Cryopreservation Of Brassidium Shooting Star Orchid Using Dropletvitrification Method
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Date
2014-07
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Universiti Sains Malaysia
Abstract
The aims of this research are to investigate the efficiency of Protocorm-like
bodies (PLBs) induction method and develop a droplet-vitrification protocol
applicable for cryopreservation of Brassidium Shooting Star orchid. The effect of
plant growth regulators such as benzylaminopurine (BAP), naphthalene acetic acid
(NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) on PLBs formation of
Brassidium Shooting Star orchid was studied. Basal corms were cultured in media
supplemented with BA alone or in combination with NAA and 2,4-D. The numbers
of PLBs induced from each explant were counted after six weeks of treatment.
Addition of 1.0 mg/L BAP to MS basal media was found to be the best media for
PLBs induction that producing an average of 25.43±5.12 PLB per explant. Seven (7)
parameters have been optimised in droplet-vitrification method such as PLB size,
sugar type, preculture concentration, preculture incubation duration, plant
vitrification solution 2 (PVS2) incubtion duration, thawing method and unloading
durations. Assessments of viability were evaluated by using TTC spectrophotometric
at 490nm absorbance reading and growth recovery the regenerated PLBs. The
highest growth recovery of 30% was obtained with 3-4 mm PLBs precultured on
media supplemented with 0.25M sucrose for 7 days, followed by loading treatment
for 20 min, dehydration with PVS2 solution for 40 min and treated with unloading
solution for 15 min. In order to reveal the lethal and non-lethal damages produced by
cryopreservation, histological, scanning electron microscopy and biochemical
analysis were carried out on both cryopreserved and non-cryopreserved PLBs of
Brassidium Shooting Star orchid comparing with the PLBs stock culture.
Histological analysis displayed cryopreservation protocol caused structural changes
in cryopreserved PLBs and non-cryopreserved PLB. Similarly scanning electron
micrograph also showed severe damages in cryopreserved and non-cryopreserved
PLBs comparative to the PLBs stock culture. Addition of ascorbic acid and α-
tocopherol which reported to improve regrowth of cryopreserved plant did not affect
the regrowth of Brassidium Shooting Star orchid PLBs. However, addition α-
tocopherol promoted faster recovery of cryopreserved PLBs.
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Keywords
Efficiency of Protocorm-like bodies , cryopreservation of Brassidium Shooting Star orchid