A new insight in desiccation tolerance and cryopreservation of mazzard cherry (Prunus avium L.) seeds

A variable response of Prunus avium L. seeds to desiccation and storage in liquid nitrogen (LN) has been reported in the literature. The majority of these experiments were conducted on initially dried seeds. The desiccation and LN exposure tolerance of fresh P. avium seeds is unknown. In the present study, fresh seeds were used to determine seed response to desiccation and cryopreservation. Desiccation of seeds from a moisture content (MC) of 19.7-20.2% to 10.1-10.9% or 07.6-8.5% reduced seedling emergence from approximately 73 to 19 and 16% for first provenance; and from approximately 89 to 10-12% for second provenance of seeds. After exposure to LN, seeds had the highest seedling emergence when seed MC was the highest (19.7 and 20.2%, respectively) prior to cryostorage. Results indicated that P. avium seeds should be classified as intermediate. For cryopreservation in seed banks, we recommend that seeds be dried directly after extraction from fruits in the range of 16.8-20.2% of MC (0.21-0.25 g·g–1 of WC) and directly immersed in LN in tightly closed cryovials

Epigenetic Marks, DNA Damage Markers, or Both? The Impact of Desiccation and Accelerated Aging on Nucleobase Modifications in Plant Genomic DNA

Modifications of DNA nucleobases are present in all forms of life. The purpose of these modifications in eukaryotic cells, however, is not always clear. Although the role of 5-methylcytosine (m5C) in epigenetic regulation and the maintenance of stability in plant genomes is becoming better understood, knowledge pertaining to the origin and function of oxidized nucleobases is still scarce. The formation of 5-hydroxymetylcytosine (hm5C) in plant genomes is especially debatable. DNA modifications, functioning as regulatory factors or serving as DNA injury markers, may have an effect on DNA structure and the interaction of genomic DNA with proteins. Thus, these modifications can influence plant development and adaptation to environmental stress. Here, for the first time, the changes in DNA global levels of m5C, hm5C, and 8-oxo-7,8-dihydroguanine (8-oxoG) measured by ELISA have been documented in recalcitrant embryonic axes subjected to desiccation and accelerated aging. We demonstrated that tissue desiccation induces a similar trend in changes in the global level of hm5C and 8-oxoG, which may suggest that they both originate from the activity of reactive oxygen species (ROS). Our study supports the premise that m5C can serve as a marker of plant tissue viability whereas oxidized nucleobases, although indicating a cellular redox state, cannot

Global changes in DNA methylation in seeds and seedlings of Pyrus communis after seed desiccation and storage.

The effects of storage and deep desiccation on structural changes of DNA in orthodox seeds are poorly characterized. In this study we analyzed the 5-methylcytosine (m(5)C) global content of DNA isolated from seeds of common pear (Pyrus communis L.) that had been subjected to extreme desiccation, and the seedlings derived from these seeds. Germination and seedling emergence tests were applied to determine seed viability after their desiccation. In parallel, analysis of the global content of m(5)C in dried seeds and DNA of seedlings obtained from such seeds was performed with a 2D TLC method. Desiccation of fresh seeds to 5.3% moisture content (mc) resulted in a slight reduction of DNA methylation, whereas severe desiccation down to 2-3% mc increased DNA methylation. Strong desiccation of seeds resulted in the subsequent generation of seedlings of shorter height. A 1-year period of seed storage induced a significant increase in the level of DNA methylation in seeds. It is possible that alterations in the m(5)C content of DNA in strongly desiccated pear seeds reflect a reaction of desiccation-tolerant (orthodox) seeds to severe desiccation. Epigenetic changes were observed not only in severely desiccated seeds but also in 3-month old seedlings obtained from these seeds. With regard to seed storage practices, epigenetic assessment could be used by gene banks for early detection of structural changes in the DNA of stored seeds

Epigenetic Integrity of Orthodox Seeds Stored under Conventional and Cryogenic Conditions

