Dormancy cycling and persistence of seeds in soil of a cold desert halophyte shrub

Background and Aims

Formation of seed banks and dormancy cycling are well known in annual species, but not in woody species. In this study it was hypothesized that the long-lived halophytic cold desert shrub Kalidium gracile has a seed bank and dormancy cycling, which help restrict germination to a favourable time for seedling survival.

Methods

Fresh seeds were buried in November 2009 and exhumed and tested for germination monthly from May 2010 to December 2011 over a range of temperatures and salinities. Germination recovery and viability were determined after exposure to salinity and water stress. Seedling emergence and dynamics of the soil seed bank were investigated in the field.

Key Results

Seeds of K. gracile had a soil seed bank of 7030 seeds m⁻² at the beginning of the growing season. About 72 % of the seeds were depleted from the soil seed bank during a growing season, and only 1·4 % of them gave rise to seedlings that germinated early enough to reach a stage of growth at which they could survive to overwinter. About 28 % of the seeds became part of a persistent soil seed bank. Buried seeds exhibited an annual non-dormancy/conditional dormancy (ND/CD) cycle, and germination varied in sensitivity to salinity during the cycle. Dormancy cycling is coordinated with seasonal environmental conditions in such a way that the seeds germinate in summer, when there is sufficient precipitation for seedling establishment.

Conclusions

Kalidium gracile has three life history traits that help ensure persistence at a site: a polycarpic perennial life cycle, a persistent seed bank and dormancy cycling. The annual ND/CD cycle in seeds of K. gracile contributes to seedling establishment of this species in the unpredictable desert environment and to maintenance of a persistent soil seed bank. This is the first report of a seed dormancy cycle in a cold desert shrub.     

Global Changes in DNA Methylation in Seeds and Seedlings of Pyrus communis after Seed Desiccation and Storage

The effects of storage and deep desiccation on structural changes of DNA in orthodox seeds are poorly characterized. In this study we analyzed the 5-methylcytosine (m⁵C) global content of DNA isolated from seeds of common pear (Pyrus communis L.) that had been subjected to extreme desiccation, and the seedlings derived from these seeds. Germination and seedling emergence tests were applied to determine seed viability after their desiccation. In parallel, analysis of the global content of m⁵C in dried seeds and DNA of seedlings obtained from such seeds was performed with a 2D TLC method. Desiccation of fresh seeds to 5.3% moisture content (mc) resulted in a slight reduction of DNA methylation, whereas severe desiccation down to 2–3% mc increased DNA methylation. Strong desiccation of seeds resulted in the subsequent generation of seedlings of shorter height. A 1-year period of seed storage induced a significant increase in the level of DNA methylation in seeds. It is possible that alterations in the m⁵C content of DNA in strongly desiccated pear seeds reflect a reaction of desiccation-tolerant (orthodox) seeds to severe desiccation. Epigenetic changes were observed not only in severely desiccated seeds but also in 3-month old seedlings obtained from these seeds. With regard to seed storage practices, epigenetic assessment could be used by gene banks for early detection of structural changes in the DNA of stored seeds.     

Comparison of germination and seed bank dynamics of dimorphic seeds of the cold desert halophyte Suaeda corniculata subsp. mongolica

Background and Aims

Differences in dormancy and germination requirements have been documented in heteromorphic seeds of many species, but it is unknown how this difference contributes to maintenance and regeneration of populations. The primary aim of this study was to compare the seed bank dynamics, including dormancy cycling, of the two seed morphs (black and brown) of the cold desert halophyte Suaeda corniculata and, if differences were found, to determine their influence on regeneration of the species.

Method

Seeds of the two seed morphs were buried, exhumed and tested monthly for 24 months over a range of temperatures and salinities, and germination recovery and viability were determined after exposure to salinity and water stress. Seedling emergence and dynamics of the soil seed bank were also investigated for the two morphs.

Key Results

Black seeds had an annual dormancy/non-dormancy cycle, while brown seeds, which were non-dormant at maturity, remained non-dormant. Black seeds also exhibited an annual cycle in sensitivity of germination to salinity. Seedlings derived from black seeds emerged in July and August and those from brown seeds in May. Seedlings were recruited from 2·6 % of the black seeds and from 2·8 % of the brown seeds in the soil, and only 0·5 % and 0·4 % of the total number of black and brown seeds in the soil, respectively, gave rise to seedlings that survived to produce seeds. Salinity and water stress induced dormancy in black seeds and decreased viability of brown seeds. Brown seeds formed only a transient soil seed bank and black seeds a persistent seed bank.

