Characterization of <Emphasis Type="Italic">HoMADS 1</Emphasis> and its induction by plant hormones during <Emphasis Type="Italic">in vitro</Emphasis> ovule development in <Emphasis Type="Italic">Hyacinthus orientalis</Emphasis> L.

To understand the molecular mechanism of ovule development, a MADS box gene,HoMADS 1, has been isolated from the ovule tissues of Hyacinthus. Sequence comparison showed that HoMADS 1 is highly homologous to both class C and D genes. Furthermore, phylogenetic analysis suggests that HoMADS 1 is most likely a class D MADS box gene. RNA hybridization revealed that HoMADS 1 was exclusively expressed in the ovules. Over-expressing HoMADS 1 in transgenic Arabidopsis plants produced ectopic carpelloid structures, including ovules, indicating that HoMADS 1 is involved in the determination of carpel and ovule identities. Interestingly, during in vitro flowering, no HoMADS 1 mRNA was detected in the floral tissues at high level hormones in the media. However, HoMADS 1 mRNA accumulated in the floral tissues when the regenerated flowers were transferred to the media containing low level hormones which could induce in vitro ovule formation. Our data suggest that the induction of HoMADS 1 by plant hormones may play important roles during ovule initiation and development in the regenerated flower. Whether HoMADS 1 expression is also regulated by cytokinin and auxin during ovule development in planta remains to be investigated.     

Impact of volcanic activity on a plant-pollinator module in an island ecosystem: the example of the association of <Emphasis Type="Italic">Camellia japonica</Emphasis> and <Emphasis Type="Italic">Zosterops japonica</Emphasis>

Volcanic activity provides an indispensable opportunity to study the ecological responses of organisms to environmental devastation. We examined the reproductive success of Camellia trees to identify how volcanic activity affects the processes of leaf survival, flowering activity, fruit and seed production, pollinator abundance, pollinator visitation frequency, pollination rate, and fruit and seed maturation at different damage sites on Miyake-jima, which experienced an eruption in the summer of 2000. Volcanic gases negatively affect leaf survival and reduce flowering activity in heavily damaged areas. Pollen transfer was sufficient to ensure that higher pollination rates (83%) occurred in heavily damaged areas than in less damaged areas (26–45%), but pollinator densities were lower in response to reduced flower resources. Fruit abortion rates were greater in heavily damaged sites (78%) than in less-damaged sites (53–63%). Consequently, fruit-set rates (16–29%) did not differ significantly among sites. Seed set rates tended to increase with increasing volcanic damage. The negative correlation between seed-set rates and seed mass suggests that the decreased seed mass in severely damaged sites was attributable to the better pollination rates observed there. These results indicate that compensation mechanisms ensure better reproductive success at sites that are more strongly affected by volcanic activity.     

Floral herbivory at different stages of flower development changes reproduction in <Emphasis Type="Italic">Iris gracilipes</Emphasis> (Iridaceae)

Floral herbivores and pollinators are major determinants of plant reproduction. Because interaction of floral herbivores and pollinators occurs when herbivores attack the flowers in the bud and flower stages and because the compensatory ability of plants is known to differ according to the timing of herbivory, the effects of herbivory may differ according to its timing. In this study, we investigated the effects of floral herbivory at different stages on fruit production and seed/ovule ratio at two sites of large populations of the perennial herb, Iris gracilipes for 2 years. Herbivory at the bud and fruit stages both tended to have negative effects on fruit production, but the former caused more severe damage. On the other hand, herbivory at the flower stage tended not to have negative effects on fruit production because the degree of flower loss was smaller in the flower stage. Although herbivory decreased fruit production, flowers did not compensate for the damage by increasing the seed/ovule ratio because reproduction of I. gracilipes was limited by pollen availability rather than resources. These results indicate that floral herbivory at different stages has different effects on plant reproduction.     

