Effect of Cryopreservation Method Supported with Biochemical Analyses in the Axillary Bud of Jewel Orchid, Ludisia discolor.

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.

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

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.

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.

Early Studies on Protoplast Isolation of Ludisia discolor, A Wild Orchid.

The terrestrial Ludisia discolor, also referred to as the jewel orchid is prized for the quality of its leaves. L. discolor is known as a medicinal herb and is touted for its heat- and pathogen-resisting qualities. L. discolor is valuable in the production of both flavonoids and anthocyanins, antioxidants that are exalted in the health industry. Plant cell cultures have emerged as alternative sources of anthocyanin production. Plant protoplast cultures are used frequently in transient gene expression studies and in the establishment of callus and cell suspension cultures. Benefits of plant protoplast system include similarity to cells found in plant tissues, reproduction under controlled conditions, and prevention of masking of stress responses to previous handling techniques. A study was conducted to assess the amenability of the stem and leaves of L. discolor to protoplast isolation. The stem and leaf segments were weighed, sliced into thin layers, immersed in a digestion medium, washed and then cultured onto a recovery medium. Results indicated that the production of plant protoplasts from L. discolor may be viewed as an alternative in the generation of cell cultures and ultimately in the production of anthocyanins from the cell cultures.

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.