从种内、种间化感作用分析入侵种薇甘菊及其近邻种的非协同进化趋势

为预测入侵植物与其邻近物种间的非协同进化趋势,分别提取薇甘菊(Mikania micrantha)及其近邻种五爪金龙(Ipomoea cairica)、葛藤(Pueraria lobata)和鸡矢藤(Paederia scandens)的叶片水提液处理幼苗,对薇甘菊及其近邻种的种内、种间化感作用进行研究。结果表明,薇甘菊对3种近邻种的化感作用呈现不同趋势(SE=0.50, 0.61,–0.16),但种内存在较强的化感促进作用(SE=0.61),说明其种内化感竞争较弱;葛藤的种内化感促进作用(SE=0.32)弱于薇甘菊,种间的化感促进作用与薇甘菊相似(SE=0.52, 0.50);五爪金龙种内化感促进作用(SE=0.06)弱于薇甘菊和葛藤(SE=0.32),种间促进作用(SE=0.24)弱于薇甘菊(SE=0.61);鸡矢藤的种内、种间化感作用均表现为抑制作用(SE=–0.18,–0.07),说明种内、种间化感竞争较强。野外调查表明4种植物分布的频度和多度均表现为薇甘菊葛藤五爪金龙鸡矢藤,这与化感竞争力结果一致。可见,种内与种间化感作用在入侵植物与近邻植物的综合竞争力中扮演着极为重要的作用,这为入侵种与近邻种之间的非协同进化趋势指明了方向。     

An efficient method for <Emphasis Type="Italic">in vitro</Emphasis> regeneration of leafy spurge (<Emphasis Type="Italic">Euphorbia esula</Emphasis> L.)

Leafy spurge (Euphorbia esula L.) is a perennial, invasive weed used as a model to study invasive plant behavior, because molecular tools (such as a deep expressed sequence tag database and deoxyribonucleic acid microarrays) have been developed for this species. However, the lack of effective in vitro regeneration and genetic transformation systems has hampered molecular approaches to study leafy spurge. In this study, we describe an efficient in vitro regeneration system. Three highly regenerative lines were selected by screening the in vitro regeneration capabilities of stem explants of 162 seedlings. The effects of various culture conditions on in vitro regeneration were then evaluated based on explant competence to form calluses and shoots. High rates of shoot regeneration can be obtained using a growth medium containing 1× woody plant basal medium and 1× Murashige and Skoog (MS) basal salts, 1× MS vitamins, 1.11 μM 6-benzylaminopurine, 1.97 μM indole-3-butyric acid, and 3% sucrose, pH 5.6–5.8. After 30 d culture, multiple shoots formed either directly from the stem or indirectly from the callus. This method is a requisite for the development of genetic transformation systems for leafy spurge and may be used to develop in vitro regeneration techniques for other species in the Euphorbiaceae.     

Some propagation methods for cloning holm oak (<Emphasis Type="Italic">Quercus ilex</Emphasis> L.) plants

Holm oak (Quercus ilex L.), a typical evergreen tree of the Mediterranean area, is very important due to its ecological and economical values. Propagation of this species is extremely difficult and traditionally carried out only by seed germination. In this work, mature acorns were germinated in vitro and in peat substrate in aseptic and non-aseptic conditions. Explants from the seedlings obtained were propagated in vitro in WPM plus 4 μM BA. Plant regeneration was achieved from hypocotyls and root segments cultured in vitro on modified Gamborg medium plus 20 μM BA and 20 μM NAA. 13.8% of the hypocotyls and approximately 30% of the root segments developed both shoots and roots after 30 days of culture. Rooting of stem segments was obtained both in vitro and ex vitro by basal dipping in IBA solutions. Within ex vitro rooting, mother plant age had major influence on the percentage of rooting of the cuttings as the younger plants showed higher ability to root. In this way, Q. ilex plants could be propagated and cloned. The procedure described here would be a very useful tool for breeding programs since vegetative propagation of selected individuals can be achieved.     

