Impact of long-term,saline flooding on condition and reproduction of the clonal wetland tree, <Emphasis Type="Italic">Melaleuca ericifolia</Emphasis> (Myrtaceae)

Although water regime modification and salinity are recognised as significant threats to wetland ecosystems worldwide, the effects of long-term saline flooding (decades) on woody tree persistence are poorly quantified. We compared the condition, growth, structure and reproduction (sexual and asexual) of mature individuals of the clonal tree, Melaleuca ericifolia (Myrtaceae), that experienced continual (>30 years) flooding with trees that were only intermittently (approximately every 5 years) flooded. An index developed to assess the condition of multi-stemmed trees found that continually flooded trees were in significantly poorer condition than intermittently flooded trees, having lower crown cover, foliar cover and foliar density, and a higher incidence of dead stags and dieback. Annual stem growth correlated strongly with condition scores. Evidence for a trade-off between sexual and asexual reproductions was found; flooded trees were constrained in their vegetative lateral spread (<12 m dia.) and flowered more than intermittently flooded trees, which were not restricted in lateral spread (~30 m dia.). Flooded trees used intensively by the colonially roosting Australian Sacred Ibis (Threskiornis molucca) were in especially poor condition. These trees flowered infrequently and produced the lowest number of infructescences, but produced many new vegetative stems (ramets) within their centre. Although chronic flooding appeared to compromise the condition of M. ericifolia trees in Dowd Morass, their existence upon woody hummocks (~40 cm high) upon which they are able to produce new stems is likely to be a key mechanism in their persistence. It is unknown, however, how long this process can maintain the existing population. Production and maintenance of a large aerial seed bank by living mature trees under flooded conditions may allow M. ericifolia to regenerate sexually upon drawdown and may be important for population persistence in the longer term.     

Influence of woody invader control methods and seed availability on native and invasive species establishment in a Hawaiian forest

When invasive woody plants become dominant, they present an extreme challenge for restoration of native plant communities. Invasive Morella faya (fire tree) forms extensive, nearly monospecific stands in wet and mesic forests on the Island of Hawai’i. We used logging, girdling, and selective girdling over time (incremental girdling) to kill stands of M. faya at different rates, with the objective of identifying a method that best promotes native forest re-establishment. We hypothesized that rapid canopy opening by logging would lead to establishment of fast-growing, non-native invaders, but that slower death of M. faya by girdling or incremental girdling would increase the establishment by native plants adapted to partial shade conditions. After applying the M. faya treatments, seed banks, seed rain, and plant recruitment were monitored over 3 years. Different plant communities developed in response to the treatments. Increased light and nitrogen availability in the logged treatment were associated with invasion by non-native species. Native species, including the dominant native forest tree, (Metrosideros polymorpha) and tree fern (Cibotium glaucum), established most frequently in the girdle and incremental girdle treatments, but short-lived non-native species were more abundant than native species. A diverse native forest is unlikely to develop following any of the treatments due to seed limitation for many native species, but girdling and incremental girdling promoted natural establishment of major components of native Hawaiian forest. Girdling may be an effective general strategy for reestablishing native vegetation in areas dominated by woody plant invaders.     

莫莫格扁秆藨草恢复湿地土壤种子库对长芒稗入侵的响应

湿地恢复过程中,时常有外来种或本地杂草入侵。土壤种子库作为未来植被的潜在种源,对湿地恢复效果具有重要的指示意义。在莫莫格国家级自然保护区,以恢复白鹤栖息地(扁秆藨草(Scirpus planiculmis)沼泽)为目的,进行了退化湿地的水文恢复;但退化湿地恢复2a后,一年生杂草长芒稗(Echinochloa caudata)在大部分区域成为建群种。以长芒稗入侵湿地和扁秆藨草自然湿地为研究对象,对比分析了长芒稗和扁秆藨草的土壤种子库及生长结实特征。结果表明,在自然湿地扁秆藨草种子库规模是长芒稗的18.42倍,而在恢复湿地长芒稗种子库大小是扁秆藨草的5.04倍。与自然湿地相比,扁秆藨草种子库密度在入侵湿地明显减少,但仍保留了一定量具有活力的种子(664.32±105.98)粒/m~2,这与研究区扁秆藨草较高的种子生产力(9210.4±1513.4)粒/m~2及种子较强的浮力(FP50=39.7d)有关,说明扁秆藨草具备通过种子库或水传播恢复的潜力。长芒稗土壤种子库密度在入侵湿地高达(3345.9±520.3)粒/m~2,明显高于自然湿地种子库规模(P0.01),说明恢复湿地受长芒稗入侵影响严重,这与长芒稗较高的种子生产力(7621.4±376.25)粒/m~2及较弱的种子浮力(FP0=5d)有关,同时也表明长芒稗通过水传播扩散的能力较弱。另外,研究区长芒稗平均高度超过1m,且盖度较大,不仅阻碍扁秆藨草种子的水播,也降低了到达地表的光照水平,从而抑制扁秆藨草更新。因此,在莫莫格受长芒稗入侵湿地,于开花结实前收获长芒稗地上植物体及凋落物应是限制长芒稗扩展、同时促进扁秆藨草恢复的有效措施。     

