The role of GA3 in the germination process of high-mountain endemic and threatened species: Leontopodium nivalePinguicula fiorii and Soldanella minima subsp. samnitica (central Apennines,Italy)

The ex situ conservation of biodiversity is an essential tool for environmental protection interventions. Germination studies of seeds that belong to endangered species are essential for ex situ conservation strategies. In this study, we investigate the germination responses of three high-altitude endemic and vulnerable species (Leontopodium nivale, Pinguicula fiorii and Soldanella minima subsp. samnitica). Specifically we identified potential dormancy mechanisms by investigating the responses of germination percentage and rate to different concentrations of gibberellic acid (GA₃), by performing a general linear model. L. nivale reached a germination percentage of 98.0 ± 2.0% (mean ± SE) under control conditions (20°C; 12/12 photoperiod; no GA₃ addition). P. fiorii showed the highest germination percentage (78.0 ± 2.0%) in the treatment with GA₃ 500 ppm. S. minima subsp. samnitica did not show sensitivity to GA₃ but responded positively to cold stratification (6 month at 5°C) with a germination percentage of 90 ± 6%. This study made it possible to acquire important information on the germination process of threatened and rare endemic taxa.     

Seed germination behavior of five Resedaceae species from arid Arabian desert: gibberellic acid and light effects

Abstract

Light and growth-promoting compounds, such as gibberellic acids (GA₃), are among the most important factors that can break physiological seed dormancy. Here, we investigate the effects of GA₃ and light on germination of five species of Resedaceae that are known to have different levels of physiological dormancy. Seeds were incubated at 20/30?°C in both 12-hr photoperiod and complete darkness. To study the effect of growth hormone on germination, seeds were soaked for 24?h in different concentrations of GA₃ before sowing. The annuals (Reseda aucheri and Oligomeris linifolia) and the perennial Ochradenus arabicus had deep physiological dormancy, and exogenous application of GA₃ enhanced their germination in the light, but not in darkness; few or no seeds germinated in the dark in these species. Ochradenus aucheri and O. baccatus had intermediate and non-deep dormancy, respectively, and application of GA₃ enhanced their germination in both light and darkness. Germination of the annual species was much slower than that of the shrubby perennials. Overall, these results indicate that conditions under which seed developed, matured and stored on maternal plants as well as incubation conditions should be taken into consideration when assessing germination behavior of the perennial species of Resedaceae.     

Seed dormancy,germination and soil seed bank of Lamiophlomis rotata and Marmoritis complanatum (Labiatae), two endemic species from Himalaya–Hengduan Mountains

Seed dormancy and germination characteristics are important factors determining plant reproductive success. In this study, we aimed to explore the characteristics of seed dormancy and germination of two endemic Labiatae species (Lamiophlomis rotata and Marmoritis complanatum) in the Himalaya–Hengduan Mountains. Germination was first tested in the light using freshly matured seeds at 25/15 and 15/5°C, and then again after dry after-ripening. Dried seeds were incubated in the light at a range of constant temperatures (1–35°C). The effects of dark and GA₃ on germination were tested at several different temperatures. Base temperature (T_b) and thermal times for 50% final germination (θ₅₀) were calculated. Seeds were also buried at the collection site to test seed persistence in the soil. Increased final germination after dry after-ripening indicated that the seeds of the two species exhibited non-deep physiological dormancy; however, they exhibited different germination characteristics and soil seed bank types. In L. rotata, GA₃ only promoted germination at 5°C, producing no significant effect at other temperatures. Dark conditions decreased germination significantly at all temperatures. T_b and θ₅₀ values were 0.6 and 82.7°C d. The soil seed bank of this species was classified as persistent. In M. complanatum, GA₃ significantly promoted germination at all temperatures except 15°C. Dark conditions depressed germination significantly at warmer temperatures (20 and 25°C) but had no effect at lower temperatures. T_b and θ₅₀ values were 0.1 and 92.3°C d. The soil seed bank was classified as transient. Our results suggest that the seed dormancy and germination of the two co-existing species share some commonalities but there are also species-specific adaptations to the harsh alpine environment.     

