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Research Project: Innovations that Improve the Efficiency and Effectiveness of Managing and Preserving Ex Situ Plant Germplasm Collections

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

Author
item ZHANG, JIN-MEI - Chinese Academy Of Agricultural Sciences
item HUANG, BIN - Fujian Agricultural & Forestry University
item ZHANG, XIAO-NING - Jiangxi Academy Of Agricultural Sciences
item Volk, Gayle
item XIN, XIA - Chinese Academy Of Agricultural Sciences
item YIN, GUANG-KUN - Chinese Academy Of Agricultural Sciences
item HE, JUAN-JUAN - Chinese Academy Of Agricultural Sciences
item LU, XIN-XIONG - Chinese Academy Of Agricultural Sciences
item ZHOU, YUAN-CHANG - Fujian Agricultural & Forestry University
item CHEN, XIAO-LING - Chinese Academy Of Agricultural Sciences

Submitted to: In Vitro Cellular and Developmental Biology - Plants
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/4/2015
Publication Date: 7/14/2015
Citation: Zhang, J., Huang, B., Zhang, X., Volk, G.M., Xin, X., Yin, G., He, J., Lu, X., Zhou, Y., Chen, X. 2015. Identification of a highly successful cryopreservation method (droplet-vitrification) for petunia. In Vitro Cellular and Developmental Biology - Plants. 51:445-451.

Interpretive Summary: Gene banks have adopted cryopreservation technologies to provide safe, secure, long-term back-ups for plant collections that are clonally maintained. Many horticultural crops, including petunia cultivars, are clonally propagated. This research uses petunia (Petunia x hybrida) shoot tips as a model system to identify and optimize key steps in a vitrification-based cryopreservation procedure. We found that optimization of the concentration of sucrose in the preculture solution, plant vitrification solution exposure duration, and recovery medium significantly affected recovery levels. Using the optimized method, shoot tip regrowth levels were as high as 80% after liquid nitrogen exposure. The method was successfully applied to three additional petunia cultivars. This research has resulted in a method that will be implemented within the National Genebank of China for long-term conservation of petunia cultivars.

Technical Abstract: Petunia (Petunia × hybrida Vilm.) is a very important crop conserved in the National Genebank of China. Petunia cultivar “Niu 2” was used to develop a droplet-vitrification protocol to cryopreserve shoot tips. Six variables (age of the in vitro plants, concentration of sucrose in the preculture solution, preculture duration, loading duration, PVS2 exposure length and recovery medium) were tested in an orthogonal array experiment, and additional experiments were performed to further optimize preculture duration, loading time, PVS2 treatment duration, and PVS2 concentration. The highest recovery levels were obtained using the following procedure: shoot tips (2-3 mm length) were dissected from in vitro plantlets, and precultured in 0.2 M sucrose solution for 2 days, incubated with 2 M glycerol + 0.4 M sucrose solution for 30 min at 25°C, cryoprotected with plant vitrification solution 2 (PVS2, 30% glycerol, 15% dimethyl sulfoxide, 15% ethylene glycol, 0.4 M sucrose) for 30 min at 0 °C and rapidly immersed in liquid nitrogen (LN). Cryopreserved shoot tips were then warmed at 25°C, and unloaded in 1.2 M sucrose for 20 min and recovered on the solidified MS basal medium supplemented with half strength of NH4 + and KNO3. Survival and recovery levels were as high as 95% and 80%, respectively. Three petunia cultivars, “Niu 1”, “Niu 3” and “Niu 4”, had average survival levels of 67%, 79% and 74%, respectively, and regrowth levels of 55%, 59% and 54%, respectively, after cryopreservation.