Cross Pollen Research Articles

Characterization and Breakdown of Self-Incompatibility in Field Bindweed (Convolvulus arvensis L.)

Field bindweed biotypes differ in glyphosate tolerance. Our previous studies have indicated quantitative genetic inheritance of this trait. In order to study the genetics of these differences we must be able to make sexual crosses, however, self-crossing and backcrossing are difficult due to self-incompatibility (SI) in field bindweed flowers. Therefore research was conducted to characterize and overcome the SI system in field bindweed. We determined SI to be of the multiallelic, sporophytic type based on observations of pollen-stigma interaction, and in the pattern of compatibility of F1 backcrosses to the parent biotypes. Pollen in incompatible crosses produced short pollen tubes that failed to penetrate the stigma surface. Backcrossing showed most F1 plants to be compatible with one parent, indicating dominance interactions between alleles. SI in field bindweed was successfully overcome by both bud pollination and heat treatment which exposed plants to 38 degrees C for 2 days before pollination, however, no seeds were produced. Treatment of stigma tips with a hot soldering iron for 2-3 s allowed abundant pollen tube growth from selfed pollen lying in a zone adjacent to the heat-injured area. This method resulted in production of viable selfed seed at rates equal to that observed in outcrosses.

Cryptic self‐incompatibility in tristylous Decodon verticillatus (Lythraceae)

There is growing evidence that many self-compatible plants control the level of self-fertilization with postpollination processes that give a siring advantage to cross pollen over self pollen through "cryptic self-incompatibility" (CSI). Previous marker-gene experiments with self-compatible, tristylous Decodon verticillatus (Lythraceae) have demonstrated a siring advantage to cross pollen, though the extent to which this advantage results from prezygotic discrimination vs. early acting inbreeding depression is not clear. Here, we provide evidence that prezygotic mechanisms are involved in this siring advantage by comparing pollen tube numbers at various times following cross- and self-pollination conducted in a natural population. In the 24 h following pollination, cross pollen yielded almost twice as many pollen tubes at various positions in the style compared to self pollen. After 36 and 48 h, the difference between pollen types had disappeared, suggesting that the advantage to cross pollen results from differences in the rate of pollen germination and;clor tube growth rather than pollen tube attrition. Comparison of tube numbers after legitimate vs. illegitimate cross-pollination did not reveal any difference, suggesting that D. verticillatus possesses CSI unrelated to heteromorphic self- and intramorph-incompatibility found in other heterostylous members of the Lythraceae. CSI resulting from differential pollen tube growth may minimize geitonogamous selfing when cross pollen is abundant, while maximizing fecundity when cross pollen is scarce due to local clonal spread.

Gene dispersal from transgenic crops

The risk of release of genetically modified oilseed rape (Brassica napus) was investigated in relation to interspecific gene flow with hoary mustard (Hirschfeldia incana). Microscopic studies showed polymorphism within the population of hoary mustard for pollen germination on oilseed rape flowers. The transgenic herbicide-resistant and a commercial cultivar of oilseed rape were not different for pollen behaviour and ovule fertilization. Pollen tube growth was slow and erratic in interspecific crosses. Fertilization efficiency of oilseed rape and hoary mustard pollen in interspecific crosses was 15% and 1.3%, respectively, of that in intraspecific crosses. This unequal efficiency in reciprocal crosses was confirmed by hybrid seed set in pods. There was no post-zygotic barrier to the development of hybrid embryos in hoary mustard pods. Up to 26 spontaneous hybrids per male sterile oilseed rape plant, and one per hoary mustard plant, were obtained in field experiments. Hybrids were identified by isozyme electrophoresis, morphology and cytology. All hybrids were triploid with 26 chromosomes, and had low fertility. They produced 0.5 seeds per plant after spontaneous backcrossing with hoary mustard. Some of these descendants were produced from unreduced gametes. Our results suggest that gene flow is likely to occur, but its actual frequency under crop growing conditions remains to be estimated.