Seed dormancy and germination in Jeffersonia dubia (Berberidaceae) as affected by temperature and gibberellic acid

The genus Jeffersonia, which contains only two species, has a trans‐Atlantic disjunct distribution. The aims of this study were to determine the requirements for breaking dormancy and germination of J. dubia seeds and to compare its dormancy characteristics with those of the congener in eastern North America. Ripe seeds of J. dubia contain an underdeveloped embryo and were permeable to water. In nature, seeds were dispersed in May, while embryos began to grow in September, and were fully elongated by late November. Germination started in March of the next year, and seeds emerged as seedlings soon after germination. In laboratory experiments, incubation at high temperatures (25 °C, 25/15 °C) for at least 8 weeks was required to initiate embryo growth, while a transfer to moderate temperatures (20/10 °C, 15/6 °C) was needed for the completion of embryo growth. At least 8 weeks at 5 °C was effective in overcoming physiological dormancy and for germination in seeds after the embryos had fully elongated. Thus, both high and low temperatures were essential to break dormancy. Gibberellic acid (GA₃) treatment could substitute for the high temperature requirement, but not for the low temperature requirement. Based on the dormancy‐breaking requirements, it is confirmed that the seeds have deep simple morphophysiological dormancy. This dormancy type is similar to that of seeds of the eastern North American species J. diphylla. Although seeds require 10–11 months from seed dispersal to germination in nature, under controlled conditions they required only 3 months after treatment with 1000 mg·l^?1 GA₃, followed by incubation at 15/6 °C. This represents practical knowledge for propagation of these plants from seed.     

Hoary cress (Cardaria draba (L.) Desv.) seed germination ecology,longevity and seedling emergence

The effects of gibberellic acid (GA₃), potassium nitrate (KNO₃), prechilling, temperature, salt stress and osmotic potential on seed germination and sowing depth on seedling emergence and burial depth on seed viability of hoary cress (Cardaria draba (L.) Desv.), were studied in a series of laboratory, glasshouse and outdoor experiments. The optimal temperature for hoary cress seed germination was 20°C, both in light/dark and darkness regimes. Seed germination of hoary cress at 400 ppm concentration of GA₃ in a light/dark regime was maximal. Potassium nitrate concentrations increased the percentage of germination in comparison with the control treatment. Increasing the duration of dry prechilling to 30 and 45 days promoted the seed germination of hoary cress. Germination of hoary cress markedly decreased as salt and drought stress increased. Seed germination of hoary cress occurred at a range of pH from 3 to 11. Seedling emergence significantly decreased as planting depth increased. Total seed viability decreased with increasing burial depth. The maximum increase in mortality occurred in seeds that were buried at 5‐cm depth.     

Gibberellins in seeds of Arabidopsis thaliana: biological activities,identification and effects of light and chilling on endogenous levels

The activities of several gibberellins in stimulating germination of wild-type and GA-deficient gal seeds of Arabidopsis thaliana were compared. Of the six compounds tested GA₄ and GA₇-isolactone had the highest activity and GA₇ and GA₉ the lowest; activities of GA₁ and GA₃ were intermediate. Combined application of pure GAs presented no indications that more than one GA receptor is involved. Four GAs were identified in extracts from wild-type and GA-insensitive gai seeds by combined gas chromatography mass spectrometry: GA₁, GA₃, GA₄ and GA₉. Effects of light and chilling on levels of GA₁, GA₄ and GA₉ were studied using deuterated standards. Light increased both GA levels and germination in unchilled wild-type and gai seeds. As a result of irradiation GA levels in gai seeds were 7–10 times as high as in wild-type seeds. In the dark germination was 0%, in the light 14% of gai seeds and 95% of wild-type seeds germinated. A chilling pre-treatment of 7 days at 2°C was required to enhance further the germination of gai seeds in the light. Light did not increase GA levels of chilled seeds of either genotype; levels of GA₄ and GA₉ of chilled gai seeds, in the light were respectively 7 and 12 times lower than in non-chilled seeds, whereas the latter seeds germinated better. Slightly elevated levels of GA₄ were detected in darkness after chilling, but germination capacity was still 0%. These results strengthened the conclusion that GAs are required for germination of A. thaliana seeds, whereby GA₄ has intrinsic biological activity. However, it is unlikely that light and chilling stimulate germination primarily by increasing levels of GA. Instead GA sensitivity is a possible alternative.     

