Cold Stratification Research Articles

Non-deep simple epicotyl morphophysiological dormancy in seeds of Helleborus thibetanus (Ranunculaceae)

Helleborus thibetanus is a perennial herbaceous plant with high potential for medicinal and landscaping purposes. However, the dormancy characteristics of seeds and the conditions that help this species break dormancy have not been investigated. This study investigated embryo growth and the emergence of radicles and epicotyls in seeds over a range of GA3 pretreatment and temperature sequences. Upon reaching maturity, the seeds had underdeveloped embryos that developed fully at 15 °C warm stratification within 6 weeks. Immediately after embryo growth, radicles emerged during warm stratification. However, the epicotyls failed to emerge soon after the radicle emergence. Epicotyl emergence was observed in over > 13 % of seeds with an emerged radicle following 4 weeks of cold stratification at 4 °C. The effects of GA3 on radicle and epicotyl emergence were used to determine the specific types of seed dormancy. GA3 pretreatment partially substituted 15 °C warm stratification to promote radicle emergence and effectively served as a substitute for the 4 °C cold stratification to break the epicotyl dormancy. The overall temperature and GA3 requirements for breaking dormancy and seed germination indicate non-deep simple epicotyl morphophysiological dormancy in H. thibetanus.

Metabolite and Transcriptomic Changes Reveal the Cold Stratification Process in Sinopodophyllum hexandrum Seeds.

Sinopodophyllum hexandrum (Royle) Ying, an endangered perennial medicinal herb, exhibits morpho-physiological dormancy in its seeds, requiring cold stratification for germination. However, the precise molecular mechanisms underlying this transition from dormancy to germination remain unclear. This study integrates transcriptome and plant hormone-targeted metabolomics techniques to unravel these intricate molecular regulatory mechanisms during cold stratification in S. hexandrum seeds. Significant alterations in the physicochemical properties (starch, soluble sugars, soluble proteins) and enzyme activities (PK, SDH, G-6-PDH) within the seeds occur during stratification. To characterize and monitor the formation and transformation of plant hormones throughout this process, extracts from S. hexandrum seeds at five stratification stages of 0 days (S0), 30 days (S1), 60 days (S2), 90 days (S3), and 120 days (S4) were analyzed using UPLC-MS/MS, revealing a total of 37 differential metabolites belonging to seven major classes of plant hormones. To investigate the biosynthetic and conversion processes of plant hormones related to seed dormancy and germination, the transcriptome of S. hexandrum seeds was monitored via RNA-seq, revealing 65,372 differentially expressed genes associated with plant hormone synthesis and signaling. Notably, cytokinins (CKs) and gibberellins (GAs) exhibited synergistic effects, while abscisic acid (ABA) displayed antagonistic effects. Furthermore, key hub genes were identified through integrated network analysis. In this rigorous scientific study, we systematically elucidate the intricate dynamic molecular regulatory mechanisms that govern the transition from dormancy to germination in S. hexandrum seeds during stratification. By meticulously examining these mechanisms, we establish a solid foundation of knowledge that serves as a scientific basis for facilitating large-scale breeding programs and advancing the artificial cultivation of this highly valued medicinal plant.

Germination characteristics of Angelica gigas and Angelica acutiloba as affected by cold stratification and hot water treatment before sowing

Germination characteristics of <i>Angelica gigas</i> and <i>Angelica acutiloba</i> as affected by cold stratification and hot water treatment before sowing

Germination ecology of Phytolacca americana L. in its invasive range

AbstractInvasive species are a worldwide problem, and the germination process is useful to understand the characteristics that allow alien species to be invasive and their projected response to global climate change. Phytolacca americana is one of the most invasive plants in Italy, and we tested, for different populations (from different altitudes) how light, temperature, and cold stratification affect seed germination. According to our analyses, P. americana produces an exceptionally high number of seeds that may potentially survive in soil for extended periods. Seeds subjected to cold stratification and exposed to warmer temperatures, both in light and darkness, exhibited faster germination, with a higher germination rate and a shorter T50. Seeds collected at the highest elevation (337 m a.s.l.) have germinated in all tested thermal conditions, albeit with a lower germination percentage and a longer T50 compared with seeds collected at lower elevations (5 and 50 m a.s.l.) and tested under warm and moderate temperatures. In general, P. americana seems to adapt to moderate‐warm temperatures (at low elevations) and moderate‐cool temperatures (at highest elevations) and appears to increase germination with seeds exposed to cold stratification. These results, in a scenario of climate change, show that the invasiveness of P. americana may increase in the future.

