Cryopreservation Of Ascocenda Wangsa Gold Orchid Using Pvs2 Vitrification Method
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Date
2015-07
Authors
Rajasegar, Arulvilee
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Abstract
An efficient plant vitrification solution 2 (PVS2) cryopreservation method was developed for protocorm-like bodies (PLBs) of Ascocenda Wangsa Gold orchid. The effects of organic additives such as coconut water, potato and banana homogenates on PLBs proliferation of Ascocenda Wangsa Gold in different media culture were studied. The numbers of PLBs induced from each explant were counted after four weeks of treatment. Addition of 15% of coconut water, 2% of potato and banana homogenate into Vacin and Went medium was found to be the best media culture for PLBs induction at 94.7%. Parameters assessed included the preculture sucrose concentration effect of PVS2 exposure periods, thawing duration, temperature, and culture conditions based on 2, 3, 5-triphenyltetrazolium chloride absorbance readings and regrowth rates. A regrowth rate of 33.3% was obtained after 2 months when the PLBs were dehydrated in PVS2 for 30 min. The growth rate was improved to 47% when thawed at 45°C for 85 s. The highest growth rate (53.3%) was obtained when the PLBs were subjected to a 7-day dark treatment before being transferred to a 16-h/8-h light/dark photoperiod. Histological and scanning electron microscopy analyses were conducted to study the lethal and non-lethal damages of cryopreserved and non-cryopreserved PLBs of Ascocenda Wangsa Gold. The studies showed that embryogenic cells only survived in cryopreserved PLBs after freezing and thawing treatments. Start codon targeted (SCoT) markers were used to study the genetic stability of cryopreserved, non-cryopreserved and control plantlets of Ascocenda Wangsa Gold. Four informative primers were selected from 16 SCoT primers based on their capability to produce clear and reproducible monomorphic bands. The SCoT analyses showed that the cryopreserved and non-cryopreserved PLBs were genetically similar as control plant. The vitrification method developed in this study is a feasible and safe method for strengthening the germplasm conservation of this orchid for commercial purposes.
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Orchids , Plant tissue culture