Book Chapter

Somatic Embryogenesis and Gene Expression Editors: Junaid Aslam, P. S. Srivastava and M. P.
Sharma Copyright 2013, Narosa Publishing House, New Delhi
Factors Regulating Somatic Embryogenesis in Plants

Khaled Elmabrouk Suliman Elmeer
Genetic Engineering Department, Biotechnology Centre, Doha Qatar Corresponding Author: elmeer@gmail.com
Abstract
One of the most spectacular achievements in plant tissue culture has been the discovery of the induction of somatic embryogenesis in cell culture by which somatic cells develop into differentiated plants through characteristic embryological stages without fusion of gametes. These steps are accurately regulated by many factors, including plant genotype, level of sugar in the medium, type and concentration of growth regulators, photoperiod, gelling agents, time exposure and induction and maturation medium which stimulates somatic embryogenesis. This article reviews the recent work carried out on the role of various factors controlling somatic embryogenesis.
Keywords: Gelling agents, Light, Plant growth regulators, Somatic embryogenesis INTRODUCTION
Somatic embryogenesis in cell culture demonstrated the persistence of totipotency in cells of higher plants, i.e., the regeneration of whole plants from cells. These embryos are bipolar, in contrast to monopolar shoots or roots and are not connected to the vascular tissue of the mother plant or the explant during initiation and development (Merkle, 1999). With monopolar buds and roots, on other hand, it is often possible to show their connection with vascular elements of the mother plant or callus (Bajaj, 1995). Somatic embryogenesis is the process by which somatic cells develop into differentiated plants through characteristic embryological stages without fusion of gametes (Cyr, 2000). Various developmental stages of somatic embryos i.e., proembryo, globular, heart- and torpedo-shaped, are all strikingly similar to those occurring after the fertilization of the egg cell (Junaid et al., 2006). Somatic embryos are similar to the zygotic embryos that
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develop from fertilized eggs (Crouch and Gray, 1982), and the mechanisms that control somatic and zygotic embryogenesis are thought to be similar (Goldberg et al., 1994). Williams and Maheswaran (1986) suggested that somatic embryos could arise either from single cells or from groups of cells. Proof of a single cell origin would be the presence of a suspensor with somatic embryos; whereas a multicellular origin could be indicated by the fusion of the somatic embryos base with a broad area of parental tissue. Somatic embryos have always been assumed to have a single cell origin, although embryogenesis has never been demonstrated conclusively from a single isolated cell. Somatic embryos have usually been described as originating from proembryonic masses of cells that develop from single cells. In some species, histological studies have confirmed the probable single cell origin of somatic embryos when they develop directly either from explants or as secondary embryos from the surface of older somatic embryos. Somatic embryos arise by a process of cloning, which does not involve meiotic recombination events associated with fertilization and seed formation (Stuart and Redenbaugh, 1987). Various terms, such as embryo-like structures, embryoids, adventitious embryos, accessory embryos, secondary embryos, asexual embryos, somatic embryos, etc., have been applied to these structures by various authors (Bajaj, 1995). Somatic embryos may arise in vitro from three sources of cultured diploid cells: 1) vegetative cells of a mature plant; 2) reproductive tissues other than the zygote and 3) hypocotyls and cotyledons of embryos and young plantlets without intervening callus development. The phenomenon of somatic embryogenesis has now been reported in over 300 plant species that includes trees, cereal and grasses, vegetables and fruits, legumes and oilseed crops, ornamental, medicinal and miscellaneous plants (Bajaj, 1995). Secondary somatic embryogenesis is a phenomenon whereby new somatic embryos are initiated from primary somatic embryos. Such cultures have been described in at least 80 Gymnosperm and Angiosperm species.
1. SOMATIC EMBRYOGENESIS AND ITS PATTERNS

Two general patterns of embryogenic development of in vitro embryogenesis have been described: i) Direct embryogenesis, in which the embryos originate directly from tissues in the absence of callus proliferation (i.e. nucellar cells of polyembryonic varieties of citrus, epidermal cells of hypocotyl of wild carrot). This occurs through pre-embryogenic determined cells (PEDC) where the cells are already committed to embryonic development and need only be released (Cyr, 2000) i.e. requiring only growth regulators or favourable condition to allow release into cell division and expression of embryogenesis (Williams and Maheswaran, 1986). ii) Indirect embryogenesis, callus proliferation is prerequisite to embryo development i.e. secondary phloem of domestic carrot, leaf tissue explants of coffee, etc. This occurs in differentiated, non-embryogenic cells or induced
58 Khaled Elmabrouk Suliman Elmeer embryogenic determined cells (IEDC) (Cyr, 2000). Growth regulators are required not only for re-entry into mitosis but also for determination of the embryogenic state (Williams and Maheswaran, 1986).
To distinguish patterns of embryogenesis as direct or indirect based simply on intercalation of mitotic cycles between explant and embryo organization is, in physiological terms, an oversimplification. The most meaningful way to define direct and indirect appears to be with reference to the epigenetic state of explant cells. Thus somatic cells, which are embryonic, or not far removed from embryonic, are generally more easily induced to undergo somatic embryogenesis than differentiated vegetative cells. Highly differentiated cells appear to require major epigenetic changes, making the initiation of embryogenesis less direct. In this term, directness of embryogenesis is measured as epigenetic distance of explant cells from the embryonic state (Yeung, 1995).Tissue associated with zygotic embryogenesis (nucellus, integuments, proembryos, megagametophytes) can all be considered as part of a pre-embryogenic determined cell (PEDC) system. One of the important distinctions between direct and indirect embryogenesis depends on the timing of differentiation and acquisition of competence. In the former case, the cells respond to experimental treatment and become determined in a short time without prior proliferation of cells. In the latter case, a longer time is needed to acquire the embryogenic competent state. This state is often preceded by cell proliferation (Yeung, 1995). Indirect somatic embryos formation from callus or suspension culture is observed more frequently than direct embryogenesis (George, 1993). In both systems the embryogenic cells from which embryoids are visibly derived show a number of common features, which are characteristic of rapidly dividing meristematic cells. These include small size, dense cytoplasmic contents, and large nuclei with prominent enlarged nucleoli, small vacuoles and profusion of starch grains. Their histochemical and ultrastructural properties are suggestive of intense RNA synthesis and metabolic activity (Williams and Maheswaran, 1986). The division of somatic embryogensis into two patterns is useful as long as it is understood that there is no a priori method of ascertaining whether or not a cell needs to be redetermined. One example of the utility of this classification is that it delineates a possible role for plant growth regulators as agents that contribute to the determination process, but not directly to embryogenesis. Cells that become embryogenic by the PEDC pattern often requires no exogenous growth regulators, and in some cases, growth regulators inhibit initiation. Growth regulators applied to PEDCs after the proliferation of embryogenic tissue often result in the formation of a callus consisting entirely of masses of proliferating proembryos. These examples are best considered as PEDC cloning, and the role of growth regulators can be considered as agents to stimulate division that results in more PEDCs. In the induced embryogenic determined cell (IEDC) system, a distinct dedifferentiation of the callus phase occurs prior to redetermination, and the presence of growth regulators ensures callus formation with attendant redetermination.
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2. APPLICATION OF SOMATIC EMBRYOGENESIS