Characteristic of epicotyl dormancy and its hormonal regulation in Chinese cork oak (Quercus variabilis)

Emergence heterogeneity caused by epicotyl dormancy contributes to variations in seedling quality during large-scale breeding. However, the mechanism of epicotyl dormancy release remains obscure. We first categorized the emergence stages of Chinese cork oak (Quercus variabilis) using the BBCH-scale. Subsequently, we identified the key stage of the epicotyl dormancy process. Our findings indicated that cold stratification significantly released epicotyl dormancy by increasing the levels of gibberellic acid 3 (GA3) and GA4. Genes associated with GA biosynthesis and signaling also exhibited altered expression patterns. Inhibition of GA biosynthesis by paclobutrazol (PAC) treatment severely inhibited emergence, with no effect on seed germination. Different concentrations (50 μM, 100 μM, and 200 μM) of GA3 and GA4+7 treatments of germinated seeds demonstrated that both can promote the emergence, with GA4 exhibiting a more pronounced effect. In conclusion, this study provides valuable insights into the characterization of epicotyl dormancy in Chinese cork oak and highlights the critical role of GA biosynthesis in seedling emergence. These findings serve as a basis for further investigations on epicotyl dormancy and advancing large-scale breeding techniques.

Diversity of Treatments in Overcoming Morphophysiological Dormancy of Paeonia peregrina Mill. Seeds.

Paeonia peregrina Mill. is a protected, herbaceous species native to Southeastern Europe and Turkey. Due to its vulnerability, it has to be protected both in its natural habitats and through cultivation. Peonies are known to have a low potential for natural regeneration due to their seed dormancy, low germination rate, and long germination period. In this study, treatments with gibberellic acid (GA3 150, 200, 250, 300, and 350 mg L-1 GA3) and warm (at 20/16 °C day/night regime) and cold stratification (at 4 °C) were used to accelerate dormancy release and increase the germination rate. The seeds of P. peregrina from four natural habitats in Serbia and the Institute's collection were collected and analyzed. They showed an underdeveloped embryo that needs to grow inside the seed before it can germinate. The application of GA3 accelerated each stage of germination (seed coat rapturing, hypocotyl dormancy release, and epicotyl dormancy release) for approximately 10 days compared to the control. It was also found that two-day imbibition with 200 mg L-1 GA3 significantly accelerated and equalized germination. Higher GA3 concentrations had a more pronounced impact on each stage but also resulted in greater seed infection after the seed coat rupture, elongated and weak seedlings, while lower concentrations did not result in obtaining uniform seedlings. There were no significant differences observed between localities. Restoring P. peregrina through seeds and nursery-produced plants is crucial for conserving the genetic diversity of the tested species.

Effects of climate change on seed germination may contribute to habitat homogenization in freshwater forested wetlands

Climate changes in temperate regions are expected to result in warmer, shorter winters in temperate latitudes. These changes may have consequences for germination of plant species that require a period of physiological dormancy. The effect of cold duration on seed germination has been investigated in a number of plant taxa, but has not been well studied in wetland and bottomland forest tree species, an ecosystem that is threatened by habitat homogenization. Our work sought to test the role of changing winter temperatures on seed germination in specialist (Nyssa aquatica and Taxodium distichum) and generalist (Acer rubrum and Liquidambar styraciflua) tree species within forested wetlands throughout the eastern U.S. The experiment was conducted in an environmental chamber in Norfolk, VA, USA. Seeds of T. distichum, N. aquatica, A. rubrum, and L. styraciflua were exposed to each of seven pre-germination cold exposure durations (0, 15, 30, 45, 60, 75, and 90 days) and observed for germination for 30 days. Cold stratification duration positively impacted total percent germination in N. aquatica (p < 0.0001) as well as A. rubrum (p = 0.0008) and T. distichum (p = 0.05). Liquidambar styraciflua seeds exhibited more rapid rates of germination with increasing cold exposure duration and greater percent germination compared to the others regardless of cold stratification duration. Our results provide insight into how community dynamics and biodiversity of wetland and bottomland trees may shift with a changing climate. Further, this work emphasizes the importance of understanding the role of plant functional traits in early life stages in community dynamics and has implications for management practices.

