A simplified method for high recovery of kiwifruit (Actinidia spp.) shoot tips after droplet vitrification cryopreservation suitable for long-term conservation

Abstract

Kiwifruit (Actinidia spp., Family Actinidiaceae) is a recently domesticated crop with highly endangered genetic resources in field genebanks and in the centre of diversity. Improved genotypes of several species in the genus have been commercialised and several other species are used in breeding programmes. Cryopreservation is considered a safe and cost-effective option for ex-situ conservation of clonal crops. In the current research we tested if the currently available method of droplet vitrification (DV) cryopreservation could be simplified whilst improving plant regeneration. Plant regeneration in the nine accessions belonging to A. chinensis var. chinensis, and A. chinensis var. deliciosa, A. arguta, A. macrosperma and A. polygama after cryopreservation ranged from 59 to 88% with the improved method where cold acclimation and antioxidant treatment steps were eliminated while sucrose pretreatment of shoot tips has been reduced to two steps in two days. Use of smaller (0.5–1 mm compared to 2 mm) in vitro shoot tips from younger (two weeks after subculture), fast-growing shoots and the use of liquid sucrose for shoot tip pretreatment are considered to be the reasons for this improved response. We also introduced the use of sodium dichloroisocyanurate as an alternative to household bleach as a sterilant for kiwifruit tissue culture establishment. The improved DV protocol is being used to cryopreserve valuable kiwifruit genetic resources with greater efficiency. Use of small shoot tips (0.5–1mm) from young shoots pretreated in liquid sucrose media in the dark significantly improved plant regeneration in five Actinidia species after droplet vitrification cryopreservation