Assessment of genetic and epigenetic changes following cryopreservation in papaya

Abstract

A vitrification based cryopreservation technique for storage of in vitro shoot tips of papaya has been tested to ensure applicability across a range of genotypes and to assess the stability of both genotype and phenotype of such clonal material following cryopreservation. Shoot tips of 12 genotypes were cryopreserved, recovery rates were determined and resultant plants were screened for genetic and epigenetic changes. Genomic DNA structure was explored using polymerase chain reaction (PCR) based randomly amplified DNA fingerprinting (RAF), and methylation patterns were monitored using the amplified DNA methylation polymorphism (AMP) PCR technique. Plantlets were recovered following cryopreservation in all but one genotype and recovery rates of 61-73% were obtained from six genotypes. The regenerated plantlets showed varying levels of genomic DNA modifications (0-10.07%), and methylation modifications (0.52-6.62%) of detected markers. These findings have not been reported previously for papaya, and indicate some genotype dependent variability in DNA modifications occur following cryopreservation which may result in somaclonal variation.

Potato Shoot Tip Cryopreservation. A Review

“…First comparative studies of epigenetic marks in plants before and after cryopreservation have already been done. Modifications in DNA methylation have been determined in almond (Channuntapipat et al 2003), papaya (Kaity et al 2008), chrysanthemum (Martín and González-Benito 2006), Ribes ), strawberry (Hao et al 2002a) and citrus (Hao et al 2002b). In potato, no studies on epigenetic modifications in connection with cryopreservation have been published so far, but first analyses at IPK Gatersleben pointed to methylation changes, although it is not clear yet, whether these changes are due to cryopreservation or long-term in vitro storage (A Kaczmarczyk, unpublished results).…”

Current Issues in Plant Cryopreservation

“…Most studies have confirmed the presence of genetic stability (Castillo et al, 2010;Helliot et al, 2002) and where changes in the genome have been found, such as in sugarcane and potato with RFLP markers, the changes could not be related to the process of cryopreservation itself (Castillo et al, 2010;Harding, 2004 (Channuntapipat et al, 2003), papaya (Kaity et al, 2008), chrysanthemum (Martín & González-Benito, 2006), Ribes , strawberry (Hao et al, 2002a), citrus (Hao et al, 2002b) and potato (Kaczmarczyk et al, 2010). Changes in methylation might be caused by stressful in vitro conditions, osmotic dehydration and the application of cryoprotective agents (Harding, 2004).…”

Cryopreservation changes the DNA methylation of embryonic axes of Quercus robur and Fagus sylvatica seeds during in vitro culture

“…The aim of effective cryopreservation is the long-term preservation of plant material, and it is important to know whether this induces permanent genetic changes. Changes in the level of DNA methylation were observed in papaya (Kaity et al 2008), chrysanthemum (Martín and González-Benito 2006;Martin et al 2011) currants (Johnston et al 2009), strawberries (Hao et al 2002a), citrus (Hao et al 2002b) and potatoes (Kaczmarczyk et al 2012). Changes in global DNA methylation may also be induced by cryovitrification agents and the conditions of in vitro culturing (Harding 2004).…”

“…3). Kaity et al (2008) studied the genetic stability of papaya shoots during cryopreservation after vitrification with PVS2. Those authors showed that the observed changes could be related to the toxicity of DMSO as a component of PVS2.…”