Size-dependent sex allocation in a monocarpic perennial herb, <Emphasis Type="Italic">Cardiocrinum cordatum</Emphasis> (Liliaceae)

The size-dependent sex allocation model predicts that the relative resource allocation to female function often increases with plant size in animal-pollinated plants. If size effects on reproductive success vary depending on the environmental conditions, however, the size dependency may differ among populations. We tried to detect site-specific variation in size-dependent sex allocation of a monocarpic hermaphrodite with reference to light availability. Multiple flowers and fruits were sampled from the individuals of Cardiocrinum cordatum, a monocarpic understory herb, and pollen, ovule and seed production were measured with reference to the plant size in two populations. Furthermore, frequency and foraging behavior of pollinator visitation was observed. Ovule production per flower increased with plant size in both populations, while pollen production per flower increased with size only in the population under sparse canopy. Therefore, proportional allocation to male function decreased with plant size in the population under closed canopy, but did not change in the population under sparse canopy. Pollinators usually visited only one flower per plant, indicating the negligible geitonogamous pollination in this species. Although seed production under closed canopy was lower than that under sparse canopy, seed-set rate per flower and seed mass per fruit were independent of plant size in either of the populations. Size-dependent sex allocation in this species was site-specific, suggesting that not only resource storage before reproduction (i.e., plant size) but also resource availability of environment throughout the reproductive process (i.e., light availability) affect reproductive performance in this species.     

<Emphasis Type="Italic">In vitro</Emphasis> germination and axillary shoot propagation of <Emphasis Type="Italic">Centaurea zeybekii</Emphasis>

In vitro culture is an important aid for ex situ conservation of rare, endemic or threatened plants. In this work, we establish an efficient method for the seed germination, seedling development, and axillary shoot propagation of Centaurea zeybekii Wagenitz. The seeds, collected from a wild population, were surface sterilised and cultured on various in vitro germination media. The effects of photoperiod and temperature on seed germination were also investigated. Germinations were obtained after 6 weeks in culture and the radicle emergence was evaluated as a main indicator. A high frequency of germination was obtained on distilled water supplemented with vitamines and 1 mg/L GA₃. Although the seed germination frequencies were not affected by photoperiod, the highest germination frequency was obtained at 24 ± 2°C. A high frequency of axillary shoot proliferation was produced on MS medium supplemented with 1 mg/L BA. Then, the axillary shoots were separated and transferred to MS medium with or without plant growth regulators for rooting. Rhizogenezis was promoted after 6 weeks only in MS and 1/2 MS media containing 0.5 mg/L IBA. The rooting process was very slow and the percentage of shoot rooting was also very low (15%). The present study not only enables reinforcement of wild plant populations using ex situ growth of individuals, but it also helps to large number of aseptic seedling to use it in clonaly micropropagation studies.     

<Emphasis Type="Italic">Gypsy embryo</Emphasis> specifies ovule curvature by regulating ovule/integument development in rice

The embryo position in a seed is stable in most plant species, indicating the existence of a strict regulatory mechanism that specifies the embryo position in the seed. To elucidate this mechanism, we analyzed the gypsy embryo (gym) mutant of rice, in which the position of the mature embryo in the seed is altered at a low frequency. Analyses of early embryogenesis and ovule development showed that the ectopic embryo was derived from an ill-positioned egg cell, which resulted from the incomplete curvature of the ovule. Although the development of both the inner and outer integuments was impaired, the ovule curvature was associated closely with the extent of inner integument growth. Therefore, inner integument development controls ovule curvature in rice. The expression patterns of OSH1 and OsMADS13 indicated that, in gym, a small number of indeterminate cells are maintained on the style side of the ovule and then in the integument primordium at a low frequency. The prolonged survival of these indeterminate cells disturbs normal integument development. The gym fon2 double mutant suggests that GYM and FON2 are involved redundantly in floral meristem determinacy. Possible functions of the GYM gene and the ovule developmental mechanism are discussed.     

