Mechanisms in mutualisms: a chemically mediated thrips pollination strategy in common elder.

MAIN CONCLUSION: This study provides first evidence of a thrips species pollinating Sambucus nigra and describes how interactions are driven by plant biochemical signalling and moderated by temporal changes in floral chemistry. The concept of flower-feeding thrips as pollinating insects in temperate regions is rarely considered as they are more frequently regarded to be destructive florivores feeding on pollen and surrounding plant tissue. Combining laboratory and field-based studies we examined interactions between Sambucus nigra (elderflower) and Thrips major within their native range to ascertain the role of thrips in the pollination of this species and to determine if floral chemicals mediated flower visits. If thrips provide a pollination service to S. nigra, then this will likely manifest in traits that attract the pollinating taxa at temporally critical points in floral development. T. major were highly abundant in inflorescences of S. nigra, entering flowers when stigmas were pollen-receptive and anthers were immature. When thrips were excluded from the inflorescences, fruit-set failed. Linalool was the major component of the inflorescence headspace with peak abundance coinciding with the highest number of adult thrips visiting flowers. Thrips were absent in buds and their numbers declined again in senescing flowers inversely correlating with the concentration of cyanogenic glycosides recorded in the floral tissue. Our data show that S. nigra floral chemistry mediates the behaviour of pollen-feeding thrips by attracting adults in high numbers to the flowers at pre-anthesis stage, while producing deterrent compounds prior to fruit development. Taking an integrative approach to studying thrips behaviour and floral biology we provide a new insight into the previously ambiguously defined pollination strategies of S. nigra and provide evidence suggesting that the relationship between T. major and S. nigra is mutualistic.

Cryopreservation of Pollen Grains of Pineapple and Other Bromeliads.

Cryopreservation of pollen grains is an efficient technique to overcome asynchronous flowering and to support actions for genetic improvement and conservation of important alleles. It can be used both by germplasm curators and plant breeders. In the case of Bromeliaceae, a family with wide diversity but also high vulnerability, the form of conservation can be crucial to prevent the increasing problem of genetic erosion. This chapter describes a method of cryopreservation of pollen grains of different Bromeliaceae species, including pineapple, after dehydration with silica and subsequent immersion in liquid nitrogen. The efficiency of this protocol has been demonstrated by the high pollen viability percentage and production of seeds after in vivo pollination with cryopreserved grains. The protocol can be used for cryopreserving pollen of many species of bromeliads and is easy to perform.

Novel computed tomography-based tools reliably quantify plant reproductive investment.

The flower is a bisexual reproductive unit where both genders compete for resources. Counting pollen and ovules in flowers is essential to understand how much is invested in each gender. Classical methods to count very numerous pollen grains and ovules are inefficient when pollen grains are tightly aggregated, and when fertilization rates of ovules are unknown. In this study we have therefore developed novel counting techniques based on computed tomography. In order to demonstrate the potential of our methods in very difficult cases, we counted pollen and ovules across inflorescences of deceptive and rewarding species of European orchids, which possess both very large numbers of pollen grains (tightly aggregated) and ovules. Pollen counts did not significantly vary across inflorescences and pollination strategies, whereas deceptive flowers had significantly more ovules than rewarding flowers. The within-inflorescence variance of pollen-to-ovule ratios in rewarding flowers was four times higher than in deceptive flowers, possibly demonstrating differences in the constraints acting on both pollination strategies. We demonstrate the inaccuracies and limitations of previously established methods, and the broad applicability of our new techniques: they allow measurement of reproductive investment without restriction on object number or aggregation, and without specimen destruction.

Yucca brevifolia fruit production, predispersal seed predation, and fruit removal by rodents during two years of contrasting reproduction.