Regeneration via somatic embryogenesis is considered pivotal for application of cell culture methods to the genetic improvement of crop plants. In addition to the use of somatic embryogenesis as a system in basic research in plants, there are other aspects, which have attracted attention due to their potential in agricultural production and crop improvement programmes. Examples include genetic transformation of somatic embryos and the production of transgenic plants, somatic embryo-derived synthetic seed (Bajaj, 1995), micrografting of somatic embryos, fluid drilling of somatic embryo-derived plants, somatic hybridisation, metabolite production, embryo rescue (Bajaj, 1995) and cryopreservation of somatic embryos for conservation of germplasm (Chee, 1995). Somatic embryos offer some potential advantages to conventional micropropagation systems including: 1) high proliferation rate as many as 1.35 million embryos per litre of suspension culture; 2) singulation each embryo being a separate package that can be handled without the physical separation required in organogenesis or axillary branching systems; and bipolarity the well developed embryo contains root and shoot meristems, indicating that conversion to seedling can be obtained in a single step (Chee, 1995). Chee and Tricoli (1988) noted that suspension cultures would allow scale-up and mass production of cucumber somatic embryos. The use of bioreactors to this end would then be a necessity, for production of cucurbit seeds of added value. Mass production of somatic embryos and their encapsulation may lead to commercialisation of synthetic seeds (Chee, 1995). Somatic embryogenesis can be utilized as a regeneration technique for cell selection of natural or induced mutation. Selection for salt tolerance and disease resistance has proven efficient in embryogenically competent callus tissue of citrus (Janick, 1993). Somatic embryos may be an economical means for automated mass production of elite plant varieties such as F1 hybrids, if true-to-type multiplication is confirmed. This technique would also enable micropropagation of sterile plants like haploids, triploids, male sterile genotypes and even interspecific hybrids where zygotic embryo development is poor. Because of the absence of vascular connections between the nucellus and other maternal tissues, polyembryonic species are generally free of infection that might have affected the parent plant. Similarly, plants derived via somatic embryogenesis from the nucellus or nucellar callus would also be free of pathogens including viruses (Janick, 1993). Somatic embryos could be valuable for the screening of their ability to grow in the presence of pathotoxin, herbicides or stress conditions (Debeaujon and Branchard, 1993).They may also be used to regenerate plants from transformed tissue or after somatic hybridization, particularly if embryos originate from a unique cell. The discovery that microspores can undergo embryogenesis to form haploid embryos has provided a method for generating haploid in large numbers. Subsequent chromosome doubling of haploids derived from diploid (i.e. monoploids) produces homozygous diploids useful for selection as inbreds in the production of F1 hybrids or for the
60 Khaled Elmabrouk Suliman Elmeer isolation and characterization of homozygous mutant cell lines. Microspore embryogenesis occurs either directly from the haploid microspore or indirectly from multicellular haploid callus (Janick,1993).The continuous proliferation of somatic embryos via secondary embryogenesis has several possible applications such as: 1) preservation of embryogenic lines of woody species, 2) rejuvenation of mature trees, 3) production of somatic embryos in which zygotic embryos contain commercially important metabolites and 4) the possibility for large scale multiplication of plant material (Raemarkers et al., 1995). Automation could enhance the use of somatic embryogenesis for micropropagation in two ways: as an effective tool for research in somatic embryogenesis, and for improving the efficiency of embryo production by reducing labour costs (Ibaraki and Kurata, 2001). 3. FACTORS REGULATING SOMATIC EMBRYOGENESIS
Most important requirements for the successful induction of embryogenic callus and suspension cultures is the capability of the plant genotype to undergo embryogenesis on the chosen system of induction (medium plus added growth regulators). In some genera most genotypes are competent, but in others there may be a wide variation in competence even between different varieties or cultivars within a species. The level of sugar (e.g. sucrose or glucose) in the medium may need to be within critical concentrations and a supply of reduced nitrogen is required (George, 1993). Cultures should be grown in the presence of an auxin for the initiation of embryogenesis stage I (induction stage). After the beginning of embryogenesis, it is usually (but not invariably) necessary for stage I tissues or cells to be subculture to a medium containing a reduced auxin concentration, or containing no auxin at all- stage II (maturation stage) (Nomura and Komamine, 1985). There may be an optimum length of time during which the stage I routine should be maintained. An extended period before subculture can result in the failure to obtain stage II embryogenesis (George, 1993).
3.1. Genotype
The growth of cultured tissues or organs, and morphogenesis in vitro are more influenced by genotype than by any other factor. There are numerous examples in the literature in which the results obtained during tissue culture have varied from one variety of plant to another (Stipp et al., 2001) and it is probably true to say that effects of genotype impose one of the greatest constraints to the tissue culture and micropropagation of the plants (George, 1993). Genotype effects may interact with media or environmental conditions (Raimondi et al., 2001). Raimondi et al. (2001) noted in Asparagus officinalis L. the interaction between genotype, explant type and time of culture initiation were significant for callus induction and growth during the two initial culture stages. Plastira and Perdikaris (1997) showed an interaction between genotype and regeneration ability in tomato. Hanzel et al. (1985) in their research on barley reported relatively strong genotype by medium interactions for callus initiation.
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Variety A may undergo morphogenesis in response to growth regulators of one kind, while variety B is unresponsive until the concentration of the regulators is changed and/or different regulatory compounds are used, Because of genotypic specificity, media and the cultural environment often need to be varied from one genus or species of plant to another. Although general methodologies can be established for plant tissue culture, even closely related varieties of plants can differ in their cultural requirements. The best method of micro-propagating a new plant must usually be determined by experiment (George,1993). Nevertheless, it must be emphasized that manipulation of exogenous factors can sometimes foster somatic embryogenesis in recalcitrant genotypes (Ammirato, 1989). The effect of genotype is also seen in differences in the texture and colour, and morphogenic capacity of callus from closely related varieties of plants. The rate of callus proliferation from genetically similar plants may not, however, always be a guide to the morphogenic capacity of the callus, the ability of closely related plants to produce somatic embryos directly from explants is also under genetic control and differences between varieties are often found (George, 1993). Inheritance studies have shown that capacity for somatic embryogenesis in some species is heritable and probably controlled by at least two loci (Brown et al., 1995). Alexandrova and Conger, (2002) identified two somatic embryogenesis related genes designated DGE1 and DGE2 in for Dactylis glomerata). RNA blot analysis showed that the DGEs were expressed in embryogenic but not in nonebryogenic leaf cultures. DGE1 transcripts were detected in leaf cultures induced by direct and indirect embryogenesis, while DGE2 was found only in leaf cultures induced by direct embryogenesis. NadolskaOrczyk and Malepszy, (1989) suggested a genetic determinism in the ability to regenerate somatic embryo-derived plants from cucumber (Cucumis sativus L.) leaf explants; the intermediately and intensively regenerating lines contained two pairs of dominant genes responsible for in vitro plant regeneration, characterized by complementary and probably additive interactions. The frequently regenerating line differed from the intermediately regenerating line due to the effect of one gene. It was supposed that these genes belonged to three different loci. Moreover, the ability of leaf explants to regenerate plants had high heritability. Oridate and Oosawa (1992) also noted a significant genotype effect in somatic embryogenesis from melon (Cucumis melo L.) seeds. Transfer of the frequency of somatic embryogenesis from superior responding cultivars to inferior cultivars was demonstrated. The mode of inheritance of embryogentic capacity was difficult to determine due to a large variation in morphogenetic response from F2 seeds, but cytoplasmic effects were observed in certain crossings. Although systematic screenings of genotypes are rare, there is no doubt that genetic factors play a major role in somatic embryogenesis competence (Oridate and and Oosawa, 1992).
3.2.
Explants Type
The choice of explant can have a great influence on the success of an embryogenesis protocol (Brown et al., 1995). Explant size, age, and the manner in which it is cultured,
62 Khaled Elmabrouk Suliman Elmeer can all affect whether tissue culture can be successfully initiated, and whether morphogenesis can be induced (George, 1993), Significant differences in embryogenic response have been observed among mature vegetative tissues (Williams and Maheswaran, 1986) and among young tissues (Chee, 1995). The relative responsiveness of various immature tissues seems to be species-specific; for example, hypocotyls produced more embryos than seeds or cotyledons of cotton (Trolinder and Xhixian, 1989), while cotyledons were more embryogenic than hypocotyls in cucumber (Chee, 1995). In general, mature vegetative tissues are more convenient and more readily available than immature or floral tissues. Among herbaceous dicots, leaves and petioles are often used, with shoot tips, stems and roots being used to a lesser degree (Brown et al., 1995). Levels of endogenous growth regulators can vary among organs, and likely affect embryogenesis (Carman, 1990). Nolan et al. (1989) observed significantly greater embryogenesis in explants taken from regenerated plantlets of Medicago truncatula than in those from the original plant. Somatic embryogenesis and plant recovery are obtained from numerous sources including protoplasts, but the best results are observed with explants coming from seedlings, especially cotyledons and hypocotyls (Debeaujon and Branchard, 1993). Diverse types of explants are leaf, cotyledon, hypocotyls, stem, shoot tip, internode, root, cultured anthers, mesophyll protoplasts, cotyledonary protoplasts, immature embryos, leaf protoplasts, cell suspensions from leaf callus, seed, zygotic embryos and transformed roots or hypocotyls were able to undergo somatic embryogenesis. 3.3. Media
One of the other important factors governing the growth and morphogenesis of plant tissue in culture is composition of the culture medium. A suitable medium for initiation and maintenance of callus can only be obtained by trial and error (Allan, 1991). Somatic embryos have been grown on a range of media from the relatively dilute Whites medium (White, 1963), to the more concentrated formulations of Gamborg et al. (1968) (B5), Schenk and Hildebrandt (1972) (SH), and Murashige and Skoog (1962) (MS). The B5, SH, and MS media are all classified as high salt media, and MS, in particular, has about ten times the salt concentration of Whites medium. In their survey of somatic embryogenesis in crop plants, Evans et al. (1981) noted that 70% of the explant was cultured on a MS medium or a modification of MS. A key element of this medium is the presence of high level of nitrogen in the form of ammonium nitrate (Brown et al., 1995), the benefit of this for both embryo initiation and maturation is well established (Wetherell and Dougall, 1976). The major components for culture media for somatic embryogenesis should provide the following: 1) a carbon and energy source, 2) inorganic macro- and micronutrients with potassium and iron EDTA, 3) a level of reduced nitrogen, 4) organic compounds vitamin and amino acids, 5) growth regulators such as auxin and cytokinin (Brown et al., 1995).
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3.4. Carbohydrate Source

