Effect of PVS2 vitrification on Brassidium shooting star orchid using protocorm-like bodies (PLBs)

A cryopreservation procedure was developed to preserve protocorm-like bodies (PLBs) of Brassidium Shooting Star using the PVS2 vitrification technique. The optimised protocol involved the preculture of 3-4mm PLBs in half-strength Murashige and Skoog (MS) semi-solid medium supplemented with 0.8M sucrose, followed by dehydration in PVS2 solution for 20 minutes at 0°C, prior to storage in liquid nitrogen. The viability of non-cryopreserved and cryopreserved PLBs was determined by the 2,3,5-triphenyltetrazolium chloride (TTC) assay, after two weeks of recovery. The chlorophyll contents, total soluble protein and peroxidase activities of both non-cryopreserved and cryopreserved PLBs were assayed after three weeks of recovery. The results from the biochemical analyses indicated that control PLBs produced the highest viability, followed by treatment on non-cryopreserved PLBs (-LN) and cryostored PLBs (+LN), except in the peroxidase activity assay. The peroxidase activity was detected as the highest in cryostored PLBs followed by treated but non-cryopreserved PLBs, and control PLBs

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.</i