Dissecting seed dormancy and germination in Aquilegia barbaricina,through thermal kinetics of embryo growth

• Threshold‐based thermal time models provide insight into the physiological switch from the dormant to the non‐dormant germinating seed.
• This approach was used to quantify the different growth responses of the embryo of seeds purported to have morphophysiological dormancy (MPD) through the complex phases of dormancy release and germination. Aquilegia barbaricina seeds were incubated at constant temperatures (10–25 °C) and 25/10 °C, without pre‐treatment, after warm+cold stratification (W+C) and GA₃ treatment. Embryo growth was assessed and the time of testa and endosperm rupture scored. Base temperatures (T_b) and thermal times for 50% (θ₅₀) of embryo growth and seed germination were calculated.
• W+C enabled slow embryo growth. W+C and GA₃ promoted rapid embryo growth and subsequent radicle emergence. The embryo internal growth base temperature (T_be) was ca. 5 °C for W+C and GA₃‐treated seeds. GA₃ treatment also resulted in similar T_b estimates for radicle emergence. The thermal times for embryo growth (θ_e50) and germination (θ_g50) were four‐ to six‐fold longer in the presence of GA₃ compared to W+C.
• A. barbaricina is characterised by a multi‐step seed germination. The slow embryo growth during W+C reflects continuation of the maternal programme of development, whilst the thermal kinetics of both embryo and radicle growth after the removal of physiological dormancy are distinctly different. The effects of W+C on the multiphasic germination response in MPD seeds are only partially mimicked by 250 mg·l^?1 GA₃. The thermal time approach could be a valid tool to model thermal kinetics of embryo growth and radicle protrusion.

     

The germination characteristics of <Emphasis Type="Italic">Scrophularia marilandica</Emphasis> L. (Scrophulariaceae) seeds

Investigations on seeds of Scrophularia marilandica L. were undertaken to determine their germination requirements. Seeds were collected from three naturally occurring sites and one greenhouse-grown population in London, Ontario in September and October of 1997. Some were set to germinate immediately after collection; others were stored in or on soil outside and/or under controlled laboratory conditions before testing. Germination was assessed under two light/temperature regimes (35°C 14 h light, 20°C 10 h dark and 25°C 14 h light, 10°C 10 h dark), in continuous darkness, and in the presence of two germination-promoting chemicals (GA₃ and KNO₃). Fresh seeds germinated best at 35/20°C, while stored seeds germinated best at 25/10°C. No differences in percent germination were found among three seed-maturity stages. All chemical treatments, except 0.01 M KNO_3, increased percent germination. Significant differences were found both among and within sites for most chemical treatments, but exposure to 3 × 10⁻⁴ M GA₃ caused almost every seed to germinate. When compared to the control, both the gibberellic acid and the soil-storage treatments contributed to faster germination. Exposure of seeds to naturally prevailing conditions on the soil surface followed by testing under the 25/10°C regime produced the highest percent germination. No seeds germinated in the dark. In summary, seeds of S. marilandica exhibit physiological dormancy, which can be alleviated by exposure to light, after-ripening and/or cold stratification. It is probable that the differences in germination response among sites can be attributed to differences in environmental conditions during seed production. These experiments indicate that the seeds of S. marilandica must be buried shortly after dispersal in order to form a persistent seed bank.     

