Endogenous gibberellin levels influence in-vitro shoot regeneration in Arabidopsis thaliana (L.) Heynh.

The regeneration of shoot buds from callus cells in vitro is an important technique in modern plant genetic manipulation. Whilst it is clear that genetic factors play a major role in determining the ability of callus cells to become organized into regenerating shoot buds, the precise nature of these factors remains unknown. Here we show that callus derived from mutants of Arabidopsis thaliana which have reduced levels of endogenous bioactive gibberellins (GAs), or reduced responsivity to GAs, regenerates shoot buds more readily than does callus derived from wild-type controls. In addition, exogenous GA reduces, and exogenous paclobutrazol (a GA-biosynthesis inhibitor) increases, the frequency of shoot bud regeneration from wild-type callus. These results show that GA levels play a role in regulating shoot bud regeneration from callus, and suggest that variation in endogenous GA levels or responsivity may account for a major component of the genetic variation in shoot bud regeneration frequency described in other species.     

Protoplast culture and plant regeneration of several species in the genus Dianthus

Summary Seventeen cultivars belonging to the genus Dianthus were examined for protoplast isolation, culture and shoot regeneration under the same conditions. These included D. caryophyllus, D. chinensis, D. barbatus, D. plumarius, D. superbus and D. japonicus as well as interspecific hybrid cultivars (D. caryophyllus x D. chinensis and D. chinensis x D. barbatus). In all cultivars, viable protoplasts were isolated at high yields from leaves of axenic shoot cultures and some of these protoplasts divided and formed colonies. However, shoot regeneration frequencies were markedly different among the species. High frequency shoot regeneration was obtained from D. chinensis and interspecific hybrid cultivars, while only low frequency or no shoot regeneration was obtained from other species.Abbreviations MS Murashige and Skoog (1962) - FW fresh weight - MES 2-N-morpholinoethane sulfonic acid - FDA fluoroscein diacetate - NAA 1-naphthaleneacetic acid     

Long‐term nitrogen additions increase likelihood of climate stress and affect recovery from wildfire in a lowland heath

Increases in the emissions and associated atmospheric deposition of nitrogen (N) have the potential to cause significant changes to the structure and function of N‐limited ecosystems. Here, we present the results of a long‐term (13 year) experiment assessing the impacts of N addition (30 kg ha^?1 yr^?1) on a UK lowland heathland under a wide range of environmental conditions, including the occurrence of prolonged natural drought episodes and a severe summer fire. Our findings indicate that elevated N deposition results in large, persistent effects on Calluna growth, phenology and chemistry, severe suppression of understorey lichen flora and changes in soil biogeochemistry. Growing season rainfall was found to be a strong driver of inter‐annual variation in Calluna growth and, although interactions between N and rainfall for shoot growth were not significant until the later phase of the experiment, N addition exacerbated the extent of drought injury to Calluna shoots following naturally occurring droughts in 2003 and 2009. Following a severe wildfire at the experimental site in 2006, heathland regeneration dynamics were significantly affected by N, with a greater abundance of pioneering moss species and suppression of the lichen flora in plots receiving N additions. Significant interactions between climate and N were also apparent post fire, with the characteristic stimulation in Calluna growth in +N plots suppressed during dry years. Carbon (C) and N budgets demonstrate large increases in both above‐ and below‐ground stocks of these elements in N‐treated plots prior to the fire, despite higher levels of soil microbial activity and organic matter turnover. Although much of the organic material was removed during the fire, pre‐existing treatment differences were still evident following the burn. Post fire accumulation of below‐ground C and N stocks was increased rapidly in N‐treated plots, highlighting the role of N deposition in ecosystem C sequestration.     

