The Development Of Encapsulationdehydration And Vitrification Protocols For Protocorm-Like Bodies (PLBs) Of Dendrobium sonia-28
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Date
2012-02
Authors
Poobathy, Ranjetta
Journal Title
Journal ISSN
Volume Title
Publisher
Universiti Sains Malaysia
Abstract
The vitrification and encapsulation-dehydration methods of cryopreservation
were applied on protocorm-like bodies (PLBs) of the orchid hybrid Dendrobium
sonia-28, with survival assessments conducted through growth observations, visualand
spectrophotometric 2,3,5-triphenyltetrazolium chloride (TTC) assays. The best
survival (16.0% regeneration) for vitrification was obtained when 3-4mm PLBs were
precultured in semi-solid half-strength MS medium supplemented with 0.4M
sucrose, loaded in a loading solution for 20 minutes, dehydrated for 50 minutes at
0°C in plant vitrification solution 2 (PVS2), cryopreserved in liquid nitrogen (LN)
for 24 hours, thawed in a 40±2°C water bath for 90 seconds, unloaded in 1.2M
sucrose for 20 minutes, and regenerated in semi-solid half-strength MS medium
containing 2g.L-1 charcoal, with all media supplemented with 0.6mM ascorbic acid.
The PLBs were gradually exposed to light (darkness in the first week of recovery,
exposure to 3.4μmol.m-2.s-1 for the subsequent three weeks and 150μmol.m-2.s-1
thereafter). The best viability rate (85.5% when observed for any signs of redness)
for encapsulation-dehydration was obtained when 3-4mm PLBs were pretreated in
semi-solid 0.5M sucrose medium, encapsulated for 30 minutes using 3% (w/v) liquid
sodium alginate medium supplemented with 0.4M sucrose and 0.1M calcium
chloride, osmoprotected in 0.75M sucrose solution for 24 hours at 25°C, 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-strengt
MS medium followed by a transfer to medium supplemented with 1mg.L-1 6-
benzyladenopurine (BAP), with gradual exposure to light (darkness in the first week
of recovery, exposure to 3.4μmol.m-2.s-1 in the second week and 150μmol.m-2.s-
1thereafter). Biochemical analyses of PLBs subjected to the vitrificationcryopreservation
experiment indicated a general decrease in the total protein content
of cryopreserved PLBs with progression of the protocol.
Description
Keywords
Encapsulation-dehydration and vitrification , protocols for protocorm-like bodies of dendrobium sonia-28