Dormancy and germination in Stachys germanica L. subsp. bithynica (Boiss.) Bhattacharjee seeds: Effects of short-time moist chilling and plant growth regulators

We investigated the germination requirements of the species Stachys germanica L. subsp. bithynica (Boiss.) Bhattacharjee (Lamiaceae). We studied the effects of scarification, short-time moist chilling (+4 °C) for 15 and 30 days, and various doses of gibberellic acid (GA₃; 0, 100, 150 and 250 ppm), Kinetin (KIN; 50 ppm) and a combination of 250 ppm GA₃ and 50 ppm KIN. The hormone and moist chilling treatments were carried out under both continuous darkness (20 °C) and photoperiodic (20/10 °C; 12/12 h, respectively) conditions. Seeds failed to germinate in response to short-time moist chilling treatments with distilled water under both continuous darkness and photoperiodic conditions. Seeds were found to have dormancy. Treatments with GA₃ or a combination of GA₃ and KIN were successful at breaking seed dormancy. A maximum of 37% of the seeds germinated after GA₃ application in all series. When only KIN was applied at a 50 ppm concentration, germination (12%) was found only with moist chilling for 30 days under continuous darkness. The highest germination rates were found in seeds treated with combination of 250 ppm GA₃ and 50 ppm KIN. In the combination treatments, while the moist chilling treatments for 15 days resulted in 68 and 73% germination, respectively, these rates were up to 95% in the moist chilling treatments for 30 days under continuous darkness and photoperiodic conditions. Mean germination time (MGT) in GA₃ and KIN combinations was lower than in other treatments. Scarification with 80% sulphuric acid did not promote germination. The characteristics of physiological dormancy of S. germanica ssp. bithynica seeds are consistent with conditions of existence in the in alpine habitat of this species.     

Germination behaviour of seeds of Withania somnifera (L.) Dunal: a high value medicinal plant

In order to evolve a quick method for smooth and optimum germination for Withania somnifera- a medicinally efficacious multipurpose plant, present investigation was carried to study the effect of physico-chemical treatments, storage, temperature, photoperiod and growth regulators (GA₃, IAA, IBA, 2–4 D and BA) on germinability. The most effective treatment is GA₃ at 150 μg/ml concentration at 25 °C. The optimal temperature for germination is 25 °C and continuous light favored germination showing that photoperiod has a significant role. The seedlings derived from seeds performed well when grown in a glasshouse. The data have implications for conservation and cultivation of the species studied.     

Nitrate,abscisic acid and gibberellin interactions on the thermoinhibition of lettuce seed germination

Germination of lettuce seeds has obvious thermoinhibition, but the mechanism for thermoinhibition of seed germination is poorly understood. Here, we investigated the interactions of nitrate, abscisic acid (ABA) and gibberellin on seed germination at high temperatures to understand further the mechanism for thermoinhibition of seed germination. Our results showed that lettuce (Lactuca sativa L. ‘Jianye Xianfeng No. 1’) seeds exhibited notable thermoinhibiton of germination at ≥17°C in darkness, and at ≥23°C in light, but the thermoinhibited seeds did not exhibit secondary dormancy. Thermoinhibition of seed germination at 23 or 25°C in light was notably decreased by 5 and 10 mM nitrate, and the stimulatory effects were markedly prevented by nitric oxide (NO) scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. The sensitivity of seed germination to exogenous ABA increased with increasing temperature. Thermoinhibition of seed germination was markedly decreased by fluridone (an inhibitor of ABA biosynthesis) and GA₃, and was increased by diniconazole (an inhibitor of the ABA-catabolizing enzyme ABA 8′-hydroxylase) and paclobutrazol (an inhibitor of GA biosynthetic pathway). The effect of fluridone in decreasing thermoinhibition of seed germination was obviously antagonized by paclobutrazol, and that of GA₃ was notably added to by fluridone, and that of nitrate was antagonized by paclobutrazol, diniconazole and ABA and was added to by GA₃ and fluridone. Our data show that thermoinhibition of lettuce seed germination is decreased by nitrate in a NO-dependent manner, which is antagonized by ABA, diniconazole and paclobutrazol and added by fluridone.     

