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  1. Cryopreservation of Brassidium Shooting Star orchid using the PVS3 method supported with preliminary histological analysis
    Mubbarakh SA, Rahmah S, Rahman ZA, Sah NN, Subramaniam S
    Appl Biochem Biotechnol, 2014 Jan;172(2):1131-45.
    PMID: 24146369 DOI: 10.1007/s12010-013-0597-0
    Cryopreservation is an alternative, safe, and cost-effective method for long-term plant genetic resource conservation. This study was conducted to optimize the conditions for cryopreserving the protocorm-like bodies (PLBs) of Brassidium Shooting Star orchid with the PVS3 vitrification method. Five parameters were assessed in this study: PLB size, sucrose concentration, preculture duration, PVS3 duration, and unloading duration. The viability of the cryopreserved PLBs was determined using the triphenytetrazolium chloride assay and growth recovery assessments. The optimum condition for the cryopreservation of the PLBs of Brassidium Shooting Star orchid is based on the size range between 3 and 4 mm precultured with half-strength semi-solid MS media supplemented with 0.25 M sucrose for 24 h, followed by treatment with loading solution mixture of 2 M glycerol and 0.4 M sucrose supplemented with half-strength liquid MS media at 25 °C for 20 min. The PLBs were then dehydrated with PVS3 at 0 °C for 20 min prior to immersion in liquid nitrogen; finally, the PLBs were immersed with half-strength liquid MS media supplemented with 1.2 M sucrose for 30 min. Histological analyses displayed denser cytoplasm and voluminous nucleus in the cryopreserved PLBs of Brassidium Shooting Star orchid.
    Matched MeSH terms: Orchidaceae/drug effects
  2. 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.

    Matched MeSH terms: Orchidaceae/drug effects
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