Triploid Production

Abstract

Double fertilization is unique to the flowering plants. The male gametophyte (pollen) produces two sperms which fertilize two different constituent cells of the female gametophyte (embryo sac). One of them fuses with the egg nucleus (syngamy) forming the diploid zygote and the other one fuses with the two nuclei in the central cell (triple fusion) resulting in a triploid primary endosperm cell. Whereas the zygote develops into a well-organized diploid embryo, the progenitor of the next generation, the primary endosperm cell forms an unorganized, short-lived triploid endosperm tissue, the main source of nutrition for the embryo. Totipotency of endosperm cells has been established. Endosperm culture offers a direct approach to regenerate triploid plants for commercial exploitation. The conventional method to produce triploids by crossing tetraploids with diploids is lengthy and laborious.

Appendix

Protocol for the production of mulberry triploids (after Thomas et al. 2000)

• (i) Collect immature seeds (20 DAP) from the donor plants and wash them thoroughly in 1 % Savlon solution. After rinsing in 70 % ethanol for 15 s surface sterilize them in 0.1 % mercuric chloride solution for 7 min.

• (ii) Excise endosperm with intact embryo from the surface sterilized seeds and culture them on MS + 5 μM BAP + 1 μM NAA. Incubate the cultures in light.

• (iii) After a week, discard the germinated embryo and incubate the cultures in dark.

• (iv) After 5 weeks, transfer the callused endosperm to MS + 5 μM 2,4-D for the maintenance of endosperm callus cultures. Maintain the cultures in dark.

• (v) To regenerate shoots from the callus, transfer it to MS + 5 μM BAP + 1 μM NAA.

• (vi) After 4 weeks, excise 3–4 cm long shoots and transfer them, individually, to 1/2 MS + 7.5 μM IBA for rooting.

• (vii) After about 4 weeks, wash the roots of the plants and transfer them to small pots or polythene bag and maintain them under high humidity chambers for hardening.