Effects of pollen selection on progeny vigor in a Cucurbita pepo x C. texana hybrid

We examined the effects of pollen selection for rapid pollen-tube growth on progeny vigor. First, we crossed a wild gourd (Cucurbita texana) to a cultivated zucchini (Cucurbita pepo cv Black Beauty) to produce an F₁ and then an F₂ generation. Half of the F₁ seeds were produced by depositing small loads of C. texana pollen onto the stigmas of C. pepo. These small pollen loads were insufficient to produce a full complement of seeds and, consequently, both the fast- and the slow-growing pollen tubes were permitted to achieve fertilization. An F₂ generation was then produced by depositing small loads of F₁ pollen onto stigmas of F₁ plants. The F₂ seeds resulting from two generations of small pollen loads are termed the non-selected line because there was little or no selection for pollen-tube growth rate on these plants. The other half of the F₁ and F₂ seeds were produced by depositing large pollen loads (>10 000 pollen grains) onto stigmas and then allowing only the first 1% or so of the pollen tubes that entered the ovary to fertilize the ovules. We did this by excising the styles at the ovary at 12–15 h after pollination. The resulting F₂ seeds are termed the selected line because they were produced by two generations of selection for only the fastest growing pollen tubes. Small pollen loads from the F₂plants, both the selected and the non-selected lines, were then deposited onto stigmas of different C. pepo flowers, and the vigor of the resulting seeds was compared under greenhouse and field conditions. The results showed that the seeds fertilized by pollen from the selected line had greater vegetative vigor as seedlings and greater flower and fruit production as mature plants than the seeds fertilized by pollen from the non-selected line. This study demonstrates that selection for fast pollen-tube growth (selection on the microgametophyte) leads to a correlated increase in sporophyte (progeny) vigor.     

Production of fertile tobacco pollen from microspores in suspension culture and its storage for in situ pollination

Summary A simple procedure is described for the in vitro production of tobacco (Nicotiana tabacum L.) pollen from microspores isolated just before entering mitosis. During a 3-day culture period in a liquid medium containing pyrimidine nucleosides these microspores develop into young pollen grains to the stage of starch deposition. Pollen maturation and transition to dormancy is achieved during a further 2- to 3-day culture period in the same medium stepwise supplemented by a concentrated solution of sucrose and l-proline. Upon transfer of the pollen to a simple germination medium containing sucrose and boric acid, up to 40% of the grains were observed to produce relatively long tubes. The in vitro-matured pollen grains can be stored at-20° C either suspended in 1.17 M sucrose and 100 mM l-proline or separated from the medium on filter paper discs. The stored pollen germinated both in vitro and on the stigma, the pollen tubes grew through the style into the ovary and pollination produced up to 300 viable seeds per pod. The procedure is of interest for pollen developmental studies and various fields of pollen manipulation, such as in vitro pollen selection.     

In-situ seed production after pollination with in-vitro-matured,isolated pollen

Immature tobacco (Nicotiana tabacum L.) pollen has been isolated from anthers in three distinct stages of development, including the microspore stage. In in-vitro cultures, fully functional, mature pollen was obtained. In a germination medium, this pollen produced pollen tubes. After application to stigmas in situ, the in-vitro-matured pollen fertilized ovules, and seeds were produced. Genetic tests with seedlings obtained from pollinations with in-vitro-matured pollen from a transgenic plant revealed normal Mendelian segregation of two marker genes, the neomycin-phosphotransferase II gene and the nopaline-synthase gene. These results are of interest with respect to the control of self-incompatibility, cytoplasmic male sterility and pollen-allergen formation, and it offers an alternative route for gene transfer in those plants which cannot be regenerated in vitro.Abbreviations cms cytoplasmic male sterility; AMGLU, MS, M2S, MR26 - GK culture media, see Material and methods     

In vitro pollen embryogenesis in Nicotiana tabacum L. and its relation to pollen sterility,sex balance,and floral induction of the pollen donor plants