<Emphasis Type="Italic">SQUA</Emphasis>-like genes in the orchid <Emphasis Type="Italic">Phalaenopsis</Emphasis> are expressed in both vegetative and reproductive tissues

The SQUA family (AP1/FUL family) of MADS-box genes plays an important role in the transition from the vegetative to the reproductive development of angiosperms, and its origin might be concurrent with fixation of floral structure in angiosperms. Here, we isolated two Phalaenopsis MADS-box genes designated ORAP11 and ORAP13, both of which belong to the monocot FUL-like clade of the SQUA family. RT-PCR showed that both genes are strongly expressed in the floral bud, and also detected in the vegetative organs. During later stages, ORAP11 was only detected in the column, but ORAP13 signal was absent from all of the floral organs. In-situ hybridization experiments detected both genes in the tips and margins of developing petals and lips, the developing column, and ovule. Over-expression of both genes in tobacco induced early flowering and changed plant architecture. Our results suggest that in Phalaenopsis, both genes might share partly redundant activities and play important roles in the process of floral transition and morphological architecture. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Dry environments promote the establishment of females in monomorphic populations of <Emphasis Type="Italic">Wurmbea biglandulosa</Emphasis> (Colchicaceae)

In flowering plants, the evolution of dimorphic breeding systems from monomorphic ancestors can be associated with dry environments. One hypothesis to explain this pattern is that seed fertility of hermaphrodites decreases more than seed fertility of females under dry conditions, so that females have greater relative fitness. This could occur if seed production of hermaphrodites is more resource-limited than that of females, or shifts in pollination increase levels of selfing and inbreeding depression in hermaphrodites. Here we assess the role of dry environments in promoting a female fitness advantage in Wurmbea biglandulosa by focusing on monomorphic and dimorphic populations that occur along a longitudinal gradient of decreasing rainfall. Dimorphic populations occurred in sites with higher temperatures, lower rainfall and lower soil moisture. Overall, females had greater seed fertility than did hermaphrodites from monomorphic populations, which in turn had greater seed fertility than hermaphrodites from dimorphic populations. Ovuliferous flower and ovule production by the three gender morphs and seed fertility of females and hermaphrodites in monomorphic populations did not vary with soil moisture. By contrast, seed fertility of hermaphrodites in dimorphic populations was positively related to soil moisture. Accordingly, female frequency was higher in those sites where hermaphrodites produced relatively fewer seeds. Taken together our results indicate that dry environments promote the establishment of females by decreasing the relative seed fitness of hermaphrodites. Moreover, because seed fertility of hermaphrodites in monomorphic populations did not vary with soil moisture, resource limitation of female function may play only a minor role in the establishment of females. Other factors such as shifts in pollination and mating patterns of hermaphrodites could be involved. Key words:breeding system evolution, environmental stress, gender dimorphism, gynodioecy, sex ratio variationCo-ordinating editor: J.F. Stuefer     

In search of the best pollinators for canola (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) production in Pakistan

To discover the pollinator community of canola (Brassica napus L.) and the best pollinators for canola production, an experiment was performed at the research farm of Bahauddin Zakariya University, Multan, Pakistan. The insect pollinator community was composed of 35 species in 3 orders and 14 families. Most of the bees (Hymenoptera) and a butterfly species (Lepidoptera) foraged for nectar, whereas all the flies (Diptera) foraged either for pollen or both nectar and pollen. Eight major pollinators were tested for their pollination efficiency. The nectar-robbing behavior of many species made it difficult to judge the efficiency of an insect on the basis of visitation rate and stay time; therefore, the amounts of pollen deposited and pollen harvested per visit were also measured. The single visit efficiency in terms of the number of seeds per pod revealed that Apis dorsata, A. florea, and Halictus sp. were superior for canola pollination, having Spears’ values of 1.62, 1.55 and 1.73, respectively. With the increase in the number of seeds per pod, seed weight per pod also increased, confirming the importance of these three pollinator species in canola production.     

The interplay of pollination,costs of reproduction and plant size in maternal fertility limitation in perennial <Emphasis Type="Italic">Paeonia officinalis</Emphasis>

Although several factors can limit female fertility in perennial plants, rarely have they been jointly studied in a single species over several years. In this study we experimentally manipulate seed production and simultaneously analyse the potential contribution of pollen limitation, costs of reproduction and plant size to variation in seed output over a 3-year period in the perennial herb Paeonia officinalis, in southern France. Since this rare species is threatened by forest closure in many sites we also examine the causes of female fertility variation in relation to habitat closure (open habitat vs. woodland). P. officinalis has a partial self-incompatibility system and only very low ability for autonomous self pollination in the absence of pollinators. However, supplementary pollination of individual plants in three consecutive years did not significantly increase seed production above natural levels. Forest closure was associated with a decline in ovule and seed production, which again was not due to pollen limitation since supplementary pollination had no significant effect on seed set in the woodland habitat. Comparison of the maternal fertility of plants which were previously excluded from reproduction with those which were hand pollinated to maximise seed set in two previous years produced no evidence that seed production in year three is limited by costs associated with prior reproduction. Likewise, flowering probability was not related to prior seed production but was however positively related to plant size. The absence of any influence of pollen limitation or prior reproduction on seed production suggests that sub-maximal seed production in long-lived perennial herbs may be part of a size-dependent strategy that maximises life-time seed production and fitness without compromising survival.     