A number of carbohydrates have been used in tissue culture. Sucrose and glucose are the best general carbon sources for growth of cultures (Evans et al., 1981). Other carbohydrates could substitute for sucrose, or glucose, depending on the species and the plant, or tissue, used (Junaid et al., 2008). Sucrose alone at 0.25M induced greater somatic embryogenesis than glucose at the same concentration and the specific sucrose effect is more beneficial to cucumber somatic embryogenesis. A specific carbon effect is one of the main and critical stimuli for cucumber embryogenesis and also the osmotic potential provided by sugar has a promotive effect but of a lesser magnitude than the specific carbon effect. Somatic embryogenesis from expanded cotyledons of melon was not induced in medium without sucrose, suggesting that the addition of sugar plays an important role as an energy source and might be essential for somatic embryogenesis. Sucrose on a molar basis was more effective than any other sugar for somatic embrygenesis from expanded cotyledons (Nakagawa et al., 2001). Eapen and George (1993) noted in peanut that amongst the different sugars, sucrose was most favourable for somatic embryogenesis, producing the highest frequency and average number of somatic embryos. Sucrose, maltose, glucose and fructose induced embryogenesis from pea apices particularly at higher concentration and a 3-4-fold increase in embryogenesis was observed when fructose was used at 504 mM (Loiseau et al., 1995). Maltose was the optimum carbon source in achieving somatic embryogenesis from barley microspores (Scott and Lyne, 1994), wheat anthers (Orshinsky et al., 1990), alfalfa petioles (Strickland et al., 1987), and Hevea brasiliensis (Blanc et al.,2002), but it was not beneficial in cucumber and peanut (Eapen and George, 1993). Measurement of sugar concentration in the culture media indicated that sucrose was more rapidly hydrolysed than maltose and the regulation of endogenous hexose contents at a low level, through slow maltose hydrolysis, was a key element of the biochemical signal-leading callus towards somatic embryogenesis (Blanc et al., 2002). In citrus, sucrose and galactose were found to be equally effective (Cabasson et al., 1995). In wild carrot suspensions, embryo number was not affected by carbohydrate source. In nature, carbohydrates are transported within plant tissue mainly as sucrose and tissue may have an inherent capacity for uptake, transport and utilisation of sucrose (Eapen and George, 1993). Other reports showed the possibility of obtaining somatic embryogenesis with various sugars, such as galactose in citrus (Cabasson et al., 1995), fructose in pea (Loiseau et al., 1995) and glucose, maltose, fructose and sucrose in alfalfa (Strickland et al., 1987). The inhibitory effect of fructose on callus formation was reported in carrot root culture (Stehsel and Caplin,1969). By increasing the sucrose concentration in Daucus carota and Sium suave somatic embryo culture, precocious germination was prevented (Ammirato,1989); elevated sucrose concentration could also change the morphogenic regeneration patterns inducible by IAA. In the presence of IAA at a given concentration (57.1M) and with sucrose at 131mM, only adventitious shoot formation occurred, whereas 263mM sucrose induced both shoot organogenesis and somatic
64 Khaled Elmabrouk Suliman Elmeer embryogenesis. Raising sucrose to 394mM resulted in somatic embryogenesis only and increased somatic embryo formation to about 70%. Giving osmotic stress without hormonal treatment could induce somatic embryogenesis in carrot. When apical meristems of carrot seedling were cultured on hormone-free MS medium with 0.7M sucrose or 0.25-1.00mM cadmium ion, then transferred to hormone-free MS medium with 0.1M sucrose, somatic embryos were formed on the surface of the explant without visible callus formation (Kamada et al., 1989). High concentrations of a given carbohydrate inducing a high frequency of somatic embryogenesis may therefore be through either specific carbon or osmotic potential effects. Osmotic potential from elevated sucrose concentration may affect somatic embryogenesis (May and Trigiano,1991); Elevated sucrose is beneficial for inducing embryogenesis and reduces the harmful effect of higher 2,4-D concentration on normal embryo formation. The role of sucrose in inducing high somatic embryogenic frequency is mainly due to the specific carbon effect, which may be related to the source of carbon supply to plant cells or to nutritional requirements. Glucose and sucrose have specific carbon effects and also act as osmoregulators. Sucrose in the medium partially acts as a carbon energy source and partly as an osmoticum (Brown et al., 1979). Both mannitol and glucose were therefore better than sucrose as osmoregulators, and from these, it is evident that when initiation media contain sucrose at high concentration, either glucose or mannitol can replace part of the sucrose. In hypocotyl explants of flax (Linum usitatissimum L.) MS medium supplemented with the monosaccarides glucose, or fructose, at high concentration (4%) consistently gave highly embryogenic cultures, with higher somatic embryo frequency and higher growth rates when compared with media supplemented with sucrose or maltose (Cunha and Fernandes-Ferreira, 1999). The combination of 5% sucrose in embryo induction medium and 5% fructose in subculture medium gave the highest germination rate (Levi and Sink, 1990). 3.5. Amino Acids and Vitamins
Cultured cells are normally capable of synthesizing all of required amino acids yet, the addition of amino acid or amino acids mixture may be used to stimulate cell growth and facilitate plant regeneration. Growth of plant callus tissue was significantly stimulated by the addition of amino acids specifically glutamine. This stimulation suggested that organic nitrogen was a growth-limiting factor in plant tissue cultures. The inclusion of glutamine decreased the culture lag phase, which indicated that glutamine was much more readily assailable than inorganic nitrogen. Glutamine plays an important role in nitrogen assimilation as it is an intermediate in the transfer of ammonia into amino acids. Supplementing date palm culture media with organic nitrogen, especially glutamine, in high concentrations improved callus induction and rate of growth and many stimulate embryogenesis in such a manner similar to other plants (Abo El-Nil, 1989). Glutamine supports the growth of cells that have high energy demands and synthesize large amounts of proteins and nucleic acids. It is an alternative energy source for rapidly dividing cells and cells that use glucose inefficiently. Cells require nitrogen
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atoms to build molecules such as nucleotides, amino acids, amino-sugars and vitamins. When glucose levels are low and energy demands are high, cells can metabolize amino acids for energy. Glutamine is one of the most readily available amino acids for use as an energy source and it is a major source of energy for many rapidly dividing cell types in vitro. L-glutamine can serve as the sole source of nitrogen which can be taken more rapidly than inorganic nitrogen (Thorn et al., 1980). Casein hydrolysates can be a source of calcium, phosphate, several microelements, vitamins and most importantly, a mixture of up to 18 amino acids. Several casein hydrolysates are available commercially but their value for plant tissue culture can vary considerably. Acid hydrolysis can denature some amino acids and so products prepared by enzymatic hydrolysis are to be preferred. The best can be excellent sources of reduced nitrogen, as they can contain a relatively large amount of glutamine. Casein hydrolysate overcomes the shortage of glutamine when there is insufficient phosphorus for adequate biosynthesis however several investigators have concluded that casein hydrolysate itself is more effective for plant culture than the addition of the major amino acids. This has led to speculation that casein hydrolysates might contain some unknown growth promoting factor (George et al., 2008). The effect of casein hydrolysate (amino acids mixture) as a sole nitrogen sources on callus growth was reported by Heimer and Filner (1970) on tobacco callus. Zenk et al., (1975) found that, when cultured medium was supplemented with casein hydrolysate at level greater that 4 gl-1, callus growth was stimulated in Morinda citrifolia. Casein hydrolysate was used as a sole nitrogen source for beans (Crocomo et al., 1976), carrot (Wetherell and Dougall, 1976) and fenugreek (Singh et al., 1981). Cardi and Monti (1993) found that, the addition of casein hydrolysate at 2gl-1 is important for callus production in pea and kidney bean. Vitamins are nitrogenous substances required in trace amounts to serve catalytic functions in enzyme systems. Plant cells grown in vitro are capable of synthesizing essential vitamins in suboptimal quantities; thus, culture media are often supplemented with vitamins to enhance growth. Various standard media formulations and modifications thereof show wide differences in vitamin composition (Bhojwani and Razdan, 1983). Thiamine (vitamin B1) is generally considered to be an essential ingredient for plant tissue cultures and is usually added at 0.1-5 mgl-1. Biotin (vitamin H) is less common in culture media and is usually added at 0.01-1 mgl-1 (Bhojwani and Razdan 1983). Thiamine is an important cofactor in carbohydrate metabolism, and biotin is important in carboxylation reactions. Both thiamine and biotin biosynthesis pathways utilize the transfer of sulfur from cysteine to cofactor precursors (Begley et al., 1999). The role of vitamins in callus induction and plant regeneration has not been defined. Instead, culture media of existing regeneration systems are supplemented with arbitrarily selected vitamins at variable concentrations, including inositol, calcium pantothenate, nicotinic acid, pyridoxine, thiamine, and biotin (Omar et al., 1992). Specific media components such as amino acids and vitamins have been found to exert a profound effect on tissue culture systems of certain species and optimization of such compounds can stimulate regeneration in recalcitrant cultivars (Benson, 2000).
66 Khaled Elmabrouk Suliman Elmeer 3.6. Growth Regulators