The use of in vitro culture to improve the propagation of Rhododendron ponticum subsp. baeticum (Boiss. & Reuter)

Rhododendron ponticum subsp. baeticum is endemic in the southern region of the Iberian Peninsula. The relict populations of this species are vulnerable, due mainly to difficult conditions for the establishment of seedlings, resulting in a virtual lack of sexual recruitment. In order to preserve the surviving populations, in vitro culture methods have been applied for both the sexual and the agamic propagation of the species. The in vitro germination of seeds was high when conducted with Anderson’s medium without plant growth regulators. The self-rooted seedlings obtained were easily transplanted to outside conditions. The presence of growth regulators in the medium interfered with the development of the seedlings, causing heavy callus formation. The in vitro growth of explants took place readily in Anderson’s medium plus 0.072 mg L⁻¹ of BA and 0.036 mg L⁻¹ of NAA although the explants did not form roots. Rooting was achieved by the basal dipping of the explants in hydroalcoholic solutions of 500 mg L⁻¹ IAA during the outside transplanting process. Therefore, the combination of in vitro grown explants together with ex vitro rooting, results in a good method for the agamic propagation of Rhododendron ponticum subsp. baeticum.     

High-frequency<Emphasis Type="Italic">in vitro</Emphasis> flowering in six species of<Emphasis Type="Italic">ceropegia</Emphasis>

High-frequencyin vitro flowering is reported here fromin vitro regenerated shoots ofin vitro-raised seedlings of rare and endemicCeropegia lawii, Ceropegia maccannii, Ceropegia oculata, andCeropegia sahyadrica, as well as the widely distributedCeropegia bulbosa var.bulbosa andCeropegia hirsuta. In our first set of experiments, the MS medium contained 87 mM sucrose and was supplemented with varying concentrations of BAP (4.4 to 26.6 μM). For the second set of trials, varying concentrations of sucrose (87 to 233 mM) were tested in MS media containing a constant 4.4 p.M BAP. Sub-cultured apical as well as axillary buds flowered with similar frequencies after 30 d of incubation. For all six species, the highest percentage of flowering shoots was obtained with either 26.6 μM BAP or 175 mM sucrose. Although smaller in size, theirin vitro flowers were morphologically comparable within wVo-derived flowers. Variations among species were noted for the number of flower buds per shoot and the percentage of flower formation. Because all six species showed similar responses in both experiments, we can suggest that this protocol is applicable across the wide range ofCeropegia species.     

Potential distribution and environmental threat of <Emphasis Type="Italic">Pueraria lobata</Emphasis>

Pueraria lobata (kudzu) is an invasive weed originating from East Asia. Local infestations have been recently observed in Switzerland and northern Italy; however, the potential for P. lobata to spread and to become abundant and damaging in the Alpine countries is not known. The aim of this study was to project the potential distribution of P. lobata under current climate in Switzerland, Austria and Slovenia and parts of northern Italy using the ecoclimatic model CLIMEX. In addition, areas at risk were identified where P. lobata may occur as a strong and aggressive competitor. This was derived from the plants’ distribution and climatic requirements in the south-eastern United States where the heaviest infestations occur. Projections show that 60.84% of the total land area of northern Italy, followed by 47.08% of Slovenia, 21.01% of Austria and only 1.97% of Switzerland are climatically suitable. P. lobata may become a troublesome weed due to very favourable climatic conditions only in some parts of northern Italy and Slovenia. In climatically suitable areas, any occurrence of the plant should be carefully observed. In infested and highly climatically suitable areas, there is a need for strategic management to prevent further spread of P. lobata.     

