Somatic embryogenesis
Somatic embryogenesis is an artificial process in which a plant or embryo is derived from a single somatic cell.[1] Somatic embryos are formed from plant cells that are not normally involved in the development of embryos, i.e. ordinary plant tissue. No endosperm or seed coat is formed around a somatic embryo.
Cells derived from competent source tissue are cultured to form an undifferentiated mass of cells called a callus. Plant growth regulators in the tissue culture medium can be manipulated to induce callus formation and subsequently changed to induce embryos to form the callus. The ratio of different plant growth regulators required to induce callus or embryo formation varies with the type of plant.[2] Somatic embryos are mainly produced in vitro and for laboratory purposes, using either solid or liquid nutrient media which contain plant growth regulators (PGR’s). The main PGRs used are auxins but can contain cytokinin in a smaller amount.[3] Shoots and roots are monopolar while somatic embryos are bipolar, allowing them to form a whole plant without culturing on multiple media types. Somatic embryogenesis has served as a model to understand the physiological and biochemical events that occur during plant developmental processes as well as a component to biotechnological advancement.[4] The first documentation of somatic embryogenesis was by Steward et al. in 1958 and Reinert in 1959 with carrot cell suspension cultures.[5][6]
Plant regeneration by somatic embryogenesis[edit]
Plant regeneration via somatic embryogenesis occurs in five steps: initiation of embryogenic cultures, proliferation of embryogenic cultures, prematuration of somatic embryos, maturation of somatic embryos and plant development on nonspecific media. Initiation and proliferation occur on a medium rich in auxin, which induces differentiation of localized meristematic cells. The auxin typically used is 2,4-D. Once transferred to a medium with low or no auxin, these cells can then develop into mature embryos. Germination of the somatic embryo can only occur when it is mature enough to have functional root and shoot apices [3]
Factors influencing[edit]
Factors and mechanisms controlling cell differentiation in somatic embryos are relatively ambiguous. Certain compounds excreted by plant tissue cultures and found in culture media have been shown necessary to coordinate cell division and morphological changes.[9] These compounds have been identified by Chung et al.[10] as various polysaccharides, amino acids, growth regulators, vitamins, low molecular weight compounds and polypeptides. Several signaling molecules known to influence or control the formation of somatic embryos have been found and include extracellular proteins, arabinogalactan proteins and lipochitooligosaccharides. Temperature and lighting can also affect the maturation of the somatic embryo.
Applications[edit]
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.[1]
Tracking and fate maps[edit]
Understanding the formation of a somatic embryo through establishment of morphological and molecular markers is important for construction of a fate map. The fate map is the foundation in which to build further research and experimentation. Two methods exist to construct a fate map: synchronous cell-division and time-lapse tracking. The latter typically works more consistently because of cell-cycle-altering chemicals and centrifuging involved in synchronous cell-division.[17]
Angiosperms[edit]
Embryo development in angiosperms is divided into several steps. The zygote is divided asymmetrically forming a small apical cell and large basal cell. The organizational pattern is formed in the globular stage and the embryo then transitions to the cotyledonary stage.[18] Embryo development differs in monocots and dicots. Dicots pass through the globular, heart-shaped, and torpedo stages while monocots pass through globular, scutellar, and coleoptilar stages.[19]
Many culture systems induce and maintain somatic embryogenesis by continuous exposure to 2,4-dichlorophenoxyacetic acid. Abscisic acid has been reported to induce somatic embryogenesis in seedlings. After callus formation, culturing on a low auxin or hormone free media will promote somatic embryo growth and root formation. In monocots, embryogenic capability is usually restricted to tissues with embryogenic or meristematic origin. Somatic cells of monocots differentiate quickly and then lose mitotic and morphogenic capability. Differences of auxin sensitivity in embryogenic callus growth between different genotypes of the same species show how variable auxin responses can be.[20]
Carrot Daucus carota was the first and most understood species with regard to developmental pathways and molecular mechanisms.[17] Time-lapse tracking by Toonen et al. (1994) showed that morphology of competent cells can vary based on shape and cytoplasm density. Five types of cells were identified from embryonic suspension: spherical cytoplasm-rich, spherical vacuolated, oval vacuolated, elongated vacuolated, and irregular shaped cells. Each type of cell multiplied with certain geometric symmetry. They developed into symmetrical, asymmetrical, and aberrantly-shaped cell clusters that eventually formed embryos at different frequencies.[21] This indicates that organized growth polarity do not always exist in somatic embryogenesis.[17]
Gymnosperms[edit]
Embryo development in gymnosperms occurs in three phases. Proembryogeny includes all stages prior to suspensor elongation. Early embryogeny includes all stages after suspensor elongation but before root meristem development. Late embryogeny includes development of root and shoot meristems.[18] Time-lapse tracking in Norway Spruce Picea abies revealed that neither single cytoplasmic-rich cells nor vacuolated cells developed into embryos. Proembryogenic masses (PEMs), an intermediate between unorganized cells and an embryo composed of cytoplasmic-rich cells next to a vacuolated cell, are stimulated with auxin and cytokinin. Gradual removal of auxin and cytokinin and introduction of abscisic acid (ABA) will allow an embryo to form.[17] Using somatic embryogenesis has been considered for mass production of vegetatively propagated conifer clones and cryopreservation of germplasm. However, the use of this technology for reforestation and tree breeding of conifers is in its infancy.[22][23]