Plant growth regulators are of major importance and a fine balance between auxin and cytokinins may be crucial in establishing cultures and maintaining callus (Allan, 1991). For culture derived from differentiated tissues growth regulators in the medium especially auxins, or auxins in combination with cytokinin, appear essential for the onset of growth and the induction of embryogenesis (Fujimura and Komamine, 1980). The processes of somatic embryogenesis can be divided into different phases; each of them probably has its own specific hormonal requirements. Auxin is known as the essential growth regulator in the induction of embryogenic callus. Of all the auxin, or auxin-like plant growth regulators, 2,4-D has proven extremely useful (Ammirato, 1989). The most characteristic effect of cytokinins on cultured plant cells is their influence on cell proliferation, and differentiation. Fujimura and Komamine (1980) demonstrated that cytokinins are important for differentiation of the embryogenic organs in Daucus carota. Although cytokinins have sometimes been incorporated into induction medium, they are probably not crucial for success. It is known that the type of morphogenesis largely depends upon the ratio and concentration of auxins and cytokinins present in the medium. Regeneration starts with a period of differentiation during which the tissue becomes competent to respond to the embryogenic stimulus (Dudits et al., 1991). This phase required a high level 2,4-D vary in concentration from 2mgl-1 in Bunium persicum to 100mgl-1 in date palm. During the next phase, induction callus became committed to form embryos, this phase required a lower concentration of 2,4-D. For development of embryos, 2,4-D had to be removed. Kwack and Fujieda (1988) obtained direct somatic embryogenesis in C. moshata nucellus without any growth regulators. They explain this striking phenomenon by occurrence of pre-embryogenic determined cells in the explant. Their hypothesis was supported by histological studies. The mode of regeneration seemed to be dependent upon 2,4-D concentration such that a high concentration led to somatic embryogenesis while a lower concentration induced caulogenesis. There were similar reports for melon (Tabei et al., 1991). Debeaujon and Branchard, (1993) observed that squash embryo abnormalities were related to the 2,4-D concentration in the induction medium. Indeed, squash embryos obtained in medium with a concentration of more than 7mgl-1 2,4-D showed development abnormalities. According to Bergervoet et al. (1989), the use of NAA in suspension media instead of continuous 2,4-D changed the pattern of morphogenesis into caulogenesis rather than embryogenesis. The combination of 1mgl-1 2,4-D and 0.1mgl-1 BAP was efficient for induction of somatic embryogenesis in melon calli (Debeaujon and Branchard, 1993) Somatic embryo maturation was commonly accomplished with growth regulator-free media, although cytokinins (Chee, 1995) or rarely, ABA or gibberellins (Tabei et al., 1991) were also used. An exception to this rule was found for squash tissue where exogenous auxin was permanently required for induction and continuous development of embryos. Debeaujon and Branchard (1993), noted that BAP reduced squash embryogenesis efficiency; BAP was the most frequently
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used cytokinin for both melon and cucumber. Nevertheless, TDZ was found to be superior to BAP for the induction phase in culture of melon (Gray et al., 1993) and watermelon (Citrullus lanatus Thunb.) Cytokinins were required, except for squash, sometimes required for growth of embryos into plantlets (Kavathekar et al., 1978).
3.7. Period of Exposure to Auxin

