Protocols for Inducing Somatic Embryogenesis in Culture
The plant material Ducus carota represents the classical example of somatic embryogenesis in culture. The protocol is described below:
1. Leaf petiole or root segments from seven day old seedlings or cambium tissue from seven day old seedling or cambium tissue from storage roots can be used as explant. The leaf petiole and root segment can be obtained from aseptically growth seedlings. Cambium tissue can be obtained from surface sterilized tap root.
2. Following aseptic technique, explants are placed individually on a semi-solid MS medium containing 0.1 mg/l, 2,4-D and 2% sucrose. Cultures are incubation in dark. In this medium explant will produce sufficient callus tissue.
3. After 4 weeks of callus growth, cell suspension culture is to be initiated by transferring 0.2g of callus to a 150 ml of Erlenmeyer flask containing 20-25 ml of liquid medium of the same composition as used for callus growth. Flasks are placed on a horizontal gyratory shaker with 125-160 rpm at 25 0C. The presence or absence of light is not critical at this stage.
4. Cell suspension are subcultured every 4 weeks by transferring 5ml to 65 ml of fresh liquid medium.
5. To induce a more uniform embryo population, cell suspension is passed through a series of stainless steel mesh sieves. For carrot, the 74 µ produce a fairly, dense suspension of single cell and small multiple clumps. To induce somatic embryogenesis, portion of sieved cell suspension are transferred to 2,4-D free liquid medium or cell suspension can be plated in semi-solid MS medium devoid of 2,4-D. for normal embryo development and to inhibit precocious germination especially root elongation , 0.1 µm ABA can be added to the culture medium. Cultures are incubated in dark.
6. After 3-4 weeks, the culture would contain numerous embryos in different stage of development.
7. Somatic embryos can be placed on a agar medium devoid of 2, 4-D for plantlet development.
8. Plantlets are finally transferred to pots or vermiculite for subsequent development.