8. • The discovery that pollen grains could develop into embryos was made by accident
with Datura by Guha and Masheshwari in 1964.
9.
10.
11.
12.
13. Collection of unopened flower buds
Surface sterilized with 70% ethanol
Anthers excised from flower bud and kept separately
Anther in first meiotic division is selected by acetocarmine test
Inoculate in the medium containing glutamine, L-Serine and inositol.
Incubate the culture at 250C for 15 days, the anthers grow into embryoids
Embryoids get transferred for rooting at 250C under 3000 lux light for 4-5
weeks.
14.
15. Anther collected from flower buds and pollen grains are isolated. These
anthers are placed in small sterile beaker containing 20ml of liquid MS/
White/ Nitsch
Anthers are pressed against the side of the beaker with sterile glass rod to
release the pollen grains
The homogenised anthers are then filtered through a nylon sieve to remove
the debris. Centrifugation is done to separate all the debris from pollen
grains
The pellet of pollen is suspended in fresh liquid medium and washed twice
by repeated centrifugation and resuspended in fresh medium
2.5ml of pollen suspension is spread over in the solid media and incubated
at 27-300C with cool, white light, 16hr photoperiod
Young embryoids shows up after 30 days. The embryoids grows into haploid
plantlets
16. Factors affecting anther and pollen culture
1. Genotype of the donor plant
2. Stage of the microspore
3. Culture medium
4. Temperature
5. Physiological status of the donor plant
