Protocol for Pollen Culture
Isolated pollen can be cultured by two methods.
Method I:
This method is described here for the culture of isolated pollen of tobacco. This technique can be considered as the basic protocol for pollen culture and involves the following procedure.
1. Selection of suitable unopened flower bud, sterilization, excision of anther without filaments are the same as described previously in anther culture.
2. About 50 anthers are placed in small sterile beaker containing 20 ml of liquid basal medium (MS or White or Nitsch and Nitsch).
3. Anthers are then pressed against the side of beaker with the sterile glass piston of a syringes to squeeze out the pollens.
4. The homogenized anthers are then filtered through a nylon sieve to remove that the anther tissue debris.
5. The filtrate or pollen suspension is then centrifuged at low speed ( 500-800 rpm/min) for five minutes. The supernant containing fine debris is discarded and pillet of pollen is suspended in fresh liquid medium and washed twice by repeated centrifugation and resuspension in fresh liquid medium.
6. Pollens are mixed finally with measured volume of liquid basal medium so that it makes the density of 10 3-10 4 pollen/ml.
7. A 2.5 ml of pollen suspension is pipetted off and is spread in 5 cm petridish. Pollens are best grown in liquid medium but, if necessary, they can be grown by plating very soft agar added medium. Each dish is sealed with cello tape to avoid dehydration.
8. Petridishes are incubated at 27-30 0C under low intensity of white cool light (500 lux,16 hrs).
9. Young embryoids can be observed after 30 days. The embryoids ultimately give rise to haploid plantlets.
10. Haploid plantlets are then incubated at 27-30 0C in a 16 hrs day light regime at about 2000 lux. Plantlets at maturity are transferred to soil as described in anther culture.
Method 2:
This method is known as nurse culture technique. Sharp et.al. ( 1972) first introduced this method. The steps are as under.
1. Selection of flower bud sterilization excision of anther, isolation of suitable pollen is the same as described previously.
2. In this method, the intact anthers are placed horizontally on the top of solid or semisolid basal medium within a conical flask.
3. A small filter paper disc is placed over the intact anther and about 10 pollen grains in the suspension are then placed on the filter paper disc. Here the intact anthers are considered as the nurse tissue. A control set is also prepared in exactly the same way except that the pollen grain on the filter paper are directly kept on solid medium. Sometimes, callus tissue derived from any part of the plant is used as nurse tissue.
4. With this method, pollen grains the control set did not grown at all. The pollen gains kept on nurse tissue grow and form a culture of green parenchymatous tissue in two weeks, such tissue ultimately form the haploid callus tissue.