Conclusions

The presence of a dormancy cycle in black but not in brown seeds of S. corniculata and differences in germination requirements of the two morphs cause them to differ in their germination dynamics. The study contributes to our limited knowledge of dormancy cycling and seed bank formation in species producing heteromorphic seeds.     

Polyamines, IAA and ABA during germination in two recalcitrant seeds: Araucaria angustifolia (Gymnosperm) and Ocotea odorifera (Angiosperm)

Background and Aims

Plant growth regulators play an important role in seed germination. However, much of the current knowledge about their function during seed germination was obtained using orthodox seeds as model systems, and there is a paucity of information about the role of plant growth regulators during germination of recalcitrant seeds. In the present work, two endangered woody species with recalcitrant seeds, Araucaria angustifolia (Gymnosperm) and Ocotea odorifera (Angiosperm), native to the Atlantic Rain Forest, Brazil, were used to study the mobilization of polyamines (PAs), indole-acetic acid (IAA) and abscisic acid (ABA) during seed germination.

Methods

Data were sampled from embryos of O. odorifera and embryos and megagametophytes of A. angustifolia throughout the germination process. Biochemical analyses were carried out in HPLC.

Key Results

During seed germination, an increase in the (Spd + Spm) : Put ratio was recorded in embryos in both species. An increase in IAA and PA levels was also observed during seed germination in both embryos, while ABA levels showed a decrease in O. odorifera and an increase in A. angustifolia embryos throughout the period studied.

Conclusions

The (Spd + Spm) : Put ratio could be used as a marker for germination completion. The increase in IAA levels, prior to germination, could be associated with variations in PA content. The ABA mobilization observed in the embryos could represent a greater resistance to this hormone in recalcitrant seeds, in comparison to orthodox seeds, opening a new perspective for studies on the effects of this regulator in recalcitrant seeds. The gymnosperm seed, though without a connective tissue between megagametophyte and embryo, seems to be able to maintain communication between the tissues, based on the likely transport of plant growth regulators.     

Catalase is a key enzyme in seed recovery from ageing during priming

Ageing induces seed deterioration expressed as the loss of seed vigour and/or viability. Priming treatment, which consists in soaking of seeds in a solution of low water potential, has been shown to reinvigorate aged seeds. We investigate the importance of catalase in oxidation protection during accelerated ageing and repair during subsequent priming treatment of sunflower (Helianthus annuus L.) seeds. Seeds equilibrated to 0.29 g H₂O g⁻¹ dry matter (DM) were aged at 35 °C for different durations and then primed by incubation for 7 days at 15 °C in a solution of polyethylene glycol 8000 at −2 MPa. Accelerated ageing affected seed germination and priming treatment reversed partially the ageing effect. The inhibition of catalase by the addition of aminotriazol during priming treatment reduced seed repair indicating that catalase plays a key role in protection and repair systems during ageing. Ageing was associated with H₂O₂ accumulation as showed by biochemical quantification and CeCl₃ staining. Catalase was reduced at the level of gene expression, protein content and affinity. Interestingly, priming induced catalase synthesis by activating expression and translation of the enzyme. Immunocytolocalization of catalase showed that the enzyme co-localized with H₂O₂ in the cytosol. These results clearly indicate that priming induce the synthesis of catalase which is involved in seed recovery during priming.     

Effect of Temperature Regimes on Germination of Dimorphic Seeds of Atriplex prostrata

Dimorphic seeds of Atriplex prostrata were removed from cold dry storage monthly over a one year period to test for fluctuations in seed dormancy and germination rate. For each seed type, four replicates of 25 seeds were exposed to four alternating night/day temperature regimes mimicking seasonal fluctuations in Ohio: 5/15 °C; 5/25 °C; 15/25 °C and 20/35 °C with a corresponding 12-h photoperiod (20 μmol m⁻² s⁻¹; 400 – 700 nm). We found a significant three-way interaction of seed size, temperature and month for both percent germination and the rate of germination. Large seeds showed the greatest germination at the 20/35 °C and 5/25 °C temperature regimes and small seeds at the 5/25 °C regime. Large seeds had greater germination at all temperatures as compared to small seeds. Large seeds had the fastest germination rates at 20/35 °C followed by 5/25 °C whereas small seeds had the fastest rates at 5/25 °C followed by 20/35 °C. This revised version was published online in July 2006 with corrections to the Cover Date.     