PREMISE OF THE STUDY: The distribution of Yucca brevifolia, a keystone species of the Mojave Desert, may contract with climate change, yet reproduction and dispersal are poorly understood. We tracked reproduction, seed predation, and fruit dispersal for two years and discuss whether Y. brevifolia is a masting species. METHODS: Fruit maturation, seed predation (larval yucca moths), and fruit dispersal (rodents) were monitored on a random sample of panicles during 2013 and 2014, which were years of high and low reproduction, respectively. Fates of fruits placed on the ground and in canopies were also tracked. Rodents were live-trapped to assess abundance and species composition. KEY RESULTS: In 2013, 66% of inflorescences produced fruit of which 53% escaped larval predation; 19.5% of seeds were destroyed in infested fruits. Total seed production was estimated to be >100 times greater in 2013 than 2014. One-third of the fruit crop fell to the ground and was removed by rodents over the course of 120 d. After ground fruits became scarce, rodents exploited canopy fruits. Rodent numbers were low in 2013, so fruits remained in canopies for 370 d. In 2014, fruit production was approximately 20% lower. Larvae infested the majority of fruits, and almost twice the number of seeds were damaged. Fruits were exploited by rodents within 65 d. CONCLUSIONS: High fertilization, prolific seed production, and low predispersal predation in 2013 suggests that pollinator attraction and satiation of seed predators influence masting in Y. brevifolia. Abundant, prolonged fruit availability to seed-dispersing rodents likely extends recruitment opportunities during mast years.

Seasonal variation of responses to herbivory and volatile communication in sagebrush (Artemisia tridentata) (Asteraceae).

Plants can respond to insect herbivory in various ways to avoid reductions in fitness. However, the effect of herbivory on plant performance can vary depending on the seasonal timing of herbivory. We investigated the effects of the seasonal timing of herbivory on the performance of sagebrush (Artemisia tridentata). Sagebrush is known to induce systemic resistance by receiving volatiles emitted from clipped leaves of the same or neighboring plants, which is called volatile communication. Resistance to leaf herbivory is known to be induced most effectively after volatile communication in spring. We experimentally clipped 25 % of leaves of sagebrush in May when leaves were expanding, or in July when inflorescences were forming. We measured the growth and flower production of clipped plants and neighboring plants which were exposed to volatiles emitted from clipped plants. The treatment conducted in spring reduced the growth of clipped plants. This suggests that early season leaf herbivory is detrimental because it reduces the opportunities for resource acquisition after herbivory, resulting in strong induction of resistance in leaves. On the other hand, the late season treatment increased flower production in plants exposed to volatiles, which was caused mainly by the increase in the number of inflorescences. Because the late season treatment occurred when sagebrush produces inflorescences, sagebrush may respond to late herbivory by increasing compensation ability and/or resistance in inflorescences rather than in leaves. Our results suggest that sagebrush can change responses to herbivory and subsequent volatile communication seasonally and that the seasonal variation in responses may reduce the cost of induced resistance.

Effects of Floral Scent, Color and Pollen on Foraging Decisions and Oocyte Development of Common Green Bottle Flies.

The common green bottle fly Lucilia sericata (Meigen) and other filth flies frequently visit pollen-rich composite flowers such as the Oxeye daisy, Leucanthemum vulgare Lam. In laboratory experiments with L. sericata, we investigated the effect of generic floral scent and color cues, and of Oxeye daisy-specific cues, on foraging decisions by recently eclosed flies. We also tested the effect of a floral pollen diet with 0-35% moisture content on the ability of females to mature their oocytes. Our data indicate that (1) young flies in the presence of generic floral scent respond more strongly to a uniformly yellow cue than to any other uniform color cue (green, white, black, blue, red) except for ultraviolet (UV); (2) the floral scent of Oxeye daisies enhances the attractiveness of a yellow cue; and (3) moisture-rich pollen provides nutrients that facilitate ovary maturation of flies. With evidence that L. sericata exploits floral cues during foraging, and that pollen can be an alternate protein source to animal feces and carrion, Pollen apparently plays a major role in the foraging ecology of L. sericata and possibly other filth flies. These flies, in turn, may play a significant role as pollinators, as supported by a recently published study.

Pollen transfer efficiency and its effect on inflorescence size in deceptive pollination strategies.