Vegetative micro-cloning to sustain biodiversity of threatened Moringa species

Efficient vegetative cloning in vitro requires definition of plant growth regulator regimes for each genotype, and therefore formulation of a uniform culture protocol for a genetically heterogeneous wild or uncultivated plant population is often impossible. The likelihood of cloning a wide array of plant genotypes by avoiding the use of plant growth regulator(s) was explored with Moringa oleifera Lamk., Moringa stenopetala (Baker f.) Cufod, and Moringa peregrina Forssk. ex Fiori tree seedlings. Propagation was achieved by multiple shoot regeneration from the cotyledonary node of decapitated seedlings, followed by axillary shoot growth from single node shoot segments and rooting of excised shoots. All steps were accomplished on basal Murashige and Skoog medium without plant growth regulator supplements. The results revealed competence for generation of multiple shoots from cotyledonary node tissue, stimulated by repeated shoot harvest, in seedlings of all three tree species. Tens of plants per seedling were regenerated in about 4 mo from culture initiation. In a given species clone size was seedling-dependent, which presumably stems from genotypic variability among seedlings in regeneration ability in vitro. By this means the laborious search for a plant growth regulator regime suitable for organogenesis induction and adapted per genotype became redundant, and biodiversity of the seed germplasm could be maintained. The approach ideally suits establishment of clones of wild plants of endangered species, like those of the Moringaceae, species with high ability for producing supplementary shoots, and without the need to add plant growth regulators, including the rooting stage.     

Cultivating <Emphasis Type="Italic">in vitro</Emphasis> coconut palms (<Emphasis Type="Italic">Cocos nucifera</Emphasis>) under glasshouse conditions with natural light,improves <Emphasis Type="Italic">in vitro</Emphasis> photosynthesis nursery survival and growth

Plantlets of coconut were cultured in vitro under three different ambient conditions including a standard culture room, a culture room inside a glasshouse with natural light but controlled temperature, and a standard glasshouse with natural light and natural fluctuations of temperature. Plantlets from the 3 treatments were compared in terms of growth, plant survival as well as net photosynthesis and efficiency of PSII (Fv/Fm ratio) both at the end of the in vitro stage and at 3 stages of ex vitro acclimatization. At the end of the in vitro stage, plantlets cultured in vitro under glasshouse conditions showed the best performance showing the highest photosynthesis rate, dry weight and number of leaves. Plantlets from the standard culture room showed the lowest photosynthesis and growth rate. After 6 months of ex vitro acclimatization, plantlets originally grown in vitro under glasshouse conditions maintained better field survival and growth rates in terms of fresh weight, dry weight and leaf number than plantlets originally grown in vitro in the standard culture room. Although more studies are required to define the reason for this effect, it is clear that the conditions of standard culture rooms are not the best for in vitro cultivation of coconut and perhaps other tropical species.     

Rosmarinic acid content in basil plants grown <Emphasis Type="Italic">in vitro</Emphasis> and in hydroponics

The accumulation of selected caffeic acid derivatives (CADs), in particular rosmarinic acid (RA), was investigated in different tissues (leaves, roots and plantlet shoots) of sweet basil (Ocimum basilicum L.) plants grown either in vitro or in hydroponic culture (floating system) under greenhouse conditions. Two cultivars with green leaves (Genovese and Superbo) and one with purple leaves (Dark Opal) were tested. The content of CADs in HCl-methanol extracts was determined by HPLC. LC-MS and LC-MS-MS were used to confirm the identification of the metabolites of interest. Apart from rosmarinic acid (RA) and a methylated form of this substance, no other CADs were detected at significant level in any of the analyzed samples. The content of RA ranged approximately from 4 to 63 mg/g DW, depending on the growing system. The highest RA content was found during the in vitro multiplication, in the acclimatized plants and in the roots of hydroponically-grown seedlings at full bloom. In vitro, 6-benzyladenine reduced the accumulation of RA in purple-leaf Dark Opal cultivar, but an opposite effect of this growth regulator was observed in the green-leaf genotypes. Our findings suggest the possibility to scale-up RA production by means of in vitro or hydroponic culture of sweet basil.     

Evaluating costs for the <Emphasis Type="Italic">in vitro</Emphasis> propagation and preservation of endangered plants

In vitro methods provide opportunities for propagating and preserving endangered plant species when seed-based methods are not adequate. Such species include those that produce few or no seeds, as well as species with recalcitrant seeds. Tissue culture propagation methods can be used to produce such plants for reintroduction, research, education, display, and commerce. They can also be the basis for tissue banking as a way to preserve genetic diversity when seeds cannot be banked. With some recalcitrant species, embryo banking, a method which also utilizes in vitro culture for recovery germination, is possible. The number of endangered species that will require in vitro methods is estimated to be at least 5,000 worldwide. Further information is needed to identify these species, and the ongoing collection of information into databases on endangered species and recalcitrant species will help provide this. The costs of these methods are higher than for traditional propagation and preservation, but they may be necessary for species under higher threat. The multiplication rate of a culture, as well as the rates of rooting and acclimatization, has a major effect on the number of transfers needed for producing plants or tissue for banking, and improvements that will increase the efficiency of these steps can help lower costs. Further research into factors affecting the growth of tissues in vitro, as well as coordination of efforts among institutions with infrastructure for in vitro work, should facilitate the application of in vitro methods to the endangered species that cannot be propagated or preserved using seeds.     

