Anthony, Jessica Jeyanthi James
(2012)
Development Of Cryopreservation In Denrobium Bobby Messina Using PVS2 Vitrification And Encapsulation-Dehydration Methods.
Masters thesis, Universiti Sains Malaysia.
Abstract
Two new cryopreservation techniques, PVS2 vitrification and encapsulationdehydration,
were applied on PLBs of Dendrobium Bobby Messina, highly-prized
orchid in Malaysia, with survival monitored through observations of growth and the
2,3,5-triphenyltetrazolium chloride (TTC) assay. In the vitrification method, the
parameters optimized were PLBs size, preculture concentration, preculture durations
and PVS2 incubation temperature and duration. In the encapsulation-dehydration
method, the parameters optimized were PLBs size, preculture concentration,
preculture duration and dehydration period with silica gel. In cryopreservation of
Dendrobium Bobby Messina by vitrification technique, the optimized protocol based
on TTC spectrophotometrical analysis and growth recovery were 3-4mm PLBs
precultured in 0.2M sucrose for 1 day, treated with a mixture of 2M glycerol and
0.4M sucrose supplemented with half strength liquid MS media at 25°C for 20
minutes and subsequently dehydrated with plant vitrification solution 2 (PVS2) at
0°C for 20 minutes prior to storage in liquid nitrogen. Following rapid warming in a
water bath at 40°C for 90 seconds, the PLBs were washed with half strength liquid
MS media supplemented with 1.2M sucrose. Subsequently, the PLBs were cultured
on half strength semi-solid MS media supplemented with 2% (w/v) sucrose without
any growth regulators. The optimized vitrification method was successful in
preserving this orchid as it produced growth recovery in non-cryopreservaed and
cryopreserved PLBs up to 100% and 40% for Dendrobium Bobby Messina
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