Self-pollen Deposition Research Articles

Male flowers are better fathers than hermaphroditic flowers in andromonoecious Passiflora incarnata

• The diversity of plant breeding systems provides the opportunity to study a range of potential reproductive adaptations. Many mechanisms remain poorly understood, among them the evolution and maintenance of male flowers in andromonoecy. Here, we studied the role of morphologically male flowers ('male morph') in andromonoecious Passiflora incarnata. • We measured morphological differences between hermaphroditic and male morph flowers in P.incarnata and explored the fruiting and siring ability of both flower types. • Male morph flowers in P.incarnata were of similar size to hermaphroditic flowers, and there was little evidence of different resource allocation to the two flower types. Male morph flowers were less capable of producing fruit, even under ample pollen and resource conditions. By contrast, male morph flowers were more successful in siring seeds. On average, male morph flowers sired twice as many seeds as hermaphroditic flowers. This difference in male fitness was driven by higher pollen export from male morph flowers as a result of greater pollen production and less self-pollen deposition. • The production of male morph flowers in P.incarnata appears to be a flexible adaptive mechanism to enhance male fitness, which might be especially beneficial when plants face temporary resource shortages for nurturing additional fruits.

Effects of pollen reward removal on fecundity in a self‐incompatible hermaphrodite plant

The pollen of hermaphrodite plants is often utilised by flower-visiting animals. While pollen production has obvious benefits for plant male fitness, its consequences for plant female fitness, especially in self-incompatible hermaphrodite species, are less certain. Pollen production could either enhance seed production though increased pollinator attraction, or reduce it if ovules are discounted by deposition of self pollen, as can occur in species with late-acting self-incompatibility. To test the effects of pollen reward provision on female fitness, we artificially emasculated flowers in two populations of the succulent Aloe maculata (Asphodelaceae), which has a late-acting self-incompatibility system, over the course of its flowering period. Flowers of this species are visited by sunbirds (for nectar) and native bees (for pollen and nectar). We measured floral visitation rates, floral rejection rates, pollen deposition on stigmas and fruit and seed set in both emasculated and non-emasculated plants. We found that flowers of emasculated plants suffered reduced visitation and increased rejection (arrival without visitation) by bees, but not by sunbirds; had fewer pollen grains deposited on stigmas and showed an overall decrease in fruit set and seed set. Rates of seed abortion were, however, greatly reduced in emasculated flowers. This study shows that pollen rewards can be important for seed set, even in self-incompatible plants, which have been assumed to rely on nectar rewards for pollinator attraction. Seed abortion was, however, increased by pollen production, a result that highlights the complexity of selection on pollen production in hermaphrodite flowers.

Pollination and late-acting self-incompatibility in Cyrtanthus breviflorus (Amaryllidaceae): implications for seed production

Animal pollination is typically an uncertain process that interacts with self-incompatibility status to determine reproductive success. Seed set is often pollen-limited, but species with late-acting self-incompatibility (SI) may be particularly vulnerable, if self-pollen deposition results in ovule discounting. Pollination is examined and the occurrence of late-acting SI and ovule discounting assessed in Cyrtanthus breviflorus. The pollination system was characterized by observing floral visitors and assessing nectar production and spectral reflectance of flowers. To assess late-acting SI and ovule discounting, growth of self- and cross-pollen tubes, and seed set following open pollination or hand pollination with varying proportions of self- and cross-pollen, were examined. Native honeybees Apis mellifera scutellata pollinated flowers as they actively collected pollen. Most flowers (≥70 %) did not contain nectar, while the rest produced minute volumes of dilute nectar. The flowers which are yellow to humans are visually conspicuous to bees with a strong contrast between UV-reflecting tepals and UV-absorbing anthers and pollen. Plants were self-incompatible, but self-rejection was late-acting and both self- and cross-pollen tubes penetrated ovules. Seed set of open-pollinated flowers was pollen-limited, despite pollen deposition exceeding ovule number by 6-fold. Open-pollinated seed set was similar to that of the cross + self-pollen treatment, but was less than that of the cross-pollen-only treatment. Flowers of C. breviflorus are pollinated primarily by pollen-collecting bees and possess a late-acting SI system, previously unknown in this clade of the Amaryllidaceae. Pollinators of C. breviflorus deposit mixtures of cross- and self-pollen and, because SI is late-acting, self-pollen disables ovules, reducing female fertility. This study thus contributes to growing evidence that seed production in plants with late-acting SI systems is frequently limited by pollen quality, even when pollinators are abundant.