Salt stress effects on growth,pigments, proteins and lipid peroxidation in <Emphasis Type="Italic">Salicornia persica</Emphasis> and <Emphasis Type="Italic">S. europaea</Emphasis>

The effects of NaCl stress on growth, water status, contents of protein, proline, malondialdehyde (MDA), various sugars and photosynthetic pigments were investigated in seedlings of Salicornia persica and S. europaea grown in vitro. Seeds were germinated under NaCl (0, 100, 200, 300, 400, 500 and 600 mM) on Murashige and Skoog medium for 45 d. The shoot growth of both species increased under low NaCl concentration (100 mM) and then decreased with increasing NaCl concentrations. In contrast to S. persica, root length in S. europaea reduced steadily with an increase in salinity. Proline content in S. persica was higher than in S. europaea at most NaCl concentrations. Proline, reducing saccharide, oligosaccharide and soluble saccharide contents increased under salinity in both species. In contrast, contents of proteins and polysaccharides reduced in both species under salt stress. MDA content remained close to control at moderate NaCl concentrations (100 and 200 mM) and increased at higher salinities. MDA content in S. europaea was significantly higher than S. persica at higher salinities. Salt treatments decreased K⁺ and P contents in seedlings of both species. Significant reduction in contents of chlorophylls and carotenoids due to NaCl stress was also observed in seedlings of both species. Some differences appeared between S. persica and S. europaea concerning proteins profile. On the basis of the data obtained, S. persica is more salt-tolerant than S. europaea.     

In vitro embryo culture of Scirpus acutus Muhl.

An in vitro embryo culture protocol was developed for Scirpus acutus Muhl. A maximum of 85.6% of germination was obtained when isolated embryos were cultured in vitro, a result similar to those reported in the literature with traditional dormancy breaking treatments. In vitro seedling development was optimal in half-strength Murashige and Skoog (1962) liquid medium. An average of 3–4 shoots were produced from the initial seedlings. Clusters of plantlets were successfully acclimatized and transferred to soil. These results corroborate the findings of previous studies stating that seed dormancy in Scirpus is caused by the seed/fruit coats. In vitro embryo culture thus allows for the production of Scirpus acutus Muhl. seedlings that can be transferred to natural or artificial wetlands.     

Mitochondrial function plasticity in <Emphasis Type="Italic">Acanthamoeba castellanii</Emphasis> during growth in batch culture

The alterations in mitochondrial bioenergetics during growth in a batch culture of Acanthamoeba castellanii were studied. The capacity of cytochrome pathway-dependent respiration measured in vitro decreased from the intermediary phase, when cell division slowed down. The pattern of the cytochrome pathway capacity changes was paralleled from the intermediary phase by alterations in the amount of total (and reducible) membranous ubiquinone. These changes were accompanied by a decrease in mitochondrial reactive oxygen species production in vitro (when no energy-dissipating system was active), and almost no change in superoxide dismutase activity and protein level, thus indicating an equivalent need for this enzyme in oxidative stress defence in A. castellanii culture. On the other hand, a decrease in the activity and protein level of alternative oxidase and uncoupling protein was observed in vitro, when cells shifted from the exponential growth phase to the stationary phase. It turned out that the contribution of both energy-dissipating systems in the prevention of mitochondrial reactive oxygen species generation in vivo could lead to its constant level throughout the growth cycle of A. castellanii batch culture. Hence, the observed functional plasticity insures survival of high quality cysts of A. castellanii cells.     

