Capsule harvesting times and various dehydration methods for cryopreservation of Paphiopedilum bellatulum (Rchb.f.) Stein seeds

Abstract

Paphiopedilum bellatulum (Rchb.f.) Stein is an attractive terrestrial lady's slipper orchid that is threatened with extinction, due to over-collection and loss of suitable habitats. Consequently, ex situ conservation of this orchid is urgently needed. This study was done to develop an effective technique to cryopreserve P. bellatulum seeds for long-term germplasm preservation. The capsules were harvested at one month interval from 5 to 9 months after pollination (MAP) and then seeds were subjected to three techniques before preservation in liquid nitrogen: 1) desiccation, 2) desiccation and wrapping with aluminum foil, and 3) vitrification. After harvesting the capsules at one month interval from 5 to 9 MAP, seed moisture content (MC) continuously decreased from 28.80% at 5 MAP to 19.74% at 9 MAP. Meanwhile, the seeds harvested at prolonged timing inclined to have more seed germination percentage. Nine MAP seeds showed the highest seed germination at 37.08%. After cryopreservation, desiccation technique and modification with aluminum foil wrap technique did not show different results. The seeds harvested at 9 MAP showed the highest germination by desiccation for 5 h at 30.58 and 28.75%, respectively. For the vitrification technique, the seeds were harvested at 9 MAP, then pretreated with loading solution for 15 min, followed by exposure to PVS2 solution for 60 min gave the highest germination rate at 30.96%. However, the three methods were not significantly different in the highest germination after cryo-storage. Cryopreservation of P. bellatulum using fully mature seed by desiccation technique is recommended as it is the most practicable and reduces the time and cost for ex situ long-term storage. These results can be used to develop protocols for cryopreservation of Paphiopedilum and play an important role in global conservation strategies.