Floral morphology mediates temporal variation in the mating system of a self‐compatible plant

The mating system of self-compatible plants may fluctuate between years in response to ecological factors that cause variation in the deposition of self pollen vs. outcross pollen on stigmas. Such temporal variation may have significant ecological and evolutionary consequences, but it has rarely been studied, and the mechanisms that mediate temporal variation have almost never been investigated. We tested for variation in the proportion of seeds self-fertilized (s) between two years within 19 populations of the short-lived herb Aquilegia canadensis. Selfing varied widely among populations (range in s = 0.17-1.00, mean s = 0.82) but was inconsistent across years, indicating significant temporal variation. Three populations exhibited especially wide swings in the mating system between years. Mean s did not decrease with increasing population size (N), nor was the fluctuation in s associated with mean N or the change in N. As expected, s declined with increasing separation between anthers and stigmas within flowers (herkogamy), and s fluctuated to a greater extent in populations with more herkogamous flowers. Self-compatible plants can experience wide temporal variation in self-fertilization, and floral traits such as herkogamy may mediate temporal variation by forestalling self-pollination and thus allowing outcrossing during periods when pollinators are frequent.

Mating system and the hybridization between self-compatible Phlox cuspidata and self-incompatible Phlox drummondii

Mating system can impact the frequency of hybridization and therefore the maintenance of species diversity. I evaluate the effects of weak self-incompatibility (SI) in Phlox cuspidata and SI in Phlox drummondii on mating success within species and on hybridization dynamics between species under controlled conditions. The effects of SI on hybridization frequency were assessed by manipulating the relatedness of conspecific pollen and the relative timing of pollen deposition in mixed-donor interspecific pollinations. Selfing as opposed to outcrossing increased hybridization by 16% in P. cuspidata maternal plants and by 48% in P. drummondii maternal plants because self pollen did not compete as well against heterospecific pollen. The relative timing of conspecific versus heterospecific pollen deposition also impacted hybridization. In self-compatible P. cuspidata, the deposition of self pollen 5 h earlier than heterospecific pollen decreased hybridization by 28%. In self-incompatible P. drummondii, a 5 h delay in the deposition of compatible conspecific pollen increased hybridization by 32%. In this hybrid system, early self-pollination can decrease hybridization (but increase inbreeding) by P. cuspidata maternal plants, and SI may increase hybridization by P. drummondii maternal plants.

Plant–pollinator interactions: comparison between an invasive and a native congeneric species

Plant–pollinator interactions determine reproductive success for animal-pollinated species and, in the case of invasive plants, they are supposed to play an important role in invasive success. We compared the invasive Senecio inaequidens to its native congener S. jacobaea in terms of interactions with pollinators. Visitor guild, visitation rate, and seed set were compared over 3 years in three sites in Belgium. Floral display (capitula number and arrangement) and phenology were quantified, and visiting insects were individually censused, i.e. number of visited capitula and time per visited capitulum. As expected from capitula resemblance, visitor guilds of both species were very similar (proportional similarity = 0.94). Senecio inaequidens was visited by 33 species, versus 36 for S. jacobaea. For both species, main visitors were Diptera, especially Syrphidae, and Hymenoptera. Visitation rate averaged 0.13 visitor per capitulum per 10 min for S. inaequidens against 0.08 for S. jacobaea. However, insects visited more capitula per plant on S. jacobaea, due to high capitula density (886 m −2 versus 206 m −2 for S. inaequidens), which is likely to increase self-pollen deposition considerably. Seed set of S. jacobaea was lower than that of S. inaequidens. We suggest that floral display is the major factor explaining the differences in insect visitation and seed set between the two Senecio species.