Pollination systems differ in pollen transfer efficiency, a variable that may influence the evolution of flower number. Here we apply a comparative approach to examine the link between pollen transfer efficiency and the evolution of inflorescence size in food and sexually deceptive orchids. We examined pollination performance in nine food-deceptive, and eight sexually deceptive orchids by recording pollen removal and deposition in the field. We calculated correlations between reproductive success and flower number (as a proxy for resources allocated during reproductive process), and directional selection differentials were estimated on flower number for four species. Results indicate that sexually deceptive species experience decreased pollen loss compared to food-deceptive species. Despite producing fewer flowers, sexually deceptive species attained levels of overall pollination success (through male and female function) similar to food-deceptive species. Furthermore, a positive correlation between flower number and pollination success was observed in food-deceptive species, but this correlation was not detected in sexually deceptive species. Directional selection differentials for flower number were significantly higher in food compared to sexually deceptive species. We suggest that pollination systems with more efficient pollen transfer and no correlation between pollination success and number of flowers produced, such as sexual deception, may allow the production of inflorescences with fewer flowers that permit the plant to allocate fewer resources to floral displays and, at the same time, limit transpiration. This strategy can be particularly important for ecological success in Mediterranean water-deprived habitats, and might explain the high frequency of sexually deceptive species in these specialised ecosystems.

Can floral display size compensate for Allee effects caused by low population abundance and density in Synthyris bullii (Plantaginaceae), a rare species?

PREMISE OF THE STUDY: Conservation seeks to address the issues of small population size, and the reproductive limitations confronting these populations. Sparse, small plant populations often suffer Allee effects such as pollinator limitation. However, some studies show that plants in sparse populations experience reduced resource competition. As a result, these plants may produce larger floral displays, which are also predicted to attract pollinators. The negative impacts from reduced floral quantity may thus be offset by improved floral quality. METHODS: In a 2-yr field study, population abundance and density were quantified for 24 populations of Synthyris bullii (Plantaginaceae), a rare prairie endemic. In each population, data were collected on inflorescence size, fruit/seed set, and seed germination. KEY RESULTS: Inflorescence size had a positive relationship with population inflorescence abundance and density. Fruit set and germination responded positively to floral quality (i.e., flower density). In comparison, seed set showed a positive relationship with only floral quantity (i.e., population abundance). CONCLUSIONS: Contrary to our predictions, inflorescence size and population size were not inversely related. While attractive floral displays in sparse populations potentially compensate in terms of fruit set, population abundance nevertheless plays an important role in seed set. Because floral quality and quantity differ in their ability to explain reproductive outcomes, studies should examine reproduction at several stages, otherwise the impacts of population size may be overlooked. Allee effects manifesting at a critical stage of reproduction, such as seed production, may act as a bottleneck impeding successful recruitment.

Pollination and floral ecology of Arundina graminifolia (Orchidaceae) at the northern border of the species' natural distribution.

Arundina graminifolia is an early successional plant on Iriomote Island, the Ryukyus, Japan, where it is endangered. Populations flower for more than half a year, and many inflorescences bloom for one to several months. The nectarless gullet flowers, which open for up to six days, are self-compatible but cannot self-pollinate spontaneously; thus they rely on pollinating agents for capsule production. Field observations at two habitats identified at least six species of bees and wasps, primarily mate-seeking males of Megachile yaeyamaensis and Thyreus takaonis, as legitimate pollinators. Thus, this orchid is a pollinator generalist, probably owing to its long blooming period and simple flower morphology. Carpenter bees, which were previously reported to pollinate this orchid, frequently visited flowers but were too large to crawl into the labellum chamber and never pollinated the flowers. Extrafloral nectaries on inflorescences attracted approximately 40 insect taxa but were not involved with pollination. Fruit-set ratios at the population level varied spatiotemporally but were generally low (5.2-12.4 %), presumably owing to infrequent flower visits by mate-seeking pollinators and the lack of food rewards to pollinators.

Inflorescence dimorphism, heterodichogamy and thrips pollination in Platycarya strobilacea (Juglandaceae).