Low levels of realized seed and pollen gene flow and strong spatial genetic structure in a small,isolated and fragmented population of the tropical tree Copaifera langsdorffii Desf

Over the past century, the Brazilian Atlantic forest has been reduced to small, isolated fragments of forest. Reproductive isolation theories predict a loss of genetic diversity and increases in inbreeding and spatial genetic structure (SGS) in such populations. We analysed eight microsatellite loci to investigate the pollen and seed dispersal patterns, genetic diversity, inbreeding and SGS of the tropical tree Copaifera langsdorffii in a small (4.8 ha), isolated population. All 112 adult trees and 128 seedlings found in the stand were sampled, mapped and genotyped. Seedlings had significantly lower levels of genetic diversity (A=16.5±0.45, mean±95% s.e.; H_e=0.838±0.006) than did adult trees (A=23.2±0.81; H_e=0.893±0.030). Parentage analysis did not indicate any seed immigration (m_seeds=0) and the pollen immigration rate was very low (m_pollen=0.047). The average distance of realized pollen dispersal within the stand was 94 m, with 81% of the pollen travelling <150 m. A significant negative correlation was found between the frequency and distance of pollen dispersal (r=−0.79, P<0.01), indicating that short-distance pollinations were more frequent. A significant SGS for both adults (∼50 m) and seedlings (∼20 m) was also found, indicating that most of the seeds were dispersed over short distances. The results suggested that the spatial isolation of populations by habitat fragmentation can restrict seed and pollen gene flow, increase SGS and affect the genetic diversity of future generations.     

Effects of Moisture Content on Germination of Seeds of Hancornia speciosa Gom. (Apocynaceae)

Seeds of Hancornia speciosa germinated best at a temperatureof 20–30 °C. The viability of the seeds during storagewas short and the best storage conditions for viability entailedkeeping the seeds in polyethylene bags. Seed viability was maintainedonly when the seeds were stored at a moisture content above30%; storage conditions which allowed dehydration resulted ina rapid loss of viability (the seeds showed recalcitrant behaviour). Low temperature during storage did not improve longevity. Arelationship between germination and moisture content was established,but when the moisture content fell below 25% there was a drasticreduction of germination. After 9 weeks of storage, even athigh moisture content, seeds lost viability. Loss of seed viability during seed dehydration was associatedwith increased leakage of electrolytes and organic solutes,and reduced tetrazolium staining during subsequent imbibition. Hancornia speciosa, germination, recalcitrant seeds, storage, moisture     

DNA methylation of Quercus robur L. plumules following cryo-pretreatment and cryopreservation

Pedunculate oak (Quercus robur) is an ecologically and economically important forest tree species which produces seeds that are classified as recalcitrant. Thus, cryopreservation of seed meristems is a method for long-term preservation of this germplasm in gene banks. During cryopreservation, many factors, such as desiccation, cryoprotection and cooling/rewarming, can induce stress in the frozen meristems. In this study, in vitro survival and the global DNA methylation level of plumules after cryoprotection, desiccation and cryostorage was evaluated. Results indicated that both desiccation and storage in liquid nitrogen have negligible influence on DNA methylation status of Q. robur plumules. These findings support the cryopreservation of plumules as an appropriate method for conservation of Q. robur germplasm.     

Thermal thresholds as predictors of seed dormancy release and germination timing: altitude-related risks from climate warming for the wild grapevine Vitis vinifera subsp. sylvestris

Background and Aims

The importance of thermal thresholds for predicting seed dormancy release and germination timing under the present climate conditions and simulated climate change scenarios was investigated. In particular, Vitis vinifera subsp. sylvestris was investigated in four Sardinian populations over the full altitudinal range of the species (from approx. 100 to 800 m a.s.l).

Methods

Dried and fresh seeds from each population were incubated in the light at a range of temperatures (10–25 and 25/10 °C), without any pre-treatment and after a warm (3 months at 25 °C) or a cold (3 months at 5 °C) stratification. A thermal time approach was then applied to the germination results for dried seeds and the seed responses were modelled according to the present climate conditions and two simulated scenarios of the Intergovernmental Panel on Climate Change (IPCC): B1 (+1·8 °C) and A2 (+3·4 °C).