Pollination of two dioecious plants:Mercurialis annuaL. (Euphorbiaceae) andBryonia dioicaL. (Cucurbitaceae)

Abstract Dioecy ensures cross pollination but pollen must travel from male to female plants, sometimes covering long distances. The present study concerns the reproductive strategy of two Italian dioecious plants: Mercurialis annua L. and Bryonia dioica L. The former is anemophilous and the latter is entomophilous; the latter has nectar and pollen as rewards. The distribution of pollen grains on the stigma is very different in the two species, reflecting the different types of pollination. In both species there is a number-dependent mechanism of pollen germination on the stigma. This mechanism could enhance male gametophytic competition.

No Evidence for Selective Fruit Abortion in the Australian Shrub Grevillea barklyana (Proteaceae)

Plants may increase the average quality of their seed crops by selectively aborting less vigorous progeny when resources are limited. Selective fruit abortion was examined in selfing and outcrossing populations of the Australian proteaceous shrub Grevillea barklyana. Inflorescences were pollinated with cross pollen, self pollen, or a combination of the two pollination types on different flowers. Fruit initiation and maturation were similar on self- and cross-pollinated inflorescences. Self-incompatibility indices indicated that all plants were fully self-compatible. When self and cross fruits competed for limited maternal resources on the same inflorescences, numbers of self and cross fruits did not deviate from a 50:50 ratio. This indicates that differences in the vigor of self and cross progeny were minimal, which may be a consequence of populations having a history of inbreeding. Most fruits aborted early in development, before substantial investment of resources. Results in the selfing and outcrossing populations were similar, indicating that neither selective abortion nor inbreeding depression can account for differences in mating patterns.

Ovule pre-emption and pollen limitation in a self-fertile perennial herb (Blandfordia grandiflora, Liliaceae).

The extent, frequency and causes of pollenlimited seed production were examined in partially selffertile populations of Blandfordia grandiflora for 2 years. Percentage seed set of open-pollinated plants (50-57%) did not differ within or between years, and was about 19% less than experimentally cross-pollinated plants (70-75%). Floral visits by honeybees did not differ through the flowering season and the number of pollen grains deposited on stigmas within 12 h of flowers opening exceeded the number of ovules per flower, indicating that the quality rather than the quantity of pollination limited seed set. Pollen limitation was caused by concurrent self- and cross-pollination and the subsequent abortion of some selfed ovules due to inbreeding depression. Natural seed set (55%) was intermediate between selfed (43%) and crossed (75%) flowers and was not increased when flowers that had been available to pollinators for 24 h were hand cross-pollinated, suggesting that ovules were already fertilized. Similarly, experimental pollination with both cross and self pollen within 24 h of flowers opening did not increase seed set relative to natural seed set, indicating that both cross- and self-fertilizations had occurred. In contrast, when selfing followed crossing by 48 h, or vice versa, seed set did not differ from crossed-only or selfed-only flowers, respectively, indicating that ovules were pre-empted by the first pollination. Collectively, these results indicate that under natural conditions self pollen pre-empts ovules, rendering them unavailable for cross-fertilization. This selfing reduces fecundity by 50%, as estimated from the natural production of cross seeds when selfing was prevented. Consequently, selection should favour floral traits, such as increased stigma-anther separation or protandry, that reduce interference between male and female functions that leads to selfing.

Postfertilization causes of differential success of pollen donors inErythronium grandiflorum (Liliaceae): nonrandom ovule abortion

Seed paternity in Erythronium grandiflorum does not fully reflect the proportion of pollen on the stigma. When two types of pollen are simultaneously applied to the stigma, outbred seeds are produced over inbred, and seeds from more distant donors are produced over seeds from donors nearby. I looked for postfertilization causes of these previously reported patterns of differential success of pollen donors. I simultaneously pollinated stigmas with pollen from two donors and observed ovule development through a window sliced in the ovary. Pollen donor pairs were self and cross, donors 1 and 100 m from the recipient, and two donors each 100 m from the recipient. Since one donor was always the alternate homozygote from the recipient at the malate dehydrogenase locus, I could determine the paternity of developing seeds. When it appeared that ovules were aborting, I removed them and determined their paternity using starch gel electrophoresis. Ovules fertilized by self pollen were more likely to abort than ovules fertilized by cross pollen, and ovules fertilized by nearby donors were more likely to abort than ovules fertilized by distant donors. Ovules fertilized by donors 100 m from the recipient were equally likely to abort. There was not a significant relationship between the proportion of ovules fertilized by a pollen donor and the probability of those fertilized ovules developing into seeds. There was no relationship between ovule position within a fruit and ovule abortion. I manipulated available resources by removing leaves and by permitting only one fruit to develop per plant. Decreasing the amount of resources increased the proportion of aborted ovules. Abortion of ovules of lesser quality appears to release resources that can then be used to develop other offspring.

