SOMATIC EMBRYOGENESIS

Objective:- Protocol for somatic embryogenesis in carrot.

Plant Material:- Hypocotyl of carrot seedling.

Procedure:-

1. Wash seeds by submerging in water with a few drops of detergent in a beaker and shake by hand, or wrap seeds in two layers of cheese cloth/muslin cloth/nylon pouch and then wash with water.

2. Submerge the seeds in 70% alcohol for 30-60s. Decant the alcohol.

3. Transfer the seeds to a flask or beaker containing 20-40% commercial sodium hypochlorite for 15-20 min. Rinse 4x with sterile distilled water.

4. Place 2-3 seeds per culture vessel on the surface of MS agar medium.

5. Incubate the cultures at 25°C under 16 h photoperiod with -1000 lux light intensity for 1-2 weeks.

6. Collect the germinated seedlings when the cotyledons are fully expanded. Place each seedling on a sterile petri dish and excise the hypocotyl from each seedling and cut them transversely into two parts.

7. Place the hypocotyl sections on the following medium: MS + 1-2 mg/l 2,4-D.

8. Incubate the cultures in dark at 25°C for 4-8 weeks.

9. Maintain the callus by subculturing small pieces on fresh medium every 3- 4 weeks. Callus will contain pro- embryo initial cells as well as minute microscopic embryos in the early stages of development.

10. Place 0.5 to 1 cm2 callus pieces on MS agar medium without growth regulators and incubate the cultures at 25°C under the 16h photoperiod with ~1000 lux light intensity. Within 2-3 weeks of cultures will exhibit embryos and green plantlets.

11. Tease out individual or group of plantlets from the callus mass and transfer on half strength MS medium under 16h photoperiod with high light intensity of ~5 lux. Within 4-5 weeks the cultures will resemble seedling carrots.

12. Transfer the plantlets to small pots containing sterile peat moss and vermiculite in a 1:1 ratio. Enclose the plantlets with plastic containers to maintain high humidity.

13. Transfer the plants to soil and follow the procedure of plant establishment and hardening.