Lecture 5 Somatic Embrygenesis

SOMATIC EMBRYOGENESIS AND ORGANOGENESIS BACKGROUND AND DISCUSSIONS

ORGANOGENESIS In organogenesis, cultured explants (e.g cotyledons, hypocotyls, stem, leaf, shoot apex, root, flowers, petioles and embryos) are induced to develop adventitious roots, shoots and other organs. Organogenesis can be induced in specific nutrient media containing balanced ratio of plant growth regulators such as auxin and cytokinin. In such cases, plant regeneration from cultured cells may occur through shoot-bud differentiation. The cells of leaf cultures of some species are able to directly differentiate shoots and roots, leading to the production of plantlets.

ORGANOGENESIS There are three organogenesis:

methods

of

plant

regeneration

via

5. Depends on adventitious organs arising from a callus culture or alternatively, 7. Directly from an explant. 9. Axillary bud formation and growth from some explants.

FACTORS AFFECTING ORGANOGENESIS Organ formation is controlled by quantitative interaction (ratio rather then absolute concentration) of constituents in growth and development medium. Generally, cytokinin (e.g. adenine or kinetin) in the medium leads to the production of shoot differentiation and development. Reports indicate that kinetin is 30,000 times more potent than adenine. The shoot-forming effect is modified by auxin (IAA and NAA), which at lower concentration (5 µM IAA) suppresses the differentiation of shoot in tobacco callus. A relatively high concentration of auxin seem to favour cell proliferation and root differentiation, while higher levels of cytokinin promote shoot differentiation.

FACTORS AFFECTING ORGANOGENESIS The inhibitory effect of auxin can be counteracted by increased levels of phosphate ions in the medium and this promotes shoot formation in the absence of cytokinin. Casein hydrolysate or tyrosine also induces kinetin type shoot formation even in the presence of higher levels of IAA in the medium. The requirement for exogenous auxin and cytokinin in the process of shoot differentiation varies with the tissue system and apparently depends on the endogenous levels of the two hormones in the tissue, viz a viz. an auxin and cytokinin. Endosperm cultures require cytokinin alone or in combination with a very level of auxin for shoot differentiation.

FACTORS AFFECTING ORGANOGENESIS Polyamines have been shown to be associated with the induction of cell division, growth and differentiation of plant cells. Other cytokinins which influence the induction of shoots include BA, 2-iP and Zeatin.

FACTORS AFFECTING ORGANOGENESIS In alfalfa and cereals, a two step process of organogenic differentiation occurs. Alfalfa callus initiated on a medium (induction medium) containing higher auxin (2,4-D) to kinetin, favours shoot-formation. Whereas, a higher proportion of kinetin to 2,4-D in the medium (regeneration medium) supports root induction. The cereal callus, on the other hand, is initiated in the induction medium containing 2,4-D and kinetin but organogenesis occurs only when pieces of callus are transferred to a hormone-free regeneration medium

PHYSICAL FACTORS AFFECTING ORGANOGENESIS Light Intensity In the case of Pelargonium callus maintained under continuous light or high light intensity remains whitish and does not exhibit organogensis. Such light intensity has also been shown to be inhibitory for shoot formation in tobacco. The quality of light also influence organogenic differentiation. Blue light promotes shoot differentiation in tobacco calus while red light stimulates rooting. In general, maintenance of callus under alternating light and dark periods (15-16h) may prove satisfactory for differentiation of shoots.

PHYSICAL FACTORS AFFECTING ORGANOGENESIS Temperature Affects the callus growth and differentiation. Increases in temperature up to 33ºC may be associated with the rise in the growth of tobacco callus but for differentiation of shoot, a lower temperature (18ºC) may be optimal. Physical state of medium Medium solidified with agar favour shoot formation. Physiology of explant Genome and the physiological state of the explant are other factors accounting for differentiation in culture.

Somatic Embryogenesis (SE) SE is the process of a single plant cell or a group of callus initiating the developmental pathway that leads to reproducible regeneration of a non-zygotic embryo capable of germinating to form complete plants. SE occurs most frequently in tissue culture and as an alternative ORGANOGENESIS for regeneration of whole plant. Adherence to SE (this pattern of morphogenesis) depends on the co-ordinated behaviour of a cell or cells to establish polarity as a unit and thereby initiate gene action sequentially specific to emerging tissue regions.

