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Droplet-vitrification for shoot tip cryopreservation of shallot (Allium cepa var. aggregatum): effects of PVS3 and PVS2 on shoot regrowth

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Abstract

The present study described a droplet-vitrification cryopreservation for shoot tips of shallot (Allium cepa var. aggregatum), a small bulb onion. Shoot tips taken from in vitro stock shoots were precultured with 0.3 M and 0.5 M of sucrose, with 1 day for each concentration. Precultured shoot tips were treated with a loading solution containing 2 M glycerol and 0.6 M sucrose for 20 min and then exposed to plant vitrification solution 3 (PVS3) at 24 °C for 3 h of dehydration. Following exposure to PVS3, shoot tips were moved onto 5.0 µl PVS3 droplets on aluminum foil strips, followed by direct immersion into liquid nitrogen for 1 h. Frozen shoot tips were thawed by incubation in liquid MS medium containing 1.2 m sucrose for 20 min at room temperature, and then post-thaw cultured for shoot regrowth. Exposure of the shoot tips to PVS3 produced shoot regrowth (58%). Differential scanning calorimetry (DSC) detected 1.8% of freezable water in the shoot tips that had been dehydrated by PVS2, and no freezable water in those by PVS3 treatment. Exposure to PVS3 provided a broader safe temperature range (− 196 °C to − 88 °C), compared to that (− 196 °C to − 116 °C) of PVS2, for cryopreserved samples. Histological observations found that PVS3 dehydration allowed many cells in the apical dome and in the leaf primordia to survive following freezing in LN, while PVS2 dehydration resulted in much fewer surviving cells in the apical dome. The droplet-vitrification cryopreservation produced 56%, 72% and 32% shoot regrowth in cryopreserved shoot tips taken from in vitro shoots, adventitious buds regenerated from stem discs and field-grown bulbs, respectively. Advantages and disadvantages of the use of different source explants for cryopreservation were discussed. The droplet-vitrification cryopreservation produced 45% and 70% shoot regrowth in the additional two shallot genotypes ‘Kverve’ and ‘Lunteviga’. The results obtained in this study provide technical supports for setting-up cryo-bankings of genetic resources of shallots and other Allium species.

Key message

Establishment of a droplet-vitrification cryopreservation of shallot shoot tips provided technical supports for long-term preservation of diverse genetic resources and cryotherapy for virus eradication in shallot plants.

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Abbreviations

ABIM:

Adventitious bud induction medium

AD:

Apical dome

6-BA:

6-Benzylaminopurine

CE:

Cryopreservation efficiency

DSC:

Differential scanning calorimetry

FAA:

Formalin-acetic acid-alcohol solution

LN:

Liquid nitrogen

LP:

Leaf primordium

MS:

Murashige and Skoog (1962) medium

NAA:

Naphthylacetic acid

PVS:

Plant vitrification solution

PVS2:

Plant vitrification solution 2

PVS3:

Plant vitrification solution 3

SCM:

Stock culture medium

SE:

Standard error

TB:

Toluidine blue

TDZ:

Thidiazuron

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Acknowledgements

We acknowledge financial supports from the Research Council of Norway (Project No. 255032/E50), NIBIO, and the Norwegian Genetic Resource Centre. Professional assistance obtained from Crop Research Institute, Czech Republic in the DSC test is appreciated as well. We also acknowledge the Image center of Norwegian University of Life Sciences for providing technical guidance and valuable courses for microscopy.

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Authors and Affiliations

  1. Division of Biotechnology and Plant Health, Norwegian Institute of Bioeconomy Research, Ås, Norway

    Min-Rui Wang, Zhibo Zhang, Dag-Ragnar Blystad & Sissel Haugslien

  2. State Key Laboratory of Crop Stress Biology for Arid Areas, College of Horticulture, Northwest A&F University, Yangling, 712100, Shaanxi, China

    Min-Rui Wang & Qiao-Chun Wang

  3. Crop Research Institute, The Czech Republic, Praha 6, Czechia

    Jiří Zámečník & Alois Bilavčík

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  1. Min-Rui Wang
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Correspondence to Zhibo Zhang, Dag-Ragnar Blystad or Qiao-Chun Wang.

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Communicated by Sergio J. Ochatt.

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Wang, MR., Zhang, Z., Zámečník, J. et al. Droplet-vitrification for shoot tip cryopreservation of shallot (Allium cepa var. aggregatum): effects of PVS3 and PVS2 on shoot regrowth. Plant Cell Tiss Organ Cult 140, 185–195 (2020). https://doi.org/10.1007/s11240-019-01721-4

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