Cryopreservation of axillary shoot tips of in vitro-grown grape (Vitis) by a two-step vitrification protocol

Abstract

Axillary shoot tips of in vitro grape(Vitis vinifera L. cv. CabernetSauvignon) were successfully cryopreservedby vitrification. Axillary shoot tipswere excised from 4- and 5-month oldplantlets were cultured onsolidified 1/2 MS medium with 1 mg l^-1BA at 25 ^°C. Shoot tips wereprecultured on solidified mediumsupplemented with 0.3 M sucrose for 3 daysand then treated with a mixture of 2 Mglycerol plus 0.4 M sucrose (LS solution)for 20 min at 25 ^°C. They were thendehydrated with PVS2 for 80 min at0 ^°C before being plunged into LN for1 hr. Samples were then warmed rapidly inwater at 40 ^°C. The recovery of theshoot tips amounted to approximately 60%.To enhance further recovery, a two-stepdehydration procedure by vitrification wasexamined. Osmo-protected shoot tips werefirst dehydrated with a 50% PVS2 (halfstrength of the solution) for 30 min,followed by PVS2 for 50 min at 0 ^°C.This revised dehydration procedure improvedshoot recovery from 60 to 80%. Thisprocedure by vitrification was applied toten other species/cultivars of Vitiswith an average recovery of 64%. Thismethod incorporates a simple and reliableapproach for providing high levels ofgrowth recovery. It also appears promisingfor the cryopreservation of Vitisgermplasm.