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Development of a new vitrification solution, VSL, and its application to the cryopreservation of gentian axillary buds

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Abstract

Vitrification methods are convenient for cryopreserving plant specimens, as the specimens are plunged directly into liquid nitrogen (LN) from ambient temperatures. However, tissues and species with poor survival are still not uncommon. The development of vitrification solutions with high survival that cover a range of materials is important. We attempted to develop new vitrification solutions using bromegrass cells and found that VSL, comprising 20% (w/v) glycerol, 30% (w/v) ethylene glycol, 5% (w/v) sucrose, 10% (w/v) DMSO and 10 mM CaCl2, gave the highest survival following cryopreservation, as determined by fluorescein diacetate staining. However, the cryopreserved cells showed little regrowth, for unknown reasons. To check its applicability, VSL was used to cryopreserve gentian axillary buds and the performance was compared with those of conventional vitrification solutions. Excised gentian stem segments with axillary buds (shoot apices) were two-step precultured with sucrose to induce osmotic tolerance prior to cryopreservation. Gentian axillary buds cryopreserved using VSL following the appropriate preculturing approach exhibited 78% survival (determined by the regrowth capacity), which was comparable to PVS2 and PVS1 and far better than PVS3. VSL had a wider optimal incubation time (20–45 min) than PVS2 and was more suitable for cryopreserving gentian buds. The optimal duration of the first step of the preculture was 7–11 days, and preculturing with sucrose and glucose gave a much higher survival than fructose and maltose. VSL was able to vitrify during cooling to LN temperatures, as glass transition and devitrification points were detected in the warming profiles from differential scanning calorimetry. VSL and its derivative, VSL+, seem to have the potential to be good alternatives to PVS2 for the cryopreservation of some materials, as exemplified by gentian buds.

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Abbreviations

DSC:

Differential scanning calorimetry

TC:

Treated control

MS medium:

Murashige and Skoog medium

LN:

Liquid nitrogen

PVS2:

Plant vitrification solution 2

VSL:

Vitrification solution L

VSL+:

Vitrification solution L+

DMSO:

Dimethylsulfoxide

FDA:

Fluorescein diacetate

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Acknowledgments

The authors would like to thank Dr. Duncan A. Vaughan (NIAS, Ibaraki) for his critical reading of the manuscript and Ms. T. Kitashima, A. Oda, H. Nakatani, R. Iwanami and Y. Koike (NIAS, Ibaraki) for their technical assistance. P.T. would like to acknowledge JSPS funds for visiting scientists, and M.S. a JSPS fellowship for young scientists.

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Author notes

  1. Pramod Tandon

    Present address: Department of Botany, North-Eastern Hill University, Shillong, 793 022, India

Authors and Affiliations

  1. Environmental Stress Research Unit, Division of Plant Sciences, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki, 305-8602, Japan

    Mitsuteru Suzuki, Pramod Tandon, Masaya Ishikawa & Takayuki Toyomasu

Authors
  1. Mitsuteru Suzuki
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  2. Pramod Tandon
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  3. Masaya Ishikawa
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  4. Takayuki Toyomasu
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Corresponding author

Correspondence to Masaya Ishikawa.

Additional information

Mitsuteru Suzuki, Pramod Tandon and Masaya Ishikawa contributed equally to the work.

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Suzuki, M., Tandon, P., Ishikawa, M. et al. Development of a new vitrification solution, VSL, and its application to the cryopreservation of gentian axillary buds. Plant Biotechnol Rep 2, 123–131 (2008). https://doi.org/10.1007/s11816-008-0056-5

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  • DOI: https://doi.org/10.1007/s11816-008-0056-5

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