Germination response to winter temperature changes with seed shape and length of temperature exposure.

In many regions, the climate is changing faster during winter than during the other seasons, and a loss of snow cover combined with increased temperature variability can expose overwintering organisms to harmful conditions. Understanding how species respond to these changes during critical developmental times, such as seed germination, helps us assess the ecological implications of winter climate change. To address this concern, we measured the breaking of seed dormancy and cold tolerance of temperate grassland species in the lab and field. In the lab, we ran germination trials testing the tolerance of 17 species to an extreme cold event. In the field, we deployed seeds of two species within a snow manipulation experiment at three locations and measured germination success biweekly from seeds subjected to ambient and reduced snow cover from winter into spring. From lab trials, cold tolerance varied among species, with seed germination decreasing <10%-100% following extreme cold events. Cold tolerance was related to seed traits, specifically less round seeds, seeds that required cold stratification, and seeds that mature later in the season tended to be more impacted by extreme cold temperatures. This variation in seed cold tolerance may contribute to altered community composition with continued winter climate change. In the field, germination increased through late winter, coinciding with the accumulation of days where temperatures were favorable for cold stratification. Through spring, germination success decreased as warm temperatures accumulated. Collectively, species-specific seed cold tolerances and mortality rates may contribute to compositional changes in grasslands under continued winter climate change.

Seed dormancy, germination requirements, and implications of herbicides for Penstemon albidus and P. nitidus

Seed‐based restoration is dependent on seed germination, and poor germination can cause restoration failure. Many restoration failures can be attributed to a lack of knowledge of germination characteristics, species‐specific seed dormancy, or the effects of widely used herbicides on germination. White penstemon (Penstemon albidus) and Waxleaf penstemon (P. nitidus) are native to the Northern Great Plains region of North America, and increased germination of these species would contribute to improved restoration in the region. We performed two concurrent experiments to determine: (1) the germination requirements and dormancy class of these species; and (2) the effects of herbicides on germination. To determine germination requirements, we applied pretreatments (scarification, smoke, and KNO3) and three durations (2, 4, and 8 weeks) of cold and warm stratification. To test the effects of herbicides on germination, three commonly used herbicides (atrazine, trifluralin, and 2,4‐D) were applied at six concentrations (100, 50, 10, 1, 0.1, and 0% of the recommended field application rate). Germination characteristics indicate both species express physiological dormancy. Physiological dormancy denotes an embryo with low growth potential that is unable to overcome mechanical constraints but can be alleviated with proper temperature cues. Both species required cold stratification, with P. nitidus needing a longer period (8 weeks) than P. albidus (4 weeks). Final germination percentage of P. albidus decreased with higher doses of 2,4‐D but was not affected by atrazine or trifluralin. These experiments help to create protocol for the use of our study species, as well as other species, in restoration plantings.

Seed dormancy types and germination response of 15 plant species in temperate montane peatlands.