Early seed fall and seedling emergence: precursors to tropical restoration

We explore processes of seed immigration and seedling recruitment before an experimental rainforest restoration matures enough to affect either. Twenty-four 30 × 30-m plots were fenced in 12 ha of pasture in 2006. Seeds were collected in ninety-six 1-m⁻² seed traps; recruits were censused in ~12,000 m² in which establishment was allowed. We tested effects of distance from forest, living trees, and stumps of trees cut during site preparation on seed rain in 2007 and effects of these and soil depth on recruits through June 2008. Seed fall and recruitment were not correlated with distance to forest 90–400 m away, nor to living shade trees outside the 160 × 485-m experimental grid. Recruitment differed for animal- and wind-dispersed species in a topographically complex landscape. Recruitment of wind-dispersed species was random with respect to soil depth or distance to recent stumps. Recruitment of animal-dispersed species was multimodal; partial correlations with number of stumps within 30 m of plots were significant with soil depth held constant (P < 0.025), as were correlations of recruitment with soil depth with number of stumps held constant (P < 0.01). Animal-dispersed recruits were often not conspecifics of adults that had been cut, indicating a legacy of attraction by fruiting trees of animals bearing seeds from distant sources. Ecological implications are that recruitment in pastures released from grazing reflects a mix of widely scattered wind-dispersed pioneers and, where animal-dispersed trees exist, multi-modal and decidedly non-random recruitment of pioneer and later successional animal-dispersed trees from seed banks.     

Effects of plant competition,seed predation,and nutrient limitation on seedling survivorship of spotted knapweed (<Emphasis Type="Italic">Centaurea stoebe</Emphasis>)

We measured seed germination and seedling survivorship of spotted knapweed, Centaurea stoebe, in a series of laboratory and field experiments to evaluate the efficacy of seed limitation as a management focus. This work was initiated 6 years after introduction of several biological control agents. The soil seed bank of the site used in this study contained a mean density of 5,848 seeds/m² (ranging from 0 to 16,364 seeds/m²), and 92% of the seeds isolated from soils were shriveled, discolored, and/or partially decayed. Additionally, none of the intact seeds germinated, suggesting that the viable seed bank at our field study site has been exhausted. Centaurea stoebe seeds were planted into pots under a range of soil nitrogen (N) availability, with half of the pots containing a single density of previously established seedlings of a native cool-season grass, slender wheatgrass (Elymus trachycaulus). A watering regime mimicking local precipitation was applied. Spotted knapweed exhibited large biomass responses to N addition, but the presence of grasses suppressed the ability to exploit this N. Surprisingly, low soil N conditions improved knapweed survivorship in the presence of grasses. Nevertheless, recruitment and biomass were still far below the levels reached in the absence of competition. To evaluate the effect of density on successful recruitment, Centaurea stoebe seed was introduced into a meadow at three densities matching reduced levels of seed production under the constraints of seed predators. These densities were sown with or without a seed mixture of native species, into an existing plant community lacking C. stoebe, and seedling recruitment was recorded over 2.5 years. Across all plots and densities sown (568–2,272 seeds m⁻² year⁻¹), seedling recruitment was less than 1%. The invasion potential of spotted knapweed was greatly diminished when realistic levels of plant competition and biological control limit seed production. We therefore conclude that a combination of seed limitation and shortage of ‘safe sites’ within undisturbed vegetation can limit densities of C. stoebe.     

Variation in response to heavy metals during vegetative growth in <Emphasis Type="Italic">Dorycnium pentaphyllum</Emphasis> Scop.

