Ranjetta Poobathy

Catalase and Superoxide Dismutase Activities and the Total Protein Content of Protocorm-Like Bodies of Dendrobium Sonia-28 Subjected to Vitrification

Dendrobium sonia-28 is an important ornamental orchid in the Malaysian flower industry. However, the genus faces both low germination rates and the risk of producing heterozygous progenies. Cryopreservation is currently the favoured long-term storage method for orchids with propagation problems. Vitrification, a frequently used cryopreservation technique, involves the application of pretreatments and cryoprotectants to protect and recover explants during and after storage in liquid nitrogen. However, cryopreservation may cause osmotic injuries and toxicity to cryopreserved explants from the use of highly concentrated additives, and cellular injuries from thawing, devitrification and ice formation. Reactive oxygen species (ROS), occurring during dehydration and cryopreservation, may also cause membrane damage. Plants possess efficient antioxidant systems such as the superoxide dismutase (SOD) and catalase (CAT) enzymes to scavenge ROS during low temperature stress. In this study, protocorm-like bodies (PLBs) of Dendrobium sonia-28 were assayed for the total protein content, and both SOD and CAT activities, at each stage of a vitrification exercise to observe for deleterious stages in the protocol. The results indicated that cryopreserved PLBs of Dendrobium sonia-28 underwent excessive post-thawing oxidative stress due to decreased levels of the CAT enzyme at the post-thawing recovery stage, which contributed to the poor survival rates of the cryopreserved PLBs.

Autofluorescence study and selected cyanidin quantification in the Jewel orchids Anoectochilus sp. and Ludisia discolor

Anoectochilus sp. and Ludisia discolor are known as Jewel orchids. Both species are terrestrial wild orchids that grow in shaded areas of forests. The Jewel orchids are renowned for the beauty of their leaves, which are dark-green laced with silvery or golden veins. The orchids are used as a cure in various parts of Asia. Overharvesting and anthropogenic disturbances threaten the existence of the Jewel orchids in the wild, necessitating human intervention in their survival. An understanding of the structure and adaptations of a plant may assist in its survival when propagated outside of its habitat. In this study, ex vitro leaves of Anoectochilus sp. and L. discolor were subjected to freehand sectioning, and then inspected through brightfield and fluorescence microscopy. The study indicated that all parts of both plants presented typical monocotyledonous characteristics except the leaves. The leaves displayed dorsiventrality with distinct palisade and spongy mesophyll layers. The spongy mesophyll layer contained cells which fluoresced a bright red when exposed to ultraviolet, blue, and green light wavelengths, hinting at the presence of anthocyanins for photoprotection. Cyanidin was detected in the leaves of L. discolor, as enumerated through high performance liquid chromatography (HPLC). The observations indicated that Anoectochilus sp. and L. discolor are well-adapted to live under shaded conditions with minimal exposure to light.

Cryopreservation of PLBs of Brassidium Fly Away Using Encapsulation-Dehydration Technique

In vitro grown protocorm-like bodies (PLBs) of Brassidium Fly Away orchid hybrid were cryopreserved using encapsulation- dehydration technique. The viability of the cryopreserved cells was determined by 2,3,5-triphenyltetrazolium chloride (TTC) assay. For the preculture treatment, the PLBs were excised into two standard sizes of 1-2 and 4-5 mm and were precultured on half-strength Murashige and Skoog (MS) semi solid medium supplemented with diff erent concentrations of sucrose (0, 0.2, 0.4, 0.6, 0.8 and 1.0M). The PLBs size 4-5 mm and 0.6 M sucrose concentration was selected based on highest viability obtained in TTC assay. The PLBs were encapsulated for 30 minutes using 3% (w/v) liquid sodium alginate medium supplemented with 0.4M sucrose and 0.1M calcium chloride and osmoprotected in 0.75M sucrose solution for 24 hours at 25°C. The beads were then dehydrated using 50g heat-sterilised silica gel for four hours, cryopreserved for 24 hours, thawed in a 40±2°C water bath for 90 seconds, and regenerated in semi-solid half-strength. Biochemical analyses were conducted and the cryopreserved PLBs had produced lower content of chlorophyll while the highest specifi c peroxidase activity was observed in cryopreserved PLBs.