Fruit abortion in the Chihuahuan-Desert endemic cactus <Emphasis Type="Italic">Opuntia microdasys</Emphasis>

Resource and pollen limitation, as well as pollen/ovule incompatibility, have been proposed as causes to explain fruit abortion. To assess whether abortion in Opuntia microdasys was due to resource and/or pollen limitation and could therefore be reversed fruit set and seed set were studied using controlled pollination experiments on 60 plants that had been randomly assigned a combination of watering and fertilization treatments. On the other hand, to test whether fruit abortion was irreversible, due to pollen/ovule incompatibility, we examined the reproductive biology of the species. This included observations on floral phenology, nectar production, flower visitors, numbers of pollen grains and ovules, and self-pollination experiments. Results showed that O. microdasys is a fully self-incompatible species and its floral biology and the activity of the main pollinator allow constant deposition of incompatible pollen onto stigmas, which may contribute to fruit abortion. Reproductive success was limited by nutrients and pollen, but the fruit set increased only by 58%, compared to 47% of the control, after the experimental addition of pollen, nutrients and water. The magnitude of pollen and resource limitation suggests that similar levels of abortion will be present in good as well as in bad years. Selfing as well as incompatibility between ramets from the same clone and between closely related plants seem plausible candidates to explain the large proportion of fruit abortion, and experimental cross pollination between genotypes identified through molecular markers are necessary to fully understand the considerable abortion rate that remains unexplained after pollen and resource addition. Interestingly, the possible reason why the abortion of energetically expensive fruits has not been eliminated by natural selection is that the aborted fruits are propagules able to root and produce new plants with the same genotype of the mother. Abortion would have a dramatic effect on cross-fertilized genotypes because they result in zero fitness, but it would have a positive effect on the fitness of the maternal genotype because a clonal offspring is produced. Evidently, the exact fitness consequences to the maternal plant will depend on the differences in survival and reproduction of these different offspring types.     

Abortive embryogenesis in hybrid swarm populations of <Emphasis Type="Italic">Pinus sylvestris</Emphasis> L. and <Emphasis Type="Italic">Pinus muga</Emphasis> Turra

Comparative study on fertilization process in Pinus sylvestris, Pinus mugo and in their putative hybrid swarm individuals was done involving pre-zygotic and post-zygotic stages. The amount of surviving ovules from open pollination reflecting the mode of interaction between pollen grains and nucellar tissue of an ovule averaged at 8.1 of sound ovules per conelet in Pinus sylvestris, 7.3 ovules in the hybrid swarm population and at 4.9 ovules in Pinus mugo. A strong correlation was observed between the number of surviving ovules and the proportion of germinating seeds in the compared species and hybrids. Normal course of embryogenesis in Pinus sylvestris and Pinus mugo contrasted with increased frequency of disturbances observed in the hybrid swarm individuals. The differential survival rates of the ovules and deviations from typical pattern of embryogenesis are discussed from the standpoint of cross-ability relationship between Pinus sylvestris and Pinus mugo.     

Breeding system of the annual Cruciferae, <Emphasis Type="Italic">Arabidopsis kamchatica</Emphasis> subsp. <Emphasis Type="Italic">kawasakiana</Emphasis>

The breeding system of an annual Cruciferae, Arabidopsis kamchatica subsp. kawasakiana, was studied in three natural populations. We applied four experimental treatments, open pollination, bagging, emasculation + bagging, and emasculation + hand-pollination + bagging. None of the emasculated flowers with bags produced fruits but we observed high fruit sets in the other three treatments. The results confirmed that A. kamchatica subsp. kawasakiana is a self-compatible, non-apomictic species that can produce seeds through auto-pollination. Considering the life cycle as an annual, increased reproductive assurance through auto-pollination should be critical for the maintenance of populations of A. kamchatica subsp. kawasakiana.     

