Protocol for Anther Culture
Tobacco is the ideal material for anther culture. So the basic protocol described below should be applicable to anther culture in general with modifications. The immature anthers containing uninucleate pollens at the time of first mitosis are the most suitable material for the induction of haploids.
1. Collect the flower buds of Nicotiana tabacum at the onset of flowering. Select the flower bud of 17-22 mm in length when the length of the sepals equals that of the petals. Reject all flower buds which are beginning to open.
2. Transfer the selected flower buds to the laminar airflow. Each flower bud contains five anther and these are normally surface sterile in closed buds. The flower buds are surface sterilized by immersion in 70% ethanol for 10 seconds followed immediately by 10 minutes in 20% sodium hypochlorite. They are washed three times with sterile distilled water. Finally transfer the buds to sterile petridish.
3. To remove the anthers, slit the side of the bud with a sharp scalpel and remove them, with a pair of forceps, place the five anthers with the filaments to another petridish. The filaments are cut gently. Damaged anthers should be discarded as they often form callus tissue the damaged parts other than the pollens.
4. Anthers are placed on agar solidified basal MS or White or Nitsch and Nitsch medium.
5. The culture is kept initially in dark. After 3-4 weeks, the anthers normally undergo pollen embryogenesis and haploid plantlets appear from the cultured anther. In some cases, anther may undergo proliferation to form callus tissue which can be induced to differentiate into haploid plants.
6. At this stage the cultures are incubated at 24-28 0C in a 14 hrs day light regime at about 2000 lux.
7. Approximately 50mm tall plantlets are freed from agar by gently washing with running tap water and then transferred to small pots containing autoclaved potting compost. Cover each plant with glass beaker to prevent desiccation and maintain in a well-lit-humid green house. After some week, remove the glass beaker and transfer the plant to larger pots when the plants will mature and finally flower.