Key Results

Cold stratification released physiological dormancy, while very few seeds germinated without treatments or after warm stratification. Fresh, cold-stratified seeds germinated significantly better (>80 %) at temperatures ≥20 °C than at lower temperatures. A base temperature for germination (T_b) of 9·0–11·3 °C and a thermal time requirement for 50 % of germination (θ₅₀) ranging from 33·6 °Cd to 68·6 °Cd were identified for non-dormant cold-stratified seeds, depending on the populations. This complex combination of thermal requirements for dormancy release and germination allowed prediction of field emergence from March to May under the present climatic conditions for the investigated populations.

Conclusions

The thermal thresholds for seed germination identified in this study (T_b and θ₅₀) explained the differences in seed germination detected among populations. Under the two simulated IPCC scenarios, an altitude-related risk from climate warming is identified, with lowland populations being more threatened due to a compromised seed dormancy release and a narrowed seed germination window.     

Inter-nucleosomal DNA fragmentation and loss of RNA integrity during seed ageing

The germination of viable seeds is the basis for new plant growth and development. Seeds lose viability during storage, but the biochemical mechanisms of seed death are not fully understood. This study aimed to investigate degradation patterns of nucleic acids during seed ageing and subsequent water uptake. Seeds of Pisum sativum L. were artificially aged at 50°C and 12% seed water content (WC). Nucleic acids degradation was studied during ageing and during imbibition of four seed lots with differential viability from highly viable to dead. As seeds lost viability during ageing, DNA was gradually degraded into internucleosomal fragments, resulting in ‘DNA laddering’, in conjunction with disintegration of 18S and 28S rRNA bands. During imbibition, non-aged controls had high levels of DNA and RNA integrity through to radicle protrusion. In an aged seed lot with 85% total germination (TG) DNA fragmentation decreased upon imbibition probably due to nucleosome degradation, while rRNA integrity did not improve. In an aged seed lot with 44% TG, neither DNA nor rRNA integrity improved upon imbibition. Dead seeds showed DNA degradation as laddering throughout imbibition along with extensive degradation of rRNA. We present a model in which interlinked programmed and non-programmed events contribute to seed ageing, and suggest that protection of nucleic acids during ageing is key to seed longevity.     

Increases in the longevity of desiccation-phase developing rice seeds: response to high-temperature drying depends on harvest moisture content

Background and Aims Previous studies have suggested that the drying conditions routinely used by genebanks may not be optimal for subsequent seed longevity. The aim of this study was to compare the effect of hot-air drying and low-temperature drying on subsequent seed longevity for 20 diverse rice accessions and to consider how factors related to seed production history might influence the results.Methods Seeds of rice, Oryza sativa, were produced according to normal regeneration procedures at IRRI. They were harvested at different times [harvest date and days after anthesis (DAA), once for each accession] and dried either in a drying room (DR; 15 % relative humidity, 15 °C) or in a flat-bed heated-air batch dryer (BD; 45 °C, 8 h d^–1) for up to six daily cycles followed by drying in the DR. Relative longevity was assessed by storage at 10·9 % moisture content and 45 °C.Key Results Initial drying in the BD resulted in significantly greater longevity compared with the DR for 14 accessions (seed lots): the period of time for viability to fall to 50 % for seeds dried in the BD as a percentage of that for seeds dried throughout in the DR varied between 1.3 and 372·2 % for these accessions. The seed lots that responded the most were those that were harvested earlier in the season and at higher moisture content. Drying in the BD did not reduce subsequent longevity compared with DR drying for any of the remaining accessions.Conclusions Seeds harvested at a moisture content where, according to the moisture desorption isotherm, they could still be metabolically active (>16·2 %) may be in the first stage of the post-mass maturity, desiccation phase of seed development and thus able to increase longevity in response to hot-air drying. The genebank standards regarding seed drying for rice and, perhaps, for other tropical species should therefore be reconsidered.     

Intermediate complex morphophysiological dormancy in seeds of the cold desert sand dune geophyte Eremurus anisopterus (Xanthorrhoeaceae; Liliaceae s.l.)

Background and Aims

Little is known about morphological (MD) or morphophysiological (MPD) dormancy in cold desert species and in particular those in Liliaceae sensu lato, an important floristic element in the cold deserts of Central Asia with underdeveloped embyos. The primary aim of this study was to determine if seeds of the cold desert liliaceous perennial ephemeral Eremurus anisopterus has MD or MPD, and, if it is MPD, then at what level.