Seedling growth variation in response to sand burial in four <Emphasis Type="Italic">Artemisia</Emphasis> species from different habitats in the semi-arid dune field

We compared seedling growth of four Artemisia species dominated at different habitats to determine whether interspecific seedling growth variation of a same genus in tolerance to burial can be used to explain plant distribution in the sand dune field. Interdune lowland species, Artemisia gmelinii, stabilized dune species, A. frigida, semi-stabilized dune species, A. halodendron, and active dune species, A. wudanica were selected. Seedlings grown for 3 weeks were treated at five burial depths for three burial times in pot experiments. Species from the habitats with little burial had smaller survival rate, dry weight and stem elongation speed than those from the habitats with intensive burial when buried. Furthermore, when buried, the former tended to adjust biomass allocation between shoot and root and produce adventitious buds, while the latter tended to maintain a constant root:shoot ratio and produce adventitious roots. We conclude that (1) seedlings of species with a long evolutionary history of exposure to sand burial (from the active sand dune), show quicker stem growth when buried than do seedlings of species from the habitats with little or no sand burial; (2) seedlings of species which do not change root:shoot ratio might be more tolerant of sand burial than those do; (3) seedlings of species from the habitats with intensive sand burial is prone to produce adventitious roots and seedlings of species from the habitats with little or no sand burial tend to produce adventitious buds when buried.     

Anatomical inferences on aerial bud protection of three Eugenia shrub species from the Cerrado

• Location and degree of protection of aerial buds are important functional traits in disturbance- or stress-prone environments since aerial buds ensure the development of new organs under favourable growing conditions. This study was carried out in a Brazilian Cerrado area under regeneration after long-term Pinus cultivation, where the trees were clear-cut in 2012 and the remaining material was burned in 2014.
• After the fire treatment, several species resprouted from belowground organs and their aboveground organs were directly exposed to full sunlight. We collected 15 terminal branches with fully expanded leaves from three individuals of each of three Eugenia species to investigate if those with well-developed belowground organs invest in bark for aboveground bud protection. The samples were analysed using light and electron microscopy.
• In addition to terminal and axillary buds, all species presented accessory buds, and the number varied according to the node analysed. None of the aerial buds were protected by bark, but all were well protected by cataphylls and densely pubescent leaf primordia. There were also inter- and intra-petiolar colleters that released a mucilaginous protein exudate. The distance between the shoot apical meristem and the outer surface was longer in the terminal bud than in axillary buds. The bud leaf primordia covering the shoot apical meristem had a thick cuticle, unicellular non-glandular trichomes that accumulate phenolic and lipophilic compounds, and secretory cavities.
• Our study shows that all three Eugenia species studied here had highly protected aerial buds allocated from belowground organs. These morphological traits may improve the chances of the species' persistence in areas subjected to frost events, low relative humidity, high irradiance and harmful UV levels.

     

Population responses of Ulex shrubs to fire in a lowland heath community

Question: How does the frequency of heathland fire events affect population growth rates of two woody shrub species, Ulex gallii and U. minor? Location: Dry heathland on the south coast of England, UK. Methods: The population dynamics of U. gallii and U. minor were modelled at each phase of the heathland cycle — pioneer, building, mature and degenerate — using periodic matrix products to investigate the response to different fire regimes. Results: Population growth rates of both Ulex species declined under annual burning. Initially, as the time between burns increased, population growth rate increased for both species. Maximum population growth rates for each Ulex species were achieved under a 16‐yr fire return interval. Fire return intervals > 16 yr resulted in declining population growth rates. Conclusions: A species‐specific critical fire frequency can be predicted, the minimum fire return intervals permitting persistence were 4 yr for U. minor and 3 yr for U. gallii. These patterns are similar to those reported for a range of woody plant species within savanna environments.     

