Genebank Conditions Research Articles

Will a plant germplasm accession conserved in a genebank change genetically over time?

The simplified question on the genetic change of a conserved plant germplasm accession over time is raised for a better understanding of the challenging mission of conserving more than 7.4 million germplasm accessions in 2000 genebanks worldwide for generations to come. Its answer will influence how these genebanks operate to ensure the continued survival and availability of the conserved plant genetic resources for future food security. Here, we explore the expected impact of evolutionary forces on plant germplasm in genebanks, search for the theoretical expectations and empirical evidence for such impacts from the literature, and discuss the ramifications of the evidence for long-term plant germplasm management and conservation. It is expected that genetic changes of long-term conserved germplasm under genebank conditions will occur commonly as an evolutionary rule, not as an exception. Incorporating evolutionary biology into the Genebank Standards and operational procedures will benefit the mission of long-term germplasm conservation.

Derivation of seed viability constants (KE and CW) and prediction of longevity of Solanum aethiopicum seeds

The seed viability equation uses species viability constants to predict seed longevity under different storage conditions. There are already established universal values of constants that account for the effect of temperature on seed longevity (CH and CQ) which can be applied to different crop species. However, constants accounting for the effect of seed moisture (CW) and genotype (KE) are species-specific. This study aimed to derive viability constants and predict seed longevity for African eggplant (Solanum aethiopicum) under genebank conditions. The seeds were subjected to experimental storage at 40°C and different seed moisture contents (6, 8, 10 and 12%) for 120 days to derive survival curves and hence, CW and KE. CW and KE values for S. aethiopicum were 3.32 and 7.4978, respectively. The validation of the derived constants show that they can be used to predict seed longevity of other seed lots within the same species. The genebank curators and other commercial seed technologists may be able to accurately predict longevity of African eggplant seeds under cold dry storage conditions using the improved viability equation and the constants established in this research.

Effect of growth medium composition on the viability and genetic stability of Chrysanthemum × morifolium Ramat. explants under in vitro cold storage conditions

Cultivation of plants at low positive temperatures is promising for the in vitro preservation of valuable Chrysanthemum × morifolium Ramat. varieties. The study aims to examine the effect of chlorocholine chloride concentrations in a specialized medium on the viability and the physiological and morpho-anatomical parameters of chrysanthemum plants (Excel, Sheer Purple, and William Seward varieties), as well as their genetic stability after 12 months of cold storage under in vitro gene bank conditions. In vitro microshoot tips (without leaves) were placed on a 1/4 Murashige and Skoog medium supplemented with 60.0 g/L sucrose and 0.2–0.8 g/L chlorocholine chloride. The explants were stored in cooling chambers at a temperature of 4 °C. After 12 months of preservation, a reduction was observed in growth kinetics with increasing concentration of chlorocholine chloride in the medium. The viability of explants decreased from 96 to 91% at higher concentrations of chlorocholine chloride. The effective photochemical quantum yield of photosystem II decreased more than twice under cold storage conditions as compared to the control. Several histological features were identified in the leaves of in vitro explants cold-stored in the gene bank: decreased in leaf thickness and mesophyll density in leaf blades, degradation of palisade tissue, and a high degree of transpiration regulation. The SSR and ISSR analyses were used to show that the microshoots of the examined chrysanthemum varieties were identical to the original plants following in vitro cold storage. The conducted studies revealed that the presence of 0.6–0.8 g/L chlorocholine chloride in the medium reduces the growth kinetics of explants while maintaining their high viability and genetic stability.

