Development Of Cryopreservation In Dendrobium Sabin Blue By PVS2 Vitrification Method
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Date
2017-08
Authors
James Antony, Jessica Jeyanthi
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Publisher
Universiti Sains Malaysia
Abstract
The aim of this study was to utilize PVS2 vitrification method in cryopreservation of Dendrobium Sabin Blue. Several factors were sequentially optimized involving PLB size with inclusion of antioxidant treatments (ascorbic acid) at the cryopreservation stages, preculture conditions, loading and PVS2 conditions, PVS2 durations and unloading durations. Parameters were optimized using the 2, 3, 5- triphenyl tetrazolium chloride (TTC) spectrophotometrical and growth recovery analyses. The optimal regeneration percentage obtained with final optimised experiment involved 3-4mm PLBs precultured on half-strength semi-solid Murashige and Skoog (1962) medium supplemented with 0.2 M sucrose and 0.3 mM ascorbic acid for 1 day, loaded for 20 minutes at 30±2°C, dehydrated with plant vitrification solution 2 (PVS2) for 20 minutes at 0°C prior to storage in liquid nitrogen and rapidly thawed at 40±2°C for 90 seconds. This was followed by treatment with unloading solution composed of half-strength liquid MS medium supplemented with 1.2 M sucrose and 0.3 mM ascorbic acid for 20 minutes at 30±2°C. Next, the PLBs were cultured on half-strength semi-solid MS medium supplemented with 20 g/L sucrose devoid of plant growth regulators. The final optimised experiment obtained regeneration percentage of 15% in cryopreserved PLBs. PLBs proliferation growth index analysis resulted in significantly highest value of 16.95 from regenerated cryopreserved PLBs. The potential damages occurred at the cryopreservation stages were confirmed through biochemical, histological, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and molecular analyses. Biochemical analyses revealed a general reduction in chlorophyll, carotenoid, porphyrin contents; increased level in proline contents and fluctuated outcomes in catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POX) enzyme activities in PLBs exposed to different cryopreservation stages. This proved the importance of addition of ascorbic acid at the cryopreservation stages of Dendrobium Sabin Blue PLBs. Alternatively, histological analysis showed increased somatic embryogenesis in PLBs at the cryopreservation stages and in regenerated cryopreserved PLBs comparative to the stock culture PLBs. This was also confirmed via scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analyses revealing the presence of actively occurring somatic embryogenesis in regenerated cryopreserved PLBs. Finally, DAMD and ISSR analyses confirmed the occurrence of 7% polymorphism and monomorphism respectively in the regenerated cryopreserved PLBs.
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Keywords
To utilize PVS2 vitrification method , in cryopreservation of Dendrobium Sabin Blue