Embryo abortion and histological features in the interspecific cross between <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L. and <Emphasis Type="Italic">P. coccineus</Emphasis> L

In this paper, the causes of early embryo abortion in the reciprocal crosses between Phaseolus vulgaris L. (a cultivar) and Phaseolus coccineus L. (a wild form) were studied. Methacrylate resin sections, 3–5 μm thick, of 3 to 14 day-old seeds were used to examine the embryo developmental stages and the state of seed tissue. It was observed that, embryos aborted at different developmental stages (globular to early cotyledon) depending on the maternal parent. The use of P. coccineus cytoplasm resulted in a higher number of abortion than in reciprocal crosses. Many of them took place between 5 and 6 days after pollination (DAP). Histological analyses permitted to observe that the embryo development was slower in the cross between P. coccineus and P. vulgaris, compared to parental seeds. It would be related to a deficient endosperm development in reciprocal crosses and, in some extent, hypertrophy of the suspensor might be the main cause of early embryo abortion. Then, it would be practical to overcome this incompatibility by rescuing the embryo at the globular stage of development.     

Effects of defoliation by horse chestnut leafminer (<Emphasis Type="Italic">Cameraria ohridella</Emphasis>) on reproduction in<Emphasis Type="Italic"> Aesculus hippocastanum</Emphasis>

In large parts of Europe horse chestnut trees ( Aesculus hippocastanum) suffer from severe defoliation by an alien invasive species, the specialist leaf mining moth Cameraria ohridella (Lepidoptera; Gracillariidae). Heavily infested trees have a drastically shortened period for photosynthesis. We quantified the effect of moth infestation on reproduction of horse chestnut trees in two different cities in central Europe, Bern and Munich. C. ohridella negatively affected seed and fruit weight of A. hippocastanum at both locations. In Munich, seed weight of heavily damaged trees was reduced by almost half. However, the number of seeds per fruit, the number of fruits per inflorescence, and the number of inflorescences per tree did not change due to herbivory. We conclude that C. ohridella mining affects seed quality but not seed quantity. The reduced seed weight may severely impair growth and survival of horse chestnut seedlings and thus may endanger the long-term persistence of A. hippocastanum in its endemic forests in south-east Europe.     

Co-dependency between a specialist <Emphasis Type="Italic">Andrena</Emphasis> bee and its death camas host, <Emphasis Type="Italic">Toxicoscordion paniculatum</Emphasis>

Among associations of plants and their pollinating bees, mutually specialized pairings are rare. Typically, either pollen specialist (oligolectic) bees are joined by polylectic bees in a flowering species’ pollinator guild, or specialized flowers are pollinated by one or more polylectic bees. The bee Andrena astragali is a narrow oligolege, collecting pollen solely from two nearly identical species of death camas (Toxicoscordion, formerly Zigadenus). Neurotoxic alkaloids of these plants are implicated in sheep and honey bee poisoning. In this study, T. paniculatum, T. venenosum and co-flowering forbs were sampled for bees at 15 sites along a 900-km-long east–west transect across the northern Great Basin plus an altitudinal gradient in northern Utah’s Bear River Range. Only A. astragali bees were regularly seen visiting flowering panicles of these Toxicoscordion. In turn, this bee was never among the 170 bee species caught at 17 species of other prevalent co-occurring wildflowers in the same five state region (38,000 plants surveyed). Our field pollination experiments show that T. paniculatum is primarily an outcrosser dependent on pollinator visitation for most capsule and seed set. Thus, both A. astragali and two sister species of Toxicoscordion are narrowly specialized and co-dependent on each other for reproduction, illustrating a rare case of obligate mutual specialization in bee–plant interactions.     