Despite their crucial role in determining the fate of seeds, the type and breaking mode of seed dormancy in peatland plants in temperate Asia with a continental monsoon climate are rarely known. Fifteen common peatland plant species were used to test their seed germination response to various dormancy-breaking treatments, including dry storage (D), gibberellin acid soaking (GA), cold stratification (CS), warm followed cold stratification (WCS), GA soaking + cold stratification (GA + CS) and GA soaking + warm followed cold stratification (GA + WCS). Germination experiment, viability and imbibition test, and morphological observation of embryos were conducted. Of the 15 species, nine showed physiological dormancy (PD), with non-deep PD being the dominant type. Four species, Angelica pubescens, Cicuta virosa, Iris laevigata, and Iris setosa exhibited morphophysiological dormancy. Two species, Lycopus uniflorus and Spiraea salicifolia, demonstrated nondormancy. Overall, the effect hierarchy of dormancy-breaking is: CS > GA > WCS > GA + CS > D > GA + WCS. Principal component analysis demonstrated that seed traits, including embryo length: seed length ratio, seed size, and monocot/eudicot divergence, are more likely to influence seed dormancy than environmental factors. Our study suggests that nearly 90% of the tested peatland plant species in the Changbai Mountains demonstrated seed dormancy, and seed traits (e.g. embryo-to-seed ratio and seed size) and abiotic environmental factors (e.g. pH and temperature seasonality) are related to germination behavior, suggesting seed dormancy being a common adaptation strategy for the peatland plants in the temperate montane environment.

Deep Simple Epicotyl Morphophysiological Dormancy in Seeds of Endemic Chinese Helleborus thibetanus

Helleborus thibetanus is an endemic species in China with important ornamental and medicinal value. However, the seeds have dormancy, and their germination percentage is low under natural conditions. This research was carried out to determine the seed germination requirements of H. thibetanus and to characterize the type of seed dormancy. The morphological post-ripening process of the seed embryo was studied according to the morphological anatomy, and the effects of temperature and gibberellic acid (GA3) on seed germination were investigated in H. thibetanus. The H. thibetanus seeds had a heart-shaped embryo at maturity. The embryo fully grew within the seed through warm stratification, and the embryo/seed ratio increased from 8.58% to 42.6%. The shortest time for a radicle to emerge (58.33 d) and the highest radicle emergence percentage (84.44%) were obtained at a temperature of 15 °C. The results showed that the H. thibetanus seeds had a morphophysiological dormancy. In addition, 300 mg/L GA3 treatments shortened the time of warm stratification and increased the radicle emergence percentage. Seeds with emerged radicles could not emerge epicotyl–plumule without cold stratification, which showed that the H. thibetanus seeds had epicotyl physiological dormancy. The length of the roots, cold stratification time, and GA3 markedly affected the release of the epicotyl physiological dormancy in H. thibetanus. The seeds with 2.5 cm roots required the shortest time to break their dormancy (50 d), and the epicotyl–plumule emergence percentage was the highest. Additionally, GA3 treatment also shortened the incubation time in cold stratification (5 °C) and successfully broke the epicotyl physiological dormancy. Our study showed that H. thibetanus seeds exhibited deep simple epicotyl morphophysiological dormancy. Temperature, GA3, and duration of stratification played vital roles in the seed germination of H. thibetanus. This research will provide valuable data for seed germination and practical dormancy-breaking techniques and will promote the cultivation and conventional crossbreeding of H. thibetanus.

Low-cost and reliable substrate-based phenotyping platform for screening salt tolerance of cutting propagation-dependent grass, paspalum vaginatum

