Cryopreservation of Habenaria radiata and Habenaria rhodocheila seeds using the aluminum cryo-plate vitrification method

The genus Habenaria consists of 600 species of terrestrial orchids distributed in Asia, America and Africa. Habenaria rhodocheila Hance is found in Thailand, Myanmar, Cambodia and Laos. Habenaria radiata (Thunb.) K. Spreng is native to Japan. The number of both species has decreased in their habitats because of overcollecting and environmental changes in the habitats. Conservation of the surviving plants is an urgent issue to maintain the genetic diversity of the species. Seeds of H. radiata and H. rhodocheila were surface sterilized, desiccated in a laminar air-flow cabinet, and then subjected to the cryo-plate vitrification method. Viability of the seeds with or without immersion in liquid nitrogen (LN) was estimated by seed germination. The seeds of H. radiata showed high viability after immersion in LN without PVS2 treatment, and the viability decreased when exposure to PVS2 exceeded 40 min. On the other hand, viability of the H. rhodocheila seeds after immersion in LN increased with PVS2 treatment for 10 to 30 min, stayed at high levels with 40 to 60 min of PVS2 treatment, and then decreased with more than 90 min of PVS2 treatment.