One hypothesis to explain how auxin may influence the ability of cells to give rise to somatic embryos was put forward by Okkels (1988), who suggested that auxin brings about the demethylation of DNA in dividing cells, and that this is a requisite for proembryo formation. In contrast, embryo development was thought to require the methylation of DNA, which becomes possible in pro-embryos when auxin is withdrawn. There may be an optimum length of time during which the stage I routine should be maintained. An extended period before subculture can result in the failure to obtain embryogenesis at stage II. Maintenance of the cultures on high auxin usually causes embryo development to arrest or a loss of embryogenic capability (George, 2008). There is a relation between the rate of growth of cells in culture and their capacity for embryogenesis. Generally, slow growing cells retain their embryogenic capacity longer than fast growing cells, and treatment which decelerates growth such as transferring tissue to a less enriched medium, or removing the hormonal components of the medium, prolong this capacity. Embryogenic callus tissue of some species can be subcultured for a prolonged period on a medium containing auxin, but still retains the capacity to produce somatic embryos, which can develop into plantlets when the tissue is transferred to an auxin-free medium. Embryogenic callus derived from Corylus avellana zygotic embryos could, for example, be subcultured over two years without losing its morphogenic potential. In other cases prolonged culture on a medium containing growth regulator levels sufficient to induce the formation of embryogenic callus can result in the callus losing its capacity to produce embryos. This incapacity is hastened by exposure to high auxin levels. Nodular embryogenic callus of Saccharum began to lose its morphogenic potential after 12 weeks culture on medium containing 7mgl-1 2,4-D. After 20 weeks on such a medium the number of plants obtained per unit weight of callus had decreased to about one third of those obtained 8 weeks previously, and after a further 20 weeks, the ability of callus to produce plants had almost disappeared (Guiderdoni and Demarly, 1988). Regeneration starts with a period of dedifferentiation during which the tissue becomes competent to respond to the embryogenic stimulus (Christianson, 1987). Prolonged culture on the stage I medium resulted in callusing of already formed embryoidal primordia. The optimum culture period on the stage II medium did not exceed 14 days, in most cases, in genus Cucumis. This period was necessary for greening and enlargement of the embryoids, but if exceeded, resulted in browning and death of the explants (Trulson and Shahin, 1986). A progressive decrease in embryogenesis usually resulted when plant tissues are maintained in vitro for prolonged periods through repeated subcultures. This decline has
68 Khaled Elmabrouk Suliman Elmeer been attributed, at least partly, to genetic changes, a reduction in the proportion of diploid cells and an eventual attainment of aneuploidy has been associated with the decline phenomenon. More research is needed to determine the optimum stage of embryo development for removal of 2,4-D (Cade et al., 1990). Reducing the period of exposure to auxin to 7 days by transferring microshoots to auxin-free medium increased the frequency of root formation (84%), and led to more rapid root formation and a reduction in basal callus formation of Quercus robur L. (Puddephat et al., 1999). In gerbera the effectiveness of shoot regeneration from petioles depended on the duration of periods on the induction medium and the duration of the regeneration period. 3.8. Gelling Agents
Gelling agent type and concentration had a significant impact on the induction of somatic embryogenic tissue. Because it is usually desirable for tissue explants to be in contact with but not submerged in the culture medium, solid or semisolid media are often used. Tissues cultured on such media maintain good contact with the media but also have good aeration. In the literature, several gelling agents have been used such as agar, gelatine, starch-derivative gels, silica gel and gelrite, however, agar remains the most commonly used gelling agent. Gelling agents were found to influence callus formation and somatic embryogenesis. In cucumber, agar was superior to gelrite in inducing somatic embryos from cotyledon explants. Gelrite was used for its superiority to other gelling agent, as it gave best results for maintenance of embryogenic lines in grape (Perl et al., 1995). In other crops gelrite had a negative effect as it produced vitrified shoots in white ash Debeaujon and Branchard, (1993) working on melon, obtained their best results on solid media. Improvement in the percentage of embryogenic calli was noted (75% instead of 33%) when gelrite was used place of agar. They reported that culture in liquid medium gave rise to vitreous plantlets, which differs from the results of Oridate and Oosawa (1986) where normal plants were obtained from melon suspension cultures. Use of 0.3% phytagel instead of agar enabled the formation of embryogenic calli in squash. Kwack and Fujieda (1988) noted an inhibitory effect of agar (proportional to its concentration) on C. moschata embryogenic response. As a means of reducing abnormal growth in cucumber, Ziv and Gadasi (1986) suggested the use of double-layer culture with activated charcoal in the lower agar layer and ABA in the top liquid layer. This technique increased the frequency of regenerated plantlets and improved their morphological development. In Poa pratensis L. on the medium solidified with gelrite, regeneration frequency of the callus was twice as high as on agar-solidified medium (Ark et al., 1991). In Pinus strobes L. regardless of the type of gelling agent used, gel strength increased with gelling agent concentration and was critical to the maturation response. High gel strength was associated with reduced water availability from the medium to the culture. The water potential of gelled maturation medium remained constant between 0.4 and 1.0% gellan gum. It was concluded that the embryogenic tissue was exposed to varying amounts of water at the onset of and during the culture period,
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and that the amount of water in the culture environment in turn influenced the maturation response. Elmeer and Hennerty (2008) found that Kinetin yielded low numbers of somatic embryos compared with BAP in the presence of agar, while in gelrite the kinetin was superior to BAP,the results indicate that there was an interaction between the cytokinin and the gelling agents in the induction stage and that BAP requires agar, while kinetin requires gelrite in order to maximise somatic embryos. Sansberro et al. (2001) found interactions between type of agar and plant regeneration of Ilex paraguariensis.
3.9. Photoperiod
The light requirements of tissue cultures are not the same as those of autotrophically developing whole plants. In tissue culture photosynthesis is not a necessary activity, except perhaps during the stage of preparation for reestablishment of plants in soil, although light is needed to regulate certain morphogenetic processes (Murashige, 1974). It has been reported to be important for the formation of shoots, the initiation of roots, the differentiation of cladophylls (Murashige, 1974), and in asexual embryogenesis. Somatic embryogenesis has occurred under a variety of light/dark regimes. In one case, Nicotiana tabacum, high light intensities were required for somatic embryogenesis. In other cases, Daucus, (Michler and Lineberger, 1987) and Carum, (Ammirato, 1989) embryo maturation proceeded more normally in complete darkness. The optimum requirement with respect to illumination of cultures varies among plants. Equal rates of asexual embryogenesis have been attained in darkness and under lowintensity light with tissue culture of some species, while other species producing asexual embryos only when provided with light (George, 1993). Callus growth does not take place in some plants unless light is excluded from cultures, for example Hosta scape explants only produce callus in the dark. In other species, light may be necessary for callus initiation, but once callus has been obtained, it can be subcultured and maintained in the dark (George, 1993). Croke and Cassells (1997) in a study on zonal geraniums (Pelargonium X hortorum Baily) found that induction in the dark resulted in a 2-3-fold increase in the percentage of explants responding and in an approximate doubling of the number of somatic embryos per explant; the response was genotype-dependent. Elmeer and Hennerty (2008) found a strong relationship between NAA and light and between the 2,4-D and the dark. 2,4-D alone added in the induction phase in the presence of light induced little embryogenesis, while, if combined with NAA, somatic embryos were produced. When the tissues were grown in the dark, the situation was completely different: concentrations of 1 and 2 mgl-1 2,4-D alone permitted the development of a greater number of somatic embryos, while the presence of NAA generally inhibited the embryogenic response. It was possible to induce somatic embryos with a combination of either NAA + light or 2,4-D + dark.
70 Khaled Elmabrouk Suliman Elmeer 3.10. Sodium Chloride