Pollen sterility, sex balance, and floral induction of the pollen donor plants were tested for a possible relation to embryogenesis from in vitro cultured tobacco pollen (Nicotiana tabacum L. var. Badischer Burley). The pollen grains destined to become embryos in culture (P-grains) were sterile for the donor plants as judged by their staining reaction with acetocarmine and fluorescin-diacetate, and by an in vitro germination test. They were produced in high frequency in flowers which exhibited a shift in sex balance towards femaleness. Sex balance could be measured by the relative length of pistil to stamens. High P-grain frequency, high pollen sterility, and a shift in sex balance towards femaleness could be induced by raising the donor plants under short days and/or low temperature (18–15° C) as compared to long days at 24° C. Short days and/or low temperature also reinforced floral induction, revealing that the tobacco variety Badischer Burley is a quantitative short day and low temperature plant and that the variety follows the rule that conditions of strong floral induction shift sex balance towards femaleness. At 12° C and short days, contabescent flowers were formed with completely sterile anthers containing a few and mostly collapsed P-grains. Based on these results, it is now possible to predict conditions by which haploids via pollen embryogenesis might be produced in high frequency from low-yielding and recalcitrant species.Abbreviations DPF dead pollen grain frequency - LD24 long days at 24° C - PD pollen dimorphism - P:S ratio of pistil to stamen length - SD15 short days at 15° C     

Experimental analyses of the evolutionary processes inTrillium

In the Japanese species ofTrillium examined here, every interspecific cross produced healthy F₁ progeny at the rate of about 50 to 60 per cent of the total inborn ovules except forT. hagae. These figures were not appreciably different from those of self-pollinated or sib-mated examples of these species.Trillium hagae, a putative natural hybrid betweenT. kamtschaticum (2n=10) andT. tschonoskii (2n=20), accepted all foreign sources of pollen grains in manual pollination. These and other crossing experiments suggest that no appreciable barrier has developed that might cause interspecific crossability and that they are habitual inbreeders. Despite theri crossability, a very low rate of actual pollen flow was detected by examining electrophoretic phenotypes of seeds taken from sympatric populations of these species.     

Effects of soil phosphorus on pollen production,pollen size,pollen phosphorus content,and the ability to sire seeds in Cucurbita pepo (Cucurbitaceae)

To determine the effects of soil phosphorus on pollen production, pollen grain size, phosphate concentration per pollen grain, and the siring ability of pollen, two cultivars of the common zucchini (Cucurbita pepo) were grown under two soil phosphorus conditions in an experimental garden. Overall, soil phosphorus availability had a significant effect on reproductive output through the female function and on traits affecting the male function of plants (staminate flower production, pollen production per flower, and pollen grain size). In addition, pollen produced by plants in the high phosphorus soils had a higher phosphate concentration than pollen produced by plants in the low phosphorus soils. A pollen mixture experiment revealed that pollen produced by plants in the high phosphorus treatment sired significantly more seeds than pollen produced by plants in the low phosphorus treatment. This study showed that growing conditions such as soil phosphorus can influence the size of a pollen grain and its chemical composition, which, in turn, can affect its ability to sire mature seeds.     

Effects of pollen-synthesized green fluorescent protein on pollen grain fitness

Genetically engineered pollen with a visible marker gene could be useful to monitor the movement of transgenic pollen provided there are no negative physiological or fitness effects of expressing such a gene. In this study, we measured the fitness of Nicotiana tabacum cv. Xanthi pollen expressing the marker gene green fluorescent protein (GFP). Average pollen tube germination frequencies and pollen tube growth rates in vitro were measured in three different types of plants: (1) plants producing GFP in pollen cells only (LAT59-GFP), (2) plants synthesizing GFP under the control of a constitutive promoter (CaMV 35S) in which no GFP was produced in pollen, and (3) non-transgenic plants. Pollen synthesizing the GFP protein did not differ significantly in average pollen germination frequencies from pollen without GFP (P=0.65). Average pollen tube growth rates over a 5-h period did not differ significantly between transgenic and non-transgenic types (R²=0.89, 0.98, and 0.95, respectively, for GFP-tagged, 35S-GFP, and wild type). Overall, GFP expression in pollen grains of tobacco was not found to have an effect on pollen fitness under the controlled experimental conditions of this study.     