Two decades of climate change alters seed longevity in an alpine herb: implications for ex situ seed conservation

Climate warming in mountain areas is increasing faster than the global average, threatening alpine plants. Climate affects many traits including seeds, the longevity of which is important for conservation, facilitating genebank storage. Seeds of alpine species are considered short-lived in storage, but their longevity increases when produced under a warmer parental environment. Consequently, with climate warming, seeds of alpine species may have increased fitness and be longer-lived in genebank storage. We assessed seed longevity under artificial ageing in 10 accessions of the arctic-alpine species Viscaria alpina stored under genebank conditions for different time periods over the last 20 years. The seed collection site was in the northern Apennines, where above average warming and variable precipitation has been recorded. The time taken for viability to fall to 50% (p50) was estimated using probit analysis; correlation and general linear regression were used to investigate the effects of length of time in storage, seed mass and climate under which seeds were produced on seed longevity. p50 varied between 7.77 and 18.49 days. There was no relationship between length of time in storage or seed mass on seed longevity. p50 was higher in years with increased temperature and lower precipitation during the growing season, with precipitation having more impact than temperature. The results suggest that seeds of alpine species are suitable for genebank storage, and inter-annual variation in precipitation induces a plastic response in seed longevity. Using genebank stored seeds provides insights into how alpine species may respond to future climate changes and could have implications for genebank storage.

Comparative seed longevity under genebank storage and artificial ageing: a case study in heteromorphic wheat wild relatives.

Seed longevity is a complex trait that depends on numerous factors. It varies among species and populations, and within different seed morphs produced by the same plant. Little is known about variation in longevity in different seed morphs or the physiological and molecular basis of these differences. We evaluated the longevity and oxidative stress status in heteromorphic seeds aged in two different storage conditions. We compared controlled ageing tests (seed storage at 45°C and 60% relative humidity; a method of accelerated ageing used to estimate longevity in genebank conditions) with storage in a genebank for up to 40 years (-18°C and 8% seed moisture content). We employed as study species two wild wheats characterized by seed heteromorphism: Aegilops tauschii and Triticum monococcum subsp. aegilopoides. We estimated the ROS content and the expression of genes coding for enzymes related to the H2 O2 scavenging pathway. Results confirmed that seed longevity varies between different seed morphs. Different storage environments resulted in different longevity and survival curves. ROS levels, even if with variable patterns, were higher in several aged seed lots. We observed consistency in the expression of two genes (GSR and CAT) related to ROS scavenging in the late phase of pre-germinative metabolism. Differences in seed longevity between morphs were observed for the first time under genebank conditions. Our results suggest also that controlled ageing tests should be used with caution to infer ranks of longevity under cold storage.

Clematis plants conservation under in vitro genebank conditions

Clematis plants conservation under in vitro genebank conditions

Does Storage under Gene Bank Conditions Affect Seed Germination and Seedling Growth? The Case of Senecio morisii (Asteraceae), a Vascular Plant Exclusive to Sardinian Water Meadows.

Understanding seed viability under long-term storage conditions provides basic and useful information to investigate the effectiveness of seed banking. Besides the germination success, seedling establishment is also an important requirement, and a decisive step to ensure plant propagation. We used comparative data of germination, seedling growth, and survival percentage between fresh and 10-years-stored seeds of Senecio morisii, a narrow endemic and vulnerable species of Sardinia (Italy), in order to evaluate if differences exist in these traits between fresh and 10-years-stored seeds. Stored seeds showed higher germination percentages than fresh ones, whereas seedling growth and survival did not present significant differences between them, except for seedling growth in plants produced from seeds germinated at 25 °C. This study allowed us to assess if seeds of S. morisii were able to germinate under controlled conditions, and if they maintained their viability and germination capacity for at least 10 years of long-term storage in the seed bank. In addition, the high seedling survival detected in both fresh and stored seeds suggests that stored seeds of S. morisii can be used to support reinforcement or reintroduction actions when fresh materials are not available.

