Protocol for Embryo Culture
The following protocol for embryo culture is based on the method used for Capsella bursapastoris. With modification, this basic protocol should be applicable to embryo culture in general.
1. The capsules in the desired stages of development are surface sterilized for 5-10 minutes in 0.1% HgCl2 in a laminar air flow.
2. Wash repeatedly in sterile water.
3. Further operations are carried out under a specially design dissecting microscope at a magnification of about 90 X. The capsules are kept in a depression slide containing few drops of liquid medium.
4. The outer wall of capsule is removed by a cut in the region of the placenta; the halves are push apart with forceps to expose the ovules.
5. A small incision in the ovule followed by slight pressure with a blunt needle is enough to free the embryos.
6. The excised embryos are transferred by micropipette or small spoon headed spatula to standard 10 cm petri dishes containing 25 ml of solidified standard medium. Usually 6-8 embryos are cultured in petridish.
7. The Petri dishes are sealed with cello tape to prevent desiccation of the culture.
8. The cultures are kept in culture room at 25+- 1 0C and given 16 hrs illumination by cool white fluorescent tube.
9. Subcultures into fresh medium are made at approximately four weaks interval.
In case of fresh seed or dry and imbided seeds, the schedule is slightly changed. Seeds are cleaned by 5% Teepol for 10 minutes and dipped in 70% ethyl alcohol for 60 seconds. Surface sterilization in 0.1 % HgCl2 is followed by washing in sterile water, then the seeds are decotylated using a sharp scalpel and embryos are transferred to solid nutrient medium.