Embryo Culture

In addition to root, shoot, and pollen culture, embryo culture has also been done for the production of haploid plants. Embryo culture is used for the recovery of plants from distinct crosses. Embryo culture is useful where embryo fails to develop due to degeneration of embryonic tissues. It is being used extensively in the extraction of haploid barley (Hordeum vulgare) from the crosses H. vulgare x H. bulbosum. Embryo culture is also a routine technique employed in orchid propagation and in breeding of those species that show dormancy. Das and Barman (1992) developed the method of regeneration of tea shoots from embryo callus. The embryo callus produced somatic embryoids within 8 weeks of culture in the second medium which differentiated into buds after 2 weeks. Several shoots with 4-6 leaves developed after 16 weeks of culture.

Culturing method
The general method of embryo culture follows the following steps.

1. Pluck healthy and mature fruits from the field and wash thoroughly in running water for about an hour.
2. 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.
3. Break seeds aseptically and isolate the embryo.
4. 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.
5. Incubate the cultures at 22-25°C under a 16 h photoperiod of 2000 lux luminous intensity.
6. After two weeks of inoculation the embryo begins to swell on callus proliferation medium. Distinct callus growth is observed after 4 weeks.
7. 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.
8. 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.