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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: Modifications to a Vitis shoot tip cryopreservation procedure: Effect of shoot tip size and use of cryoplates

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

Submitted to: CryoLetters
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/1/2019
Publication Date: 5/1/2019
Citation: Bettoni, J.C., Bonnart, R.M., Shepherd, A.N., Kretzschmar, A.A., Volk, G.M. 2019. Modifications to a Vitis shoot tip cryopreservation procedure: Effect of shoot tip size and use of cryoplates. CryoLetters. 40(2):103-112.

Interpretive Summary: The USDA-ARS National Plant Germplasm System maintains grape (Vitis) collections in field genebanks located in Geneva, NY and Davis, CA. These field collections are at risk of loss due to abiotic and biotic threats. This manuscript provides a method whereby vegetative shoot tips of Vitis can be preserved in liquid nitrogen for the long-term. It demonstrates that the use of 1 mm shoot tips is critical for the success of the described procedure and that high regrowth levels were obtained when shoot tips were placed onto aluminum cryoplates for solution processing and liquid nitrogen exposure. These results demonstrate that Vitis cryopreservation is ready to be implemented to secure Vitis collections in the National Plant Germplasm System.

Technical Abstract: BACKGROUND: Cryopreservation has been considered a preferred method for the long-term storage of plant germplasm, especially to efficiently conserve and maintain the genetic integrity of genebank materials. Droplet vitrification procedures have been developed to cryopreserve Vitis shoot tips from in vitro-grown plants. OBJECTIVE: This research tested modifications of the previously described Vitis droplet vitrification procedure by comparing shoot tip sizes of 1.0, 1.5. and 2.0 mm and by performing the cryopreservation procedure using shoot tips mounted on cryo-plates. MATERIALS AND METHODS: Uniform shoot tips were obtained from nodal sections cultured from in vitro grown stock plants of V. aestivalis and V. afghanistan. Shoot tips were precultured for 3 days on medium containing 0.3 M sucrose, salicylic acid, glutathione (reduced form), and ascorbic acid. They were cryopreserved using either droplet vitrification on aluminum foil strips or by placement in calcium alginate gel in the wells of aluminium cryo-plates. Shoot tips were then treated with loading solution followed by PVS2 treatment prior to liquid nitrogen (LN) exposure. Shoot tips were warmed in unloading solution and placed on recovery medium. RESULTS: The highest regrowth levels of cryopreserved shoot tips were obtained using 1 mm shoot tips and a PVS2 exposure for 90 minutes at 0oC with the droplet vitrification method on aluminum foil strips or by using 30 minutes of PVS2 at 22oC using V cryo-plates. CONCLUSION: Shoot tip size is an important factor in the cryopreservability of Vitis shoot tips; 1 mm shoot tips were the most successful for the droplet vitrification cryopreservation method that was tested. In addition, the V cryo-plate cryopreservation technique described herein can be easily executed and results in high regrowth levels (=70%) with quality plants obtained from cryo-exposed shoot tips, making it a practical and promising Vitis cryopreservation methodology.