Additive effects of herbivory,nectar robbing and seed predation on male and female fitness estimates of the host plant <Emphasis Type="Italic">Ipomopsis aggregata</Emphasis>

Many antagonistic species attack plants and consume specific plant parts. Understanding how these antagonists affect plant fitness individually and in combination is an important research focus in ecology and evolution. We examined the individual and combined effects of herbivory, nectar robbing, and pre-dispersal seed predation on male and female estimates of fitness in the host plant Ipomopsis aggregata. By examining the effects of antagonists on plant traits, we were able to tease apart the direct consumptive effects of antagonists versus the indirect effects mediated through changes in traits important to pollination. In a three-way factorial field experiment, we manipulated herbivory, nectar robbing, and seed predation. Herbivory and seed predation reduced some male and female fitness estimates, whereas plants tolerated the effects of robbing. The effects of herbivory, robbing, and seed predation were primarily additive, and we found little evidence for non-additive effects of multiple antagonists on plant reproduction. Herbivory affected plant reproduction through both direct consumptive effects and indirectly through changes in traits important to pollination (i.e., nectar and phenological traits). Conversely, seed predators primarily had direct consumptive effects on plants. Our results suggest that the effects of multiple antagonists on estimates of plant fitness can be additive, and investigating which traits respond to damage can provide insight into how antagonists shape plant performance.     

Exploring the boundary between pollination syndromes: bats and hummingbirds as pollinators of<Emphasis Type="Italic"> Burmeistera cyclostigmata</Emphasis> and<Emphasis Type="Italic"> B. tenuiflora</Emphasis> (Campanulaceae)

In this study I documented the degree of specialization in the pollination systems of Burmeistera cyclostigmata and B. tenuiflora (Campanulaceae) to explore the potential role of floral isolation in the diversification of the genus. I asked which floral characteristics are important in specializing on either bat or hummingbird pollination, and whether overlap between these floral syndromes can exist. I examined nocturnal and diurnal pollen deposition, pollinator visitation rates, and single visit effectiveness and related them to intra- and interspecific variation in Burmeistera floral characteristics at Monteverde, Costa Rica. Bats and hummingbirds visited both Burmeistera species, and bats pollinated both species. Owing to differences in floral morphology, however, hummingbirds effectively pollinated only B. tenuiflora. The generalized pollination system of B. tenuiflora demonstrates that there can be overlap in the boundary between ornithophily and chiropterophily, and that nectar production and timing of anthesis do not serve as barriers between these syndromes. The high intraspecific variation in floral color from green to red or purple did not correlate with either nocturnal or diurnal pollen deposition. Degree of flower accessibility did affect pollination; nocturnal pollen deposition significantly decreased as flowers become more obstructed. In Burmeistera, floral morphology and accessibility appear to be the most important floral characteristics for specialization at the boundary between ornithophily and chiropterophily.     

<Emphasis Type="Italic">In vitro</Emphasis> culture of the resurrection plant <Emphasis Type="Italic">Haberlea rhodopensis</Emphasis>

The small group of resurrection plants is a unique model which could help us in further understanding of abiotic stress tolerance. The most frequently used approach for investigations on gene functions in plant systems is genetic transformation. In this respect, the establishment of in vitro systems for regeneration and micro propagation is necessary. On the other hand, in vitro cultures of such rare plants could preserve their natural populations. Here, we present our procedure for in vitro regeneration and propagation of Haberlea rhodopensis – a resurrection plant species, endemic for the Balkan region.     

Inbreeding influences herbivory in <Emphasis Type="Italic">Cucurbita pepo</Emphasis> ssp. <Emphasis Type="Italic">texana</Emphasis> (Cucurbitaceae)

In a series of field experiments Diabrotica beetle herbivory was found to influence the magnitude of inbreeding depression in Cucurbita pepo ssp. texana, an annual monoecious vine. Beetles damage flowers and fruits and chew dime-sized holes in leaf tissue between major veins. Inbred plants were found to be more likely to be damaged by beetles and to have more leaves damaged per plant than outcrossed plants. A positive linear association was found between the coefficient of inbreeding and the magnitude of leaf damage, whereas a negative association was found between coefficient of inbreeding and several male and female fitness traits. When pesticides were used to control beetle herbivory, the interaction between coefficient of inbreeding and pesticide treatment was significant for fruit production and marginally significant for pollen quantity per anther. Therefore, the magnitude of inbreeding depression in C. pepo ssp . texana varies depending on the severity of beetle herbivory.