Postfertilization Causes of Differential Success of Pollen Donors in Erythronium grandiflorum (Liliaceae): Nonrandom Ovule Abortion

Seed paternity in Erythronium grandiflorum does not fully reflect the proportion of pollen on the stigma. When two types of pollen are simultaneously applied to the stigma, outbred seeds are produced over inbred, and seeds from more distant donors are produced over seeds from donors nearby. I looked for postfertilization causes of these previously reported patterns of differential success of pollen donors. I simultaneously pollinated stigmas with pollen from two donors and observed ovule development through a window sliced in the ovary. Pollen donor pairs were self and cross, donors 1 and 100 m from the recipient, and two donors each 100 m from the recipient. Since one donor was always the alternate homozygote from the recipient at the malate dehydrogenase locus, I could determine the paternity of developing seeds. When it appeared that ovules were aborting, I removed them and determined their paternity using starch gel electrophoresis. Ovules fertilized by self pollen were more likely to abort than ovules fertilized by cross pollen, and ovules fertilized by nearby donors were more likely to abort than ovules fertilized by distant donors. Ovules fertilized by donors 100 m from the recipient were equally likely to abort. There was not a significant relationship between the proportion of ovules fertilized by a pollen donor and the probability of those fertilized ovules developing into seeds. There was no relationship between ovule position within a fruit and ovule abortion. I manipulated available resources by removing leaves and by permitting only one fruit to develop per plant. Decreasing the amount of resources increased the proportion of aborted ovules. Abortion of ovules of lesser quality appears to release resources that can then be used to develop other offspring.

Evidence for selective fruit abortion in Banksia spinulosa (Proteaceae)

Abstract Fruit set is consistently low in the genus Banksia (Proteaceae). One explanation for low fruit:flower ratios is that excess flowers allow plants to selectively abort inferior progeny thereby increasing the average quality of the seed crop. We examined whether self-pollinated fruits were aborted selectively in a partially self-compatible population of Banksia spinulosa. Inflorescences were divided in half and pollinated with cross pollen on both sides, self pollen on both sides or cross pollen on one side and self pollen on the other. Seed production was reduced significantly by 38% on fully self-pollinated compared to fully cross-pollinated inflorescences, indicating partial self-incompatibility or inbreeding depression. On inflorescences pollinated with both cross and self pollen, selfed seed set was reduced by 63% compared to crossed seed set. On the selfed half of these inflorescences, fewer fruits and seeds were produced, and more fruits had aborted seeds than on randomly selected halves of fully self-pollinated inflorescences. These results suggest that self-fertilized embryos were developed in the absence of outcrossed progeny but were selectively aborted when outcrossed progeny were present on inflorescences. To determine the amount of outcrossing occurring in the study population, outcrossing rates ( t ) of naturally pollinated plants were examined by electrophoretic assessment of progeny arrays. Values of t did not differ significantly from one, indicating complete outcrossing. High outcrossing, despite the high probability of self pollen receipt under natural conditions, is consistent with the conclusion that selective abortion occurs in B. spinulosa.