Grasses seed storage under genbank conditions

The seed viability of 86 acessions of fodder grasses: awnless brome (Bromopsis inermis (Leyss.) Holub.), orchardgrass (Dactylis glomerata L.), Timothy grass (Phleum pretense L.), crested wheat grass (Agropyron cristatum (L.) Gaertn.), clustered wheat grass (Agropyron desertorum (Fisch. ex Link) Schult. et Scult.f.), annual ryegrass (Lolium multiflorum Lam.), perennial ryegrass (L. perenne L.), red fescue (Festuca rubra L.), meadow fescue (Festuca pratensis Huds.), gigantea fescue (Festuca gigantea (L.) Vill.), blue fescue (Festuca inarmata Schur. (Festuca amethystina (Hack. ex Boiss.) St.-Yves)), tall fescue (Festuca arundinacea Schreb.), tall oat-grass (Arrhenatherum elatius (L.) P. Beauv. ex J. S. et K. B. Presl.), slender wheat grass (Roеgneria trachycaulon (Link) Nevski), creeping bent grass (Agrostis alba L.), redtop (Agrostis gigantea Roth.), which were stored under controlled temperature and moisture content 3 – 6 % was investigated. It has been determined that the seeds of these species have different longevity at different storage temperatures under the genebank conditions. Phleum pretense, Phleum pretense, Agropyron cristatum, Lolium multiflorum Festuca rubra seed viability was without changing after storage at temperature 4oC for 10 years and longer. The best seed longevity of grasses were achieved when the seeds are kept at –20oC. Accessions features of different grasses species seed storage are discussed.

Genetic diversity of Bolivian wild potato germplasm: changes during ex situ conservation management and comparisons with resampled in situ populations

Potato wild relatives are important sources of novel variation for the genetic improvement of the cultivated potato. Consequently, many natural populations have been sampled and were deposited as accessions in gene banks around the world. Here we investigate to what extent the genetic variation of Bolivian wild potato species is maintained under gene bank conditions and how this diversity relates to that of current in situ populations. For this purpose, materials from seven potato species were screened for microsatellite variation. Genetic changes between different generations of ex situ germplasm were not observed for Solanum leptophyes and S. megistacrolobum, but were detected for S. neocardenasii and S. okadae, while each of the species S. acaule, S. avilesii and S. berthaultii showed stability in some cases and genetic change in others. The observed changes were ascribed to genetic drift and contamination resulting from human error during regeneration. Re-collected populations of six of the studied species showed highly significant genetic differences with the ex situ accessions that, apart from changes during ex situ maintenance, are most likely to be attributed to sampling effects during collecting and in situ genetic changes over time. The implications of the results for ex situ and in situ conservation strategies of wild potato species are discussed.

One-step fitting of seed viability constants for two Australian plant species, Eucalyptus erythrocorys (Myrtaceae) and Xanthorrhoea preissii (Xanthorrhoeacea)

Long-term ex-situ seed storage under controlled conditions in gene banks has become an important tool for conserving threatened Australian plants; however, there is scant information about the seed longevity of most species. The aim of the present study was to determine whether the seed longevity of two contrasting Australian species could be modelled using the seed viability equation, and whether the universal temperature constants are applicable to these species. Seeds of Eucalyptus erythrocorys F.Muell. (Myrtaceae) and Xanthorrhoea preissii Endl. (Xanthorrhoeaceae) were aged at moisture contents ranging from 3.9 to 15.7% and temperatures between –20 and 60°C. Survival data were fitted to the seed viability equation in one step and the species constants for each species determined. Both E. erythrocorys and X. preissii seeds exhibited orthodox seed storage behaviour whose longevity could be modelled using the seed viability equation. The viability constants were KE = 8.81, CW = 4.97, CH = 0.0412 and CQ = 0.000379 for E. erythrocorys and KE = 8.77, CW = 5.29, CH = 0.0382 and CQ = 0.000473 for X. preissii. The universal temperature constants could not be used without a significant increase in error. The storage behaviour of these two Australian species is in keeping with that of orthodox species from around the world. Predictions are that E. erythrocorys will be long-lived under gene bank conditions, whereas X. preissii would be moderately long-lived. Current long-term gene bank storage conditions appear suitable for storage of these species; however, recommendations for short-term storage need to be re-evaluated.