Selection for tolerance to copper during pollen formation in Mimulus guttatus Fischer ex DC

In Mimulus guttatus, copper tolerance is determined largely by a single gene and is expressed in both the sporophyte and microgametophyte. This study explores the extent to which selection during pollen formation affects copper tolerance in the sporophytic generation. Two sets of plants heterozygous for copper tolerance, produced by reciprocal crosses between different copper-tolerant or sensitive families, and the plant on which the original observations were based, were cloned and grown in control or copper-supplemented solutions. Pollen viability and the number of tolerant progeny produced in backcrosses to sensitive plants were compared. In addition, the effect of copper treatment on pollen viability in vitro was compared for plants tolerant, sensitive and heterozygous for copper tolerance. The extent to which in vitro pollen viability decreased in response to copper treatment corresponded to the copper tolerance of the pollen source. When grown with added copper, four of the five plants showed significant reductions in pollen viability, ranging from 18% to 48% of control values. The reductions in pollen viability were correlated with an increase in tolerant progeny (r= 0.679, p=0.004). Increases in tolerant progeny could be large, ranging from 119% to 170% of that of controls, but were usually smaller than was predicted from the reductions in viable pollen. In addition, plants derived from reciprocal crosses differed significantly in the extent to which pollen viability was decreased and sporophytic tolerance increased. Thus, while selection during pollen formation could increase sporophytic tolerance, sporophytic factors, perhaps including cytoplasmic or epigenetic ones, moderated the effectiveness of pollen selection for copper tolerance.     

Early in vitro flowering and seed production in culture in <Emphasis Type="Italic">Dendrobium</Emphasis> Chao Praya Smile (Orchidaceae)

Plantlets of Dendrobium Chao Praya Smile maintained in vitro were induced to flower, which produced viable seeds within about 11 months. A two-layer (Gelrite-solidified layer topped with a layer of liquid medium of the same volume and composition) culture system containing benzyladenine (BA) at 11.1 μM induced the highest percent of flowering (45%) in plantlets within 6 months from germination. The percentage of inflorescence induction was increased to 72% by pre-selecting morphologically normal seedlings prior to two-layer culture. Plantlets in culture produced both complete (developmentally normal but smaller than flowers of field grown plants) and incomplete flowers. Pollen and female reproductive organs of in vitro-developed complete flowers were morphologically and anatomically similar to flowers of field grown plants. In addition, 65% of the pollen grains derived from in vitro-developed flower were tetrad suggesting that regular meiosis occurred during microsporogenesis. The percentage of germination of pollen grains derived from in vitro-developed flowers and flowers of field grown plants, incubated on modified Knops’ medium for 8 days, were 18.2 and 52.8%, respectively. Despite a lower percentage of germination of the pollen grains derived from in vitro-developed flowers, flowers induced in culture could be self-pollinated and developed seedpods with viable seeds. Nearly 90% of these seeds developed into protocorms on germination in vitro. These seedlings were grown in culture and induced to flower in vitro again using the same procedure.     

Analysis of protein content in pollen loads produced in north-west Spain

An analysis was made of the protein content of pollen loads produced by the bees in a hive situated in Viana do Bolo (Ourense, north-west Spain), to establish whether or not the relative quantity of protein in the pollen of each plant species influences the preference made by the bee of the flowers that supply pollen to the hive. This analysis was performed on all types of pollen that formed more than 5% of the pollen spectrum. Pollen load samples were collected directly from the hive from March to September. Pollen loads were separated by colour, and their specific homogeneity was confirmed microscopically. The Bradford method has been used for protein extraction and spectrophotometry was used for the determination of protein content. The results show that the different pollen loads have high protein content. Pollen of the plant species that reached relatively higher percentages in the pollen spectrum are also those that have the highest protein content. These were Cytisus scoparius type, uncultivated Poaceae, Quercus robur type, Sanguisorba minor, Salix fragilis and Spergularia rubra type. The pollen of the systematic units, which had pollen loads that could be identified at the level of species, maintained a constant value of protein content independently of the date the samples were obtained. The pollen of the systematic units, which had pollen loads that could be identified at the level of pollen type, has varied in protein content in the analyses performed on samples obtained on different dates. This result is due to the fact that the different species that integrate the pollen type flower on different dates, and thus have a pollenkitt with different characteristics.     