Evidence for selective fruit abortion in Banksia spinulosa (Proteaceae)

Fruit set is consistently low in the genus Banksia (Proteaceae). One explanation for low fruit:flower ratios is that excess flowers allow plants to selectively abort inferior progeny thereby increasing the average quality of the seed crop. We examined whether self-pollinated fruits were aborted selectively in a partially self-compatible population of Banksia spinulosa. Inflorescences were divided in half and pollinated with cross pollen on both sides, self pollen on both sides or cross pollen on one side and self pollen on the other. Seed production was reduced significantly by 38% on fully self-pollinated compared to fully cross-pollinated inflorescences, indicating partial self-incompatibility or inbreeding depression. On inflorescences pollinated with both cross and self pollen, selfed seed set was reduced by 63% compared to crossed seed set. On the selfed half of these inflorescences, fewer fruits and seeds were produced, and more fruits had aborted seeds than on randomly selected halves of fully self-pollinated inflorescences. These results suggest that self-fertilized embryos were developed in the absence of outcrossed progeny but were selectively aborted when outcrossed progeny were present on inflorescences. To determine the amount of outcrossing occurring in the study population, outcrossing rates (t) of naturally pollinated plants were examined by electrophoretic assessment of progeny arrays. Values of t did not differ significantly from one, indicating complete outcrossing. High outcrossing, despite the high probability of self pollen receipt under natural conditions, is consistent with the conclusion that selective abortion occurs in B. spinulosa.

Pedigree analysis of the transmission of a double-stranded RNA in barley cultivars

The barley cultivar Barsoy ( Hordeum vulgare L.) contains an unusual 13.1 kb cellular double-stranded ribonucleic acid (dsRNA). This non-viral dsRNA is efficiently transmitted by both egg and pollen in crosses with dsRNA-free cultivars. The pedigree of Barsoy was reconstructed and tested for dsRNA occurrence and sequence similarity in an effort to determine the origin and distribution of this molecule. The ancestors of Barsoy barley were tested for the presence of dsRNA by CF-11 cellulose chromatography and agarose electrophoresis. Of 31 ancestral cultivars tested, eight contained a dsRNA with electrophoretic properties similar to the one from Barsoy. A cDNA probe made with Barsoy dsRNA revealed that the dsRNAs isolated from related cultivars have similar nucleotide sequences. The dsRNA was found in four out of five of the oldest ancestors of Barsoy tested. One of these cultivars, Nakano Wase, was introduced to the US from Japan for breeding purposes in 1911. These results support a theory for vertical transmission of the dsRNA from generation to generation and suggest that the molecule has been maintained in barley cultivars for over 80 years.

Reproductive biology ofCistus ladanifer (Cistaceae)

The phenology, major floral characteristics, fruiting levels, and breeding system ofCistus ladanifer L. (Cistaceae), a common western Mediterranean shrub species, were studied in a southern Spanish population. The white, large (64 mm in diameter) flowers of this shrub appear during spring (March–May) and produce abundant pollen and nectar. In the year of study, flowers lasted up to three days, during which they were visited by a diverse array of insects including beetles, flies, and bees. Hand-pollinations revealed that flowers do not set any seed unless cross pollen is applied to the stigma. Microscopical observations indicate that self pollen tubes grow down the stigma but invariably fail to induce fruit maturation. At the plant level, all estimates of fecundity investigated (number of seeds per capsule, proportion of ovules developing into seed, and proportion of flowers setting fruit) were highly dependent on nearest neighbour distance, with isolated plants setting as little as 0% fruit. In contrast, plants within a clump often transformed into fruit as much as 90% of the flowers. At the population level, seed output was estimated to range between 3,000 and 270,000 seeds per plant during 1991.

Breeding Systems of Disjunct Populations of Christmas Bells (Blandfordia grandiflora R.Br., Liliaceae): Variation in Self-Fertility and an Ovular Mechanism Regulating Self-Fertilisation

