(i) |
Pluck healthy and mature fruits from the field and wash thoroughly in running water for about an hour. |
(ii) |
Surface sterilize with 0.01% Tween-20 for 15 min, rinse seeds several times with distilled water and finally treat with 0.01% HgCl2 solution for 10-15 min. Finally rinse it for six times with sterile distilled water. |
(iii) |
Break seeds aseptically and isolate the embryo. |
(iv) |
Culture embryo on callus proliferation medium. Supplement the basal medium of Murashige and Skoog (1962) with different combinations and concentrations of sugar, vitamins, hormones and other growth adjuvants for callus proliferation and shoot regeneration. |
(v) |
Incubate the cultures at 22-25°C under a 16 h photoperiod of 2000 lux luminous intensity. |
(vi) |
After two weeks of inoculation the embryo begins to swell on callus proliferation medium. Distinct callus growth is observed after 4 weeks. |
(vii) |
After 8 weeks of inoculation transfer the callus on shoot regeneration medium. Within 4 weeks of transfer into second medium the callus turns green and produces soft spongy tissue. Some of these tissues are differentiated into embryoids. |
(viii) |
The embryoids produce cluster of budlets when subcultured onto shoot regeneration medium. The budlets grow into shoots and produce 2-3 leaf appendages within 12 weeks. Thereafter, they are separated into individual shoots and then subcultured into a fresh medium of the same composition until shoots develop. |