Influence of carbon source and concentration on the in vitro development of olive zygotic embryos and explants raised from them

The influence of sucrose or mannitol on in vitro zygotic embryo germination, seedling development and explant propagation of olive tree (Olea europaea L.) was compared. Embryos germinated without sucrose in the medium but for adequate development of the seedlings to yield viable plants, a carbohydrate supply was necessary; both sucrose and mannitol were equally suitable for this purpose. However, when explants obtained from in vitro germinated embryos were cultured with mannitol or sucrose, then the polyalcohol promoted significantly more growth than sucrose by increasing shoot length, pairs of leaves formed, and breaking apical dominance. This improved the in vitro culture of olive plant material, thus allowing new olive clonal lines to be obtained in shorter times. This will assist in future breeding experiments with the species.     

EFFECTS OF SOIL WATER DISTRIBUTION ON THE LATERAL ROOT DEVELOPMENT OF THREE SPECIES OF CALIFORNIA OAKS

The responses of seedling root systems of three species of oaks in California to two experimental soil moisture regimes were studied by comparing lateral root development, root and shoot weights, and root: shoot ratios. In the first soil moisture treatment the taproot was allowed to extend into moist soil throughout the duration of the experiment (control), while in the second treatment (shallow) the taproot grew into a dry substrate below 30 cm of moist soil. The treatments were intended to approximate soil moisture conditions experienced by oak seedlings in the field when deep soil water sources vary in their accessibility (control: accessible, shallow: inaccessible). Lateral root growth of Quercus agrifolia did not increase significantly when the primary root tip died in the shallow treatment, resulting in an overall decrease in the percent of the root system composed of lateral roots. Q. douglasii and Q. lobata increased lateral root weights by 80% and 70%, respectively, on the upper 30 cm of the primary root when the primary root tip died. Q. lobata was the only species that decreased in shoot and root weight (25% and 21%, respectively) with the loss of the root tip, indicating that, unlike the other species, it was dependent on the primary root for maximum growth. The morphological responses of these species correspond with their distributions and also may be a factor that influences their interactions with other species.     

Effects of sugars and growth regulators on <Emphasis Type="Italic">in vitro</Emphasis> growth of <Emphasis Type="Italic">Dactylorhiza</Emphasis> species

The influence of sugars and growth regulators on shoot and root growth of Dactylorhiza species was studied under in vitro conditions. The seedling development was stimulated with the application of glucose and sucrose at concentration of 10 g dm⁻³ each. The improvement of shoot growth rate and shoot length was enhanced by cytokinins N ⁶-(2-isopentenyl)adenine or N ⁶-benzyladenine and their combination with auxin indolebutyric acid (IBA). The root growth rate and root length of seedlings increased in the presence of IBA and α-naphthaleneacetic acid. Individual Dactylorhiza species showed statistically significant differences in shoot and root development depending on sugar and growth regulator combinations.     

Species specificity in settling-plant selection by foundress ant queens in <Emphasis Type="Italic">Macaranga</Emphasis>–<Emphasis Type="Italic">Crematogaster</Emphasis> myrmecophytism in a Bornean dipterocarp forest

Previous studies have demonstrated that the obligate myrmecophytism between Macaranga ant-plants and Crematogaster plant-ants is highly species specific, although multiple Macaranga species can coexist in a microhabitat. However, the species specificity has been described based on the study of trees with established plant-ant colonies. We studied how the process of settling into the partner Macaranga seedlings by single foundress Crematogaster queens contributes to species specificity. By sampling seedlings of three sympatric Macaranga myrmecophytes species in the field, we tested two hypotheses. The first is that foundresses correctly select their specific partner plant species when they settle into seedlings. The second hypothesis is that the seasons in which seedlings available for settling by foundresses appear are segregated among the Macaranga species, and the seasons in which foundress queens settle are synchronized to the appearance of seedlings of specific partner species; thus species specificity is consequently generated. Our results support the former hypothesis but not the latter: we always observed foundresses settling species-specific host plants, and seedlings suitable for settling were always available in each Macaranga species. Electronic Publication     

The possibilities and challenges of <Emphasis Type="Italic">in vitro</Emphasis> methods for plant conservation

