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Identification of a highly successful cryopreservation method (droplet-vitrification) for petunia
- 作者:Jin-Mei Zhang ; Bin Huang ; Xiao-Ning Zhang…
- 关键词:Droplet ; vitrification ; Genetic stability ; One ; factor experiment ; Orthogonal array ; Petunia
- 刊名:In Vitro Cellular & Developmental Biology - Plant
- 出版年:2015
- 出版时间:August 2015
- 年:2015
- 卷:51
- 期:4
- 页码:445-451
- 全文大小:463 KB
- 参考文献:Benson EE (2008) Cryopreservation theory. In: Reed BM (ed) Plant cryopreservation: a practical guide. Springer, New York, pp 15-0View Article
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- 作者单位:Jin-Mei Zhang (1)
Bin Huang (2) Xiao-Ning Zhang (3) Gayle M. Volk (4) Yuan-Chang Zhou (2) Xiao-Ling Chen (1)
1. National Genebank, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100081, China 2. Fujian Agriculture and Forestry University, Fuzhou, 350002, China 3. Institute of Rice Research, JiangXi Academy of Agricultural Sciences, Nanchang, 330200, China 4. USDA-ARS National Center for Genetic Resources Preservation, 1111 S. Mason St., Fort Collins, CO, 80521, USA
- 刊物主题:Plant Sciences; Cell Biology; Developmental Biology; Plant Breeding/Biotechnology; Plant Genetics & Genomics;
- 出版者:Springer US
- ISSN:1475-2689
文摘
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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