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

Poobathy R, Zakaria R, Murugaiyah V, Subramaniam S

PLoS One, 2018;13(4):e0195642.
PMID: 29649288 DOI: 10.1371/journal.pone.0195642

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.
Development of a PVS2 droplet-vitrification cryopreservation technique for Aranda Broga Blue orchid protocorm-like bodies (PLBs)

Ping KS, Poobathy RR, Zakaria R, Subramaniam S

Cryo Letters, 2018 5 8;38(4):290-298.
PMID: 29734430

　　BACKGROUND: Conservation of commercially important ornamental plants is important to maintain its unique beauty to cater the market demands.
OBJECTIVE: The main objective is to develop an efficient cryopreservation technique for Aranda Broga Blue orchid PLBs using droplet-vitrification method.
MATERIALS AND METHODS: Several critical factors in cryopreservation were accessed such as preculture concentrations and durations, choice of vitrification solutions, two-step or three-step vitrification, growth recovery medium and PVS2 exposure duration.
RESULTS: The best growth regeneration percentage (5%) was obtained when 3-4mm PLBs were precultured in 0.2M sucrose for 3 days, followed by osmoprotection for 20 minutes, dehydration in PVS2 for 20 minutes at 0 degree C, LN storage, thawed and unloading for 20 minutes, and growth regeneration in VW10 medium. PLBs were found to be very sensitive to osmotic stress imposed by high molecular weight cryoprotectant such as sucrose and glycerol. Osmotic potential of growth recovery medium is one of the main factors that affect growth recovery in cryopreserved PLBs.
CONCLUSION: Current report showed possibilities in cryopreserving Aranda Broga Blue PLBs using droplet-vitrification technique. However, further improvement of growth recovery can be done by focussing on approaches that facilitate sufficient water removal from PLBs without causing severe osmotic injuries to the plant cells.
Catalase and superoxide dismutase activities and the total protein content of protocorm-like bodies of Dendrobium sonia-28 subjected to vitrification

Poobathy R, Sinniah UR, Xavier R, Subramaniam S

Appl Biochem Biotechnol, 2013 Jul;170(5):1066-79.
PMID: 23640259 DOI: 10.1007/s12010-013-0241-z

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.
Fulltext Droplet-vitrification of Aranda Broga Blue orchid: Role of ascorbic acid on the antioxidant system and genetic fidelity assessments via RAPD and SCoT markers

Khor SP, Yeow LC, Poobathy R, Zakaria R, Chew BL, Subramaniam S

Biotechnol Rep (Amst), 2020 Jun;26:e00448.
PMID: 32368510 DOI: 10.1016/j.btre.2020.e00448

A droplet-vitrification cryopreservation protocol has been successfully developed for Aranda Broga Blue orchid hybrid using protocorm-like bodies (PLBs). However, maximum growth regeneration percentage was recorded at 5% only based on previous report. Thus, to improve growth recovery of cryopreserved PLBs, cryopreservation stages were supplemented with ascorbic acid, tested at 50, 100 and 150 mg/L. However, results demonstrated that exogenous ascorbic acid was not favorable in regeneration of cryopreserved explants (maximum value of 1.67 % with 50 mg/L ascorbic acid supplementation). Total soluble protein and various antioxidant enzyme activities such as catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX) were evaluated after each cryopreservation stages in conjunction with the application of exogenous ascorbic acid. Addition of antioxidant must be carefully evaluated and its application may not guarantee successful growth recovery. RAPD and SCoT molecular analysis confirmed the genetic stability of regenerated cryopreserved PLBs as no polymorphism was detected compared to control PLBs culture.