Influence of heat on seed germination of seven Mediterranean Leguminosae species

The influence of high temperatures (dry heat and hot water) on germination of seven Mediterranean Leguminosae species typical of fire-prone ecosystems in southern Spain is analyzed, in order to know the response of seeds to wildfires and the possible implications in their regeneration after this disturbance. Seeds were heated to a range of temperatures (50 °–150 °C) and exposure times (1–60 min) similar to those registered in the upper soil layers during wildfires. Germination tests were carried out in plastic Petri dishes over 60 days. In general, the degree of seed germination promotion by dry heat treatments showed a wide interspecific variation, although the final germination level was increased in all the studied species except for Scorpiurus muricatus. The thermal pretreatment of 50 °C, however, was not effective for germination in any species, and rising the temperature to 70 °C only slightly enhanced the germination in Cytisus patens. The preheatings of 90 °C (5 and 10 min), 120 °C (5 and 10 min), and 150 °C (1 min) were the most effective in promoting seed germination. Hot water (100 °C) scarification also increased the final germination level in all cases, with the exception of C. patens. The germination rates after preheating were much lower than in mechanically scarified seeds and closely resembled those of the untreated seeds, except for C. reverchonii, whose seed germination rate decreased with heat. The response of species to heat shock had no clear relationship with life trait or with the specific post-fire regeneration strategy (obligate seeder or facultative resprouter). Those species coexisting in the same habitats had different heat optimal requirements for seed germination, an strategy suggested by some authors as minimizing interspecific competition in the secondary succession started after fire.     

Efficient plant regeneration from leaves of rapeseed (Brassica napus L.): the influence of AgNO3 and genotype

Factors influencing reliable shoot regeneration from leaf explants of rapeseed (Brassica napus L.) were examined. Addition of AgNO₃ to callus induction medium was significantly effective for shoot regeneration in all three genotypes initially tested. When 48 genotypes subsequently were surveyed, a large variation of shoot regenerability was observed, ranging from 100 to 0% in frequency of bud formation and from 7.5 to 0 in the number of buds per explant. A significant correlation (r=0.84) was observed between the frequency of bud formation and the number of buds per explant. The shoot regenerability from leaf explants was not related to that from cotyledonary explants (r=0.28). Histological observations showed that an organized structure developed from calluses produced at vascular bundle tissues after 7 days of culture on callus induction medium, and they developed shoot apical meristems one week after transfer onto shoot induction medium. Regenerated plantlets were obtained 2 months after the initiation of culture and they normally flowered and set seeds. No alterations of morphology or DNA contents were observed in regenerated plants and their S1 progenies.     

The age of Calluna stands moderates post‐fire regeneration rate and trends in northern Calluna heathlands

Questions: Does stand age influence the direction and rate of post‐fire successional dynamics in coastal Calluna heaths and can old degraded heath vegetation be restored through reintroduction of fire? Location: Coastal heaths in the Tarva archipelago, central Norway. Methods: We investigated revegetation dynamics after experimental fires set in young (8 years since last fire) and old (>50 years since last fire) grazed heath stands. A repeated measures design was used, with floristic data recorded in permanent plots in the post‐fire successions (n=12) over a 7‐year period. The data were analysed using multivariate ordination techniques (PCA, RDA and PRC) and mixed effects models. Results: The age of Calluna stands strongly influenced post‐fire succession, different trends due to age explained 10.4% of variation in floristic data. Young heath showed faster succession towards pre‐fire community composition than old heath, and this could partially be explained by succession‐related factors: young heath had lower cover of mosses and lichens in the pre‐burned vegetation, and lower cover of litter early in succession. Young heath had a less pronounced overall community response to fire than old heath. Vegetative regeneration of C. vulgaris was absent in both old and young heath, but Calluna still re‐established as the dominant species within 5–7 years in both young and old stands. Regeneration dynamics were also affected by habitat conditions, different trends due to habitat explained 6% of variation. Conclusions: Our study demonstrates that old stands do develop characteristic heathland vegetation and structure after fire, and while potential invasives into the system such as trees and rhizomatous species are present, they do not impair Calluna regeneration or vegetation development towards the target heathland community composition and structure. Further, as our young stands are only in their second fire rotation after restoration, we suggest that characteristic dynamics of managed heathlands can re‐establish relatively rapidly, even in severely degenerated sites (>50 years since last fire). Site‐specific factors also need to be considered. We conclude that there is restoration potential in old heaths, despite slow dynamics in the first rotation.     

