ORGANOGENESIS AND SOMATIC EMBRYOGENESIS AND SYNTHETIC SEED PRODUCTION TECHNOLOGY
In culture, the explant develops into callus tissue in a medium containing either a particular concentration of auxin or a definite auxincytokinin ratio.
Such medium is known as callus inducing or initiation medium. Proliferation of callus mass in a relatively unorganised way will continue for a prolonged period, if the callus tissue is maintained in the same medium through a number of subcultures. But the main objective in plant tissue culture is to regenerate a plant or plant organ from the callus culture.
The regeneration of plant or plant organ only takes place by the expression of cellular totipotency of the callus tissue. The callus tissue during its growth in callus inducing medium shows an extremely limited expression of totipotency, but in a certain number of plant species, this potentiality can be enhanced and extended by the adjustment of nutritional and hormonal conditions in culture.
Scattered areas of actively dividing cells, known as meristematic centres, develop as a result of differentiation and their further activity results in the production of root and shoot primordia. Skoog and his co-workers at Wisconsin, in their studies with tobacco stem pith culture, demonstrated that the initiation and the type of organ primordia formed from the resulting callus culture could be controlled by appropriate adjustment of the relative levels of the auxins and cytokinins.
With high auxin—low cytokinin roots develop, with low auxin—high cytokinin shoot buds develop; at intermediate levels undifferentiated callus tissue develops (Skoog and Miller, 1957). The expanded expression of totipotency of the callus tissue offers considerable potential for tissue culture technique as it is possible to grow the root or shoot or both. The production of adventitious roots and shoots from cells of tissue culture is called organogenesis.
Somatic embryogenesis
Development of embryo- like structures from somatic tissues, which can develop into whole plants is called somatic embryogenesis. The initiation and development of embryos from somatic tissues in plant culture was first recognized by Steward et. al., (1958) and Reinert (1958,1959) in cultures of Daucus carota. Somatic embryos may develop from sequential sages of somatic embryo development. Somatic embryos may develop from a single cell or
from group of cells. Repeated cell divisions may lead to the production of a group of cells that develop into an organized structure known as a “globular –stage embryo”. Further development results in heart and torpedo-stages. Polarity is established early in embryo development. Signs of tissue differentiation become apparent in the globular stage and apical meristems are apparent in heart-stage embryos. There are two routes of somatic embryogenesis:
Direct embryogenesis:
The embryo is formed directly from the explants tissue without the production of intervening callus. This occurs through pre-embryogenic determined cells (PEDC), where the cells are committed to embryonic development and need only to be released. Such cells are found in embryonic tissues (e.g scutellum of cereals), certain tissues of young in vitro grown plantlets (e.g. hypocotyls in Daucus carota, Ranunculus scleratus, Linum usitatissimum, Brassica napus), nucellus and embryo-sac (within ovules of mature plants).
Indirect embryogenesis
In indirect somatic embryogenesis, callus is first produced from the explants. Embryos can then be produced from the callus tissue or from a cell suspension produced from the callus. A callus can be established from explants from a wide range of carrot tissues by placing the explants on solid medium (e.g Murashige and Skoog) containing 2,4-D (1mgl-1). This callus can be used to produce a cell suspension by placing it in agitated liquid MS medim containing 2,4-D (1mgl-1). This cell suspension can be maintained by repeated subculturing into 2,4-D containing medium. Removal of the old 2,4-D containing medium and replacement with fresh medium containing abscisic acid(0.025 mgl-1) results in the production of embryos