Modifications to a Vitis Shoot Tip Cryopreservation Procedure: Effect of Shoot Tip Size and Use of Cryoplates.

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 (DV) procedures have been developed to cryopreserve Vitis shoot tips from in vitro-grown plants. OBJECTIVE: This research focused on optimizing shoot tip sizes for DV and the feasibility of using cryo-plates for Vitis cryopreservation. MATERIALS AND METHODS: Uniform shoot tips were obtained from nodal sections cultured from in vitro-grown stock plants of Vitis aestivalis and Vitis jacquemontii (PI 135726). 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 DV on aluminum foil strips or by placement in calcium alginate gel in the wells of aluminium cryo-plates (V cryo-plate method). 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. The effect of extraction or non-extraction of the cryopreserved shoot tips from alginate beads was also tested. RESULTS: The highest regrowth levels of cryopreserved shoot tips were obtained using 1 mm shoot tips and a PVS2 exposure for 90 min at 0°C with the DV method on aluminum foil strips or by using 30 min of PVS2 at 22°C 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 DV 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.