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Effects of Ascorbic Acid on PVS2 Cryopreservation of Dendrobium Bobby Messina’s PLBs Supported with SEM Analysis

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Abstract

Regrowth of the cryopreserved protocorm-like bodies (PLBs) of Dendrobium Bobby Messina was assessed based on the plant vitrification solution 2 (PVS2) optimisation conditions. The optimized protocol obtained based on TTC spectrophotometrical analysis and growth recovery were 3–4 mm of PLBs size precultured in 0.2 M sucrose for 1 day, treated with a mixture of 2 M glycerol and 0.4 M sucrose supplemented with half-strength liquid MS media at 25 °C for 20 min and subsequently dehydrated with PVS2 at 0 °C for 20 min prior to storage in liquid nitrogen. Following rapid warming in a water bath at 40 °C for 90 s, PLBs were treated with unloading solution containing half-strength liquid MS media supplemented with 1.2 M sucrose. Subsequently, the PLBs were cultured on half-strength semi-solid MS media supplemented with 2 % (w/v) sucrose without any growth regulators and resulted in 40 % growth recovery. In addition, ascorbic acid treatment was used to evaluate the regeneration process of cryopreserved PLBs. However, growth recovery rates of non-cryopreserved and cryopreserved PLBs were 30 and 10 % when 0.6 mM ascorbic acid was added. Scanning electron microscopy analysis indicates that there are not much damages observed on both cryopreserved and non-cryopreserved PLBs in comparison to PLBs stock culture.

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Abbreviations

PLB:

Protocorm-like bodies

PVS2:

Plant vitrification solution

+ LN:

Cryopreserved PLBs

− LN:

Non-cryopreserved PLB

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Acknowledgments

This work was supported by Universiti Sains Malaysia Research Grant 2011 (USM) and National Science Fellowship (NSF).

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Authors and Affiliations

  1. School of Biological Sciences, Universiti Sains Malaysia (USM), Minden Heights, 11800, Penang, Malaysia

    Jessica Jeyanthi James Antony, Chan Lai Keng & Sreeramanan Subramaniam

  2. Department of Biochemistry, Faculty of Biotechnology and Molecular Sciences, Universiti Putra Malaysia, 43400, Serdang, Selangor Darul Ehsan, Malaysia

    Maziah Mahmood

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  1. Jessica Jeyanthi James Antony
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  2. Chan Lai Keng
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  4. Sreeramanan Subramaniam
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Correspondence to Sreeramanan Subramaniam.

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Antony, J.J.J., Keng, C.L., Mahmood, M. et al. Effects of Ascorbic Acid on PVS2 Cryopreservation of Dendrobium Bobby Messina’s PLBs Supported with SEM Analysis. Appl Biochem Biotechnol 171, 315–329 (2013). https://doi.org/10.1007/s12010-013-0369-x

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