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High-efficiency vitrification protocols for cryopreservation of in vitro grown shoot tips of transgenic papaya lines

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Abstract

In vitro grown shoot tips of transgenic papaya lines (Carica papaya L.) were successfully cryopreserved by vitrification. Shoot tips were excised from stock shoots that were preconditioned in vitro for 45–50-day-old and placed on hormone-free MS medium with 0.09 M sucrose. After loading for 60 min with a mixture of 2 M glycerol and 0.4 M sucrose at 25°C, shoot tips were dehydrated with a highly concentrated vitrification solution (PVS2) for 80 min at 0°C and plunged directly into liquid nitrogen. The regeneration rate was approximately 90% after 2 months post-thawing. Successfully vitrified and warmed shoot tips of three non-transgenic varieties and 13 transgenic lines resumed growth within 2 months and developed shoots in the absence of intermediate callus formation. Dehydration with PVS2 was important for the cryopreservation of transgenic papaya lines. This vitrification procedure for cryopreservation appears to be promising as a routine method for cryopreserving shoot tips of transgenic papaya line germplasm.

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Abbreviations

ABA:

Abscisic acid

BA:

6-Benzylaminopurine

DMSO:

Dimethylsulfoxide

IBA:

Indole-3-butyric acid

LN:

Liquid nitrogen

LS:

Loading solution

MS:

Murashinge and Skoog medium

PLDMV:

Papaya leaf-distortion mosaic virus

PRSV:

Papaya ringspot virus

PVS:

Plant vitrification solution

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Acknowledgments

The authors thank the Council of Agriculture, Executive Yuan, ROC. for financial support. Stock materials were kindly provided by Professor S. D. Yeh of the laboratory, Department of Plant Pathology, National Chung-Hsing University of Taichung, ROC. Also, the authors greatly thank Professor Hungchen-Emilie Yen, Department of Life Science, National Chung-Hsing University of Taichung, ROC, for her suggestions on improvements to this manuscript.

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Authors and Affiliations

  1. Department of Life Sciences, National Chung-Hsing University, 250, Kuo-Kuang Road, Taichung, 40227, Taiwan, ROC

    Shu-Fen Tsai, Chin-Feng Chan & Song-Iuan Liaw

  2. Department of Plant Pathology, National Chung-Hsing University, Taichung, Taiwan, ROC

    Shyi-Dong Yeh

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  1. Shu-Fen Tsai
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  2. Shyi-Dong Yeh
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  4. Song-Iuan Liaw
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Correspondence to Song-Iuan Liaw.

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Tsai, SF., Yeh, SD., Chan, CF. et al. High-efficiency vitrification protocols for cryopreservation of in vitro grown shoot tips of transgenic papaya lines. Plant Cell Tiss Organ Cult 98, 157–164 (2009). https://doi.org/10.1007/s11240-009-9548-4

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