The level of 5-methylcytosine (m5C) in DNA has been observed to change in plants in response to biotic and abiotic stress factors. Little information has been reported on alterations in DNA methylation in orthodox tree seeds in response to storage conditions. In the current study, epigenetic integrity was analyzed in seeds of Pyrus communis L. in response to conventional and cryogenic storage. The results indicate that conventional storage under optimal conditions resulted in a significant increase in m5C. In contrast, a decrease in m5C level after cryostorage at high water content (WC) was observed, not only in seeds but also in 3-month-old seedlings which were smaller than seedlings obtained from seeds cryostored at optimal WC. This shows that non-optimal cryostorage conditions increase epigenetic instability in seeds and seedlings. Optimal procedures for germplasm conservation are very important for germplasm banking since they have serious implications for the quality of stored collections. Maintaining epigenetic integrity during WC adjustment and optimal storage is a characteristic feature of orthodox seeds. The current results underline the importance of proper protocols and techniques for conventional storage and particularly cryopreservation as a method for conservation of true-to-type germplasm for long periods

Optimal seed water content and storage temperature for preservation of Populus nigra L. germplasm

International audienceContextBlack poplar (Populus nigra L.) is an alluvial forest tree species whose genetic pool is decreasing in Europe. Poplar trees produce short-lived seeds that do not store well.AimThe feasibility of seed storage in conventional and cryogenic conditions after their desiccation from water content (WC) of 0.15 to 0.07 g H2O g−1 dry mass (g g−1) was investigated.MethodsSeed germinability was evaluated (seeds with a radicle and green cotyledons were counted) after storage of seeds for a period of 3 to 24 months at different temperatures: 20°, 10°, 3°, −3°, −10°, −20° or −196°C.ResultsSeeds desiccated to a 0.07 g g−1 WC can be stored successfully at −10 °C and −20 °C for at least 2 years. A significant decrease in germination was observed only after 12 months of seed storage (WC 0.15 g g−1) at temperatures above 0 °C. We demonstrated that both fresh (0.15 g g−1 WC) and desiccated (0.07 g g−1 WC) seeds can be preserved at −196 °C for at least 2 years.ConclusionsSeed storage temperature and time of storage were statistically significant factors affecting seed storability. The presented data provide a foundation for the successful gene banking of P. nigra seeds

Epigenetic Integrity of Orthodox Seeds Stored under Conventional and Cryogenic Conditions

The level of 5-methylcytosine (m5C) in DNA has been observed to change in plants in response to biotic and abiotic stress factors. Little information has been reported on alterations in DNA methylation in orthodox tree seeds in response to storage conditions. In the current study, epigenetic integrity was analyzed in seeds of Pyrus communis L. in response to conventional and cryogenic storage. The results indicate that conventional storage under optimal conditions resulted in a significant increase in m5C. In contrast, a decrease in m5C level after cryostorage at high water content (WC) was observed, not only in seeds but also in 3-month-old seedlings which were smaller than seedlings obtained from seeds cryostored at optimal WC. This shows that non-optimal cryostorage conditions increase epigenetic instability in seeds and seedlings. Optimal procedures for germplasm conservation are very important for germplasm banking since they have serious implications for the quality of stored collections. Maintaining epigenetic integrity during WC adjustment and optimal storage is a characteristic feature of orthodox seeds. The current results underline the importance of proper protocols and techniques for conventional storage and particularly cryopreservation as a method for conservation of true-to-type germplasm for long periods

Percentage 5-methylcytosine methylation of DNA in seedlings derived from desiccated pear seeds.

<p>Values marked with the same letter are not significantly different at <i>P</i><0.05, ANOVA, Tukey`s test. Seeds were collected from Borkowice and not stored before desiccation, error bars indicate standard deviations.</p

Effects of pear seed desiccation and storage on seedling emergence.

<p>(A) Unstored seeds from Borkowice. (B) Seeds from Borkowice that had been stored for 1 year. (C) Seeds from Łopuchówko that had been stored for 2 years. Values marked with the same letter are not significantly different at <i>P</i><0.05, ANOVA, Tukey`s test, error bars indicate standard deviations.</p

Assessment of global DNA methylation of seeds from Borkowice, stored for 1 year.

<p>2′deoxynucleotides derived from DNA hydrolysis (labelled spots) and RNA contamination (unlabelled spots) are clearly separated. A- adenine, m⁵C -5-methylcytosine, C-cytosine, T-thymine, G- guanine.</p