Seed-based methods are generally the most efficient for propagating and storing plant germplasm, but these methods are not always adequate, and some species can benefit from in vitro methods for conservation. For species that produce few or no seeds in the wild, plants may be propagated in vitro, and in vitro shoot tips can provide material for cryostorage when seeds are not available or are recalcitrant. In vitro propagated plants may also serve as subjects for research, without depleting the genetic resources of the species. Clonal plants can be used to test out suitable habitat and can be used for basic research on endangered species, without disturbing the wild population. Despite the effectiveness of widely used techniques, however, there are still species that resist initiation into culture or that may be difficult to root or acclimatise. Similarly, tissue cryopreservation methods may be restrained by cost, particularly in maintaining multiple genotypes of many species. Maintaining such genotypes in vitro is also costly and runs the risk of loss or change over time. Examples of the successful use of in vitro methods will illustrate the variety of applications of these techniques, but costs and specific challenges will also be discussed to help define areas where further research is needed to realise the potential of in vitro methods as a tool for conservation.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of eight species or subspecies of <Emphasis Type="Italic">Turbinicarpus (Cactaceae)</Emphasis>

Summary In vitro propagation systems by means of areole activation were developed for Turbinicarpus laui, T. lophophoroides, T. pseudopectinatus, T. schmiedickeanus subsp. flaviflorus, T. schmiedickeanus subsp. klinkerianus, T. schmiedickeanus subsp. schmiedickeanus, T. subterraneus, and T. valdezianus. In vitro-germinated seedlings were used as a primary source of explants. Multiple shoot formation from areoles was achieved for three explant types (apical, lateral, and transverse), cultured on Murashige and Skoog (MS) basal medium supplemented with 3% sucrose, 10 gl⁻¹ agar and several treatments with cytokinins. Efficiencies were in the range from 7.8 shoots per explant in T. valdezianus up to 19.7 shoots per explant in T. pseudopectinatus, using the best treatment for each species and in a single proliferation cycle. Four of the studied species responded best when 6-benzylaminopurine (3.3–8.8μM) was used, while 6-(γ,γ-dimethylallylamino)purine (19.7–24.6μM) showed better results in two species. The two remaining species showed no significant differences in their response to both cytokinins. Regarding explant type, the best results were obtained with transverse cuts for five species, with apical explants for one species, and the two remaining species showed no significant differences among the explants tested. Rooting of the in vitro-generated shoots was achieved most efficiently on half- or full-strength MS basal medium. Rooting frequencies were in the range from 54.2 to 94.2%, and the frequency of survival of the plants once transferred to soil was 91.6% on average.     

Tissue culture and wetland establishment of the freshwater monocots <Emphasis Type="Italic">Carex,Juncus, Scirpus</Emphasis>, and <Emphasis Type="Italic">Typha</Emphasis>

Summary Cell cultures of freshwater wetland monocots were regenerated, plants were grown in the greenhouse, and then established and evaluated in wetlands. Typha (cattail), Juncus (rushes), Scirpus (bulrushes), and Carex (sedges) were studied because they are common, dominant, high biomass wetland-adapted plants, tolerant of chemically diverse ecosystems. The goal was to define micropropagation and wetland establishment protocols. Tissue culture systems defined for numerous monocot crop species can be readily applied to wetland plants, with a few modifications. Issues addressed were selection of explant material, shoot and root regeneration conditions, culture age verses regenerability, greenhouse acclimatization needs, plant uniformity and requirements for wetland establishment. In vitro-germinated seedlings were an excellent source of pathogen-free regenerable tissue. T. latifolia, T. angustifolia, and J. accuminatus were regenerated from callus induced in the dark with picloram, then transferred to medium with benzyladenine in the light to promote shoot organogenesis. J. effusus, S. polyphyllus, and C. lurida could not be regenerated from callus, which turned black. They could be regenerated directly by culturing intact seedlings directly on cytokinin media in the light. Shoots rooted with little or no auxin. J. effusus rooting was promoted by the addition of charcoal to the medium. Covering plants for the first 2 wk with plastic facilitated greenhouse establishment. There were high rates of greenhouse and wetland survival. No abnormal plants were observed. These regeneration systems could be utilized for the production of wetland plants for potential application in habitat restoration and wetland creation, and would provide an alternative to field collection.