Abstract
A technique for micropropagation and cryopreservation of an endangered plant Nymphoides indica from
subcultured multiple shoot primordia is described. Excised tissues from leaves cultured on Murashige and
Skoog medium (MS medium) supplemented with 3 % (w/v) sucrose, 10 μmol L-1 Thidiazuron (TDZ) and 0.1
μmol L-1 1-naphthaleneacetic acid (NAA) formed multiple shoot primordia. These shoot primordia were
subcultured repeatedly at 3-week intervals. Subcultured shoot primordia were cryopreserved in liquid nitrogen.
Cell clumps of multiple shoot primordia treated with 5% (v/v) dimethyl sulfoxide (DMSO) and 10% (w/v)
glucose were cooled at a rate of 1°C/min to -40°C in a programmed freezer, then immersed in liquid nitrogen.
Pre-culture of shoot primordia on a medium containing 5% (w/v) sucrose for 2 days enhanced survival rate of
cryopreserved tissues. Plants with developed shoots and roots were obtained by transferring the survived shoot
primordia to MS medium lacking growth regulators.