BackgroundSalt tolerance in plants is defined as their ability to grow and complete their life cycle under saline conditions. Staple crops have limited salt tolerance, but forage grass can survive in large unexploited saline areas of costal or desert land. However, due to the restriction of self-incompatible fertilization in many grass species, vegetative propagation via stem cuttings is the dominant practice; this is incompatible with current methodologies of salt-tolerance phenotyping, which have been developed for germination-based seedling growth. Therefore, the performance of seedlings from cuttings under salt stress is still fuzzy. Moreover, the morphological traits involved in salt tolerance are still mostly unknown, especially under experimental conditions with varying levels of stress.ResultsTo estimate the salt tolerance of cutting propagation-dependent grasses, a reliable and low-cost workflow was established with multiple saline treatments, using Paspalum vaginatum as the material and substrate as medium, where cold stratification and selection of stem segments were the two variables used to control for experimental errors. Average leaf number (ALN) was designated as the best criterion for evaluating ion-accumulated salt tolerance. The reliability of ALN was revealed by the consistent results among four P. vaginatum genotypes, and three warm-season (pearl millet, sweet sorghum, and wild maize) and four cold-season (barley, oat, rye, and ryegrass) forage cultivars. Dynamic curves simulated by sigmoidal mathematical models were well-depicted for the calculation of the key parameter, Salt50. The reliability of the integrated platform was further validated by screening 48 additional recombinants, which were previously generated from a self-fertile mutant of P. vaginatum. The genotypes displaying extreme ALN-based Salt50 also exhibited variations in biomass and ion content, which not only confirmed the reliability of our phenotyping platform but also the representativeness of the aerial ALN trait for salt tolerance.ConclusionsOur phenotyping platform is proved to be compatible with estimations in both germination-based and cutting propagation-dependent seedling tolerance under salt stresses. ALN and its derived parameters are prone to overcome the species barriers when comparing salt tolerance of different species together. The accuracy and reliability of the developed phenotyping platform is expected to benefit breeding programs in saline agriculture.

Sumac (Rhus coriaria L.) sprouts: from in vitro seed germination to phenolic content and antioxidant activity for biotechnological application

The sumac plant (Rhus coriaria L.), native to the Middle East, but also growing in Italy, is used for the medicinal properties of its fruits and leaves. Recently, sumac plant have been characterized in terms of their bioactive compound content and biological activity. Regrettably, there is a lack of information on the chemical composition of sumac sprouts, which, on the contrary, could be a rich source of interesting compounds, as demonstrated for other plant species. To make the production of sumac sprouts faster and independent of environmental conditions, in vitro tissue culture can be an efficient solution. This study first established a valid protocol for in vitro sumac seed germination; secondly, the obtained sprouts were characterized, in terms of phenolic content and antioxidant activity. The results showed that mechanical scarification is mandatory to trigger in vitro germination of sumac seeds. Moreover, a significant final germination percentage and a reasonable mean germination time was obtained combining scarification, cold stratification and gibberellic acid-enriched culture media. Chemical characterization of sumac sprouts has shown that they are a source of potentially bioactive compounds, as they are rich in polyphenols and have appreciable antioxidant activity.Graphical

Seed Dormancy Class and Germination Characteristics of Berberis amurensis var. latifolia Nakai, Native to Korea

Berberis amurensis var. latifolia Nakai is a plant native to the Ulleung Island in Korea. In this study, we aimed to identify seed dormancy-breaking and germination requirements of this species using water imbibition experiments, gibberellic acid (GA3) treatment (0, 10, 100, or 1000 mg/L), cold stratification (0, 2, 4, 8, or 12 weeks at 5 °C), move-along experiments, and phenological studies. In the water imbibition experiment, the seed weight increased by more than 120% after 24 h. Analysis of the internal morphological characteristics of the seeds revealed that the embryo in freshly matured seeds was fully grown and did not grow thereafter. The final germination percentages after 12 weeks of cold stratification at 5 °C were 49 ± 6.4% and 63 ± 3.4% under light and dark conditions, respectively. In move-along and phenological studies, a longer cold stratification treatment period resulted in a higher germination percentage; however, the warm stratification treatment did not affect germination significantly. The GA3 treatment had little effect on seed germination. Therefore, we concluded that B. amurensis var. latifolia seeds have intermediate physiological dormancy, and pre-treatment with cold stratification for 12 weeks and incubation in the dark are required for effective seed propagation.