BACKGROUND AND AIMS: Unlike other taxa in Juglandaceae or in closely related families, which are anemophilous, Platycarya strobilacea has been suggested to be entomophilous. In Juglandaceae, Juglans and Carya show heterodichogamy, a reproductive strategy in which two morphs coexist in a population and undergo synchronous reciprocal sex changes. However, there has been no study focusing on heterodichogamy in the other six or seven genera, including Platycarya. METHODS: Inflorescence architecture, sexual expression and pollination biology were examined in a P. strobilacea population in Japan. Flowering phenology was monitored daily for 24 trees in 2008 and 27 in 2009. Flower visitors and inhabitants were recorded or collected from different sexes and stages. KEY RESULTS: The population of P. strobilacea showed heterodichogamous phenology with protogynous and duodichogamous-protandrous morphs. This dimorphism in dichogamy was associated with distinct inflorescence morphologies. Thrips pollination was suggested by the frequent presence of thrips with attached pollen grains, the scarcity of other insect visitors, the synchronicity of thrips number in male spikes with the maturation of female flowers, and morphological characters shared with previously reported thrips-pollinated plants. Male spikes went through two consecutive stages: bright yellow and strong-scented M1 stage, and brownish and little-scented M2 stage. The latter contained more thrips, synchronized better with the receptive stage of female flowers of the reciprocal morph and is probably the main period of pollen export. CONCLUSIONS: Platycarya strobilacea is heterodichogamous and thrips-pollinated, both of which are relatively rare conditions in angiosperms. In male spikes of P. strobilacea, there is probably a temporal decoupling of pollinator attraction and pollen export.

Evaluation of inflorescence visitors as pollinators of Echinacea angustifolia (Asteraceae): comparison of techniques.

Inflorescences (heads or capitula) of the putative self-incompatible species, purple coneflower (Echinacea angustifolia (DC) Cronq. (Asteraceae)), were visited by insects representing the Coleoptera, Diptera, Hymenoptera, and Lepidoptera, in accordance with a generalist pollination syndrome. Measurement of the effectiveness of insect species as pollinators was accomplished by permitting solitary visits to receptive, central disc florets of virgin (previously bagged) heads. Four parameters were quantified: total stigmatic pollen load and proportion of pollen grains germinated, numbers of pollen tubes at style bases, and percentages of total receptive florets that had retracted (shrivelled) styles. Quantifying total and germinated pollen grains proved ineffective, partly owing to the tendency of self-pollen to initiate pollen tubes. The most effective pollinators were Apidae, especially bumble bees (Bombus spp.) and the European honey bee (Apis mellifera L.) (mean: 39 - 61% of styles retracted). Other noteworthy pollinators were cloudless sulfur butterflies (Phoebis sennae L.--Pieridae; mean 47% of style bases with pollen tubes), golden blister beetles (Epicauta ferruginea Say--Meloidae; 44%), and grasshopper bee flies (Systoechus vulgaris Loew--Bombyliidae; 22%). Sunflower leafcutter bees (Megachile pugnata Say) were less effective (4% of styles retracted). Promisingly, analysis of the proportion of retracted styles provided similar results to the established technique of pollen-tube quantification, but had the significant advantages of being completed more rapidly, without a microscope, and in the field. The quantitative technique of retracted-style analysis appears well suited for prompt measurement of inflorescence-visiting insects as pollinators of many asteraceans.

Cell wall pectic arabinans influence the mechanical properties of Arabidopsis thaliana inflorescence stems and their response to mechanical stress.

Little is known of the dynamics of plant cell wall matrix polysaccharides in response to the impact of mechanical stress on plant organs. The capacity of the imposition of a mechanical stress (periodic brushing) to reduce the height of the inflorescence stem of Arabidopsis thaliana seedlings has been used to study the role of pectic arabinans in the mechanical properties and stress responsiveness of a plant organ. The arabinan-deficient-1 (arad1) mutation that affects arabinan structures in epidermal cell walls of inflorescence stems is demonstrated to reduce the impact on inflorescence stem heights caused by mechanical stress. The arabinan-deficient-2 (arad2) mutation, that does not have detectable impact on arabinan structures, is also shown to reduce the impact on stem heights caused by mechanical stress. The LM13 linear arabinan epitope is specifically detected in epidermal cell walls of the younger, flexible regions of inflorescence stems and increases in abundance at the base of inflorescence stems in response to an imposed mechanical stress. The strain (percentage deformation) of stem epidermal cells in the double mutant arad1 × arad2 is lower in unbrushed plants than in wild-type plants, but rises to wild-type levels in response to brushing. The study demonstrates the complexity of arabinan structures within plant cell walls and also that their contribution to cell wall mechanical properties is a factor influencing responsiveness to mechanical stress.