Several experiments have been carried out to determine the effect of sodium chloride (NaCl) on tissue multiplication and somatic embryo regeneration. Osmotic stress provided by mannitol or by NaCl, can be an important factor for conditioning embryogenic suspension cultures of the hybrid Carica papaya X C. cauliflora. Initially exposing embryogenic cultures to 300mM mannitol or 180mM NaCl, and then subculturing these suspensions into media of lower osmolarities can double the efficiency of somatic embryogenesis in comparison with untreated cultures. The presence of NaCl during embryogenesis of Shamouti orange (Citrus sinensis (L.) Osbeck) affects normal growth regulator balance. First, NaCl can replace cytokinin or abscisic acid in the greening process and second, it enhances the requirement of gibberellic acid for normal heart-shape embryo formation. The replacement of abscisic acid could be explained on the basis of NaCl-induced increased internal abscisic acid concentration (Ben-Hayyim and Goffer, 1989). The frequency of somatic embryo formation in wheat increased with increasing NaCl concentration and showed a maximum formation rate at 40mM. No differentiation was observed above 85mM (Galiba and Yamada, 1988). NaCl (10mM) consistently enhanced the quantity of cucumber tissue produced during multiplication (22.8g/100mg primary tissue compared to 15g/100mg primary tissue) and significantly enhanced the number of embryos that regenerated and grew well in soil (46 seedling/100mg primary tissue compared to 7 seedling/100mg primary tissue). Both callus induction and the growth of the primary callus in wheat tissue were suppressed by NaCl concentrations in excess of 170mM in the medium. In addition the NaCl suppressed the differentiation of shoot apices, but did not prevent the enlargement of the somatic embryoids, which resulted in the formation of more typical embryoids. The salts inhibited plantlet formation from somatic embryoids so the salts supplement should be omitted (Galiba and Yamada, 1988). In rice cultivars NaCl strongly decreased the regeneration frequency but slightly increased the survival rate of regenerated plantlets. Addition of up to 40mM NaCl to basal callus proliferation medium of sweetpotato (Ipomoea batatas (L.) Lam., did not affect callus proliferation, the development of embryos from calli subcultured to embryo production basal medium was unaffected by the KCl or NaCl treatments of the callus proliferation phase (Chee,1995).
4. CROSS FEEDING AND CO-CULTURE

