Genetic and epigenetic integrity assessment of acclimatised papaya plants regenerated directly from shoot-tips following short- and long-term cryopreservation

Abstract

A vitrification-based cryopreservation protocol was applied to in vitro sourced shoot-tips of four genotypes of Carica papaya; two female (70 and Z6) and two male (B2 and B4). Regeneration of ~58 % (70) and ~59 % (Z6) was recorded for the female genotypes confirming previously published results. Regeneration was at ~77 and ~53 % for the two male genotypes B2 and B4 respectively. Cryo-tube storage and regeneration was tested after 2–18 months storage in one male (B2) and one female (70) genotype. Regeneration post cryo-storage was similar to 1 h exposure to liquid nitrogen. Individual shoot-tips from the two female and two male genotypes were grown into complete in vitro plants, potted and acclimatised without micropropagation to provide material for randomly amplified DNA fingerprinting (RAF) and amplified DNA methylation polymorphism (AMP) analysis of multiple individuals from in vitro control, plant vitrification solution 2 (PVS2) cryoprotectant control and short (1 h) and long-term cryopreservation treatment plants. No variations were detected for genotype Z6 control and treatment individuals and no RAF variations were detected in any individuals of genotype B2. Small numbers of RAF and AMP variations were detected in some individuals from genotypes B2 (AMP variation only), B4 and 70, but these were also found in controls. Genotype 70 showed the greatest level of variation; genomic DNA variation (RAF) was detected in control and cryopreservation treatment individuals, and the PVS2 control group was the only treatment group without variations for the respective AMP analysis. The variations observed could not be correlated with any phenotypic characteristics 2 months after acclimatisation.