Adding perches for cross-pollination ensures the reproduction of a self-incompatible orchid.

BACKGROUND: Outcrossing is known to carry genetic advantages in comparison with inbreeding. In many cases, flowering plants develop a self-incompatibility mechanism, along with a floral component adaptation mechanism, to avoid self-pollination and to promote outbreeding. Orchids commonly have a lip in their flower that functions as the a visiting plate for insect pollinators. Aside from the lip, however, many species (including Coelogyne rigida) have sheaths around the axis of inflorescence. The function of these sheaths remains unknown, and has long been a puzzle to researchers. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the function of these sheaths in relation to the lip and the pollinators, as well as their role in the modes of pollination and reproduction of Coelogyne rigida in 30 flowering populations of orchids in the limestone area of Southeast Yunnan, China. We found that self-incompatible C. rigida developed specialized bird perches around the basal axis of inflorescence to attract sunbirds and to complement their behavioral tendency to change foraging locations frequently. This self-incompatibility mechanism operates separately from the floral component adaptation mechanism. This mechanism thus prevents bees from repeatedly visiting the floral lip of the same plant which, in turn, results in autogamy. In this way, instead of preventing autogamy, C. rigida responds to these negative effects through a highly efficient cross-pollination method that successfully transfers pollen to different plants. CONCLUSIONS: The proposed method ensures reproductive success, while offsetting the infertile self-pollination by insects, thereby reducing mating costs and addressing the lack of cross-pollination. The adaptation provides a novel and striking example of structural adaptation that promotes cross-pollination in angiosperms.

Behaviour of pollinator insects within inflorescences of Scrophularia species from Iberian Peninsula.

Several hypotheses have been proposed to explain the pattern of pollinator visits to vertical inflorescences. These range from a response to a pattern of resources to merely instinctive behaviour. In dichogamous plants, such behaviour has been associated with promoting outcrossing and avoiding geitonogamy. We here analyse behaviour of the principal pollinator groups in five protogynous species of Scrophularia with different flower sizes (S. sambucifolia, S. grandiflora, S. lyrata, S. scorodonia and S. canina), and the distribution of sexual phases along the inflorescences. The results in all cases show that pollinators follow a pattern of ascending visits accompanied by movements between flowers of the same whorl (horizontal movements). The relative frequency of these horizontal movements depends on the flower size, with a higher frequency in species with large flowers. In vertical movements of the three more common pollinator groups to several plant species (bumblebees, wasps and small bees), the behaviour was essentially independent of flower size, with bumblebees having the highest ratio of ascents to descents. Behaviour of the pollinators, together with the absence of a definite pattern of distribution of the sexual phases along the inflorescence, implies that geitonogamy is not avoided in any of the Scrophularia species studied.

Structure and expression profiling of a novel calcium-dependent protein kinase gene, CDPK3a, in leaves, stems, grapes, and cell cultures of wild-growing grapevine Vitis amurensis Rupr.

