Protocol for Pollen Culture - 1. Flower buds are collected from the plants after all preparations for culturing in the laboratory have been made.

2. Buds are collected in a nonsterile petri dish and the length of each bud measured using a cm scale. Buds of corolla length 21-23 mm are chosen as the pollen in them would have usually completed the first mitosis.

3. The buds are chilled for 12 days (7 to 8°C) in a refrigeration unit.

4. They are then surface sterilized in a petri dish containing a suitable sterilizing agent, for instance 0.01 % solution of mercuric chloride.

10. After regular intervals of 6, 10 and 14 days of culture, the anthers are removed from the culture and discarded. That ensures the dehiscence of all anthers and release of pollen into the culture medium.

11. The dishes are then sealed with parafilm and incubated at 28°C in darkness for the first 14 days of culture.

12. After 14 days, the cultures are transferred to an illuminated growth chamber (500 lux, 12-h daylength, 25°C).

13. The growth of haploid embryos to plantlets developed from released pollen should be observed.

14. Follow steps 13 and 14 given in the protocol for anther culture for growing the plantlets to maturity.

After removing the debris either by sieving or filtration, the suspension is layered onto a sucrose solution (30% or 55% percoll and 4% sucrose) and centrifuged at 1200 g for 5 min. The pollen (vacuolated and starch filled) with embryogenic potential forms a band at the top of the sucrose solution while non embryogenic pollen (starch-filled) settles to the bottom.

The upper band containing embryogenic or sporophytic pollen is now pipetted out and suspended in the washing medium, centrifuged again, and the supernatant removed. Finally, the pellet containing these grains is resuspended in the culture medium. Such a pollen suspension gives a better response than control in terms of induction of cell division and continued growth of the callus.