Ovule Culture – Meaning, Principle and Protocol
Ovule culture is an elegant experimental system by which ovules are aseptically isolated from the ovary and are grown aseptically on chemically defined nutrient medium under controlled conditions.
Principle:
An ovule is a megasporangium covered by integument. An ovule contains a megaspore or an egg cell. After fertilization a single cell zygote is formed which ultimately leads to form a mature embryo possessing shoots and root primordia.
In Vitro ovule culture helps to understand the factors that regulate the development of zygote through organised stages to a mature embryo. Alternatively, it may be possible to germinate pollen in the same culture as the excised and to induce in vitro fertilization and subsequent embryo production.
Protocol of Ovule Culture:
1. Collect the open flower. If fertilized ovules are desired, collect the open flowers where anthers are dehisced and pollination has taken place. To ensure the fertilization, collect the flower after 48 hrs of anther dehiscence.
2. Remove sepals, petals, androecium, etc from the overies containing either fertilized or unfertilized ovules.
3. Soak the overies in 6% NaOCL solution.
4. Rinse the overies 3-4 times with sterile distilled water.
5. Using sterile technique, ovules are gently prodded with the help of spoon shaped statula by breaking the funicules at its junction placental tissue.
6. The spatula with ovules is gently lowered into the sterile solid or liquid medium as the culture vial is slanted about 45 0 C.
7. Damaged or unorganised ovules are rejected when possible during transfer.
8. Incubate the culture in either dark or light at 25 0C.