In vitro germination and micropropagation of water chestnut (Trapa sp.)

Abstract

The water chestnut (Trapa sp.) is a minor food crop of India, southeast Asia and China. Trapa seeds are recalcitrant and their germplasm storage is problematic. The aim of the present work was to study germination and develop a protocol for micropropagation. In vitro plants were raised after a rigorous surface sterilisation procedure, using "embryo explants" (embryonal axis plus a portion of the large cotyledon), in Nitsch's basal semi-solid medium (NBS). During germination, the hypocotyl elongates in a negatively geotropic manner and the plumule rapidly develops into a shoot, bearing three or four pairs of linear, juvenile leaves, followed by spirally arranged adult leaves. Shoot tips and nodal cuttings were removed from the aseptically raised seedlings and cultured on Nitsch's basal liquid medium (NBL). Explants obtained from the regenerated shoots after 4 weeks growth were subcultured on NBL supplemented with several plant growth regulators to ascertain their effect on primary shoot length, number of nodes, production of axillary buds/secondary branches and roots. In general, anchoring roots were not formed under in vitro conditions and only green roots developed. Auxin suppressed axillary bud production but enhanced the formation of green roots. Abscisic acid inhibited juvenile leaf development. Among cytokinins, 6-benzylaminopurine (BAP) at 10^-6 M was found most effective in enhancing axillary branch production. When individual axillary branches from cultures raised on BAP (10^-6 M) were transferred to NBL, stem elongation and rooting occurred. The resulting plantlets were successfully transferred to ponds with a survival rate of 80-85%. Cultures have been maintained in NBL through 32 subcultures for 3 years. This procedure can be used for clonal propagation as well as germplasm exchange of Trapa. The cost of production is substantially reduced by using market grade sugar and jam bottles.