Comparison of the bud morphology and shoot growth dynamics of four species of Mediterranean subshrub growing along an altitudinal gradient

Bud morphology, bud activity, and shoot growth dynamics were studied over 13 months in four species of Mediterranean subshrubs growing along an altitudinal gradient in north-east Spain. The species selected were representative of the main functional types of subshrubs that grow naturally along altitudinal gradients on Mediterranean mountains. All species bore naked buds as their renewal structures, but the degree of protection of the buds varied between species, increasing with altitude. The period of morphogenesis and the level of shoot pre-formation also varied along the gradient. The species growing at low altitude had partially neoformed shoots, longer shoot growth periods, and started morphogenesis earlier than those growing at high altitude. These differences may indicate different morphological and phenological adaptations of Mediterranean subshrubs in response to increased winter cold along the gradient. At low altitudes, brachyblast-bearing species with partially neoformed shoots may be favoured because of the plastic shoot growth these structures confer. However, as the climate becomes colder and more predictable, brachyblast-bearing species may be replaced by species without brachyblasts, with tightly protected buds and more constrained patterns of shoot development.  © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society , 2006, 151 , 527–539.     

A soil heat and water transfer model to predict belowground grass rhizome bud death in a grass fire

Question: Grasses often resprout from surviving belowground buds following a fire in which aboveground matter is consumed. We used a soil heat and water transport model to present a general method for determining the potential mortality of rhizome buds due to fire for three tallgrass species (Andropogon gerardii, Sorghastrum nutans, and Panicum virgatum). Methods: Soil heating was described by physical processes that include heat conduction through the soil and heating and evaporation of soil water. We considered the following factors: soil moisture, texture, mineral thermal conductivity, maximum surface temperature, and fire residence time. Simulated soil temperature profiles were combined with measured belowground bud distributions to determine the proportion of buds expected to be heated to lethal temperatures under various conditions. Location: Wisconsin, USA. Results: Lethal temperatures for buds do not occur below ～2 cm, and at least 30% of rhizome buds remain below lethal temperatures, even under extreme conditions. Conclusions: The model explains the possible mechanisms for grass belowground rhizome bud survival in fires. Changes in fire and soil conditions do not notably impact soil temperatures and rhizome bud survival.     

A simple plant regeneration-ability assay in a range of Lycopersicon species

Plant growth regulator-dependent (PGR-dependent) in vitro shoot organogenesis has been extensively studied in tomato (Lycopersicon esculentum), whereas PGR-independent adventitious shoot organogenesis received marginal attention in L. esculentum and no consideration at all in other Lycopersicon species. In the present study, induction of PGR-independent adventitious shoots was by decapitation of the apex and removal of preexisting shoot meristems of the seedling, and seedling culture on a medium with no PGR supplements. The existence of PGR-independent regeneration-ability was verified in L. esculentum genotypes (high pigment photomorphogenic mutants and wild-type counterparts) and was uncover amongst L. cheesmanii, L. chilense, L. chmielewskii, L. hirsutum, L. parviflorum, L.␣peruvianum and L. pimpinellifolium. Compared to species other than L. esculentum, high pigment photomorphogenic mutants displayed the weakest PGR-independent regeneration-ability. Our results imply that decapitated seedlings cultured on a medium without PGRs can serve as a convenient assay system for genotypic variation in self-controlled, PGR-independent, shoot regeneration-ability in a wide range of Lycopersicon species. Using transverse thin slices of the hypocotyl placed onto a medium supplemented with 0.2 μM zeatin reboside and 0.04 μM IAA, we assessed PGR-mediated shoot regeneration in L. esculentum genotypes. In a given genotype, more plants per seedling were established by PGR-mediated than by PGR-independent regeneration. However, with both modes of organogenesis, only a fraction of shoot buds eventually grew into normal plants, while others developed into abnormal regenerants having no stem. Percentage of stem-deficiency, in a given genotype, was higher in PGR-treated cultures, which indicates that PGRs amplify the formation frequency of imperfect adventitious apical shoot meristems. Unlike L. esculentum, adventitious shoot buds of other Lycopersicon species, induced by wounding seedlings that were not treated with PGRs, rarely formed regenerants lacking a stem.     