Effects of genotype on pollen performance in Cucurbita pepo

Summary This study examines the assumption of the pollen competition hypothesis that genetic differences among microgametophytes lead to differences in pollen performance and result in non-random fertilization. In addition, we examined the assumption that pollen performance is genetically correlated with sporophyte vigor due to an overlap in gene expression between the two stages of the life cycle. The results from a pollen mixture experiment in which two cultivars of common zucchini were used show that the ability to sire seeds is nonrandom with respect to the cultivar of the pollen donor plant. The proportion of the progeny sired by the two cultivars is not independent of the region of the fruit where the seeds are produced. The progeny sired by the yellow cultivar outperformed the progeny sired by the green cultivar in a greenhouse study. In addition, the progeny sired by the yellow cultivar from the stylar region of the fruit germinated faster and had more leaf area than the progeny sired by the same cultivar from the peduncular end of the fruit. Thus, the most vigorous progeny are obtained from the stylar region of the fruit where the ovules are fertilized by the most vigorous microgametophytes.     

Movement herkogamy in Kosteletzkya virginica: Effect on reproductive success and contribution to pollen receipt and reproductive assurance

Herkogamy may act as a mechanism to reduce interference between the reproductive functions of female and male organs, but too great a distance between stigmas and anthers may also decrease the outcross pollen transfer and male and female fitness. Our goal was to quantify the effect of movement herkogamy in Kosteletzkya virginica on reproductive success and its contribution to pollen receipt and reproductive assurance. Field investigations and hand-pollination experiments were performed to test: (1) the relationship between herkogamy before curvature of un-pollinated lobes and reproductive success under pollinator exclusion; (2) the contribution of movement herkogamy to pollen receipt; and (3) the relationship between pollen load and seed set. We noted that variation in herkogamy before curvature of un-pollinated lobes did not affect the outcome of autogamous self-pollination measured by fruit set and mean number of seeds per fruit. Under natural conditions, 39% of pollen grains deposited per flower were from autogamous self-pollination via movement herkogamy resulting in contact between anthers and stigmas. The relationship between the amount of pollen grains and the number of seeds per capsule was highly significant. Flowers producing four or five seeds received more pollen grains than those producing only one seed. Our data provides experimental evidence that movement herkogamy, which decreases the separation between anthers and stigmas and results in delayed self-pollination, contributes to pollen receipt and further enhances seed set when pollinators are scarce or absent.     

Appearance and interaction of pollen and pistil pathway proteins in Gasteria verrucosa (Mill.) H. Duval

Patterns of proteins excreted during pollen germination, of germinating pollen and of the stylar and placental fluids excreted by cells along the pistil pathway of Gasteria verrucosa were analyzed by electrophoresis. The medium from germinating pollen contains several pollen exudate proteins as well as glycoproteins from the sticky pollen wall coating. No protein could be detected in the stigmatic exudate. The stylar fluid shows a protein pattern different from that of the placental fluid. The placental fluid contains some glycoproteins. After pollination, three pollen proteins begin to appear in the stylar fluid. Two of these pollen proteins remain present in the placental fluid. Some placental fluid proteins and glycoproteins are modified after pollination. The difference in protein patterns demonstrates the heterogeneity of proteins in the pollen tube pathway and suggests that proteins excreted by the pollen tube interact with other proteins in the pistil pathway, especially those in the placental fluid.     