Methods

Embryo growth and germination was monitored in seeds subjected to natural and simulated natural temperature regimes and the effects of after-ripening and GA₃ on dormancy break were tested. In addition, the temperature requirements for embryo growth and dormancy break were investigated.

Key Results

At the time of seed dispersal in summer, the embryo length:seed length (E:S) ratio was 0·73, but it increased to 0·87 before germination. Fresh seeds did not germinate during 1 month of incubation in either light or darkness over a range of temperatures. Thus, seeds have MPD, and, after >12 weeks incubation at 5/2 °C, both embryo growth and germination occurred, showing that they have a complex level of MPD. Since both after-ripening and GA₃ increase the germination percentage, seeds have intermediate complex MPD.

Conclusions

Embryos in after-ripened seeds of E. anisopterus can grow at low temperatures in late autumn, but if the soil is dry in autumn then growth is delayed until snowmelt wets the soil in early spring. The ecological advantage of embryo growth phenology is that seeds can germinate at a time (spring) when sand moisture conditions in the desert are suitable for seedling establishment.     

Seed dispersal by carnivores in temperate and tropical dry forests

The seed dispersal mechanisms and regeneration of various forest ecosystems can benefit from the actions of carnivores via endozoochory. This study was aimed to evaluate the role of carnivores in endozoochory and diploendozoochory, as well as their effect on seed viability, scarification, and germination in two forest ecosystems: temperate and tropical dry forest. We collected carnivore scat in the Protected Natural Area of Sierra Fría in Aguascalientes, Mexico, for 2 years to determine the abundance and richness of seeds dispersed by each carnivore species, through scat analysis. We assessed seed viability through optical densitometry using X‐rays, analyzed seed scarification by measuring seed coat thickness using a scanning electron microscope, and evaluated seed germination in an experiment as the percentage of seeds germinated per carnivore disperser, plant species, and forest type. In the temperate forest, four plant species (but mainly Arctostaphylos pungens) were dispersed by four mammal species. The gray fox dispersed the highest average number of seeds per scat (66.8 seeds). Bobcat dispersed seeds through diploendozoochory, which was inferred from rabbit (Sylvilagus floridanus) hair detected in their scats. The tropical dry forest presented higher abundance of seeds and richness of dispersed plant species (four species) than in the temperate forest, and the coati dispersed the highest number of seeds (8,639 seeds). Endozoochory and diploendozoochory did not affect viability in thick‐testa seeds (1,480 µm) in temperate forest and thin‐testa seeds (281 µm) in tropical dry forest. Endozoochory improved the selective germination of seeds. Nine plant species were dispersed by endozoochory, but only one species (Juniperus sp.) by diploendozoochory. These results suggest that carnivores can perform an important ecological function by dispersing a great abundance of seeds, scarifying these seeds causing the formation of holes and cracks in the testas without affecting viability, and promoting the selective germination of seeds.     

Cell death and reactive oxygen species metabolism during accelerated ageing of soybean axes

In this paper, Glycine max L. seeds under accelerated ageing condition (40°C and 100% relative humidity) were used as experimental material to study the relationships between seed viability and cell death, production and scavenging of reactive oxygen species (ROS) during accelerated ageing. Water content of seeds gradually increased, while the final germination percentage, germination rate of seeds and fresh weight of seedlings produced decreased with increasing accelerated ageing time. The accelerated ageing time (T ₅₀) when final seed germination decreased to 50% was about 10.5 days. During the period of accelerated ageing, the viability of root cells was lost gradually as manifested by the increase in staining with Evans blue. The respiration rate of seeds, ^·O₂ production rate, and H₂O₂ content of axes increased, peaked at the 10 days of accelerated ageing, and then decreased. Activities of superoxide dismutase, ascorbate peroxidase, catalase, and glutathione reductase of axes decreased; and malondialdehyde contents of axes markedly increased. A sceme to explain relationships between seed vigor, cell death, and production and scavenging of ROS during accelerated ageing was suggested. This text was submitted by the authors in English.     

Ecological significance of seed desiccation sensitivity in Quercus ilex

Background and Aims

Several widespread tree species of temperate forests, such as species of the genus Quercus, produce recalcitrant (desiccation-sensitive) seeds. However, the ecological significance of seed desiccation sensitivity in temperate regions is largely unknown. Do seeds of such species suffer from drying during the period when they remain on the soil, between shedding in autumn and the return of conditions required for germination in spring?