Cellular alteration and differential protein profile explain effects of GA3 and ABA and their inhibitor on Trichocline catharinensis (Asteraceae) seed germination

Seed physiology of wild species has not been studied as deeply as that of domesticated crop species. Trichocline catharinensis (Asteraceae) is an endemic wildflower species from the high-altitude fields of southern Brazil. This species is of interest as a source of genes to improve cultivated Asteraceae because of its ornamental features, disease resistance and ability to tolerate drought and poor soil conditions. We studied the effects of abscisic acid (ABA) and gibberellic acid (GA₃) and their inhibitors, fluridone (FLU) and paclobutrazol (PAC), on seed germination. We individually assessed ultrastructural changes and differential protein accumulation. The principal component analysis explained 69.66% of differential accumulation for 32 proteins at phase II of seed germination in response to hormone and inhibitor treatment. GA₃-imbibed seed germination (98.75%) resulted in increased protein accumulation to meet energy demand, redox regulation, and reserve metabolism activation. FLU-imbibed seeds showed a higher germination speed index as a consequence of metabolism activation. ABA-imbibed seeds (58.75%) showed osmotolerance and flattened cells in the hypocotyl-radicular axis, suggesting that ABA inhibits cell expansion. PAC-imbibed seeds remained at phase II for 300 h, and germination was suppressed (7.5%) because of the increased signaling proteins and halted reserve mobilization. Therefore, our findings provide insight into the behavior of Asteraceae non-dormant seed germination, which broadens our knowledge of seed germination in a wild and endemic plant species from a threatened ecosystem.     

Relationship between Position on the Parent Plant and Dormancy Characteristics of Seeds of Three Cultivars of Celery (Apium graveolens)

Germination and seedling emergence studies were made on seeds harvested from four different umbel positions of three cultivars of celery (Apium graveolens L.). Although heavier seeds were produced from primary umbels than from other umbels, these were less viable as measured by the germination percentage at I8°C in the light. However, germination of viable seeds from quaternary umbels was lower than that of seeds from primary umbels at 18°C in the dark when incubated with GA₄, (2 × 10 ^?4M) and seed from secondary and tertiary umbels tended to be intermediate in response. All viable seeds germinated when N⁶-benzyladenine (10^?2M) was used in combination with GA₄. Seeds from quaternary umbels of two of the cultivars had a lower high-temperature limit for germination in the dark than did seeds from other umbels. In glasshouse experiments the emergence of viable ‘quaternary’ seeds of these cultivars was higher than that of ‘primary’ seeds. Under these conditions the time to 50% of the final emergence as determined after 42 days was similar for seeds from all umbel positions within each cultivar. In two varieties seedling weights were greater from seeds of primary as compared to quaternary umbels, and in general, the largest seedlings arose from the heaviest seeds and the smallest from the lightest seeds in all three cultivars.     

Germination responses of <Emphasis Type="Italic">Erica andevalensis</Emphasis> to different chemical and physical treatments

Erica andevalensis Cabezudo & Rivera is a threatened edaphic endemic species of Andalusia (SW Spain). Under natural conditions, the plants produce a very large number of small seeds (0.3–0.4 mm) but very few seedlings survive. Different treatments (high temperature, cold pre-treatment, nitrogen salts, and gibberellic acid applications) were tested to assess germination patterns in different populations and to determinate the most favorable conditions for germination. Gibberellic acid was provided in five different concentrations from 0 to 400 ppm GA₃, while nitrogen was applied as 10 mM of either KNO₃ or NH₄NO₃. The effect of pH on germination was also tested. The species always showed a low germination rate (6.50–22%) that was not stimulated either by 1 or 4 months in dry cold pre-treatment, nitrogen application, acid pH medium, or by high temperature (80°C for 10 min); although gibberellic acid application (100–400 ppm) significantly enhanced germination. The highest percentage of germination (41.6%) was achieved with a mean germination time to start germination (t ₀) of 7.6 ± 0.54 days when the seeds were subjected to 400 ppm gibberellic acid treatment. The population origin did not have a significant effect on germination percentage.     