Spatial Segregation of the Sexes and Nutrients affect Reproductive Success in a Dioecious Wind-pollinated Grass

In many dioecious plant species in which spatial distributions of males and females have been examined, the sexes are spatially segregated – usually along an environmental gradient. Unless pollen is uniformly distributed in a population, spatial segregation of the sexes should reduce the average mating success of individuals. In three Californian populations of Distichlis spicata – a wind-pollinated grass species that exhibits spatial segregation of the sexes – I examined patterns of pollen movement and the effects of pollen load and nutrient availability on seed set to determine whether spatial segregation of the sexes actually reduces mating success for both males and females. In two of the populations, pollen dispersal was restricted, and pollen augmentation consistently, significantly increased seed set. However, in the third population – which had the lowest seed set – I found that although there were some indications of pollen limitation, pollen dispersal was not restricted, and seed production was limited primarily by nutrient availability. These results imply that in some populations of D. spicata nutrient limitation on the production of seeds by females may be sufficiently strong that spatial segregation of the sexes causes a fairly low cost to reproductive success compared with a more random distribution of the sexes. However, in other populations, pollen does limit mating success, and the spatial segregation of males and females in these populations is reducing the fecundity of both males and females.     

Pollen proteins after two-dimensional gel electrophoresis and pollen morphology of the amphiploids Aegilops kotschyi and Ae. variabilis with Secale cereale

Proteins from pollen of parent forms and amphiploids Aegilops variabilis ×Secale cereale and Ae. kotschyi×S. cereale, obtained by in vitro propagation or colchicine treatment of F₁ hybrids, were subjected to a study by two-dimensional (2-D) electrophoresis. Qualitative and quantitative diversities of protein patterns were revealed for the amphiploid pollen. The majority of peptides found in the parent forms were also present in the patterns of the amphiploid pollen; however, some of the parent-form-peptides were not expressed and proteins characteristic only of the amphiploids appeared. In the 2-D combined protein pattern obtained for the parent forms, amphiploids Ae. variabilis × S. cereale produced pollen with a poorer spectrum of proteins. In amphiploid 408B, obtained from treated the F₁ generation with colchicine, the 2-D pattern revealed the presence of less than 50% of the proteins recorded for the parent forms. Pollen grain morphology was studied under a scanning microscope. The structure and shape of exines differed from those of the parents. In the parent forms the pollen grains had only one pore, while in amphiploid pollen, one, two or three pores were observed. Possible explanations for the differences in the 2-D patterns of amphiploids and their parent forms (impoverishment of the protein spectrum and appearance of new peptides) are (1) somaclonal variation and mutagenic activity of colchicine, (2) suppression of structural genes, (3) activity of regulators and (4) translocations. Pollen grains with two or even three pores could appear as a result of the independent activity of the genes from three amphiploidal genomes. Received: 6 March 2001 / Accepted: 26 June 2001     

The relationship between pollen grain size and progeny gender in dioecious Silene latifolia (Caryophyllaceae)

Summary Experiments and observations conducted during the past 90 years have provided conflicting evidence concerning the existence of a size difference between pollen grains containing an X chromosome (female-determining) and those containing a Y chromosome (maledetermining) in dioecious Silene latifolia. Were such a size difference to exist, this might explain, at least in part, the observation that X-bearing pollen tubes reach the ovary more quickly, on average, than Y-bearing pollen tubes. We tested for such a size difference by separating pollen collected from single anthers into three size classes: small, large, and random. Fruit set (number of pollinated flowers that set fruit) and seed set (number of seeds per capsule) did not differ for these three pollination treatments. Progeny sex ratios resulting from these three pollen size classes also did not differ significantly. Thus, pollen grain size is not affected by which of the two sex chromosomes is present. Our experiment is the first direct test of this relationship. Based on our results, size differences should not be invoked to explain competitive differences in male- and female-determining microgametophytes.     