Seeds, young plants and adult plants of the perennial Mediterranean leguminous shrub Dorycnium pentaphyllum Scop. were exposed to Cd (1–100 μM) or Zn (10–10,000 μM) on nutrient solution. This species is resistant to Cd and Zn at different phenological stages. The lowest doses of Zn and Cd improved seed germination and young seedling growth, while only the highest doses of both heavy metals inhibited germination and decreased growth. High doses of Cd reduced seed imbibition and young seedling water content, while Zn did not. Osmotic adjustment was more efficient in Zn-treated young plants than in Cd-treated ones, while chlorophyll concentrations decreased in the former but not in the latter. Those differences were not observed anymore in adult plants. Exclusion processes were more efficient at the adult stage than at the young seedling stage and were more marked in response to Zn than to Cd. It is concluded that D. pentaphyllum could be used for phytostabilization of heavy metal-contaminated areas. The physiological strategies of tolerance, however, differ according to the age of the plants and the nature of the metal.     

Effect of seed predation on seed bank size and seedling recruitment of bush lupine (Lupinus arboreus)

Whether seed consumers affect plant establishment is an important unresolved question in plant population biology. Seed consumption is ubiquitous; at issue is whether seedling recruitment is limited by safe-sites or seeds. If most seeds inhabit sites unsuitable for germination, post-dispersal seed consumption primarily removes seeds that would otherwise never contribute to the population and granivory has minimal impacts on plant abundance. Alternatively, if most seeds ultimately germinate before they lose viability, there is greater potential for seed consumption to affect plant recruitment. Of the many studies on seed consumption, few ask how seed loss affects seedling recruitment for species with long-lived seed banks. We examined post-dispersal seed predation and seedling emergence in bush lupine (Lupinus arboreus), a woody leguminous shrub of coastal grasslands and dunes in California. We followed the fate of seeds in paired experimental seed plots that were either protected or exposed to rodent granivores in grassland and dune habitats. Significantly more seeds were removed by rodents in dunes than grasslands. In dunes, where rodent granivory was greatest (65% and 86% of seeds removed from plots by rodents in two successive years), there is a sparse seed bank (6.6 seeds m⁻²), and granivory significantly reduced seedling emergence (in the same two years, 18% and 19.4% fewer seedlings emerged from exposed versus protected plots), suggesting seed rather than safe-site limited seedling recruitment. In contrast, rodents removed an average of 6% and 56% of seeds from grassland plots during the same two years, and the grassland seed bank is 43-fold that of the dunes (288 seeds m⁻²). Even high seed consumption in the second year of the study only marginally influenced recruitment because seeds that escaped predation remained dormant. Burial of seeds in both habitats significantly reduced the percentage of seeds removed by rodents. Results suggest that granivores exert strong but habitat-dependent effects on lupine seed survival and seedling emergence. Received: 24 October 1996 / Accepted: 4 February 1997     

Photosystem II photochemistry and physiological parameters of three fodder shrubs, <Emphasis Type="Italic">Nitraria retusa</Emphasis>, <Emphasis Type="Italic">Atriplex halimus</Emphasis> and <Emphasis Type="Italic">Medicago arborea</Emphasis> under salt stress

Nitraria retusa and Atriplex halimus (xero-halophytes) plants were grown in the range 0–800 mM NaCl while Medicago arborea (glycophyte) in 0–300 mM NaCl. Salt stress caused a marked decrease in osmotic potential and a significant accumulation of Na⁺ and Cl⁻ in leaves of both species. Moderate salinity had a stimulating effect on growth rate, net CO₂ assimilation, transpiration and stomatal conductance for the xero-halophytic species. At higher salinities, these physiological parameters decreased significantly, and their percentages of reduction were higher in A. halimus than in N. retusa whereas, in M. arborea they decreased linearly with salinity. Nitraria retusa PSII photochemistry and carotenoid content were unaffected by salinity, but a reduction in chlorophyll content was observed at 800 mM NaCl. Similar results were found in A. halimus, but with a decrease in the efficiency of PSII (F′v/F′m) occurred at 800 mM. Conversely, in M. arborea plants we observed a significant reduction in pigment concentrations and chlorophyll fluorescence parameters. The marked toxic effect of Na⁺ and/or Cl⁻ observed in M. arborea indicates that salt damage effect could be attributed to ions’ toxicity, and that the reduction in photosynthesis is most probably due to damages in the photosynthetic apparatus rather than factors affecting stomatal closure. For the two halophyte species, it appears that there is occurrence of co-limitation of photosynthesis by stomatal and non-stomatal factors. Our results suggest that both N. retusa and A. halimus show high tolerance to both high salinity and photoinhibition while M. arborea was considered as a slightly salt tolerant species.     