Several lines of evidence suggest that cells in a plant use cell-cell communication to control the differentiation process. For example, a cell in a suspensor can develop into an embryo if the embryo proper is aborted (Yeung, 1995), and a cell that enters a new cell layer differentiates according to its new position (Van den Berg et al., 1995). The mechanism that controls cell differentiation in plants is not understood, although there is evidence that soluble signal molecules are involved (Dudits et al., 1991; Nishiwaki et al.,
71
2000). Somatic embryogenesis is the process whereby cells in a suspension culture redifferentiate and give rise to cells that can form somatic embryos, and cell-cell interactions also occur during this developmental process. For example, cells cultured at low cell density cannot form somatic embryos, but cells cultured at high density, or at low density in the presence of a cell-free growth medium preconditioned by highdensity suspension culture, can undergo somatic embryogenesis (de Vries et al., 1988). Conditioned growth medium from an embryogenic suspension culture can also stimulate somatic embryogenesis in a nonembryogenic suspension culture (Hari, 1980), also, the non embryogenic cells present within a mixed culture system were essential to the formation of embryogenic aggregates (Ogita et al., 2001). Mixed cultures may initiate hereditary changes. This ability of conditioned medium to sustain or stimulate somatic embryogenesis shows that soluble signal molecules are involved with cell-cell interactions in suspension cultures (McCabe et al., 1997). Cross-feeding experiments in which arabinogalactan proteins (AGPs) from embryogenic suspension cultures cause cells in nonembryogenic suspension cultures to become embryogenic (Kreuger et al., 1995) or allow immature somatic embryos to progress and develop into mature somatic embryos (Egertsdotter and von Arnold, 1995) suggest that the AGPs may have signaling properties (McCabe et al., 1997), and that the cell wall may play an important role in this process (Wojtaszek, 2000). Dyachok et al. (2002) suggested that endogenous lipophilic chitin oligosaccharide (LCO) acts as a signal molecule stimulating PEM and early embryo development in norway spruce. During zygotic embryogenesis, SERK (somatic embryogenesis receptor kinase) expression was detectable transiently in young zygotic embryos of up to 100 cells. These results demonstrate that competent cell formation and early somatic embryogenesis require a highly specific signal transduction chain also found during zygotic embryogenesis. Whether both examples of signal transduction chains are related is not known (Vries, 1999). Elmeer and Hennerty (2008) concluded that one cultivar of cucumber can influence the performance of another when they are co-cultured in a single medium The Mascot explants when cultured alone in the liquid medium gave a mean weight of 160.2 mg callus, while when cultured with Profito explants in the same medium they gave a mean weight of 225.5 mg callus, on increase of 40%. When profito explants were cultured in the liquid medium alone they had a mean weight of 257.2 mg callus, while when cultured in the same medium with Mascot explants, they gave a mean weight of 230.3 mg callus, which was a decrease of 10%.
5. REGENERATION THROUGH BIOREACTORS

Producing plantlets from suspension culture via somatic embryogenesis may be a useful alternative to traditional propagation systems. Bioreactor systems are well suited to this purpose as they are designed to precisely regulate several environmental parameters such as temperature, pH and dissolved oxygen, or other gases (Jay et al., 1994).The application of bioreactors in plant propagation is still a novel field. Recent experience is
72 Khaled Elmabrouk Suliman Elmeer restricted to a small number of species that, however, demonstrate the feasibility of this technology (Preil, 1991). Bioreactor culture offers promising aspects for mass propagation of species, which are able to develop somatic embryos in suspension culture and express low somaclonal variability (Preil et al., 1991). Other reports on generation of somatic embryos in bioreactors include carrot (Jay et al., 1994; Teng et al., 1994), alfalfa (Stuart and Readenbough, 1987), potato mini tubers (Akita and Takayama, 1988) and lettuce shoots (Teng et al., 1994). 6. SYNTHETIC SEED TECHNOLOGY
The synthetic seed (also referred to as artificial or somatic seed) technology is one of the most rapidly developing fields of plant science in the last decade. The synthetic seeds are generally prepared by encasing the somatic embryos obtained from tissue culture in a protective jelly capsule, which is usually prepared with sodium alginate, or by desiccating the somatic embryos with or without a coating (Kumar, 2000). To use artificial seeds as a cost-effective propagation method, high-frequency conversion of somatic embryos to vigorous plants and good stand establishment must be achieved under non-sterile conditions (Fuji et al., 1993). Alternatively it must be possible to produce transplants under controlled environment situations (Janick et al., 1993). The conversion rate of synthetic seeds to plants, which has generally been rather poor thus far, must be improved and brought to acceptable levels before they can become a commercial reality (Vasil, 1994). In order for somatic embryos to be useful as a low-cost, high volume propagation system, a delivery method is required. Four delivery system for somatic embryos have been proposed (Janick et al., 1993): 1) fluid drilling of somatic embryos, 2) encapsulation of somatic embryos in an alginate gel 3) desiccation of somatic embryos and (4) desiccation of encapsulated somatic embryos using water soluble resin. Potential applications of synthetic seeds vary from crop to crop depending upon the relative sophistication of existing production systems and the opportunities for improvement (Gray et al., 1995). Artificial seed technology can be very useful for the propagation of a variety of crop plants, especially crops for which true seeds are not used or readily available for multiplication (e.g. potato) or the true seeds are expensive (e.g. hybrid seeds) (Kumar, 2000).
7. SOMACLONAL VARIATION
The term somaclonal variation was coined by Larkin and Scowcroft (1981) and refers to the phenotypic and genotypic variation observed in plants regenerated from any form of cell culture. These variations are heritable, i.e., transmitted through meiosis and are usually irreversible. Somaclonal variation can be assessed by analysis of phenotype, chromosome number and structure, proteins, or direct DNA evaluation of plants (De Klerk, 1990). Morphological evaluation of black spruce (Picea mariana) and white spruce (P. glauca) derived from somatic embryogenesis showed several types of variation.
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The degree of variation has been shown to depend on the tissue being cultured (Murashige, 1974), to increase with the length of time that the cell or tissues have been maintained in vitro and to be similar in magnitude to variation in cells exposed to a mutagenic agent and then regenerated (Burk and Matzinger, 1976). Plant regeneration in vitro may cause phenotypic changes. These changes may be short-term and limited to the R0 generation, or they may be heritable (Plader et al., 1998). Somaclonal variation is of practical interest due to its potential uses in plant breeding. On the other hand if clonal in vitro propagation or transformation is the goal, it is an unwelcome phenomenon (Plader et al., 1998). Many factors have been suggested to play a role in causing somaclonal variation, e.g., the genotype, explant type (De Klerk, 1990), the culture duration (De Klerk, 1990; Muller et al., 1999), concentration of 2,4-D in the medium (Wernick and Milkovits, 1986), or the regeneration system. Somaclonal variation may be monitored via morphological changes, physiological changes or molecular changes (Peschke and Phillips, 1993). The latter may include changes in DNA (Cecchini et al., 1992).
8. ASSESSMENT OF GENETIC VARIABILITY USING MOLECULAR MARKERS

