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Determination of glassy state by cryo-SEM and DSC in cryopreservation of mint shoot tips by encapsulation–dehydration

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Abstract

Cryopreservation of mint shoot tips grown in vitro (Mentha × piperita) was performed after encapsulation in alginate beads. Encapsulated shoot tips were first precultured in sucrose enriched medium (0.75 M) and then dried under a sterile air flow (0–6 h). After cooling in liquid nitrogen and warming in a warm water bath, alginate beads were transferred to solid culture medium for 4 weeks. The effect of dehydration time of the encapsulated shoots was evaluated for water content, cooling and warming rates, ice crystal formation and cellular vitrification, by using low temperature scanning electron microscopy and differential scanning calorimetry. Viability of the recovered material showed a close relation between the dehydration time, cooling and warming rates, ice formation avoidance and tissue vitrification. At short drying periods (up to 3 h), ice crystals were formed and the viability was low or absent. After longer drying periods (5 and 6 h), both beads and specimens became vitrified.

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Acknowledgments

This work has been carried out thanks to project “CRYODYMINT” (AGL2010-21989-C02-02) of the Spanish Ministry of Science and Innovation. A.S. Teixeira was supported by the CSIC, within the JAE-Pre program, partially funded from the European Social Fund. It is also acknowledged the expert microscopic work of F. Pinto, who sadly passed away, shortly after this work was completed.

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Correspondence to Antonio D. Molina-García.

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Teixeira, A.S., González-Benito, M.E. & Molina-García, A.D. Determination of glassy state by cryo-SEM and DSC in cryopreservation of mint shoot tips by encapsulation–dehydration. Plant Cell Tiss Organ Cult 119, 269–280 (2014). https://doi.org/10.1007/s11240-014-0531-3

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Keywords

  • Intracellular ice formation
  • Glass transition
  • Cooling rate
  • Viability
  • Calorimetry