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Research Project: Innovative Strategies and Methods for Improving the Management, Availability, and Utility of Plant Genetic Resource Collections

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Title: Cryopreservation of 12 Vitis species using apical shoot tips derived from plants grown in vitro

item BETTONI, JEAN - University Of Santa Catarina
item Bonnart, Remi
item Shepherd, Ashley
item KRETZSCHMAR, AIKE ANNELIESE - University Of Santa Catarina
item Volk, Gayle

Submitted to: HortScience
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/19/2019
Publication Date: 6/1/2019
Citation: Bettoni, J.C., Bonnart, R.M., Shepherd, A.N., Kretzschmar, A., Volk, G.M. 2019. Cryopreservation of 12 Vitis species using apical shoot tips derived from plants grown in vitro. HortScience. 54(6):976-981.

Interpretive Summary: The USDA-ARS National Plant Germplasm System (NPGS) maintains clonally propagated field collections of grapes (Vitis) at the Davis, CA and Geneva, NY repositories. These field collections are vulnerable to biotic and abiotic threats. Cryopreservation technologies have been successfully used to secure field collections of clonally propagated collections which are then safely housed at the National Laboratory for Genetic Resources Preservation (NLGRP) in Fort Collins, CO. Here, we report a method to cryopreserve Vitis clones. The method uses in vitro sourced plant materials (i.e., shoot tips) that were processed, cryoprotected, plunged into liquid nitrogen and recovered in vitro. We show this new method is broadly applicable across 12 diverse Vitis species, giving regrowth levels of at least 43%.

Technical Abstract: We report a method to cryopreserve twelve Vitis species using shoot tips excised from in vitro-grown plants. Uniform 1 mm shoot tips were obtained from nodal sections cultured from in vitro stock plants. Shoot tips were precultured for 3 days on medium containing 0.3 M sucrose, salicylic acid, glutathione (reduced form), and ascorbic acid. They were then treated with loading solution for 20 minutes at 22 degrees C followed by half-strength PVS2 for 30 minutes, and full-strength PVS2 treatments ranging from 60 to105 minutes at 0 degrees C. After exposure to LN on foil strips, shoot tips were warmed/diluted in unloading solution for 20 minutes and placed on recovery medium. Our results demonstrate a wide applicability of this technique with regrowth levels at least of 43 % in 13 genotypes representing 12 Vitis species. Histological observations revealed that cryopreserved shoot tips appear to have a high degree of cellular preservation in both the meristematic regions and at the base of leaf primordia. Some treatments were independently replicated by two technicians to determine the transferability of the methods. We demonstrate that the droplet-vitrification procedure can be successfully replicated by technical staff and suggest that this Vitis cryopreservation method may soon be ready for implementation in genebanks.