Morphogenic response of the alginate-encapsulated axillary buds from in vitro shoot cultures of six mulberries

Axillary buds obtained from in vitro shoot cultures of six mulberries (Morus alba L., M. australis Poir., M. bombycis Koidz., M. cathyana Hemsl., M. latifolia Poir., and M. nigra L.) were encapsulated in calcium alginate hydrogel containing Murashige and Skoog (1962) nutrients (MS) and 4.4 μM benzyladenine (BA). Morphogenic response of encapsulated buds to various planting media such as MS medium + 4.4 μM BA, MS basal medium, soilrite mix + half-strength MS medium, garden soil + half-strength MS medium, soilrite mix + tap water and garden soil + tap water was evaluated. Encapsulated buds of M. alba, M. bombycis, M. latifolia and M. nigra exhibited shoot development in each of the six media tested whereas that of M. australis and M. cathyana responded only to the first four media. Analysis of variance revealed that the planting medium exhibited the greatest influence on shoot development. Of the six planting media evaluated, shoot development was highest in MS medium containing 4.4 μM BA and lowest in garden soil moistened with water. Of the six Morus species studied, one-step regeneration, i.e. both shoot and root formation, was recorded in M. alba, M. bombycis and M. latifolia. Rooted shoots were retrieved from encapsulated buds of these species on all planting media tested except the one that contained BA. Root development was significantly affected by the planting medium and the plant species with planting medium contributing the maximum amount (82%) of the total variation observed. Of the five planting media tested, the percentage of root development was highest in MS basal medium. Of the six Morus species studied, the best shoot and root development was observed in M. alba. Encapsulated buds of M. bombycis, M. latifolia and M. nigra stored for 90 days and those of M. alba, M. australis and M. cathyana for 60 days at 4 °C still regenerated shoots. Plants regenerated from the encapsulated buds were hardened off and transferred to soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

Post‐fire regeneration in alpine heathland: Does fire severity matter?

Fire severity is thought to be an important determinant of landscape patterns of post‐fire regeneration, yet there have been few studies of the effects of variation in fire severity at landscape scales on floristic diversity and composition, and none within alpine vegetation. Understanding how fire severity affects alpine vegetation is important because fire is relatively infrequent in alpine environments. Globally, alpine ecosystems are at risk from climate change, which, in addition to warming, is likely to increase the severity and frequency of fire in south‐eastern Australia. Here we examine the effects of variation in fire severity on plant diversity and vegetation composition, 5 years after the widespread fires of 2003. We used floristic data from two wide‐spread vegetation types on the Bogong High Plains: open heathland and closed heathland. Three alternative models were tested relating variation in plant community attributes (e.g. diversity, ground cover of dominant species, amount of bare ground) to variation in fire severity. The models were (i) ‘linear’, attributes vary linearly with fire severity; (ii) ‘intermediate disturbance’, attributes are highest at intermediate fire severity and lowest at both low‐ and high‐severity; and (iii) ‘null’, attributes are unaffected by fire severity. In both heathlands, there were few differences in floristic diversity, cover of dominant species and community composition, across the strong fire severity gradient. The null model was most supported in the vast majority of cases, with only limited support for either the linear and intermediate disturbance models. Our data indicate that in both heathlands, vegetation attributes in burnt vegetation were converging towards that of the unburnt state. We conclude that fire severity had little impact on post‐fire regeneration, and that both closed and open alpine heathlands are resilient to variation in fire severity during landscape scale fires.     