Preservation of seed viability during 25 years of storage under standard genebank conditions

Maintaining sufficient viability is critical to the sustainability of ex situ conserved seed collections. For this reason, accessions are regenerated when viability falls below a predefined threshold. Viability is monitored by determining the germination ability of accessions at predefined time intervals. Optimizing the frequency of these germination tests, in order to avoid waste of resources, is hampered by the scarce availability of data about seed longevity, particularly for material maintained under genebank conditions. Here we report on the analysis of nearly 40,000 germination test results collected for a wide range of crop species over a 25-years period by the centre for genetic resources, the Netherlands (CGN), where seeds of genebank accessions are dried to 3–7 % moisture content and stored for the long term under near-vacuum in aluminium foil bags at −20 °C. The results indicate that seed viability is well maintained for the large majority of seed lots during the first 25 years after regeneration as only 3.3 % of the monitoring tests revealed below-threshold germination values. It is argued that the majority of these sub-standard seed lots are due to other causes than seed ageing, including dormancy problems and estimation error in germination testing. For material, maintained under the seed management procedures and storage conditions practiced by CGN, it is therefore recommended to delay the first germination monitoring tests to 25 years after regeneration.

25. The influence of desiccation, cryopreservation and rehydration on the survival of polyembryonic Citrus seeds

Three categories of seed storage behavior are generally recognized among plant species: orthodox, intermediate and recalcitrant. Intermediate seeds, such as Citrus, can withstand partial dehydration, but they cannot be stored under conventional genebank conditions because they are cold-sensitive and desiccation does not increase their longevity. Citrus seeds that are immersed in liquid nitrogen (LN) without a previous dehydration process show a very low viability; therefore the water content when they are immersed in LN is considered to be the most critical factor in cryopreservation process. The purpose of this study was to investigate the basis of the optimal hydration status for cryopreservation of intermediate oily Citrus seeds: Citrus sinensis (sweet orange) and Citrus paradise (grapefruit). In the present work two conditions were tested: (a) seed desiccation, (b) seed desiccation followed by LN treatment with thawing under controlled conditions. In addition two pre-sowing procedures (preheating and prehumidification prior to the germinability test) were applied and their influences on the viability of the seeds were analyzed. To study the tolerance to dehydration and LN exposure, seeds were equilibrated at relative humidities (ERH) between 11% and 95% using saturated salts (LiCl, MgCl 2 , K 2 CO 3 , NaNO 2 , NaCl, KCl, KNO 3 ). Sorption isotherms were determined and modeled. Seed viability was analyzed by germination experiments; seeds were sown in hermetic controlled germination conditions and the percentage of normal seedling was evaluated 4–6 weeks after sowing. Seed desiccation sensitivity was quantified by the quantal response model. Differential scanning calorimetry thermograms between −120 and 100 °C were determined on cotyledon tissue that was previously dehydrated reaching different moisture contents; ice melting enthalpies and unfrozen water contents were measured from these thermograms. In order to analyze the effect of pre-sowing treatment on seed viability, experiments were carried out in a narrower range of equilibrium relative humidities (ERH: 59–85%). All the seeds subjected to conditions of seed desiccation, or seed desiccation followed by LN treatment were either directly sown under germination conditions or subjected to pre-sowing rehydration procedures; the tested procedures were preheating (40 °C during 4 h) and pre-humidification (equilibrium at 100% RH, 20 °C). In the cases of seeds that were only dried and in seeds that were dried and treated with LN, the tested pre-sowing treatments (pre-humidification or heating) did not significantly improve the viability of the seeds with respect to the control samples ( P < 0.05); in fact preheating significantly deteriorated the viability of the LN treated seeds. The survival of C. sinenis and C. paradise seeds, subjected to cryopreservation in LN was examined and seed desiccation sensitivity following rehydration procedures was quantified. Results showed that in order to reach the maximum viability, the seeds exposed to LN, must be first dehydrated to a range of ERH 69–81%(0.16–0.31 gH 2 O g −1 dry basis) for C. sinensis and 69–75% (0.09–0.11 gH 2 O g −1 dry basis) for C. paradise. The limit of hydration for LN Citrus seeds treatment corresponded to the unfrozen water content in the tissue, confirming that seed moisture should be reduced to such an extent to avoid the formation of intra-cellular ice crystals during ultra-rapid freezing, thus preserving the integrity of seed tissues.

