Genetic and Epigenetic Stability in Rye Seeds under Different Storage Conditions: Ageing and Oxygen Effect.

Seed ageing is a complex process and can be described as the loss of viability or quality with time. It is important to elucidate whether genetic and epigenetic stability is altered in stored seeds and in seedlings produced from them. Non-stored and stored rye seeds at different stages of ageing were compared, as well as the seedlings obtained from them. Seeds were stored at 35 °C and 15% water content, under vacuum or air atmosphere. DNA of seeds and seedlings was isolated at three stages of the deterioration curve: P75 (13 days), P20 (29 days), and P0 (36 days). Genetic stability was assessed by RAPD technique, and epigenetic changes by MSAP markers. While seeds showed genetic stability after storage, the similarity of seedlings obtained from seeds stored for 29 days was lower (95%) when compared to seedlings from control seeds. Epigenetic changes were between 15% and 30% (both de novo methylation and demethylation) in the stored seeds compared to control seeds, with no differences between 13 and 29 days of storage with either air or vacuum atmospheres. In seedlings, epigenetic changes significantly increased with storage time. In conclusion, ageing increased epigenetic instability in both seeds and seedlings, when compared to controls.

Volatile emission in dry seeds as a way to probe chemical reactions during initial asymptomatic deterioration.

The nature and kinetics of reactions in dry seeds determines how long the seeds survive. We used gas chromatography to assay volatile organic compounds (VOCs) emitted from seeds of three unrelated species as a means to non-invasively probe chemical changes during very dry, dry, and humid storage (seeds were dried to 5.5, 33, and 75% relative humidity at room temperature). VOCs emitted from seeds stored in humid conditions reflected fermentation-type reactions, with methanol and ethanol being predominant in Lactuca sativa and Carum carvi, and acetaldehyde and acetone being predominant in Eruca vesicaria. Dried C. carvi seeds continued to emit fermentation-type products, although at slower rates than the seeds stored in humid conditions. In contrast, drying caused a switch in VOC emission in L. sativa and E. vesicaria seeds towards higher emission of pentane and hexanal, molecules considered to be byproducts from the peroxidation of polyunsaturated fatty acids. Longevity correlated best with the rate of fermentation-type reactions and appeared unrelated to the rate of lipid peroxidation. Emission of VOCs decreased when seed species were mixed together, indicating that seeds adsorbed VOCs. Adsorption of VOCs did not appear to damage seeds, as longevity was not affected in seed mixtures. Collectively, the study shows similarity among species in the types of reactions that occur in dry seeds, but high diversity in the substrates, and hence the byproducts, of the reactions. Moreover, the study suggests that the most abundant VOCs arise from degradation of storage reserves within seed cells, and that these reactions and their byproducts are not, in themselves, damaging.

Measurement of cooling and warming rates in vitrification-based plant cryopreservation protocols.

Cryopreservation protocols include the use of additives and pretreatments aimed to reduce the probability of ice nucleation at all temperatures, mainly through micro-viscosity increase. Still, there is a risk of ice formation in the temperature region comprised between the equilibrium freezing (Tf ) and the glass transition (TG ) temperatures. Consequently, fast cooling and warming, especially in this region, is a must to avoid ice-derived damage. Vitrification and droplet-vitrification techniques, frequently used cryopreservation protocols based in fast cooling, were studied, alongside with the corresponding warming procedures. A very fast data acquisition system, able to read very low temperatures, down to that of liquid nitrogen, was employed. Cooling rates, measured between -20°C and -120°C, ranged from ca. 5°C s(-1) to 400°C s(-1) , while warming rates spanned from ca. 2°C s(-1) to 280°C s(-1) , for the different protocols and conditions studied. A wider measuring window (0°C to -150°C) produced lower rates for all cases. The cooling and warming rates were also related to the survival observed after the different procedures. Those protocols with the faster rates yielded the highest survival percentages.

Glassy state and cryopreservation of mint shoot tips.

Vitrification refers to the physical process by which a liquid supercools to very low temperatures and finally solidifies into a metastable glass, without undergoing crystallization at a practical cooling rate. Thus, vitrification is an effective freeze-avoidance mechanism and living tissue cryopreservation is, in most cases, relying on it. As a glass is exceedingly viscous and stops all chemical reactions that require molecular diffusion, its formation leads to metabolic inactivity and stability over time. To investigate glassy state in cryopreserved plant material, mint shoot tips were submitted to the different stages of a frequently used cryopreservation protocol (droplet-vitrification) and evaluated for water content reduction and sucrose content, as determined by ion chromatography, frozen water fraction and glass transitions occurrence by differential scanning calorimetry, and investigated by low-temperature scanning electron microscopy, as a way to ascertain if their cellular content was vitrified. Results show how tissues at intermediate treatment steps develop ice crystals during liquid nitrogen cooling, while specimens whose treatment was completed become vitrified, with no evidence of ice formation. The agreement between calorimetric and microscopic observations was perfect. Besides finding a higher sucrose concentration in tissues at the more advanced protocol steps, this level was also higher in plants precultured at 25/-1°C than in plants cultivated at 25°C.