Indirect interactions between browsers and seed predators affect the seed bank dynamics of a chaparral shrub

Interactions between herbivores and seed predators may have long-term consequences for plant populations that rely on persistent seed banks for recovery after unpredictable fires. We assessed the effects of browsing by deer and seed predation by rodents, ants and birds on the densities of seeds entering the seed bank of Ceanothus cuneatus var. rigidus, a maritime chaparral shrub in coastal California. Ceanothus produced many more seeds when protected from browsers in long-term experimental exclosures than did browsed plants, but the seed densities in the soil beneath browsed and unbrowsed Ceanothus were the same at the start of an intensive one-year study. The density of seeds in the soil initially increased in both treatments following summer seed drop: while densities returned to pre-drop levels within a few weeks under browsed plants, soil seed densities remained high for 5–8 months beneath unbrowsed plants. Rodent abundance (especially deer mice) was higher near unbrowsed plants than >30 m away, and rodents removed Ceanothus seeds from dishes in the experimental plots. At least in the short term, rodent density and rates of seed removal were inversely related to the intensity of browsing. Our data have management implications for maintaining viable Ceanothus populations by regulating the intensity of browsing and the timing, intensity and frequency of fires.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

The potential of soil seed banks of a eucalypt wetland forest to aid restoration

Soil seed banks can play an important role in the regeneration of wetland vegetation. However, their potential role in the restoration of degraded wetland forests is less certain. I surveyed the soil seed bank and extant floras of four sites across a eucalypt wetland forest of variable vegetation condition. At each site, the extant vegetation was surveyed within two 5 × 5 m² quadrats, each from which five composite soil seed bank samples were collected. Across the four sites, 57 (including 18 exotic) species were identified in the extant vegetation, while from the seed bank samples 6379 seedlings emerged from 80 taxa, 33 of which were exotic species. The soil seed bank was dominated by native and exotic monocots, and contained very few seeds of wetland tree or shrub species. Overall, the similarity between the extant and seed bank floras was very low (~24 %). Soil seed banks are likely to be of limited use in the restoration of degraded wetland forests, because the dominant species in such systems—woody and clonal plants—are typically absent from the soil seed bank. Wetland soil seed banks may contribute to the maintenance and diversity of understorey vegetation, however, they may also act as a source of exotic plant invasions, particularly when a wetland is degraded.     

Evaluation of methods for celery (<Emphasis Type="Italic">Apium Graveolens</Emphasis> L.) transformation using <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> and the <Emphasis Type="Italic">bar</Emphasis> gene as selectable marker

Callus selection (CS) and the flamingo-bill explant (FB) methods were evaluated for efficacy in transformation for celery. Agrobacterium tumefaciens strains EHA105 and GV3101, each with the bar gene under the promoters NOS (pGPTV-BAR) or 35S (pDHB321.1), were used. Leaf explants were inoculated and co-cultivated for 2 d in the dark. Calluses emerged on the explants on callus medium (C), Murashige and Skoog (MS) medium + 2,4-Dichlorophenoxyacetic acid (2,4-D) (2.3 μM) + kinetin (2.8 μM) + timentin (300 mg·l⁻¹). Calluses 4- to 6-wk-old were selected for glufosinate (GS) resistance by a two step method. First, calluses were transferred to C medium + GS 0.35, 0.5, 1, 2, 5, or 10 mg·l⁻¹; calluses formed only with 0, 0.35 and 0.5 mg·l⁻¹ GS. All growing calluses from 0 and 0.35 mg·l⁻¹ and a few from 0.5 mg·l⁻¹, were divided and placed back on C + GS 0.35–0.5 mg·l⁻¹ for another 5–6 wk. Second, tolerant clones were again divided and placed on C + GS 1–50 mg·l⁻¹. When cultivar XP85 was inoculated with both strains, using pGPTVBAR, 19 glufosinate resistant (GR) callus clones were selected, but shoots regenerated only for strain EHA105 inoculations. When both of the strains (each with pDHB321.1) were inoculated on cv. XP166, 3 and 12 GR calluses occurred for EHA105 and GV3101, respectively. Using CS, a total of 34 GR callus clones were selected, and shoots were regenerated from over 50% of them on Gamborg B5 medium + 6-(γ, γ-dimethylallylamino) purine 2ip (4.9 μM) + naphthaleneacetic acid (NAA; 1.6 μM) and rooted on MS in 5–6 mo total time. Conversely, using FB with inoculation by GV3101/pDHB321.1 on cv. XP166 yielded putative transgenic celery plants confirmed by polymerase chain reaction (PCR) in just 6 wk. Transformation of the bar gene into celery was confirmed by PCR for 5 and 6 CS and FB lines, respectively. Southern blot analyses indicated 1–2 copies in CS lines and 1 copy in FB lines. Herbicide assays on whole plants with 100 and 300 mg·l⁻¹ glufosinate indicated a range of low to high tolerance for lines derived by both methods. The bar gene was found to be Mendelian inherited in one self-fertile CS derived line.     