The breeding systems of disjunct tableland and coastal populations of Christmas bells were determined using hand-pollination experiments. In both populations, 90% or more of self-pollinated plants produced seeds. Tableland plants were significantly more self-fertile than coastal plants (ratio of self seed set to cross seed set: tableland, 0.55 ± 0.03; coast, 0.08 ± 0.02). Significant variation among plants for self-fertility was found in both populations. Autofertility was 1.6% or less in both populations indicating that pollen vectors are necessary for seed set. Seed set by agamospermy in both populations was less than 0.1%. Percentage seed abortion was greater in self-pollinated plants than cross-pollinated plants in both populations. In both self- and cross-pollinated plants, seed abortion was twice as great in the coastal population than in the tableland population. No evidence was found for stigmatic or stylar self-incompatibility. Self and cross pollen adhered to and germinated equally well on stigmas in both populations (72 - 77% germination). Similarly, there were no differences between pollination treatments or populations in the percentage of ovules penetrated by pollen tubes (82 - 89% penetration). When self-pollination preceded cross-pollination by 24 h or longer seed set was significantly reduced compared to flowers that were cross-pollinated only, suggesting ovules were pre-empted by self pollen tubes. Collectively these results strongly suggest that self seed set was reduced by a mechanism operating at the ovule level, such as early-acting inbreeding depression due to recessive seed-aborting genes, although incomplete late-acting self-incompatibility cannot be ruled out. For coastal plants, this ovular mechanism largely prevents selfing, indicating plants were predominantly outcrossing although most produced some self seed. For tableland plants, substantial seed set by selfing may occur under natural conditions.

The ovular incompatibility system in Gasteria verrucosa (Mill.) H. Duval

The nature and process of ovular incompatibility in Gasteria verrucosa was studied. Using different crossing programs and following the pollen germination and ovule penetration histochemically, an ovular gametophitic incompatibility system based on two or more loci is affirmed. Pollen tubes after self-and cross-pollination penetrate the micropyle. Although the percentage of seed setting reached about 60% after cross pollination no clear differences could be seen between the germination and ovule penetration of cross and self pollen. In the ovules, multiple penetration of micropyles and a preference for the first position ovules was observed. Results of semi-compatible crossings and mixed crossings indicated a gametophytic two or more loci incompatibility system. Ovular incompatibility was manifested by the degeneration of the sporophytic ovular tissues. In Gasteria verrucosa the system of ovular incompatibility does not seem to be restricted to the ovule.

Breeding System and Pollination Levels of Banksia ericifolia

Experiments were carefully designed to determine the breeding system of Banksia ericifolia L.f. An equivalent percentage of flowers (78%) contained pollen tubes following self-pollination and open-pollination while a significantly smaller percentage of flowers in an autogamy treatment (44%) and cross-pollination treatment (55%) contained pollen tubes. Significantly more of the inflorescences in the open-pollination (60%) and cross-pollination (33%) treatments produced fruit compared with those in the self-pollination (11%) and autogamy treatments (13%). We suggest that B. ericifolia is largely self-incompatible because fruit production did not reflect pollen tube abundances. The influence of pollination levels on fruit production was determined by reducing the number of flowers on an inflorescence to 100 (i.e. 10% of original) or fewer and hand-pollinating these with cross pollen. There was no difference in fruit production between inflorescences with reduced flower number and open-pollinated inflorescences which had their full complement of flowers. Thus, the ability of an inflorescence to produce fruit appears more likely to be determined by the type of pollen received (cross versus self) rather than by the number of pollinated flowers it contains.

The foraging behaviour of a nectar feeding marsupial, Petaurus australis.

The foraging behaviour of non-flying nectar feeding mammals has been examined rarely. The exudivorous yellow-bellied glider (Petaurus australis) was observed to feed extensively (70% of the total feeding observation time) on the nectar of all species of Eucalyptus present at a site in southeastern Australia. Gliders harvested nectar, and presumably pollen also, whenever eucalypt flowers were available and selected trees with 2-3 times as many flowers as that on trees randomly selected along a transect. The abundance of flowering trees varied temporally and, at times when few flowering trees were present, gliders chose trees with fewer flowers than at times when flowering trees were abundant. When flowering trees were superabundant or scarce, there was no relationship between the number of flowers in a tree and the duration of visits by gliders. However, at intermediate levels of abundance, the amount of time a glider spent in a tree was related to the number of flowers in a tree. Gliders devoted 90% of the time outside their dens to foraging and the above relationship is suggested to reflect two foraging options which maximize net energy gain for different abundances of flowering trees. Although gliders spent considerable lengths of time in individual trees feeding, initial deposition of cross pollen when gliders first arrive in a tree may be substantial and thus, may provide significant amounts of outcrossing for these eucalypts.

