Establishment of an efficient and wide-spectrum droplet-vitrification cryopreservation for raspberry (Rubus idaeus L.) germplasm and assessments of genetic integrity and vegetative growth in the regenerants

Abstract

By optimizing size of shoot tips, preculture medium and exposure duration to PVS2, we established an efficient and wide-spectrum droplet-vitrification cryopreservation of shoot tips of raspberry (Rubus idaeus L.). This protocol yielded 80–100% and 67–100% of survival and shoot regrowth levels in cryopreserved shoot tips across 30 raspberry genotypes. Genetic integrity was assessed in cryo-derived regenerants after 3 months of post-cryopreservation using inter simple sequence repeat (ISSR), single nucleotide polymorphism (SNPs), and insertions and deletions (InDels). ISSR did not detect any polymorphic bands in the cryo-derived regenerants. Although the number of SNPs and InDels decreased in the cryo-derived regenerants, variation trends were similar between the cryo-derived regenerants and the control. Plant vegetative growth and root growth were assessed in the cryo-derived plants after 9 weeks of growth in greenhouse. There were no significant differences in plant vegetative growth measured by plant height, number of fully-opened leaved, leaf area, and fresh and dry weight between the cryo-derived plants and the control, although significant differences were observed in root growth measured by root total length, root average diameter and root volume between the two types of plants. The results obtained in the present study indicate that the droplet-vitrification method has great potential for cryopreservation of raspberry germplasm.