The following points highlight the top seven steps involved in anther culture.
Step 1.
Collect the flower buds of Nicotiana tabacum at the onset of flowering (normally 3- 4 months old plant). Measure the length of each individual with a cm ruler. 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 (Fig 11.2).
Step 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 2% (v/v) sodium hypochlorite. They are washed three times with sterile distilled water. Finally, transfer the buds to a sterile petridish.
Step 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. During excision of anthers, special care should be taken to ensure that they are not injured in any way. Damaged anthers should be discarded as they often form callus tissue from the damaged parts other than the pollen.
Step 4.
Anthers are placed on agar solidified basal MS or White or Nitsch and Nitsch medium.
Step 5.
The culture are 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.
Step 6.
At this stage, the cultures are incubated at 24-28° C in a 14 hrs. daylight regime at about 2,000 lux.
Step 7.
Approximately 50 mm tall plantlets are freed from agar by gently washing with running tap water and then transferred to small pot containing autoclaved potting compost. Cover each plantlet with a glass beaker to prevent desiccation and maintain in a well- lit humid green-house. After one week, remove the glass beakers and transfer to larger pots when the plants will mature and finally flower.