Effect of Cryopreservation Method Supported with Biochemical Analyses in the Axillary Bud of Jewel Orchid, <i>Ludisia discolor</i>

This study investigated conserving an endangered terrestrial jewel orchid Ludisia discolor, using in vitro grown axillary buds. Excised segments of axillary buds (4–5 mm in length) were precultured on a modified Murashige and Skoog (MS) medium supplemented with 0.2 M sucrose for 24 h and osmoprotected in a loading solution for 20 min. Then, axillary buds were dehydrated in plant vitrification solution 2 (PVS2) for 10 min at 0 °C and incubated in liquid nitrogen for 1 h. Subsequently, axillary buds were rewarmed rapidly by dilution solution and transferred to a growth recovery medium supplemented with 0.05 µM melatonin, which led to an improved survival chance (16.67%) for cryopreserved L. discolor. The osmotic stress and the overproduction of reactive oxygen species (ROS) during cryopreservation stages may result in cryoinjuries and poor survival as increased levels of proline (5.51 µmol/g), catalase (85.64 U/g), peroxidase (565.37 U/g), and ascorbate peroxidase activities (12.19 U/g) were detected after dehydration, preculture, rewarming, and loading stage, respectively. Results obtained from this study indicate that further experimental designs which apply different PVS and exogenous antioxidants are needed for improved survival and regrowth of L. discolor.