In vitro regeneration via caulogenesis and brassin-induced shoot conversion of dormant buds from plumular explants of peanut (Arachis hypogaea L. cv `Okrun')

Shoot buds were induced from plumular explants of peanut (Arachis hypogaea L., cv `Okrun') preconditioned on medium containing 2,4-dichlorophenoxyacetic acid and kinetin and then transferred to regeneration medium containing benzylaminopurine and β-naphthoxyacetic acid. Buds differentiated 25 days following transfer to regeneration medium. Each explant produced 30 to 40 buds, but only 4 shoots. The remaining buds were dormant and did not produce shoots when maintained on regeneration medium. Shoots were regenerated continuously, however, when explants were subsequently transferred to shoot conversion medium containing 1 μM brassin, benzylaminopurine and β-naphthoxyacetic acid, respectively. Approximately 5 shoots were harvested every 30 days after transfer to shoot conversion medium for up to 7 months. No further shoot production was observed from explants maintained on regeneration medium without brassin. Regenerated shoots could be rooted and produced viable seeds. This procedure provides an efficient and reliable system for regeneration and transformation studies using cv `Okrun'. Received: 9 April 1997 / Revision received: 27 August 1997 / Accepted: 20 September 1997     

Cytokinins antagonize the jasmonates action on the regulation of potato (Solanum tuberosum) tuber formation in vitro

Cucurbita pepo L. (squash, pumpkin) is a highly polymorphic vegetable species of major importance. Our study characterized a spectrum of C. pepo germplasm for the ability to regenerate in vitro by direct organogenesis from cotyledon explants. Cultivars tested included both cultivated subspecies, texana and pepo, and nearly all of their respective cultivar-groups. Direct shoot regeneration occurred in all accessions, and was generally high (56–94%), with a single exception of 22% (‘Bolognese’). There was no significant difference between the percentage regeneration of the two subspecies. Shoot regeneration per responding explant was uniform (1.2–1.6 shoots per explant). Only ‘True French’ produced statistically more shoots (3.9 per explant) than other accessions. The morphology of regeneration varied. Most cultivars produced long shoots, often fasciated, amid a few small buds. Some subspecies pepo cultivars (Beirut, Yugoslavia 7, Ma’yan and True French) produced short, massive, hollow shoots, sometimes accompanied by shoots that were more normal. Two subspecies texana cultivars (Creamy Straightneck and Small Bicolor) produced single (sometimes double) shoots without other buds. The production of chimeric (mixoploid) regenerants varied and there was a tendency to regenerate chimeric plants from the widest-fruited accessions (i.e. lowest length-to-width ratio) in each subspecies. Subspecies pepo Pumpkin Group ‘Tondo di Nizza’ showed significantly greater production of chimeric regenerants. In comparison with the great range of variation observed in fruit shape, the variation of in vitro responses (mostly less than 2-fold in regeneration and shoot production) was less than expected.     

Organogenesis and embryogenesis inHelianthus tuberosus and in the interspecific hybridHelianthus annuus ×Helianthus tuberosus

A method of plant regeneration from cotyledons ofHelianthus tuberosus, Helianthus annuus ×Helianthus tuberosus and for the backcross of the interspecific hybrids onH. annuus was developed. Induction of somatic embryogenesis and plantlet regeneration from anther culture of the interspecific hybridsH. annuus ×H. tuberosus is reported.Cotyledons were cultured on Murashige and Skoog basal medium (MS) supplemented with indole-3-acetic acid (IAA) and 6-furfurylaminopurine (kinetin) or N⁶-benzylaminopurine (BAP). Shoot regeneration occurred on most of the media tested, but the best results were obtained on media with a high concentration of cytokinins (BAP or kinetin: 4 mg l^–1) and lower concentration of auxin (IAA: 0.5–1 mg l^–1).Embryogenic callus and adventitious buds were initiated from only two anthers of the hybridH. annuus ×H. tuberosus cultured on the MS medium containing BAP (0.2 mg l^–1) and 1-naphtalenacetic acid (NAA: 0.1 mg l^–1). Prolonged culture of these embryogenic calli and buds on the original medium with successive subculture on MS basal medium without growth regulators resulted in embryo formation and shoot differentiation. The plantlets, after rooting, were established in soil.     