Effects of various pre-treatments on dormancy-break and seed germination of Saussurea veitchiana (Asteraceae), an endemic medicinal herb of southwest China

Subalpine Saussurea veitchiana (Asteraceae) is useful for the treatment of various diseases and is vulnerable to extinction. Methods to break seed dormancy and enhance germination of this species, as well as the response of seeds to different temperatures, were investigated. Freshly harvested seeds germinated to very low percentages at ten temperature regimes in light conditions. Gibberellic acid (10 - 200 μM) partially broke the dormancy of fresh seeds, and the highest germination was 54% when seeds were treated with 200 μM GA3 and incubated at 20°C or 25/15°C. Cold stratification significantly increased germination over that of the control (i. e., seeds dry stored at room temperature, approximately 18°C). Seeds cold stratified at 4°C for 60 or 90 days germinated to 83 and 87%, respectively, at 25/15°C and had a MGT of 7.11 and 6.37 days, respectively. Taken together, the seeds of S. veitchiana have non-deep physiological dormancy, and cold stratification for 60 days or longer is recommended as a practical dormancy-breaking treatment for propagation of this species from seeds.

Introduction of Salicornia europaea L into in vitro culture

The goal of the research was to develop a technology for introducing Salicornia europaea L.into in vitro culture. Methods of cultivating meristems and callus cultures were studied. To cultivate meristems, the tip of the apical bud were used as an explant. Fragments of stems and leaves were used toobtain callus tissue. To study the influence of various factors on germination, seeds were soaked in sterile tap water and in solutions of gibberellin, cytokinin, auxin and NaCl, and were also subjected to cold stratification (independently and with subsequent placement in Knop’s agarized medium). Salicornia seeds were sterilized with various antiseptics: 70% alcohol, 10% aqueous solution of sodium hypochlorite («Belizna»), amoxicillinand 3% hydrogen peroxide. The ability of the culture to form callus was studied in MS medium. As a result, it was determined that the highest germination of seeds was observed in Knop medium after treating theseeds with a suspension of green algae of the Scenedesmus genus, as well as after preliminary cold stratification, and slightly less after treating the seeds with a solution of NaCl and gibberellic acid. The most effective method of seed sterilization turned out to be treatment with alcohol followed by treatment with sodium hypochlorite. A comparative analysis of seed germination in filter paper in Petri dishes, Knop agar medium, Murashige and Skoog (hormone-free) was carried out. The ability of S. europaea L. to form callusin MS medium with phytohormones was assessed. Conclusion. To improve germination, it is recommended to subject the seeds to cold stratification. To obtain aseptic explants quickly, it is recommended to germinate the seeds in the Knop nutrient medium, having previously sterilized them with alcohol, then with sodiumhypochlorite, followed by washing with distilled water. The most suitable type of microclonal propagationfor S. europaea L. is the production of callus tissue followed by induction of organogenesis or embryogenesis.

Germination Kinetics of Ferula communis L. Seeds, a Potentially Multipurpose-Use Wild Species

Despite exhibiting intriguing features associated with its multipurpose applications and drought tolerance, Ferula communis remains a wild and uncultivated species, with limited experimental research on its biology, starting from seed germination and extending to its ecology. The purpose of this study was to investigate potential germination and kinetics in F. communis seeds in response to four cold stratification periods (0, 15, 45, and 90 days at a constant temperature of 5 °C) and four temperatures (5, 10, 15, and 20 °C) under continuous darkness. F. communis exhibited a pronounced germination potential exceeding 90%, with the optimal temperature for germination falling within the range of 5 °C to 15 °C, without necessitating cold stratification. A dramatic drop of the germination percentage was observed at 20 °C (&lt;10%), suggesting a form of conditional dormancy attributed to the higher temperature tested.