Viability of Oryza sativa L. seeds stored under genebank conditions for up to 30 years

Germination ability, equilibrium relative humidity (eRH), and moisture content of ‘control’ seed samples representing 183 rice accessions stored in the active (2–4 °C) and base (−10 °C until 1993, then −20 °C) collections of the T. T. Chang Genetic Resources Center were determined after storage for 20.5–30.5 years. Germination of seeds that had been stored in the base collection was generally high (>70 %), whereas germination was more variable for seeds stored in the active collection. Samples with lower viability after storage in the active collection were likely to have lower viability after storage in the base collection. There were significant differences in the moisture content-eRH relationship of the seeds depending on whether the seeds had been stored in the active or base collection. Based on re-test data for regular seed samples regenerated in 1979–1980 and stored in the active collection for up to 31 years, estimates of the time for ability to germinate to fall to 50 % (p50) ranged from 54 to 997 years. For the same seed samples stored in the base collection for approximately 31 years, ability to germinate has been maintained and germination increased due to improved procedures. The ability to germinate of base collection samples was also generally higher than that of ‘safety duplicate’ samples of the same seed lots that had been sent to the National Center for Genetic Resources Preservation, USA in 1981 and stored at −18 °C. This may have been due to uptake of moisture either during processing for dispatch or as a consequence of poor packaging material. The results are discussed in relation to long-term seed storage and genebank management.

Studies on genetic changes in rye samples (Secale cereale L.) maintained in a seed bank

The aim of this study was to identify genetic changes in rye seeds induced by natural ageing during long-term storage and consecutive regeneration cycles under gene bank conditions. Genomic DNA from four rye samples varying in their initial viability after one and three cycles of reproduction was analyzed by AFLP (amplified fragment length polymorphism) fingerprinting. Seven EcoRI/MseI primer combinations defined 663 fragments, and seven PstI/MseI primer combinations defined 551 fragments. The variation in the frequency of the seventy-four EcoRI/MseI bands was statistically significant between samples. These changes could be attributed to genetic changes occurring during storage and regeneration. However, the PstI/MseI fragments appeared to be uninfluenced by seed ageing, regeneration and propagation. A combined Principle Coordinate Analysis revealed differences between samples with different initial viability. We showed that materials with low initial viability differ in their response from highly viable ones, and that the changes exhibited in the former case are preserved through regeneration cycles.

Changes in germination ability during genebank storage at some medicinal plant seeds belonging to the Solanaceae family

In the present work we have examined the changes of germination ability of some medicinal plant species belonging to the Solanaceae family (Datura stramonium L., Datura innoxia Mill. and Hyoscyamus niger L.) during 5-6 years' storage period. According to our results, all the three species showed an after-ripening behavior. Potassium nitrate and gibberellic acid increased significantly the rate of germination in the case of Datura species. During the storage period tested (1995-2001), the species maintained their germination ability which is favourably but not significantly influenced by the cooled gene bank conditions. The degree of ripeness affected considerably the proportion of germinated seeds at all the three species. As a consequence of our results we emphasise that propagation with completely ripe seeds is proposed, however, half-ripe seeds developped in an unadvantageous vegetation period can be also utilized in the practice. The effect of vegetation year on stramony seeds manifested in the length of after-ripening period, while in the case of henbane the germination ability was also influenced.