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Title: Successful cryopreservation of Vitis shoot tips: Novel pre-treatment combinations applied to nine species

item Volk, Gayle
item Shepherd, Ashley
item Bonnart, Remi

Submitted to: CryoLetters
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/30/2018
Publication Date: 9/1/2018
Citation: Volk, G.M., Shepherd, A.N., Bonnart, R.M. 2018. Successful cryopreservation of Vitis shoot tips: Novel pre-treatment combinations applied to nine species. CryoLetters. 39(5):322-330.

Interpretive Summary: The USDA-ARS National Plant Germplasm System (NPGS) maintains collections of grapevine (Vitis sp.) cultivars and wild species at National Clonal Repositories in Davis, California and Geneva, NY. These collections are vulnerable to pathogens and environmental threats because they are maintained as plants in the field, and not backed up at a second location. We developed a method whereby shoot tips of grapevine could be excised from in vitro-grown plants, treated with cryoprotectants, and regrown into plants after liquid nitrogen exposure. We demonstrate that the use of pretreatments, optimized duration of cryoprotectant exposure, and improved post-culture conditions enhance survival of accessions representing nine Vitis species, including 'Chardonnay' cultivars of V. vinifera. The new methods will facilitate secure back-up of NPGS Vitis collections at the National Center for Genetic Resources Preservation's secured cryogenic facility.

Technical Abstract: BACKGROUND: Secure back-up of Vitis genetic resource collections require cryopreservation methods that give long-term survival of clonal germplasm having diverse genetic backgrounds. OBJECTIVE: This work sought to increase survival of Vitis shoot tips exposed to liquid nitrogen using combinations of pretreatments and cryoprotection procedures. The new procedure should give high survival of shoot tips from a wide range of Vitis species. MATERIALS AND METHODS: In vitro plants from nine Vitis species were used as source material for nodal sections. Shoot tips were then excised from nodal sections that were grown on medium containing benzyladenine, salicylic acid, glutathione, and ascorbic acid. The shoot tips were treated with loading solution, and then half-strength PVS2 for 30 minutes, prior to full-strength PVS2 treatments for between 60 and 90 minutes prior to liquid nitrogen (LN) exposure. RESULTS: Shoot tip regrowth levels were highest 90 minutes in PVS2+LN and ranged from 24-43% and averaged 35±2% across the nine Vitis species. CONCLUSION: The pretreatment, cryopreservation, and recovery methods yielded successful regrowth for multiple Vitis species using a droplet-vitrification procedure.