Rajasegar, Arulvilee and Poobathy, Ranjetta and Rathinam, Xavier and Oyunbileg, Yungeree and Subramaniam, Sreeramanan
(2015)
Cryopreservation of PLBs of Brassidium Fly Away Using
Encapsulation-Dehydration Techniqu.
Mongolian Journal of Biological Sciences, 13 (1-2).
pp. 19-23.
ISSN 1684-3908
Abstract
In vitro grown protocorm-like bodies (PLBs) of Brassidium Fly Away orchid hybrid
were cryopreserved using encapsulation- dehydration technique. The viability of the
cryopreserved cells was determined by 2,3,5-triphenyltetrazolium chloride (TTC)
assay. For the preculture treatment, the PLBs were excised into two standard sizes of
1-2 and 4-5 mm and were precultured on half-strength Murashige and Skoog (MS)
semi solid medium supplemented with diff erent concentrations of sucrose (0, 0.2,
0.4, 0.6, 0.8 and 1.0M). The PLBs size 4-5 mm and 0.6 M sucrose concentration
was selected based on highest viability obtained in TTC assay. The PLBs were
encapsulated for 30 minutes using 3% (w/v) liquid sodium alginate medium
supplemented with 0.4M sucrose and 0.1M calcium chloride and osmoprotected
in 0.75M sucrose solution for 24 hours at 25°C. The beads were then dehydrated
using 50g heat-sterilised silica gel for four hours, cryopreserved for 24 hours,
thawed in a 40±2°C water bath for 90 seconds, and regenerated in semi-solid
half-strength. Biochemical analyses were conducted and the cryopreserved PLBs
had produced lower content of chlorophyll while the highest specifi c peroxidase
activity was observed in cryopreserved PLBs.
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