Pollen flow of cultivated rice measured under experimental conditions

The pollen flow pattern of a cultivated rice variety, Minghui-63, was studied at horizontal and vertical levels under experimental conditions. Data obtained from pollen traps for six designed populations (as pollen sources) at different intervals showed that the dispersal of rice pollen decreased with the increase of distance from pollen sources and that the rice pollen flow was significantly influenced by weather conditions, particularly by wind direction and speed. For a mean wind speed of 2.52 m/s in a downwind direction, the observed distance of rice pollen dispersal was 38.4 m, indicating that rice pollen grains normally disperse at a relatively small range. However, the maximum distance of rice pollen flow could be up to 110 m, using regression analysis of pollen flow and wind speed, when the wind speed reached 10 m/s in this study. The frequency of pollen flow was positively correlated with pollen source size within a given range, suggesting that pollen flow will occur effectively at a considerable rate in rice fields with sufficiently large pollen sources. In addition, many more pollen grains were detected at the height of 1.0–1.5 m than at 2.0 m, indicating that rice pollen mainly disperses at relatively low heights. Results from this study are useful both for minimizing transgene escape from transgenic rice and in situ conservation of wild relatives of rice, as well as for hybrid seed production, where an effective isolation buffer zone needs to be established.     

Effects of pollination intensity on the vigor of the sporophytic and gametophytic generation of Cucurbita texana

 We examined the effects of pollen competition (pollen load size) on sporophytic vigor and gametophytic performance in Cucurbita texana, a wild gourd, while controlling for alternative interpretations of the data. Under field conditions we compared the vigor of progeny produced from large and small pollen loads and examined the in vitro performance of the pollen produced by the progeny. We found that the progeny from large pollen loads germinated faster and had a greater reproductive output (male flowers and fruits) than progeny produced from small pollen loads. In addition, we found that the pollen produced on plants derived from large pollen loads grew faster in vitro than the pollen produced on plants derived from small pollen loads. These findings indicate that pollen competition affects the performance of the resulting sporophytic generation and the microgametophytes they produce. Received: 26 January 1997 / Revision accepted: 25 June 1997     

A histological comparison of the development of pollen and female gametophytes in fertile and sterile Cryptomeria japonica

To determine a possible mechanism causing male and female sterility in Cryptomeria japonica male and female cones were collected from a C. japonica, tree, ShinDai2, that lacks pollen release and fertile seeds and specimens were processed to examine the development of pollen and female gametophytes using light microscopy and field emission scanning electron microscopy. Pre-meiotic development proceeded normally, but the formation of aberrant meiotic products was observed in cones of both sexes. In sterile microsporangia, heterogeneous microspore populations ranging from monads to polyads gave rise to mature pollen grains of non-uniform size. These pollen grains were covered with an amorphous layer and adhered to each other. In addition, they remained in the microsporangia and were not released even after the onset of pollen dissemination from fertile trees. In the ovules of sterile female cones, megaspores with abnormal shapes, numbers, and sizes formed, and the development of female gametophytes was arrested at the free nuclear or archegonium formation stages. These gametophytes collapsed, and no fertile embryo was generated. Results indicate that meiotic defects are important in the sterility mechanism.     

The reproductive assurance benefit of selfing: importance of flower size and population size

Autonomous selfing can provide reproductive assurance (RA) for flowering plants that are unattractive to pollinators or in environments that are pollen limited. Pollen limitation may result from the breakdown of once-continuous habitat into smaller, more isolated patches (habitat fragmentation) if fragmentation negatively impacts pollinator populations. Here we quantify the levels of pollen limitation and RA among large and small populations of Collinsia parviflora, a wildflower with inter-population variation in flower size. We found that none of the populations were pollen limited, as pollen-supplemented and intact flowers did not differ in seed production. There was a significant effect of flower size on RA; intact flowers (can self) produced significantly more seeds than emasculated flowers (require pollen delivery) in small-flowered plants but not large-flowered plants. Population size nested within flower size did not significantly affect RA, but there was a large difference between our two replicate populations for large-flowered, small populations and small-flowered, large populations that appears related to a more variable pollination environment under these conditions. In fact, levels of RA were strongly negatively correlated with rates of pollinator visitation, whereby infrequent visitation by pollinators yielded high levels of RA via autonomous selfing, but there was no benefit of autonomous selfing when visitation rates were high. These results suggest that autonomous selfing may be adaptive in fragmented habitats or other ecological circumstances that affect pollinator visitation rates.