It should be stressed that the process of in vitro propagation via tissue culture may cause genetic and epigenetic alterations inducing somaclonal variation (Kaeppler et al., 2000). For example, variegation, variation in leaf structure and in overall plant growth pattern, trees that do not form inflorescences, or trees that produce seedless parthenocarpic fruits. Most of these phenotypes are detected in the early stages of plant growth, while some off-types can only be detected in the field, several years after planting. The degree of diversity among individuals or populations can be determined using morphological and molecular markers. Phenotypic characteristics have a limited importance since they are influenced by environmental factors. In some species adequate levels of phenotypic polymorphism are not available, cotton being one example (Tatineni et al., 1996). In contrast, molecular markers based on DNA sequence polymorphism can be used. Molecular markers such as RAPD, AFLP, and microsatellites are ideal to identify genetic and epigenetic somaclones, each marker system has inherent advantages and disadvantages. RAPDs have been widely used for their simplicity and their capacity to detect genetic variation among very closely related genotypes in a number of species. Molecular markers can be used to characterize somaclonal variation with greater precision and less effort than phenotypic and karyologic analyses and may be a means of evaluating the genetic stability of plants regenerated through tissue culture. For example, somaclonal variants in peach regenerates initiate from two different embryo callus cultures using RAPD and suggested that genetic changes ocurred during tissue culture. Similarly, Brown et al. (1995) were able to genetically distinguish among wheat suspension culture lines and also among regenerated plants using RAPD. However, molecular techniques such as RAPD have been problematic or inconclusive in some cases. Isabel et al. (1993) used RAPD to evaluate the genetic stability of somatic embryos obtained from three different cell lines of Picea mariana (Mill.).
74 Khaled Elmabrouk Suliman Elmeer Genetic variability within a cell line could not be detected. Fourr et al. (1997) evaluated somaclonal variation in embryogenic clones of Norway spruce, Picea abies (L.) Karst. Although variation was detected by cytogenetic and morphogenetic analyses, none was observed using RAPD analysis, in spite of using several primers. Munthali et al. (1996). Molecular markers, such as amplified fragment length polymorphisms (AFLPs) (Vos et al., 1995), allow the analysis of genomic DNA using a multilocus approach with high sensitivity. The AFLP method combines the reproducibility of restriction fragment analysis and the power of the polymerase chain reaction (PCR) in a straightforward technique (Mueller and Wolfenbarger, 1999). AFLP markers have been used to detect somaclonal variation in plants. The sensitivity of the restriction analysis and the use of stringently controlled PCRs often allow the detection of point mutations (insertions/deletions), and are proving useful in detecting somaclonal variation during somatic embryogenesis (Cervera et al., 2002). In P. dactylifera, AFLPs were more sensitive than random amplification of polymorphic DNA (RAPD) markers to detect genomic alterations in regenerated plantlets (Saker et al., 2006); in E. guineensis, for example, the RAPD technique did not detect differences between groups of somaclonal variants (Rival et al., 1998). Also in E. guineensis the AFLP techniquewas used successfully with methylation sensitive restriction enzymes to detect variants from in vitro culture (Matthes et al., 2001). The AFLP technique has already been used for distinguishing peach palm landraces (Clement et al., 2002). Conformity of the derived plants constitutes the main criteria for large scale use particularly for new groves establishment. Therefore, certification of the derived plants conformity is required. For this purpose, Molecular marker techniques such as randomly amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) have been employed to detect the genetic fidelity of micropropagated plants (Joshi and Dhawan, 2007). AFLP has been recognized as a more reliable and efficient DNA marker system, compared with RFLP, RAPD, ISSR or microsatellites (Zhang et al., 2006). AFLP markers have been used extensively for studying genomic variations in different plant species because of their high reproducibility and multiplex ratio (Zhang et al., 2006). Conclusions
Somatic embryogenesis is an asexual form of plant propagation in nature that mimics many of the events of sexual reproduction. Also, this process may be reproduced artificially by the manipulation of tissues and cells in vitro. In vitro somatic embryogenesis is an important prerequisite for the use of many biotechnological tools for genetic improvement, as well as for mass propagation. Applications of this process include: clonal propagation of genetically uniform plant material; elimination of viruses; provision of source tissue for genetic transformation; generation of whole plants from single cells called protoplasts; development of synthetic seed technology.
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Some of the most important factors for successful plant somatic embryogenesis are plant genotype, culture medium and environmental incubation conditions. The changes in the growth regulators concentration and photoperiod lead to differences in the response of cultivars and explants to produce somatic embryos. For each specific genotype there was an optimum explant, concentrations of growth regulators, photoperiod, gelling agents, and exposed time of callus to the induction and maturation media. Although general methodologies can be established for plant tissue culture, even closely related varieties of plants can differ in their cultural requirements. The best method of micro-propagating a new plant must usually be determined by experiment. Somaclonal variation, which is a welcome source of genetic variation for crop breeding, is unwanted when direct regenerants have to be used in tissue culture mass propagation, or in the regeneration of genetically transformed plants.
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Factors Regulating Somatic Embryogenesis in Plants

Factors Regulating Somatic Embryogenesis in Plants

1. SOMATIC EMBRYOGENESIS AND ITS PATTERNS

Factors Regulating Somatic Embryogenesis in Plants

2. APPLICATION OF SOMATIC EMBRYOGENESIS

Factors Regulating Somatic Embryogenesis in Plants

Factors Regulating Somatic Embryogenesis in Plants

3.4. Carbohydrate Source

Factors Regulating Somatic Embryogenesis in Plants

66 Khaled Elmabrouk Suliman Elmeer 3.6. Growth Regulators

Factors Regulating Somatic Embryogenesis in Plants

3.7. Period of Exposure to Auxin

Factors Regulating Somatic Embryogenesis in Plants

70 Khaled Elmabrouk Suliman Elmeer 3.10. Sodium Chloride

4. CROSS FEEDING AND CO-CULTURE

Factors Regulating Somatic Embryogenesis in Plants

5. REGENERATION THROUGH BIOREACTORS

Factors Regulating Somatic Embryogenesis in Plants

8. ASSESSMENT OF GENETIC VARIABILITY USING MOLECULAR MARKERS

Factors Regulating Somatic Embryogenesis in Plants

76 Khaled Elmabrouk Suliman Elmeer

Factors Regulating Somatic Embryogenesis in Plants

78 Khaled Elmabrouk Suliman Elmeer

Factors Regulating Somatic Embryogenesis in Plants

80 Khaled Elmabrouk Suliman Elmeer

Factors Regulating Somatic Embryogenesis in Plants

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