Identification of a highly successful cryopreservation method (droplet-vitrification) for petunia


Petunia (Petunia × hybrida Vilm.) is an important horticultural crop conserved in the National Genebank of China. Here, a droplet-vitrification cryopreservation protocol was developed for petunia shoot tips. An orthogonal array experiment and additional one-factor experiments were performed to optimize key variables, including the age of in vitro plants, the concentration of sucrose in the preculture solution, the preculture duration, the duration of osmoprotection (2.0 M glycerol and 0.4 M sucrose), the length of exposure to and concentration of plant vitrification solution 2 (30% glycerol, 15% dimethyl sulfoxide, 15% ethylene glycol, and 0.4 M sucrose), and the recovery medium. By using the combined results of the orthogonal and one-factor experiments, the droplet-vitrification procedure for petunia cultivar Niu 2 was formulated efficiently and effectively. The highest regrowth levels were obtained using the following procedure: shoot tips were dissected from in vitro plantlets that were 20 d old, precultured in MS liquid medium with 0.2 M sucrose solution for 2 d, incubated with osmoprotection solution for 30 min at 25°C, cryoprotected with PVS2 for 30 min at 0°C, and rapidly immersed in liquid nitrogen. Cryopreserved shoot tips were then diluted in MS liquid medium with 1.2 M sucrose for 20 min at 25°C and regrown on solidified MS basal medium with half concentration of NH4NO3, KH2PO4, KNO3, and sucrose. Regrowth levels were as high as 80%. No morphological changes were observed and amplification of 15 simple sequence repeats revealed no genetic alterations after cryopreservation.

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This study was supported by Core Research Budget of the Non-profit Governmental Research Institution (ICS, CAAS, 2014JB02-002), the Crop Germplasm Resources Protection and Utilization Special Grant from the Ministry of Agriculture (2014NWB030-11), and the Agricultural Science and Technology Innovation Program/Crop Germplasm Resources Preservation and Sharing Innovation Team. The authors would like to acknowledge Remi Bonnart (National Center for Genetic Resources Preservation, United States Department of Agriculture, Fort Collins, Colorado, USA) for assistance in revision of the paper.

Author Contributions

Jin-Mei Zhang and Bin Huang designed and performed research, analyzed data, and wrote the manuscript, and JMZ and BH contributed equally to this work. XNZ and GMV participated in the analysis of the data. GMV participated in the revision of the manuscript. YCZ and XLC designed research and revised the manuscript, and they contributed equally to this work. All the authors have read and approved the final manuscript.

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Correspondence to Yuan-Chang Zhou or Xiao-Ling Chen.

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Jin-Mei Zhang and Bin Huang are co-first authors

Editor: David Duncan

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Zhang, JM., Huang, B., Zhang, XN. et al. Identification of a highly successful cryopreservation method (droplet-vitrification) for petunia. In Vitro Cell.Dev.Biol.-Plant 51, 445–451 (2015).

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  • Droplet-vitrification
  • Genetic stability
  • One-factor experiment
  • Orthogonal array
  • Petunia