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Culture Medium for Embryo Culture - The zygotic embryos develop through the following stage: proembryo, globular, heart-shaped, and torpedo-shaped to cotyledonary stage. A fully developed embryo undergoes a period of maturation, during which the embryo becomes hardy. Up to a certain stage, e.g., up to globular stage in Capsella, the embryo is heterotrophic as it derives some part of its nutrition from endosperm.

Beyond this stage, the embryo becomes autotrophic and is able to synthesize its biochemical needs from simple nutrients like salt and sugar. In general, the older is an embryo, the simpler are its nutritional needs.

Careful studies have shown that embryos are quite sensitive to the salt solutions of culture media. Further, embryo survival and growth have varied requirements in that salt solutions good for growth are bad for survival and vice-versa. Capsella embryos show improved survival when iron level of the MS medium is lowered to 2/3 of the normal, and CaCl2 concentration is doubled.

In addition, increased K and micronutrient (x2) concentration promotes embryo growth without affecting survival. In general reduced nitrogen in form of glutamine (400 mg/I) or asparagine is beneficial particularly for younger embryos.

Sucrose is the best carbon source; 8- I 2% sucrose is generally used, which approximates the osmotic potential of young embryo sac. High osmolarity of medium prevents precocious germination of embryos; mannitol at 120 g/l is suitable for heart-shaped embryos. Generally, GRs are not used since embryos are usually autotrophic in this respect. But in some species, low levels of GRs have marked beneficial effects. ABA checks precocious germination, and promotes orderly embryo development and maturation.

Earlier studies used complex additives like coconut milk, and various plant extracts, e.g., seed extracts; these generally had beneficial effects. The sucrose requirement of embryos declines sharply with embryo growth so that for best results embryos need to transfer to new media as they grow during culture.

A specially devised culture system has the following arrangement of two media in a petri dish the medium in centre (central medium) has 180 g/l sucrose, high Ca2+, 'no Fe2+, low salts and plenty of amino acids, while the medium at peripheri (outer medium) has high salt concentration, high Fe2+ but no sucrose or amino acids. Embryos are cultured on the central medium which becomes gradually modified due to the diffusion of constituents from and into the outer medium.

This system allows the culture of embryos on the same medium without any need for a subsequent transfer.
 
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