KEY MESSAGE : VaCDPK3a is actively expressed in leaves, stems, inflorescences, and berries of Vitis amurensis and may act as a positive growth regulator, but is not involved in the regulation of resveratrol biosynthesis. Calcium-dependent protein kinases (CDPKs) are known to play important roles in plant development and defense against biotic and abiotic stresses. It has previously been shown that CDPK3a is the predominant CDPK transcript in cell cultures of wild-growing grapevine Vitis amurensis Rupr., which is known to possess high resistance against environmental stresses and to produce resveratrol, a polyphenol with valuable pharmacological effects. In this study, we aimed to define the full cDNA sequence of VaCDPK3a and analyze its organ-specific expression, responses to plant hormones, temperature stress and exogenous NaCl, and the effects of VaCDPK3a overexpression on biomass accumulation and resveratrol content in V. amurensis calli. VaCDPK3a was actively expressed in all analyzed V. amurensis organs and tissues and was not transcriptionally regulated by salt and temperature stresses. The highest VaCDPK3a expression was detected in young leaves and the lowest in stems. A reduction in the VaCDPK3a expression correlated with a lower rate of biomass accumulation and higher resveratrol content in calli of V. amurensis under different growth conditions. Overexpression of the VaCDPK3a gene in the V. amurensis calli significantly increased cell growth for a short period of time but did not have an effect on resveratrol production. Further subculturing of the transformed calli resulted in cell death and a decrease in expression of the endogenous VaCDPK3a. The data suggest that while VaCDPK3a acts as a positive regulator of V. amurensis cell growth, it is not involved in the signaling pathway regulating resveratrol biosynthesis and resistance to salt and temperature stresses.

Micropropagation of onion (Allium cepa L.) from immature inflorescences.

In vitro plant production by direct organogenesis from immature flower heads is an ideal approach for clonal propagation of onions (Allium cepa L.). This technique ensures genetic stability, high propagation rate, and maintains donor plant of explants with an advantage over other means of in vitro regeneration. Onion micropropagation is usually applied in breeding programs, maintenance, and multiplication of cytoplasmic-male sterile lines for hybrid production, germplasm conservation, and as a tool for the application of other biotechnologies. For in vitro culture, mature onion bulbs are induced to reproductive phase by vernalization and forced to inflorescence initiation. Immature umbels are dissected from bulbs or cut directly when they appear from the pseudostem among the leaves. Disinfected inflorescences are cultivated in BDS basal medium supplemented with 30 g/L sucrose, 0.1 mg/L naphthalene acetic acid, 1 mg/L N (6)-benzyladenine, and 8 g/L agar, pH 5.5, under 16 h photoperiod white fluorescent light (PPD: 50-70 µmol/m(2)s) for 35 days. The regenerated shoot clumps are divided and subculture under the same conditions. For bulbification phase, the individual shoots are cultured in BDS basal medium containing 90 g/L sucrose, without plant growth regulators, pH 5.5, under 16 h photoperiod. Microbulbs can be directly cultivated ex vitro without acclimation.

Micropropagation and cryopreservation of garlic (Allium sativum L.).

Garlic (Allium sativum L.) is a very important medicinal and spice plant. It is conventionally propagated by daughter bulbs ("cloves") and bulbils from the flower head. Micropropagation is used for speeding up the vegetative propagation mainly using the advantage to produce higher numbers of healthy plants free of viruses, which have higher yield than infected material. Using primary explants from bulbs and/or bulbils (shoot tips) or unripe inflorescence bases, in vitro cultures are initiated on MS-based media containing auxins, e.g., naphthalene acetic acid, and cytokinins, e.g., 6-γ-γ-(dimethylallylaminopurine) (2iP). Rooting is accompanying leaf formation. It does not need special culture phases. The main micropropagation methods rely on growth of already formed meristems. Long-term storage of micropropagated material, cryopreservation, is well-developed to maintain germplasm. The main method is vitrification using the cryoprotectant mixture PVS3.

Effects of population size on synchronous display of female and male flowers and reproductive output in two monoecious Sagittaria species.

BACKGROUND: Flowering synchrony and floral sex ratio have the potential to influence the mating opportunities and reproductive success through female function. Here, we examine the variances in synchronous display of female and male function, ratio of male to female flowers per day and subsequently reproductive output in small populations of two monoecious plants, Sagittaria trifolia and Sagittaria graminea. METHODOLOGY/PRINCIPAL FINDING: We created plant populations of size 2, 4, 10 and 20 and recorded the daily number of blooming male and female flowers per plant to determine daily floral display, flowering synchrony index and ratio of male to female flowers per day. We also harvested the fruits, counted the seeds and calculated the number of fruits and seeds per flower to measure reproductive success through female function. There is less overlap in flowering time of female and male function in smaller populations than in larger populations. Most importantly, we found that male-biased floral sex ratio and imbalanced display period of female and male function for individual plant can lead to a population-size-dependent ratio of male to female flowers per day. Increasing ratio of male to female flowers per day was generally associated with a greater percentage of fruit production. CONCLUSIONS/SIGNIFICANCE: Our results highlight the importance of flowering synchrony of female and male function and population-size-dependent ratio of male to female flowers per day for female reproductive success. This finding improves our understanding of a mechanism that reduces reproductive success in small populations.