In vitro morphogenesis of organogenic nodules derived from <Emphasis Type="Italic">Sclerocarya birrea</Emphasis> subsp. <Emphasis Type="Italic">caffra</Emphasis> leaf explants

Sclerocarya birrea (marula) is an indigenous South African tree with highly valued medicinal and nutritional properties. Induction of nodular meristemoids from leaf explants was achieved on Murashige and Skoog (MS) and woody plant medium (WPM) supplemented with 6-benzyladenine (BA) in combination with naphthalene acetic acid (NAA), indole-3-butryric acid (IBA) and indole-3-acetic acid (IAA). Induction of nodular meristemoids from 86% of the leaf cultures was achieved on MS medium with 4.0 μM BA and 1.0 μM NAA. High levels (78–100%) of induction were also achieved on WPM with different concentrations of BA (1.0–4.0 μM) and IBA (1.0–4.0 μM). The highest conversion of meristemoids into shoots was only 22% for 4.0 μM BA and 1.0 μM NAA on MS initiation medium. This was improved to 62% when nodular clusters were cultured in a MS liquid medium. Histological studies revealed the globular stage of the nodular meristemoids. This protocol has potential for application in mass micropropagation and plant breeding of S. birrea.     

Biological control of root rot fungus <Emphasis Type="Italic">Macrophomina phaseolina</Emphasis> and growth enhancement of <Emphasis Type="Italic">Pinus roxburghii</Emphasis> (Sarg.) by rhizosphere competent <Emphasis Type="Italic">Bacillus subtilis</Emphasis> BN1

Bacterial isolates having antifungal and good plant growth-promoting attributes were isolated from chir-pine (Pinus roxburghii) rhizosphere. An isolate, Bacillus subtilis BN1 exhibited strong antagonistic activity against Macrophomina phaseolina, and other phytopathogens including Fusarium oxysporum and Rhizoctonia solani. It was characterized and selected for the present studies. BN1 resulted in vacuolation, hyphal squeezing, swelling, abnormal branching and lysis of mycelia. The cell-free culture filtrate of BN1 inhibited the growth of M. phaseolina. Pot trial study resulted in statistically significant increase in seedling biomass besides reduction in root rot symptoms in chir-pine seedlings. BN1 treatment resulted in 43.6% and 93.54% increases in root and shoot dry weights respectively, as compared to control. Also, 80–85% seed viability was recorded in treatments receiving BN1 either alone or in the presence of M. phaseolina, compared to 54.5% with M. phaseolina. Bioinoculant formulation study suggested that maximum viability of bacteria was in a sawdust-based carrier. B. subtilis BN1 produced lytic enzymes, chitinase and β-1,3-glucanase, which are known to cause hyphal degradation and digestion of the cell wall component of M. phaseolina. In the presence of M. phaseolina, population of B1 was 1.5 × 10⁴ c.f.u. g⁻¹ root after one month, which increased to 4.5 × 10⁴ c.f.u. g⁻¹ root in three months. Positive root colonization capability of B. subtilis BN1 proved it as a potent biocontrol agent.     