Characterization of volatile production during storage of lettuce (Lactuca sativa) seed.

The duration that seeds stay vigorous during storage is difficult to predict but critical to seed industry and conservation communities. Production of volatile compounds from lettuce seeds during storage was investigated as a non-invasive and early detection method of seed ageing rates. Over 30 volatile compounds were detected from lettuce seeds during storage at 35 degrees C at water contents ranging from 0.03 to 0.09 g H(2)O g(-1) dw. Both qualitative and quantitative differences in volatile composition were noted as a function of water content, and these differences were apparent before signs of deterioration were visible. Seeds stored at high water content (L >or=0.06 g H(2)O g(-1) dw) emitted molecular species indicative of glycolysis (methanol+ethanol), and evidence of peroxidation was apparent subsequent to viability loss. Seeds containing less water (0.03-0.05 g H(2)O g(-1) dw) produced volatiles indicative of peroxidation and survived longer compared with seeds stored under more humid conditions. Production of glycolysis-related by-products correlated strongly with deterioration rate when measured as a function of water content. This correlation may provide a valuable non-invasive means to predict the duration of the early, asymptomatic stage of seed deterioration.

Germination and embryo rescue from Passiflora species seeds post-cryopreservation.

Seeds of Passiflora species have been reported to have intermediate or orthodox storage behavior. The development of cryopreservation protocols for recalcitrant or intermediate seeds can provide a feasible way for long term germplasm conservation. Seed germination of three Passiflora species (P. pinnatistipula, P. tarminiana and P. mollissima) was studied after desiccation and cryopreservation. The three species showed an intermediate response to desiccation: at 3-3.5% water content their germination was reduced to 23-45%, with P. pinnatistipula showing the maximum desiccation tolerance (62% germination after drying to 4.2% water content). The safest seed water contents for cryopreservation: 4.5-4.8% for P. pinnatistipula and P. mollissima, and 9% for P. tarminiana, resulting in 84%, 73%, and 63% germination, respectively. This is the first report of seed cryopreservation for these species.

Seed cryopreservation of Halimium and Helianthemum species.

Seed germination of four Halimium species [H. atriplicifolium, H.hHalimifolium, H. ocymoides, H. umbellatum] and eight Helianthemum species [H. almeriense pau, H. appeninum.,H. cinereum, H. hirtum, H. marifolium, H. nummularium, H. syriacum, H. squamatum], all cistaceae species from the mediterranean region, was studied after seed storage in liquid nitrogen (Ln, -196C) for four months. In all samples assayed, mechanical scarification of the seed coat was carried out to enhance seed germination. For most samples studied, final germination percentages were unaffected by storage of seeds in Ln, both for intact seeds and for scarified seeds. The germination rate of cryopreserved seeds, expressed as days to reach 50 percent of the final germination percentage, was lower only for four samples of intact seeds and for three samples of scarified seeds. Therefore, this study shows that seed cryopreservation could be a suitable procedure for the long-term seed conservation of several Halimium and Helianthemum species.

Survival and genetic stability of Dendranthema grandiflora Tzvelev shoot apices after cryopreservation by vitrification and encapsulation-dehydration.

The aim of this study was to compare the genetic stability of chrysanthemum (cv. Pasodoble) apices cryopreserved using two different methods: encapsulation-dehydration and vitrification. The assessment of the genetic stability was developed using RAPDs markers. Assessment of stability was evaluated in pot-cultivated mother plants (from which buds were excised for micropropagation), in shoots (leave tissue) from which apices were extracted for cryopreservation, and in shoots regenerated from cryopreserved apices 30 days after recovery and after further 3 months in culture. Throughout the process the origin of the apices (in vitro-shoot from which they were excised) was recorded. Twenty one regenerants cryopreserved by vitrification and 25 by encapsulation-dehydration were assessed. Only one cryopreserved regenerant from the encapsulation-dehydration method showed a different band pattern. These results support the necessity of monitoring the genetic stability of the regenerants obtained after cryopreservation, as this is a very useful technique for the conservation of plant genetic resources.

Cryopreservation of Quercus suber and Quercus ilex embryonic axes: in vitro culture, desiccation and cooling factors.

This study examines different factors included in the cryopreservation protocols for Quercus ilex and Q. suber embryonic axes. In vitro incubation temperature played an important role in the appropriate development of Q. ilex axes, as 15 degrees C was superior to 25 degrees C. Q. suber axes proved to be more sensitive to desiccation and cooling. Poor survival (35%) was observed when axes were included into cryovials and then in liquid nitrogen, and none when immersed in sub-cooled liquid nitrogen (-210 degrees C). Q. ilex axes showed poorly organised development in vitro (c. 50% of non-cooled axes showed shoot development). However, c. 80% survival was observed after cryopreservation (either in liquid nitrogen or sub-cooled liquid nitrogen at 0.34 g water / g dry weight), of which c. 15% showed shoot development.