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Optimization of droplet-vitrification protocol for carnation genotypes and ultrastructural studies on shoot tips during cryopreservation

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Abstract

This study was carried out to optimize a modified droplet-vitrification procedure for the cryopreservation of shoot tips from different carnation genotypes. The best procedure was developed by applying orthogonal tests to the experimental data and by further investigation of the effects on the regrowth percentage. It consisted in preculturing shoot tips in liquid Murashige and Skoog (MS) medium with 0.3 M sucrose for 2 days, pretreating them in liquid MS medium with 5 % Dimethyl sulfoxide +5 % glycerol + 0.3 M sucrose for 10 min, osmoprotecting in Loading solution for 20 min at 25 °C, cryoprotecting with Plant vitrification solution No.2 (PVS2) for 60 min at 0 °C, transferring in drops of fresh PVS2 over aluminum strips and finally storing them in Liquid nitrogen. With the application of the optimized protocol, four carnation cultivars (‘Master’, ‘Calibra’, ‘Lamour’ and ‘Ofcar’) achieved regrowth percentage after cryopreservation ranging from 41 to 73 %. Ultrastructural observations investigated by using transmission electron microscopy showed that the cells encountered the stress during cryopreservation and the main damages occurred during the dehydration step. For surviving cells, the most of the damaged cells could be repaired after recovery growth. This modified protocol will aid in the long-term conservation of carnation germplasm and the ultrastructural studies will benefit for understanding the damage and recovery of the cells during cryopreservation.

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Abbreviations

AC:

Activated carbon

BA:

Benzylaminopurine

DS:

Dilution solution

DMSO:

Dimethyl sulfoxide

GA3 :

Gibberellin A3

IAA:

Indole acetic acid

LN:

Liquid nitrogen

LS:

Loading solution

MS:

Murashige and Skoog medium

PVS2:

Plant vitrification solution No. 2

TEM:

Transmission electron microscopy

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Acknowledgments

This study was supported by Core Research Budget of the Non-profit Governmental Research Institution (ICS, CAAS, 2011006), the Crop Germplasm Resources Protection and Utilization Special Grant from the Ministry of Agriculture, People’s Republic of China (NB2012-2130135-25-11) and National Key Technology R&D Program in accordance with the 12th five-year plan of People’s Republic of China (2013BAD01B01). The authors would like to acknowledge Professor Ge Hong of Institute of Vegetables and Flowers (CAAS) for supplying materials of Dianthus species. The authors also would like to acknowledge Barbara Reed (National Center for Genetic Resources Preservation, United State Department of Agriculture, Corvallis, Oregon, U.S.A.) for assistance in revision of the paper.

Conflict of interest

The authors declare that they have no competing interests.

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Authors and Affiliations

  1. National Genebank, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100081, China

    Jin-Mei Zhang, Xin-Xiong Lu, Xia Xin, Guang-Kun Yin, Juan-Juan He & Xiao-Ling Chen

  2. College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan, 430070, China

    Xiao-Ning Zhang & You-Ming Xu

  3. Institute of Rice Research, JiangXi Academy of Agricultural Sciences, Nanchang, 330200, China

    Xiao-Ning Zhang

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  1. Jin-Mei Zhang
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  3. Xin-Xiong Lu
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  4. Xia Xin
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  5. Guang-Kun Yin
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Correspondence to You-Ming Xu or Xiao-Ling Chen.

Additional information

Communicated by M. Lambardi.

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Zhang, JM., Zhang, XN., Lu, XX. et al. Optimization of droplet-vitrification protocol for carnation genotypes and ultrastructural studies on shoot tips during cryopreservation. Acta Physiol Plant 36, 3189–3198 (2014). https://doi.org/10.1007/s11738-014-1685-7

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  • DOI: https://doi.org/10.1007/s11738-014-1685-7

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