Phenotypic changes and DNA methylation status in cryopreserved seeds of rye (Secale cereale L.)

Abstract

Conserving genetic diversity is a major priority of the National Laboratory for Genetic Resources Preservation (NLGRP), operated by the U.S. Department of Agriculture, Agricultural Research Service. There are two long-term preservation methods employed in the NLGRP: storage in a -18 °C freezer (conventional storage) and storage in liquid nitrogen vapor phase at -135 to -180 °C (cryopreservation). To test the phenotypic and epigenetic effects of long-term cryopreservation of orthodox seeds, we evaluated 40 cereal rye accessions (20 spring habit and 20 winter habit) stored for 25 years under both conventional storage and cryogenic conditions. In laboratory evaluations of winter habit rye, seeds from cryopreserved samples had significantly higher normal germination percentage (P < 0.05) and lower abnormal germination percentage (P < 0.05) than those stored under conventional conditions. Cryopreserved spring habit rye also had higher normal germination percentage (P < 0.01) than conventionally stored samples. In addition, winter rye seedlings from cryopreserved seeds had longer roots and smaller root diameter (P < 0.05) than seedlings from conventionally stored seeds. In field evaluations conducted in Fort Collins, Colorado in 2014-15, spikes of plants grown from conventionally stored seeds of the winter accessions were slightly longer than those from cryopreserved seeds (P = 0.045). To detect DNA methylation changes, a methylation-sensitive amplified fragment length polymorphism (metAFLP) technique was applied to two accessions. After false discovery rate adjustment, no differences in methylation were detected between storage treatments on an individual locus basis. Our study indicated that cryopreservation slowed seed deterioration as evidenced by higher germination rates compared to conventional storage, had only minimal effects on other phenotypic traits, and had no significant effects on DNA methylation status.

Epigenetic Integrity of Orthodox Seeds Stored under Conventional and Cryogenic Conditions

“…Changes in the level of methylation were also considered to be due to the effect of a cold acclimating treatment (hardening at −1 • C) prior to cryopreservation or in vitro culture following cryostorage rather than storage in LN per se [41,58]. For this reason, it was decided to take an advantage of the orthodox characteristics and investigate the direct effect of LN storage on seed epigenetic integrity concerning seed WC status since the procedure of cryogenic immersion of orthodox seeds is devoid of cryoprotectants (chemical treatment) and explant isolation (mechanical injuries), possibly affecting the epigenetic response.…”

“…This means that cryo-related epigenetic instabilities can be transferred through mitotic cell divisions into regenerated plants. Previously, Lu et al [41] found that there was no significant effect on DNA methylation status or phenotypic traits in seedlings derived from orthodox Secale cereale L. seeds, although they were cryostored only at optimal WC.…”

“…It was shown that in Swietenia macrophylla King seed germplasm changes in chromatin conformation after recovery from LN were observed, possibly due to changes in the methylation status of the DNA [39]. Significantly, although cryostorage of whole seeds is commonly used in many gene banks, there is insufficient information on the influence of this storage on their epigenetic integrity [40,41]. To address this issue, the present study was designed to investigate the epigenetic changes in seeds of Pyrus communis L. which is an important crop-related wild species.…”

Application of the MSAP Technique to Evaluate Epigenetic Changes in Plant Conservation

“…By means of metAFLP, no differences were found in the methylation level of 2-week old rye plants derived from seeds stored for 25 years either under conventional seed banking or cryopreserved, although cryopreserved seeds showed higher percentage of normal germination [ 66 ].…”

Choosing the Right Path for the Successful Storage of Seeds

“…However, a warm water bath is preferred due to the assurance of a more rapid and even thawing, which limits the formation of lethal ice crystals that tend form during a slow thawing procedure. So far, successful cryopreservation protocols have been established for many economically important species or their wild relatives such as Secale cereale L. [ 121 ], Solanum lycopersicum Mill. [ 122 ], Medicago sativa L., Beta vulgaris L., Allium cepa L., Oryza sativa L., Zea mays L. [ 118 ].…”