Evidence for physiological seed dormancy cycling in the woody shrub Asterolasia buxifolia and its ecological significance in fire‐prone systems

• Dormancy cycling is a key mechanism that contributes to the maintenance of long‐term persistent soil seed banks, but has not been recorded in long‐lived woody shrub species from fire‐prone environments. Such species rely on seed banks and dormancy break as important processes for post‐fire recruitment and recovery.
• We used germination experiments with smoke treatments on fresh seeds and those buried for 1 year (retrieved in spring) and 1.5 years (retrieved the following late autumn) to investigate whether Asterolasia buxifolia, a shrub from fire‐prone south‐eastern Australia with physiologically dormant seeds, exhibited dormancy cycling.
• All seeds had an obligation for winter seasonal temperatures and smoke to promote germination, even after ageing in the soil. A high proportion of germination was recorded from fresh seeds. but germination after the first retrieval was significantly lower, despite high seed viability. After the second retrieval, germination returned to the initial level. This indicates a pattern of annual dormancy cycling; one of the few observations, to our knowledge, for a perennial species. Additionally, A. buxifolia’s winter temperature and smoke requirements did not change over time, highlighting the potential for seeds to remain conditionally dormant (i.e. restricted to a narrow range of germination conditions) for long periods.
• For physiologically dormant species, such as A. buxifolia, we conclude that dormancy cycling is an important driver of successful regeneration, allowing seed bank persistence, sometimes for decades, during fire‐free periods unsuitable for successful recruitment, while ensuring that a large proportion of seeds are available for recruitment when a fire occurs.

     

Phenylpropanoid production in callus and cell suspension cultures of <Emphasis Type="Italic">Buddleja cordata</Emphasis> Kunth

Plant tissue cultures represent a potential source for producing secondary metabolites. In this work, Buddleja cordata tissue cultures were established in order to produce phenylpropanoids (verbascoside, linarin and hydroxycinnamic acids), as these metabolites are credited with therapeutic properties. Highest callus induction (76.4–84.3%) was obtained in five treatments containing 2,4-Dichlorophenoxyacetic acid (2,4-d: 0.45–9.05 μM) with Kinetin (KIN: 2.32, 4.65 μM), whereas highest root induction (79.6%) corresponded to the α-Naphthaleneacetic acid (9.05 μM) with KIN (2.32 μM) treatment. Verbascoside was the major phenylpropanoid produced in in vitro cultures (root, white and green callus) [66.24–86.26 mg g⁻¹ dry weight (DW)], while linarin and hydroxycinnamic acid production was low (0.95–3.01 mg g⁻¹ DW). Verbascoside and linarin production were improved in cell suspension culture (116 mg g⁻¹ DW and 8.12 mg g⁻¹ DW, respectively).     

Cadmium-induced stress on the seed germination and seedling growth of <Emphasis Type="Italic">Brassica napus</Emphasis> L., and its alleviation through exogenous plant growth regulators

Because of its prolific growth, oilseed rape (Brassica napus L.) can be grown advantageously for phytoremediation of the lands contaminated by industrial wastes. Therefore, toxic effect of cadmium on the germination of oilseed rape, the capability of plants for cadmium phytoextraction, and the effect of exogenous application of plant growth regulators to mitigate phytotoxicity of cadmium were investigated. For the lab study of seedlings at early stage, seeds were grown on filter papers soaked in different solutions of Cd²⁺ (0, 10, 50, 100, 200 and 400 μM). In greenhouse study, seedlings were grown in soil for 8 weeks, transferred to hydroponic pots for another 6 weeks growth, and then treated with plant growth regulators and cadmium. Four plant growth regulators viz. jasmonic acid (12.5 μM), abscisic acid (10 μM), gibberellin (50 μM) and salicylic acid (50 μM); and three levels of Cd²⁺ (0, 50 and 100 μM) were applied. Data indicated that lower concentration of Cd²⁺ (10 μM) promoted the root growth, whereas the severe stresses (200 or 400 μM) had negative effect on the establishment of germinating seedlings. Plants treated with any of the tested plant growth regulators alleviated cadmium toxicity symptoms, which were reflected by more fresh weight, less malondialdehyde concentration in leaves and lower antioxidant enzyme activities. The application of abscisic acid to the plants cultivated in the medium containing 100 μM Cd²⁺ resulted in significantly lower plant internal cadmium accumulation. Huabing Meng and Shujin Hua contributed equally to this paper.     