Methods

To test this hypothesis, the Mediterranean holm oak (Quercus ilex) forest was used as a model system. The relationships between the climate in winter, the characteristics of microhabitats, acorn morphological traits, and the water status and viability of seeds after winter were then investigated in 42 woodlands sampled over the entire French distribution of the species.

Key Results

The percentages of germination and normal seedling development were tightly linked to the water content of seeds after the winter period, revealing that in situ desiccation is a major cause of mortality. The homogeneity of seed response to drying suggests that neither intraspecific genetic variation nor environmental conditions had a significant impact on the level of desiccation sensitivity of seeds. In contrast, the water and viability status of seeds at the time of collection were dramatically influenced by cumulative rainfall and maximum temperatures during winter. A significant effect of shade and of the type of soil cover was also evidenced.

Conclusions

The findings establish that seed desiccation sensitivity is a key functional trait which may influence the success of recruitment in temperate recalcitrant seed species. Considering that most models of climate change predict changes in rainfall and temperature in the Mediterranean basin, the present work could help foresee changes in the distribution of Q. ilex and other oak species, and hence plant community alterations.     

Increased rate of drying reduces metabolic inequity and critical water content in radicles of Cicer arietinum L.

Orthodox seed serves as easily accessible model to study desiccation-sensitivity in plant tissues because once they undergo germination, they become sensitive to desiccation imposed injuries. In the proposed study, effects of rate of drying on the viability, electrolyte leakage, superoxide accumulation, lipid-protein oxidation and antioxidant enzymes were explored in excised radicles of Cicer arietinum L. under dehydration and wet storage. For both the drying conditions, desiccation could be explained by exponential and inverse functions. Under rapid drying tissue viability as scored by germination efficiency and tetrazolium staining remained 100 % all through the analysis (24 h) but declined remarkably after 0.30 g g⁻¹ fresh mass water content (4 days) under slow drying. Moreover, precipitous fall in tissue viability was observed after 2 weeks of wet storage. Rapid drying was also accompanied with limited amounts of electrolyte leakage, superoxide radical, malondialdehyde and protein hydroperoxide, together with enhanced level of protein. Additionally, activities of both superoxide dismutase and ascorbate peroxidase were increased in rapidly dried radicles, but guaiacol peroxidase was declined. In contrary, above referred biomarkers were observed to perform either inversely or poorly during slow drying and wet storage suggesting that above documented alterations might be the resultant of ageing and not desiccation. Gathered data demonstrated that increased drying lowers the critical water content for tissue survival and also reduces the risk of damage resulting from aqueous-based deleterious reactions. Additionally, it also showed that growing radicles are a popular model to explore desiccation-sensitivity in plant tissues and/or seeds.     

Selection of suitable propagation method for consistent plantlets production in Stevia rebaudiana (Bertoni)

Stevia rebaudiana (Bert.) is an emerging sugar alternative and anti-diabetic plant in Pakistan. That is why people did not know the exact time of propagation. The main objective of the present study was to establish feasible propagation methods for healthy biomass production. In the present study, seed germination, stem cuttings and micropropagation were investigated for higher productivity. Fresh seeds showed better germination (25.51–40%) but lost viability after a few days of storage. In order to improve the germination percentage, seeds were irradiated with 2.5, 5.0, 7.5 and 10 Gy gamma doses. But gamma irradiation did not show any significant change in seed germination. A great variation in survival of stem cutting was observed in each month of 2012. October and November were found the most suitable months for stem cutting survival (60%). In order to enhance survival, stem cuttings were also dipped in different plant growth regulators (PGRs) solution. Only indole butyric acid (IBA; 1000 ppm) treated cutting showed a higher survival (33%) than control (11.1%). Furthermore, simple and feasible indirect regeneration system was established from leaf explants. Best callus induction (84.6%) was observed on MS-medium augmented with 6-benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-D; 2.0 mg l⁻¹). For the first time, we obtained the highest number of shoots (106) on a medium containing BA (1.5 mg l⁻¹) and gibberellic acid (GA₃; 0.5 mg l⁻¹). Plantlets were successfully acclimatized in plastic pots. The current results preferred micropropagation (85%) over seed germination (25.51–40%) and stem cutting (60%).