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Protocol for Anther Culture in Laboratory - A simple stepwise procedure to isolate haploid plants from cultured anthers of tobacco is given below. ,

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.

5. Using sterile, double distilled water, the buds are rinsed 3-4 times in a sterile air cabinet.

6. Using forceps and a dissecting needle, the buds are carefully teased open and the anthers removed.

7. The anthers dissected out from each bud are grouped separately on removal.

8. One anther from each group is then removed and squashed in acetocarmine stain in order to determine the stage of pollen development. The pollen should be in the first pollen division.

9. Filaments are cut from their anthers to avoid callusing from the cut ends in vitro.

10. Anthers from the group showing pollen in the first division are placed on a suitable culture medium and the cultures are incubated at 25°C in darkness.

11. After about 2 weeks in culture, young embryos emerging from cultured anthers can be detected by gently bursting the anthers on a microscope slide in a drop of acetocarmine stain and observed under a microscope.

12. The cultures are shifted to light (intensity of about 300 lux) after the specified period of approximately 2 weeks. Complete plantlets are observed after 4-5 weeks of culture.

13, Plantlets emerging out of the anthers are separated using a pair of forceps and the remaining anther tissues are discarded.

14. For quicker development of the plantlets, they may be transferred to a root inducing medium after they are about 15 mm long.

During this period the plants are placed on a 12-h day length provided with 500 lux illumination from white fluorescent tubelights. The plantlets can be transferred to soil after they attain a height of about 50 mm.

 

 
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