Somatic Embryogenesis SE is initiated by ‘pre-embryogenic determined cells’ (PEDC) or by ‘induced embryogenic determined cells’ (IEDC). In PEDCs the embryogenic pathway is predetermined and the cells appear to only wait for the synthesis of an inducer (or removal of an inhibitor) to resume independent mitotic divisions to express their potential. Such cells are formed in embryogenic tissues (young tissues) of in vitro plants e.g. embryo-sac tissues within ovules. In SE, embryo-like structures, which can develop into whole plants, are formed from somatic tissues.

Somatic Embryogenesis IEDCs require redetermination to the embryogenic state by exposure to specific growth regulators (2,4-D). These cells are differentiated generally in microspore callus cultures. Once the embryogenic state has been reached, both cell type proliferate in a similar manner as embryogenic determined cells (EDCs). Plantlets are then produced directly by following the full embryogenic pathway as a co-ordinated group of EDCs. Somatic embryos may develop form single cells or form a small group of cells. Repeated cell divisions lead to the production of a group of cells that develop into organised structure known as a ‘globularstage embryo’.

Somatic Embryogenesis Further development results in heart- and torpedo-stage embryos, from which plants can be regenerated. Polarity is established early in embryoid development. Signs of tissue differentiation become apparent at the globular stage and apical meristems are appparent in heart-stage or cotyledonstage embryoids.

Somatic Embryogenesis SE can be of direct and indirect types. In direct, SE, the embryoids are formed directly form a cell or small group of cells without the production of an intervening callus (common among reproductive tissues). It is rare in comparison with indirect SE. e.g. Young leaf explants from alfalfa (Medicago falcata), washed in plant growth regulator-free medium and placed in liquid medium (B5) supplemented with 2,4-D (4 mgl-1), kinetin (0.2 mgl1 ), adenine (1 mgl-1) and glutathione (10 mg-1). The cultures are maintained in agitated liquid medium for about 10-15 days. Washing the explants and replacing the old medium with B5 medium supplemented with maltose and polyethylene glycol results in the development of somatic embryoids. These somatic embryoids can be matured on solid medium containing abscisic acid.

Somatic Embryogenesis In Indirect SE, callus is first produced from the explant. Embryoids can then be produced from the callus tissue or from cell suspension produced from the callus. e.g. Callus can be established from explants from a wide range of carrot tissues by placing the explant on solid medium (M&S) containing 2,4-D (1 mgl-1). This callus can be used to produce cell suspension by placing it in agitated liquid MS meduim containing 2,4-D (1 mg-1). Replacing old medium and replacing with fresh medium containing abscisic acid (0.025 mgl-1) results in the production of embryoids.

SE IN CALLUS CULTURES SE is achieved in two steps: V. Callus is initiated, multiplied on a medium rich in auxin (Proliferation) Medium-PM, e.g. with 2,4-D) which induces differentiation of localised groups of the meristematic cells ‘embryogenic clumps’ (ECs). •

The ECs then develop into mature embryoids when transferred to a medium with a very low level of auxin or no auxin at all (Embryoid Development Medium-EDM). Thus embyogenic callus consist of proembryoids and these embryo-like structures are normally bipolar units and may (capable) germinate into full plantlets under suitable culture conditions. Non-zygotic

embryos

are

somatic

embryoids

or

OTHER FACTORS ASSOCIATED IN SE Nitrogen source A substantial amount of nitrogen, usually in reduced form such as ammonium salts, is required for both embryo initiation and maturation. However, a combination of reduced nitrogen and nitrate in the medium appears beneficial for a number of culture systems. The source of nitrogen can be in the form of complex addenda (coconut milk, casein hydrolysate, a mixture of amino acids and ammonium ions).

OTHER FACTORS ASSOCIATED IN SE Media Components Half normal concentration of media components and in some cases lacking phytohormones. Light Light intensities, particular wavelengths and photoperiods, moderate and stimulate the formation and development of embryoids. Exudates Extracts (tomato, banana, yeast / coconut water)

OTHER FACTORS ASSOCIATED IN S.E. Other Constituents The amount of dissolved oxygen (DO). The effect of carbon source can influence the osmotic value. Activated charcoal is reported to increase embryogenesis. As certain tissues release volatile substances which can inhibit SE in the callus.

APPLICATION OF SE Clonal propagation (large-scale propagation of plantlets) Raising somoclonal variants (new trait of plant-new variety) Cloning zygotic embryos for repetitive SE Synthesis of artificial seeds (by encapsulation of somatic embryos) Genetic transformation or plant improvement via SE

FLOW SHEET SUMMARISING THE INTER-RELATIOSHIPS BETWEEN THE DIFFERENT LINES OF EXPERIMENTATION OPENED UP BY THE TECHNIQUES OF PLANT TISSUE AND CELL CULTURE