Deep and intermediate complex morphophysiological dormancy in seeds of Ferula gummosa (Apiaceae)

We tested the hypothesis that seeds of the monocarpic perennial Ferula gummosa from the Mediterranean area and central Asia have deep complex morphophysiological dormancy. We determined the water permeability of seeds, embryo morphology, temperature requirements for embryo growth and seed germination and responses of seeds to warm and cold stratification and to different concentrations of GA₃. The embryo has differentiated organs, but it is small (underdeveloped) and must grow inside the seed, reaching a critical embryo length, seed length ratio of 0.65–0.7, before the seed can germinate. Seeds required 9 weeks of cold stratification at <10°C for embryo growth, dormancy break and germination to occur. Thus, seeds have morphophysiological dormancy (MPD). Furthermore, GA₃ improved the germination percentage and rate at 5°C and promoted 20 and 5% germination of seeds incubated at 15 and 20°C, respectively. Thus, about 20% of the seeds had intermediate complex MPD. For the other seeds in the seed lot, cold stratification (5°C) was the only requirement for dormancy break and germination and GA₃ could not substitute for cold stratification. Thus, about 80% of the seeds had deep complex MPD.     

Effects of seed chilling or GA3 supply on dormancy breaking and plantlet growth in Cercis siliquastrum L.

Germination and post-germination events have been compared in seeds of Cercis siliquastrum whose dormancy was removed by fulfilling the natural chilling requirement or by exogenous GA₃ application.Compared to the chilled ones, the GA₃ treated seeds showed precocious embryo growth and an earlier reserve mobilization, which started before radicle emergence.Although the hormonal application was interrupted at seed germination, the plantlets of Cercis siliquastrum that originated from GA₃-supplied seeds were taller than those from chilled ones. Moreover, they produced a greater number of leaves but a reduced root mass and had some difficulty in maintaining a good water balance.Thus, the treatment of Cercis siliquastrum seeds with exogenous GA₃ broke dormancy and induced germination, but also caused long-lasting consequences on morphogenesis of the growing plantlet.     

Effects of microhabitat,light and temperature on seed germination of a critically endangered Himalayan medicinal herb,Swertia chirayita: Conservation implications

Abstract

Swertia chirayita, a critically endangered medicinal herb, is being over-harvested in the wild. Understanding seed germination is a pre-requisite to ensure species conservation. The germination of seeds collected from six microhabitats was studied at 20°C, 25°C, and 30°C, both under a 14/10 h light/dark photoperiod and in continuous darkness. Two-way ANOVA indicated that microhabitat and temperature significantly affect seed germination, germination rate, germination recovery (GR), and GR rate. Overall, the seeds collected from under canopy showed a significantly (p < 0.05) higher germination than those from open habitats, at 20°C, 25°C, and 30°C (14/10 h light/dark photoperiod). Germination was negligible in continuous darkness but after transfer to a 14/10 h light/dark photoperiod, the seeds from under canopy significantly recovered at 20°C and at 25°C (p < 0.05), and showed the highest germination percentage compared to seeds collected from tree base, stump base, shrubberies, and grassy slope. Similarly, at 30°C, seeds from under canopy recorded the highest GR percentage. In general, seed germination, mean germination rate, seed GR, and GR rate were significantly greater (p < 0.05) at 25°C. Among the microhabitats tested, variation in GR rate was significant (p < 0.05). Seeds were confirmed to be positively photoblastic.     

Germination studies on three critically endangered endemic angiosperm species of the Kashmir Himalaya,India