Direct plant regeneration from leaf explants of Plumbago species and its genetic fidelity through RAPD markers

In vitro plant regeneration was achieved from leaf explants of Plumbago rosea and Plumbago zeylanica on Murashige & Skoog (1962) medium supplemented with 1.5 mg litre^?1 6‐benzylaminopurine, 0.25 mg litre^?1 indole‐3‐acetic acid, 50 mg litre^?1 adenine sulfate and 3% (w/v) sucrose. The shoot initials developed within 2–3 wk on the leaf margin as well as from the wounds of the leaf. High frequency shoot‐bud regeneration was achieved on similar medium in subsequent subcultures. The semi‐mature leaves produced more shoot‐buds as compared to the younger leaves. Mature leaves did not show any response for shoot bud initiation. More than 85% of the semi‐mature explants produced shoot‐buds per leaf explant within 4 wk of culture. Shoots rooted easily on medium having half‐strength basal Murashige & Skoog (1962) medium supplemented with 0.25 mg litre^?1 indole‐3‐butyric acid and 2% (w/v) sucrose; 84–92% of the in vitro rooted plantlets survived in the greenhouse. The regenerated plantlets appeared morphologically similar to the mother plants. No variation was detected among the regenerated plants by the use of Randomly Amplified Polymorphic DNA (RAPD) markers. This method might be useful for assessing plant improvement programmes.     

Spatio-temporal patterns in post-burn succession on Brittany heathlands

Heathlands in the Paimpont area, Brittany, were ravaged by numerous fires in the summer of 1976. The period in which the fires occurred, and their intensity had important consequences for recolonization processes. In the tall Ulex europaeus heathland described in this paper, fire occurred relatively late (mid-July), and was of high intensity. Colonisation was mainly from propagules, often from species foreign to the original heathland. Plant succession together with vertical and horizontal structure was studied in permanent plots over 13 yr. Three stages were distinguished. Vertical structure varied greatly among the three stages. It was very simple in the colonisation phase, became more complicated during the competition phase, but became simpler again in the stabilisation phase. This was particularly evident for the moss layer, which, 13 yr after the fire, was made up of Polytrichum commune alone. Mosses showed interspecific competition with size as the decisive factor: the large mosses gradually eliminated the smaller ones. Horizontal structure was studied using spatial autocorrelation, which provided some indication of the distinctive -ness and scale of spatial pattern. Some species were weakly aggregated such as Ceratodon purpureus, Polytrichum pili-ferum, and P. juniperinum, whileothers, like the P. formosum-P. commune complex, Agrostis curtisii and Ulex minor were strongly aggregated. In the current community, 13 yr after the fire, a mosaic can be distinguished with three components: (1) a moss layer of Polytrichum commune on which a herbaceous layer, dominated by Agrostis curtisii has developed; (2) a birch wood (both Betulapéndula and B. pubescens), which established on the Polytrichum - Agrostis combination; (3) patches of Ulex minor heathland, with a few Frica and Calluna plants. Extreme simplicity and stability are the two prominent features of the present vegetation state.     

High-frequency plant regeneration from leaf-disc cultures of Jatropha curcas L.: an important biodiesel plant

A simple, high-frequency and reproducible protocol for induction of adventitious shoot buds and plant regeneration from leaf-disc cultures of Jatropha curcas L. has been developed. Adventitious shoot buds were induced from very young leaf explants of in vitro germinated seedlings as well as mature field-grown plants cultured on Murashige and Skoog’s (MS) medium supplemented with thidiazuron (TDZ) (2.27 μM), 6-benzylaminopurine (BA) (2.22 μM) and indole-3-butyric acid (IBA) (0.49 μM). The presence of TDZ in the induction medium has greater influence on the induction of adventitious shoot buds, whereas BA in the absence of TDZ promoted callus induction rather than shoot buds. Induced shoot buds were multiplied and elongated into shoots following transfer to the MS medium supplemented with BA (4.44 μM), kinetin (Kn) (2.33 μM), indole-3-acetic acid (IAA) (1.43 μM), and gibberellic acid (GA₃) (0.72 μM). Well-developed shoots were rooted on MS medium supplemented with IBA (0.5 μM) after 30 days. Regenerated plants after 2 months of acclimatization were successfully transferred to the field without visible morphological variation. This protocol might find use in mass production of true-to-type plants and in production of transgenic plants through Agrobacterium/biolistic-mediated transformation.