Technique of Embryo Culture
1. Surface Sterilization:
Embryos of seed plants normally develop inside the ovule which in turn is covered by overies. Since they already exist in a sterile environment, disinfection of the embryo surface is unnecessary unless the seed coats are injured or systemic infection is present. Instead, mature seeds, entire ovule or fruits are surface sterilized. Surface sterilization is carried out by immersing the material in hypo chorine- containing commercial bleach ( 5-10% Clorox, 0.45% Sodium or Calcium hypochlorite) for 5- 10 min or ethanol ( 70-75%) for 5 min. A small amount ( 0.01-0.1%) of a surfactant may be added to disinfection solution. In case of infected seeds, the excised embryos may be immersed in 70% alcohol plus 5-10 min exposure to 2.6% sodium hypochlorite solution.
2. Excision of Embryo:
Embryo excision operation is carried out aseptically in a laminar airflow hood. A steremicroscope equipped with cool-ray flurescent lamp is required for excision of small embryo. The commonly used dissecting tools are foreceps, dissecting needles, scalpels, razor blades and Pasteur pipettes. Mature embryo can be isolated with relative ease by splitting open the seeds. Soaking a hard-coat seeds for few hours to a few days before sterilization makes its dissection easier. In case of embryos embedded in liquid endosperms, the incision is made at micropolar end of young ovule and pressure applied at opposite end to force the embryo out through the incision.
3. Embryo-endosperm Transplant:
It is very difficult to culture embryo in vitro abort at very early stages of development because of lack of knowledge of nutritional requirements. The chances of development of immature or abortive embryos increases if they are surrounded by endosperm tissue excised from another seed of same species. Generally, endosperm older than the embryo by 5 days was more efficient as a nurse tissue than one of the same age as the embryo.
4. Nutritional Requirement:
The nutritional requirements of an embryo during its development in vitro consisting two phase: a) Heterotropic phase- an early phase wherein the embryo is dependent and draws upon the endosperms and materal tissues and b) The autotrophic phase- a later phase in which the embryo is metabolically capable of synthesizing substances required for its growth, thus becoming fairly independent for nutrition.
The media constituents for in vitro growth of young or immature embryos also differ from those of mature embryos. This often necessitates the transfer of embryos from one medium to another for their orderly growth.
i) Mineral Salts:
Inorganic nutrients of MS, B5 and White’s media with certain degree of modification are the most widely used basal media for embryo culture. Monnier (1978) modified the MS medium for immature embryo culture of Capsella which contains higher levels of potassium and calcium and reduced levels of ammonium (NH4NO3) and FeEDTA and double concentration of MS micronutrients.
ii) Carbohydrates:
Sucrose is the most commonly used source of energy for embryo culture. Addition of maltose, lactose, raffinose or mannitol may be required in embryo culture of some species. In some cases glucose is found to be better than sucrose. Mature embryos grow fairly well at low sucrose concentration but younger embryos require higher level of carbohydrates.
iii) Nitrogen and Vitamins:
Ammonium nitrate is better than KNO3, NaNO3 and (NH4)2 HPO4. especially the presence of NH4+ in the medium has been found essential for proper growth and differentiation of embryos. Various Amino acids and their amides like aspargine, glutamine, and casein hydrolysate have been widely used in embryo culture.
iv) Natural Plant Extract:
The coconut milk ( CM) effectively stimulates the growth of excised young embryos of sugarcane, barley, tomato, carrot, Interspecific hybrids of Vigna and fern species. Van Overbeek et.al ( 1941) suggested that the coconut milk contains some ‘ Embryofactor’ which presumably makes up for deficiencies of certain sugars, amino acids, growth hormones and other critical metabolites of the culture medium.
In addition to coconut milk, water extracts from dates, bananas, hydrolysate of wheat-gluten and tomato juice were also effective.
v) Growth Regulators:
Auxin and cytokinins are not generally used in embryo culture since they induce callus formation. At very low concentration GA promotes embryogenesis of young barley embryos without inducing precocious germination. ABA also has a similar effect on barley and Phaseolus embryos.
vi) PH of Medium:
Excised embryos grows well in a medium with a PH 5 to 7.5. Generally the medium PH is adjusted 0.5 units higher than the desired PH in order to compensate for uncontrollable change in its value during the autoclaving process.
vii) Incubation Conditions:
The embryo cultures are incubated at 25+-2 0C. whereas in case of species to warm temperature requires 27-30 0C incubation temperature and species occurring in cold regions or seasons require incubation temperature of 17-22 0C.
Generally, an initial dark incubation ( 4 days) of embryo in culture is essential , following which they can grow to a mature stage even under continuous light regime.
5. Role of Suspensor in Embryo Culture:
Suspensor is actively involved in embryo development. The suspensor is an ephemeral structures found at the radicular end of the proembryo and attains maximum development by the time embryo reaches globular stage. In cultures the presence of a suspensor is critical , particularly for the survival of young embryos. The requirement of the suspensor may be substituted by the addition of GA or ABA to the culture medium.