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

Abstract

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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References

  1. Benson EE (2008) Cryopreservation theory. In: Reed BM (ed) Plant cryopreservation: a practical guide. Springer, New York, pp 15–30

    Google Scholar 

  2. Berke MJ (1986) The glass state and survival of anhydrous biological systems. In: Leopold AC (ed) Membrane, metabolism and dry organisms. Cornell Univ. Press, Ithaca, pp 358–364

    Google Scholar 

  3. Cellarova E, Skyba M, Urbanova M, Kapchina-Toteva V, Kosuth J, Harding K (2010) Physiological, biochemical and molecular characteristics of cryopreserved Hypericum perforatum L. shoot Tips. CryoLetters 31:249–260

    PubMed  Google Scholar 

  4. Engelmann F (2011) Use of biotechnologies for the conservation of plant biodiversity. In Vitro Cell Dev Biol Plant 47:5–16

    Article  Google Scholar 

  5. Engelmann F, Sakai A (2007) Vitrification, encapsulation-vitrification and droplet-vitrification: a review. CryoLetters 28:151–172

    PubMed  Google Scholar 

  6. Fei YL, Liu QL, Ge H (2008) Chinese crops and their wild relatives-flower. China Agriculture Press, Beijing, pp 505–508

    Google Scholar 

  7. Kong D, Shang H, Guo K, Liu Y, Zhang J, Wei H (2012) A study on optimizing the cryopreservation methods for Bama miniature pig semen. Exp Anim 61(5):533–542

    CAS  Article  PubMed  Google Scholar 

  8. Kriedt RA, Ramos-Fregonezi AMC, Beheregaray LB, Bonatto SL, Freitas LB (2011) Isolation, characterization, and cross-amplification of microsatellite markers for the Petunia integrifolia (Solanaceae) complex. Am Bot 98:e277–e279

    CAS  Article  Google Scholar 

  9. Kuriyama A, Watanabe K, Ueno S (1989) Inhibitory effect of ammoniumion on recovery of cryopreserved rice cells. Plant Sci 64:231–235

    CAS  Article  Google Scholar 

  10. Lyu SR, Wu WT, Hou CC, Hsieh WH (2010) Study of cryopreservation of articular chondrocytes using the Taguchi method. Cryobiology 60:165–176

    CAS  Article  PubMed  Google Scholar 

  11. Mandal BB, Ahuja-Ghosh S, Srivastava PS (2008) Cryopreservation of Dioscorea rotundata poir.: a comparative study with two cryogenic procedures and assessment of true-to-type of regenerants by RAPD analysis. CryoLetters 29:399–408

    CAS  PubMed  Google Scholar 

  12. Marco-Medina A, Luis Casas J, Swennen R, Panis B (2010) Cryopreservation of Thymus moroderi by droplet vitrification. CryoLetters 31:14–23

    CAS  PubMed  Google Scholar 

  13. Matsumoto T, Sakai A, Yamada K (1994) Cryopreservation of in vitro-grown apical meristems of wasabi (Wasabia japonica) by vitrification and subsequent high plant regeneration. Plant Cell Rep 13:442–446

    CAS  Article  PubMed  Google Scholar 

  14. Matsumoto T, Akihiro T, Maki S, Mochida K, Kitagawa M, Tanaka D, Yamamoto S, Niino T (2013) Genetic stability assessment of wasabi plants regenerated from long-term cryopreserved shoot tips using morphological, biochemical and molecular analysis. CryoLetters 34:128–136

    CAS  PubMed  Google Scholar 

  15. McLellan MR (1995) Methods in molecular biology, vol. 38. In: Cryopreservation and freeze-drying protocols. Humana Press, Totowa, NJ, pp 133-144

  16. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497

    CAS  Article  Google Scholar 

  17. Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321–4325

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  18. Nadarajan J, Staines HJ, Benson EE, Marzalina M, Krishnapillay B, Harding K (2006) Optimization of cryopreservation protocol for Sterculia cordata zygotic embryos using Taguchi experiments. J Trop For Sci 18:222–230

    Google Scholar 

  19. Pinker I, Halmagyi A, Olbricht K (2009) Effects of sucrose preculture on cryopreservation by droplet-vitrification of strawberry cultivars and morphological stability of cryopreserved plants. CryoLetters 30:202–211

    CAS  PubMed  Google Scholar 

  20. Reed BM (2008) Plant cryopreservation: a practical guide. Springer, New York, pp 1–513

    Google Scholar 

  21. Reed BM, Castillo NRF, Bassil NV, Wada S (2010) Genetic stability of cryopreserved shoot tips of Rubus germplasm. In Vitro Cell Dev Biol Plant 46:246–256

    Article  Google Scholar 

  22. Sakai A, Kobayashi S, Oiyama I (1990) Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification. Plant Cell Rep 9:30–33

    CAS  Article  PubMed  Google Scholar 

  23. Sekizawa K, Yamamoto SI, Rafique T, Fukui K, Niino T (2011) Cryopreservation of in vitro-grown shoot tips of carnation (Dianthus caryophyllus L.) by vitrification method using aluminium cryo-plates. Plant Biotechnol 28:401–405

    Article  Google Scholar 

  24. Teixeira AS, Gonzalez-Benito ME, Molina-Garcia AD (2013) Glassy state and cryopreservation of mint shoot tips. Biotechnol Prog 29:707–717

    CAS  Article  PubMed  Google Scholar 

  25. Vallejo-Marin M, Solis-Montero L, Bacies CFE, Lepais O (2011) Thirteen micro-satellites developed by SSR-enriched pyrosequencing for Solanum Rostratum (Slolanaceae) and related species. Am Bot 98:e296–e299

    CAS  Article  Google Scholar 

  26. Volk GM, Bonnart R, Krueger R, Lee RR (2012) Cryopreservation of citrus shoot tips using micrografting for recovery. CryoLetters 33:418–426

    CAS  PubMed  Google Scholar 

  27. Zhang JM, Zhang XN, Lu XX, Xin X, Yin GK, He JJ, Xu YM, Chen XL (2014) Optimization of droplet-vitrification protocol for carnation genotypes and ultrastructual studies on shoot tips during cryopreservation. Acta Physiol Plant 36:3189–3198

    CAS  Article  Google Scholar 

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Acknowledgments

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). https://doi.org/10.1007/s11627-015-9704-y

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Keywords

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