Phenotypic Changes and DNA Methylation Status of Cryopreserved Rye Seeds.

Cryopreservation of seed in liquid nitrogen is a method for long-term conservation of plant germplasm. However, there are concerns about cryopreservation, because long-term storage under those conditions has the possibility of affecting the viability and genetic stability of seeds. The USDA-ARS National Laboratory for Genetic Resource Preservation (NLGRP) pioneered cryopreservation procedures in the early 1980s, but the long-term effects of this method have not been adequately evaluated. Our goal was to determine whether cryopreservation is a reliable method for routine conservation of rye (Secale cereale L.) seeds. The specific objectives of this research were (1) to determine phenotypic differences between plants grown from seed stored by standard and cryopreservation methods for 25 years; and (2) to determine if changes in DNA methylation pattern are associated with preservation method. Twenty accessions each of cryopreserved and conventionally stored spring and winter habit rye were evaluated phenotypically in replicated field trials in Fort Collins, CO in 2015. The accessions were also evaluated for germination percentage and seedling morphological traits. DNA was extracted from a subset of accessions and a methylation sensitive amplified fragment length polymorphism (AFLP) assay was conducted to detect differences in methylation. A minor difference was observed in spike length of winter rye plants (mean of 12.27 cm in plants from cryopreserved seeds vs. 12.44 cm in the conventional treatment, P=0.045). Significant differences were not detected for other traits. Results of the methylation-sensitive AFLP analysis will be presented and discussed. Results of this research will contribute to future long-term conservation strategies of the NLGRP.