Little evidence for negative effects of an invasive alien plant on pollinator services

Many invasive alien plants occur in large populations with abundant flowers which are highly attractive to pollinators, and thus might affect pollination of co-occurring native species. This study focuses on the invasive Heracleum mantegazzianum and distance-dependent effects on pollination of Mimulus guttatus in abandoned grassland over 2 years. First, we examined pollinator abundance in yellow traps at 0, 10, 30 and 60–200 m from H. mantegazzianum. We then placed M. guttatus plants at the same distances to monitor effects of the invasive species on pollinator visitation and seed set of neighbouring plants. Finally, we conducted a garden experiment to test if deposition of H. mantegazzianum pollen reduces seed set in M. guttatus. No distance effect was found for the number of bumblebees in traps, although the invasive species attracted a diverse assemblage of insects, and visitation of M. guttatus was enhanced close to H. mantegazzianum. This positive effect was not reflected by seed set of M. guttatus, and heterospecific pollen decreased seed set in these plants. Overall there is little evidence for negative effects of the invasive species on pollination of neighbouring plants, and flower visitation even increases close to the invaded patches. The functional role of the invader and suitable control strategies need further clarification, since removal of H. mantegazzianum may actually damage local pollinator populations. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of mature <Emphasis Type="Italic">Prunus serotina</Emphasis> (black cherry) and regeneration of transgenic shoots

A protocol for Agrobacterium-mediated transformation was developed for in vitro leaf explants of an elite, mature Prunus serotina tree. Agrobacterium tumefaciens strain EHA105 harboring an RNAi plasmid with the black cherry AGAMOUS (AG) gene was used. Bacteria were induced for 12 h with 200 μM acetosyringone for vir gene induction before leaf explant inoculation. Explants were co-cultured for 3 days, and then cultured on woody plant medium supplemented with 9.08 μM thidiazuron, 1.07 μM napthaleneacetic acid, 60 μM silver thiosulphate, 3% sucrose, plus 200 mg l⁻¹ timentin in darkness for 3 weeks. Regenerating shoots were selected 27 days after initial co-culture, on Murashige and Skoog medium with 3% sucrose, 8.88 μM 6-benzylaminopurine, 0.49 μM indole-3-butyric acid, 0.29 μM gibberellic acid, 200 mg l⁻¹ timentin, and 30 mg l⁻¹ kanamycin for five subcultures. After 5–6 months of selection, transformation efficiencies were determined, based on polymerase chain reaction (PCR) analysis of individual putative transformed shoots relative to the initial number of leaf explants tested. The transformation efficiency was 1.2%. Southern blot analysis of three out of four PCR-positive shoots confirmed the presence of the neomycin phosphotransferase and AG genes. Transgenic shoots were rooted (37.5%), but some shoot tips and leaves deteriorated or died, making acclimatization of rooted transgenic plants difficult. This transformation, regeneration, and rooting protocol for developing transgenic black cherry will continue to be evaluated in future experiments, in order to optimize the system for several mature black cherry genotypes.     

A stable and reproducible transformation system for the wetland monocot <Emphasis Type="Italic">Juncus accuminatus</Emphasis> (bulrush) mediated by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

A highly reproducible Agrobacterium-mediated transformation system was developed for the wetland monocot Juncus accuminatus. Three Agrobacterium tumefaciens binary plasmid vectors, LBA4404/pTOK233, EHA105/pCAMBIA1201, and EHA105/pCAMBIA1301 were used. All vectors contained the 35SCaMV promoter driven, intron containing, β-glucuronidase (gus), and hygromycin phosphotransferase (hptII) genes within their T-DNA. After 48 h of cocultivation, 21-d-old seedling derived calli were placed on medium containing timentin at 400 mg l⁻¹, to eliminate the bacteria. Calli were selected on MS medium containing 40 or 80 mg l⁻¹ hygromycin, for 3 mo. Resistant calli were regenerated and rooted on MS medium containing hygromycin, 5 mg l⁻¹(22.2 μM) of 6-benzylamino-purine (BA) and 0.1 mg l⁻¹(0.54 μM) of alpha-naphthaleneacetic acid (NAA), respectively. Seventy-one transgenic cell culture lines were obtained and 39 plant lines were established in the greenhouse. All the plants were fertile, phenotypically normal, and set viable seed. Both transient and stable expression of the gus gene were demonstrated by histochemical GUS assays of resistant calli, transgenic leaf, root, inflorescence, seeds, and whole plants. The integration of gus and hptII genes were confirmed by polymerase chain reaction (PCR) and Southern analysis of both F₀ and F₁ progenies. The integrated genes segregated to the subsequent generation in Mendelian pattern. To our knowledge, this is the first report of the generation of transgenic J. accuminatus plants.