Proteomic and metabolomic insights into seed germination of Ferula assa-foetida

Cold stratification is known to affect the speed of seed germination; however, its regulation at the molecular level in Ferula assa-foetida remains ambiguous. Here, we used cold stratification (4 °C in the dark) to induce germination in F. assa-foetida and adopted a proteomic and metabolomic approach to understand the molecular mechanism of germination. Compared to the control, we identified 209 non-redundant proteins and 96 metabolites in germinated F. assa-foetida seed. Results highlight the common and unique regulatory mechanisms like signaling cascade, reactivation of energy metabolism, activation of ROS scavenging system, DNA repair, gene expression cascade, cytoskeleton, and cell wall modulation in F. assa-foetida germination. A protein-protein interaction network identifies 18 hub protein species central to the interactome and could be a key player in F. assa-foetida germination. Further, the predominant metabolic pathways like glucosinolate biosynthesis, arginine and proline metabolism, cysteine and methionine metabolism, aminoacyl-tRNA biosynthesis, and carotenoid biosynthesis in germinating seed may indicate the regulation of carbon and nitrogen metabolism is prime essential to maintain the physiology of germinating seedlings. The findings of this study provide a better understanding of cold stratification-induced seed germination, which might be utilized for genetic modification and traditional breeding of Ferula assa-foetida. SignificanceSeed germination is the fundamental checkpoint for plant growth and development, which has ecological significance. Ferula assa-foetida L., commonly known as “asafoetida,” is a medicinal and food crop with huge therapeutic potential. To date, our understanding of F. assa-foetida seed germination is rudimentary. Therefore, studying the molecular mechanism that governs dormancy decay and the onset of germination in F. assa-foetida is essential for understanding the basic principle of seed germination, which could offer to improve genetic modification and traditional breeding.

Factors controlling germination and seedling growth of an endangered Saussurea species (Asteraceae) endemic to serpentine areas in Japan

AbstractSaussurea ochiaiana Kadota (Asteraceae) is a perennial herb endemic to serpentine areas in western Japan. To date, only two locations are known habitats for this species of unknown ecological requirements. Therefore, this study aimed to clarify the environmental factors that limit the germination and seedling growth of this species, which is an essential requirement for designing a suitable conservation strategy. Our germination experiments, in which the achenes were subjected to a gradually increasing temperature and a gradually decreasing temperature after short (1 month) or long (4 months) storage, indicated that the achenes required a long cold stratification for germination. In an experiment where plants collected at the study site were potted individually, they did not grow under strongly shaded conditions consisting of only 10% relative photosynthetic photon flux density (RPPFD), but grew well under moderately shaded conditions (50% RPPFD). No significant differences in seedling growth were observed among the three pH conditions tested (pH 4, 6, and 8). Although average seedling shoot weight was significantly larger at low Ca:Mg molar ratios (0.6 and 0.3) than at high substrate Ca:Mg molar ratios (15 and 1.5) in pot culture experiments, the difference between treatments was not very large (&lt;20%). Habitat conditions suitable for the regeneration of this species are decreasing because of climate change and the depopulation of rural communities (reduction in management); therefore, urgent conservation strategies, including ex situ conservation, are needed.

Influence of shade and cold stratification on germination success of vegetative propagules from multiple Butomus umbellatus genotypes

Three experimental trials were conducted to investigate the influence of shade and cold stratification on germination success of vegetative propagules from multiple Butomus umbellatus genotypes. Shade level did not significantly impact germination of propagules in trial 1 (p=0.16); however, significant differences (p<0.0001) in germination percentages at the conclusion of the study were detected between genotypes. Rhizome segments of the triploid genotype one (G1) had the highest mean germination (95±1%); whereas bulbils of the diploid genotypes three (G3), four (G4), and five (G5) germinated to 9±2%, 1±1%, and 15±2%, respectively. Trial 2 focused on bulbils from G3, G4, and G5 diploid plants that were stratified at 4℃ for 35d. Like Trial 1, shade level was not significant (p=0.19) relative to the overall germination of cold-stratified bulbils. However, cold-stratified bulbils exhibited a much higher mean germination (≥93%) for all three genotypes. In Trial 3, the cold stratification treatments were significant and positively correlated to overall germination for G4 (p=0.005, r=0.77) and G5 (p=0.002, r=0.82), but not G3 (p=0.22, r=0.40) bulbils. Germination time significantly differed between genotypes in all cold-stratification treatments except for the 0, 120, and 180-day treatments. These studies demonstrate that a high percentage of vegetative propagules produced by B. umbellatus are capable of successfully germinating under laboratory conditions, but some require extended periods of cold exposure. Given that a single diploid bulbil can produce thousands of bulbils within a growing season; long term management of this species will need to be focused towards limiting bulbil production.