Droplet-vitrification cryopreservation of in vitro-grown shoot tips of grapevine (Vitis spp.)

Authors: BI, WENLU - Northwest Agriculture And Forestry University; HAO, XINYI - Northwest Agriculture And Forestry University; CUI, ZHENHUA - Northwest Agriculture And Forestry University; Volk, Gayle; WANG, QIAOCHUN - Northwest Agriculture And Forestry University

Submitted to: In Vitro Cellular and Developmental Biology - Plants
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/25/2018
Publication Date: 8/2/2018
Citation: Bi, W., Hao, X., Cui, Z., Volk, G.M., Wang, Q. 2018. Droplet-vitrification cryopreservation of in vitro-grown shoot tips of grapevine (Vitis spp.). In Vitro Cellular and Developmental Biology - Plants. 54:590-599. https://doi.org/10.1007/s11627-018-9931-0.
DOI: https://doi.org/10.1007/s11627-018-9931-0

Interpretive Summary: The USDA-ARS National Plant Germplasm System (NPGS) maintains grapes (Vitis) as field plantings; as such they are vulnerable to biotic and abiotic stresses. Herein we report a method whereby one millimeter shoot tips of in vitro grown Vitis plants can be excised, precultured in medium containing sucrose, glutathione, and ascorbic acid, and then successfully cryopreserved using a droplet vitrification procedure. The method was tested for three wine cultivars, two table grape cultivars, and one rootstock, and one wild species and had an average regrowth level of 51%. The results obtained in this US-Chinese research collaboration were similar to those obtained at the USDA-ARS National Laboratory for Genetic Resources Preservation in Fort Collins, Colorado for diverse Vitis species. This demonstrates that cryopreservation methods will soon be ready to be implemented within the NPGS to ensure the long-term security of Vitis field collections.

Technical Abstract: We report an efficient and broad-spectrum protocol for cryopreservation of Vitis shoot tips by droplet-vitrification. Shoot tips (1.0 mm in size) containing 5-6 leaf primordia (LPs) were precultured for 3 days with a preculture medium containing 0.3 M sucrose, 0.16 µM glutathione and 0.14 µM ascorbic acid. Precultured shoot tips were treated for 20 min at room temperature with a loading solution comprised of 2 M glycerol and 0.4 M sucrose, followed by exposure to half-strength plant vitrification solution 2 (PVS2) for 30 min and then full-strength PVS2 for 50 min at 0 C. Dehydrated shoot tips were transferred onto 2.5-µL droplets of PVS2 carried on aluminum foils, prior to a direct immersion in liquid nitrogen. With this method, an average shoot regrowth level of 51 % was obtained from cryopreserved shoot tips in six Vitis vinifera genotypes (three wine cultivars, two table cultivars and one rootstock) and two V. pseudoreticulata. Vegetative growth of the regenerants recovered after cryopreservation significantly increased as subculture cycles increased and was greater than that of the control after the third subculture following cryopreservation. Inter-simple sequence repeats (ISSR) and random amplification of polymorphic DNA (RAPD) did not identify any novel alleles in the plants recovered after cryopreservation compared to the original cultures. Establishment of this droplet-vitrification cryopreservation provides a technical platform for setting-up cryobanks and production of virus-free plant materials in Vitis.