Breeding System and Tests for Pollen-Limitation in Two Species of Banksia

Three factors that potentially influence fruit set (breeding system, inadequate cross-pollination and variable floral display) were examined for two species of Banksia. Firstly, self-pollination and autogamy treatments failed to produce any fruit in B. spinulosa and produced very few fruit in B. paludosa. This suggests that cross pollen is required to produce the level of fruit set observed in the field. Secondly, when plants were given abundant cross pollen, fruit set was not altered in B. spinulosa but was in B. paludosa. Although the number of follicles per inflorescence was was not different for open- pollinated (control) and manipulated B. paludosa inflorescences, the latter produced twice the proportion of inflorescences with follicles produced by control plants and twice the total number of follicles per plant. Thirdly, larger floral display in open-pollinated B. spinulosa plants was associated with a greater production of fruit. However, larger floral display was not associated with a greater reproductive output in B. paludosa, and we suggest that recurrent pollen-limitation in this species may be responsible for this result.

ROLE OF CALCIUM IN THE CALLOSE RESPONSE OF SELF‐POLLINATED BRASSICA STIGMAS

In Brassica oleracea, sporophytic self‐incompatibility prevents germination of self pollen, or normal growth of self pollen tubes. After self‐pollination, the papillae of stigmas synthesize callose. The role of Ca++ in the formation of stigmatic callose was tested by adding compounds that interact with Ca++ to suspensions of pollen that were known to induce callose formation in self stigmas. The calcium channel antagonist, lanthanum, and the calcium chelating agent, EGTA, reduced or abolished the callose response to self‐pollen suspensions. In the presence of Ca++, the calcium ionophore, A23187, induced callose in stigmatic papillae when added to pollen suspensions, or alone. Therefore, callose deposition in response to incompatible pollinations appears to be a calcium‐dependent process. Pretreatment of pistils with 100 μm 2‐deoxy‐D‐glucose abolished the callose response to self‐pollination, while self pollen remained inhibited and cross pollen grew normally in treated pistils. Thus, callose formation in the stigma is not an essential part of the self‐incompatibility mechanism preventing the growth of self pollen in Brassica.

Role of Calcium in the Callose Response of Self-Pollinated Brassica Stigmas

In Brassica oleracea, sporophytic self-incompatibility prevents germination of self pollen, or normal growth of self pollen tubes. After self-pollination, the papillae of stigmas synthesize callose. The role of Ca++ in the formation of stigmatic callose was tested by adding compounds that interact with Ca++ to suspensions of pollen that were known to induce callose formation in self stigmas. The calcium channel antagonist, lanthanum, and the calcium chelating agent, EGTA, reduced or abolished the callose response to self-pollen suspensions. In the presence of Ca++, the calcium ionophore, A23187, induced callose in stigmatic papillae when added to pollen suspensions, or alone. Therefore, callose deposition in response to incompatible pollinations appears to be a calcium-dependent process. Pretreatment of pistils with 100 μm 2-deoxy-D-glucose abolished the callose response to self-pollination, while self pollen remained inhibited and cross pollen grew normally in treated pistils. Thus, callose formation in the stigma is not an essential part of the self-incompatibility mechanism preventing the growth of self pollen in Brassica.

Towards an in vitro bioassay for the self-incompatibility response in Brassica oleracea

Eluates of stigmas of Brassica oleracea that were known to contain S locus-specific glycoproteins (SLSG) discriminated between self and cross pollen in vitro in three different media. Discrimination was equally evident in experiments that were the in vitro equivalents of reciprocal pollinations. In a TAPS-buffered medium, self eluates depressed pollen germination in a dose-dependent manner. TAPS medium allowed a bioassay of the effects of SLSG in eluates because it optimized germination in a way that eliminated the complicating features of the stimulatory substances in the eluates. Stigma eluates affected percentage pollen germination and optimum calcium concentrations in vitro whether or not SLSG were present in the eluates, but differently in different media, and depending on whether the eluates were cross or self with respect to the pollen tested. Thus, the effect of stigma eluates on the in vitro germination of pollen in Brassica depends on the balance of stimulatory versus inhibitory substances in the eluates.