The critically endangered and perennial alpine endemic angiosperms, namely, Aquilegia nivalis, Lagotis cashmeriana and Meconopsis latifolia inhabit such habitats in the Kashmir Himalaya that are characterised by short growing season and heavy snow cover for about 3–4 months during winter season. The seeds of these species under natural conditions experience a long period of pre-chilling during winter prior to their germination in following spring season. Taking cue from such a requirement, present study investigated the effect of chilling and exogenous application of growth hormones, NO₃ ⁻ and NH₄ ⁺ on total percent germination of otherwise deep-dormant seeds of these species, under alternate light/dark and continuous dark light regimes. Prolonged pre-chilling followed by treatment of seeds with different doses of GA₃ had a pronounced stimulatory effect on the total germination percentage in all the three species. In fact, highest germination percentage in A. nivalis was recorded only when pre-chilled seeds were treated with 1.5 mM GA₃ under alternate light/dark conditions. Likewise, germination of pre-chilled seeds of L. cashmeriana with ruptured seed coats was improved when treated with various concentrations of GA₃. Seed germination in M. latifolia was also favourably influenced by treatment of pre-chilled seeds with GA₃ or nitrogen applied either as NO₃ ⁻ or as NH₄ ⁺ under alternate light/dark conditions. Treatment of seeds with kinetin (6-furfuryl-aminopurine) had no significant influence on germination percentage in any of the three species. Thus, prolonged chilling of seeds followed by their treatment with GA₃ under alternate light/dark conditions are the requirements necessary for seed germination in these species.     

GIS-facilitated ex situ conservation of the rare Greek endemic Campanula incurva Aucher: Seed germination requirements and effect of growth regulators on in vitro proliferation and rooting

In order to develop conservation protocols for Campanula incurva, the geographical information systems (GIS) were used to unveil its ecological requirements; this facilitated the selection of substrates and of appropriate temperatures for cultivation and guided propagation experiments and acclimatization. Seed germination was tested under (i) dark, (ii) 16-h photoperiod, (iii) immersion in 400 ppm gibberellic acid (GA₃) followed by incubation at dark, and (iv) immersion in 400 ppm GA₃ followed by incubation at 16-h photoperiod (all at 21 ± 1°C). Dormancy was not detected. Germination exceeded 85% in 10 days. Shoot tips were established in vitro in Murashige and Skoog (MS) medium with 1 μM 6-benzyladenine (BA) and 0.1 μM indole-3-butyric acid (IBA). The effect of 1–8 μM BA and 1–8 μM kinetin on shoot proliferation was studied. Moreover, 8 μM BA was combined with 0, 1, 5, and 10 μM IBA to investigate effects of cytokinin/auxin. The highest number of microshoots/explant (4.03) was obtained with 8 μM BA. Microshoots were transferred to half strength MS and full strength MS media with 0, 0.5, 1, 5, and 10 μM IBA to evaluate their root induction ability. Half strength MS medium with 5 μM IBA resulted in 100% rooting (16.80 average number of roots/microshoot). Plantlets produced were successfully acclimatized.     

New findings on seed ecology of Ribes sardoum: can it provide a new opportunity to prevent the extinction of a threatened plant species?

Ribes sardoum, the most threatened endemic plant of Sardinia, is included in the Habitats Directive (92/43/EEC) and it was considered Critically Endangered in the global IUCN Red Lists. This species has been reported to have an extremely low fertility, scarce fruit production, low seed viability and a general inability to reproduce sexually. Fruits were collected for the first time from the remnant population, and the requirements for seed germination were investigated in the laboratory. Seeds were incubated at different temperatures (10, 15, and 20°C) and, in addition, they were exposed to a warm stratification (W) or a move-along treatment characterized by three cold temperature regimes (CCC). Seeds were also sown on the surface of 1% agar water with 250 mg·L^?1 of GA₃. At maturity, seeds have a linear underdeveloped embryo. Germination percentage between 35% and 65% were detected in the control and W groups. A low germination percentage occurred after CCC and during GA₃ treatment. W treatment speeds up seed germination. Our results demonstrate that fruits of R. sardoum produce viable seeds, that are able to germinate under controlled conditions, with the assumption that the seeds have morphophysiological dormancy (MPD), and that propagation from the seeds is possible. Although the ability of seed germination was demonstrated, the lack of seedlings in the natural population seems to be a consequence of unfavourable climatic conditions for recruitment. However, our results indicate that seedlings obtained under controlled conditions could be useful for future translocation reducing and/or mitigating the extinction likelihood of this highly threatened plant.