In vitro adventitious shoot regeneration via indirect organogenesis from inflorescence explants and peroxidase involvement in morphogenesis of Populus euphratica Olivier.

The inflorescences as explants for rapid propagation in vitro remained unknown in Populus euphratica Olivier. Here, we reported that multiple shoots were initiation from calli of both male and female inflorescences. The optimum medium for shoot induction from male inflorescences was lactose sulfite medium containing 1.0 mg L(-1) 6-benzylaminopurine (BA) and 0.5 mg L(-1) α-naphthalene acetic acid (NAA) or Murashige and Skoog (MS) medium containing 0.5 mg L(-1) BA and 0.2 mg L(-1) NAA. The optimum medium of shoot induction from female inflorescence calli was the MS medium containing 0.5 mg L(-1) BA and 0.2 mg L(-1) NAA. Rooting of regenerated shoots was obtained on 1/2 MS medium supplemented with 0.5∼1.0 mg L(-1) indole-3-butyric acid (IBA) and the highest frequency rooting was on medium containing 0.5 mg L(-1) IBA. No shoots were obtained on medium without BA and NAA. Peroxidase (POD) activity was measured by polyacrylamide gel electrophoresis during shoot induction and differentiation stages. The results showed that two bands of POD (2a and 2b) activity appeared lowest during the early 8 days at the dedifferentiation phase of leaves inducing calli, whereas POD 2a, 2b activity appeared to be increasing at the homeochronous dedifferentiation phase of inflorescence. Five most intensive bands, POD 1a, 1b, 1c, 2a, and ab, appeared in 8th and 28th days at the redifferentiation phase during shoot morphogenesis. These results demonstrated that the POD was involved in shoot morphogenesis from both leaf and inflorescence explants of Populus euphratica.

Floral features, pollination biology and breeding system of Chloraea membranacea Lindl. (Orchidaceae: Chloraeinae).

BACKGROUND AND AIMS: The pollination biology of very few Chloraeinae orchids has been studied to date, and most of these studies have focused on breeding systems and fruiting success. Chloraea membranacea Lindl. is one of the few non-Andean species in this group, and the aim of the present contribution is to elucidate the pollination biology, functional floral morphology and breeding system in native populations of this species from Argentina (Buenos Aires) and Brazil (Rio Grande do Sul State). METHODS: Floral features were examined using light microscopy, and scanning and transmission electron microscopy. The breeding system was studied by means of controlled pollinations applied to plants, either bagged in the field or cultivated in a glasshouse. Pollination observations were made on natural populations, and pollinator behaviour was recorded by means of photography and video. KEY RESULTS: Both Argentinean and Brazilian plants were very consistent regarding all studied features. Flowers are nectarless but scented and anatomical analysis indicates that the dark, clavate projections on the adaxial labellar surface are osmophores (scent-producing glands). The plants are self-compatible but pollinator-dependent. The fruit-set obtained through cross-pollination and manual self-pollination was almost identical. The main pollinators are male and female Halictidae bees that withdraw the pollinarium when leaving the flower. Remarkably, the bees tend to visit more than one flower per inflorescence, thus promoting self-pollination (geitonogamy). Fruiting success in Brazilian plants reached 60·78 % in 2010 and 46 % in 2011. Some pollinarium-laden female bees were observed transferring pollen from the carried pollinarium to their hind legs. The use of pollen by pollinators is a rare record for Orchidaceae in general. CONCLUSIONS: Chloraea membrancea is pollinated by deceit. Together, self-compatibility, pollinarium texture, pollinator abundance and behaviour may account for the observed high fruiting success. It is suggested that a reappraisal and re-analysis of important flower features in Chloraeinae orchids is necessary.