., 1985). In general, the production cost of plant propagation via somatic embryogenesis is potentially lower than that of microcuttings especially when bioreactors and automation procedures are introduced in the production process. The production advantages using somatic embryogenesis include: 1) a large number of uniform plantlets can be produced inexpensively; 2) production of both root and shoot meristems occur in the same propagation step; 3) easy and quick scale-up can be achieved via liquid culture; 4) long term storage via cryopreservation can be utilized; 5) there are opportunities for use of manufactured seeds (synthetic seeds) for easy handling and direct delivery to nursery. In forestry, the production of manufactured seeds throughout the year provides a complementary technology, which will reduce risks relative to seed orchards where seed production is limited and uncertain.

one of the main limitations for successful commercial application of this technology is transferring these embryos under greenhouse or field conditions. Somatic embryos do not survive if transplanted directly from in vitro to harsh ex vitro. Germinating the embryos in vitro is yet another costly step. The production of quality somatic embryos, which have the ability to survive ex vitro, is essential for the application of somatic embryogenesis technology. Efforts can be taken while embryos are still in culture to acclimatize them for transfer ex vitro. This chapter presents a review of somatic embryogenesis for the large-scale plant propagation with emphasis on the quality improvement of somatic embryos via photoautotrophic culture. Coffee (Coffea arabusta) will be the focus of this account, since much information on the mechanism of photoautotrophic transformation of somatic embryos is available in this species (Afreen et al., 2002a and b).

2. PROCESS OF DEVELOPMENT OF SOMATIC EMBRYOS Somatic embryogenesis is a multi-step regeneration process starting with formation of proembryogenic masses, followed by somatic embryo formation, maturation, desiccation and plant regeneration. Production of somatic embryos from cell, tissue and organ cultures may occur directly which involves the formation of an asexual embryo from a single cell or a group of cells on a part of the explant tissue with or without an intervening callus phase. The embryo usually germinates and grows as a normal plant, however, functionally is a clone of the parent (same genotype). There are two systems currently used for the production of somatic embryos, they are the use of gelled medium and the use of bioreactors. Both can be used in conjunction for the production of large numbers of propagules.

Somatic embryos originate from somatic cells that contain all the necessary genes to create a complete plant. Production of somatic embryos from a single cell or a group of cells is a result of down regulating somatic gene expression and turning on embryogenic genes. Such occurrences are notable in many species for instance in coffee (Figure 1) where globular, heart-shape, torpedo shape, precotyledonary, cotyledonary and germinated somatic embryos developed from leaf disc after 14 weeks of culture. Plant regeneration via somatic embryogenesis inch

2.1. Initiation of embryogenic cultures

Culturing the primary explant on medium supplemented with plant growth regulators, mainly auxin but often also cytokinin, is the first step where embryogenic cultures can be initiated. Somatic cells within the plant contain all the genetic information necessary to create a complete and functional plant. It has been proposed that plant growth regulators and stress play a central role in mediating the signal transduction cascade leading to the reprogramming of gene expression. This results in a series of cell divisions that induce either unorganized callus growth or polarized growth leading to somatic embryogenesis.

2.2. Proliferation of embryogenic cultures

On solidified or liquid medium supplemented with plant growth regulators, proliferation of embryogenic cultures is the next step to develop somatic embryos. Once embryogenic cells are formed, they continue to proliferate, forming

Globular Heart-shape Torpedo shape

Precotyledonary Cotyledonary somatic embryos somatic embryos

Figure 1. Different stages of coffee somatic embryos

Globular Heart-shape Torpedo shape

Precotyledonary Cotyledonary somatic embryos somatic embryos

Figure 1. Different stages of coffee somatic embryos pre-embryonic masses. Auxin is required for proliferation of pre-embryonic masses but is inhibitory for the conversion of pre-embryonic masses into somatic embryos (de Vries et al., 1988a and b; Nomura and Komamine, 1985). The degree of somatic embryo differentiation which takes place in the presence of auxin varies in different species. Globular, heart-shape, torpedo and cotyledonary are the different developmental stages of somatic embryogenesis (Figure 1).

2.3. Maturation of somatic embryos

During the maturation stage, somatic embryos undergo various morphological and biochemical changes. The storage organs, the cotyledons, expand concomitantly with the deposition of storage materials, the repression of germination and the acquisition of desiccation tolerance. Somatic embryos accumulate storage products that exhibit the same characteristics as those of the zygotic embryos.

2.4. Germination or embryo-to-plantlet conversion

Somatic embryogenesis is a complex process in which the quality of the final product, i.e. the survival and growth of regenerated plants, depend on the conditions provided at earlier stages, when mature somatic embryos are formed and germinate. Therefore, in order to develop mass propagation of somatic embryo plants, a better understanding of critical factors that might contribute to ex vitro performance of plants is required. only mature embryos with a normal morphology and which have accumulated enough storage materials and acquired desiccation tolerance at the end of maturation develop into normal plants. Somatic embryos usually develop into small plants, comparable to seedlings. When the plants have reached a suitable size they can be transferred ex vitro. In many reports, it has been shown that somatic embryo derived plants grow as seed plants do.

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