Reproductive biology of the morning glory Merremia macrocalyx (Ruiz & Pavon) O'Donnell (Convolvulaceae)1

Abstract Merremia macrocalyx is a vine that grows over other plants or as a low creeper and is considered a weed of important crops. Study of its reproductive biology showed that its flowers comprise the typical features of the melittophilous pollination syndrome. Anthophoridae, Apidae, and Halictidae were its main floral visitors. Merremia macrocalyx showed a mixed mating system with features that promote outcrossing while allowing some autogamy. Floral biomass was allocated mainly to the corolla (attraction) which, together with the presence of herkogamy and protandry, favors out-crossing. Nevertheless, it is self-compatible and partially autogamous due to the position of anthers above the stigma. Foraging behavior of pollinators could also facilitate deposition of self pollen on the stigma. Ovule abortion was the main predispersal loss of potential seed production, closely followed by flower and fruit abortion. Predispersal seed predation and seed abortion were relatively low. Specimens of Megacerus fl...

Does selfing provide reproductive assurance in the perennial herbBulbine vagans(Asphodelaceae)?

Selection for selfing to provide reproductive assurance depends on the balance between increased reproductive output when pollinators or potential mates are scarce and the extent that inbreeding depression erodes such fertility gains. We use glasshouse and field experiments to examine the benefits of autonomous and facilitated selfing inBulbine vagans. Autonomous selfing was delayed until after opportunities for outcrossing and reproductive output was 0.67 relative to manual selfing and open pollination. Values less than one probably reflected insufficient autonomous deposition of self pollen. In the field, reproductive output of emasculated flowers was 0.50 relative to intact flowers that could both outcross and self, indicating that outcross pollen was limited and that selfing boosted reproductive output. Because all pollen was removed from anthers before intact flowers closed, facilitated selfing rather than autonomous selfing occurred. In the glasshouse, inbreeding depression was 0.45, but under natural conditions would probably exceed 0.5. Values greater than 0.5 negate the automatic gene transmission advantage afforded by selfing and increasingly erode the benefits of reproductive assurance. We conclude that inB. vagansdelayed and facilitated selfing can confer reproductive assurance, providing the latter does not usurp ovules that could be outcrossed.

Pollination biology of a disjunct population of the endangered sandhills endemic Penstemon haydenii S. Wats. (Scrophulariaceae) in Wyoming, USA

We studied the breeding system and flower visitors of the endangered plant, Penstemon haydenii, at several south-central Wyoming, USA occurrences. In agreement with earlier studies of the species 300 km to the east in Nebraska, we found Wyoming plants to be self-incompatible and pollinator-dependent for sexual reproduction. Flower visitors were several species of native bees in the families Apidae (particularly bumblebees), Halictidae (small sweat bees), and Megachilidae (especially in the genus Osmia); and the masarid wasp Pseudomasaris vespoides. Especially important was Osmia brevis, an abundant megachilid bee, and one of only two species (the sweat bee Lasioglossum (Dialictus) pruinosum was the other) present at all five sites. As in Nebraska, fruit set did not differ between our experimental cross-pollination treatment and an open-pollinated control. However, unlike Nebraska, open-pollinated treatments in Wyoming produced significantly fewer seeds per fruit than the experimental out-crossing treatment. We discuss several possible explanations for seed limitation: (1) a scarcity of pollinators early in the flowering season; (2) resource competition for developing ovules on open-pollinated inflorescences but not on experimental inflorescences; (3) the deposition of self pollen through intra-inflorescence and intra-genet pollinator movements; (4) few S-alleles and mating types in the Wyoming metapopulation compared to the Nebraska metapopulation, from which it likely derives.

Ecological and selective effects of stigma-anther separation in the self-incompatible tropical tree Ipomoea wolcottiana (Convolvulaceae)

We evaluated the effect of stigma-anther separation (SAS) in flowers of the self-incompatible tropical tree Ipomoea wolcottiana (Convolvulaceae). We measured the deposition of self pollen on the stigma and the growth of pollen tubes. We also evaluated phenotypic selection on female fertility using univariate regressions of SAS with fruit production. SAS varied among individuals in two years, with a range of 0–6 mm. There was a negative correlation between SAS and the number of self pollen grains on the stigma; trees with SAS of 0–2 mm received averages of ca 80–130 self grains. The effect of loading the stigma with self-pollen was tested by allowing isolated flowers to self-pollinate, then cross-pollinating by hand: there was a negative correlation between the pollen load and the proportion of grains forming pollen tubes. The highest proportion of grains forming tubes was found with loads of 50 grains. The results suggest that more self pollen deposition leads to fewer tubes from outcross pollen. There was a significant effect of SAS on fruit production in both years. The analyses suggested stabilizing selection; very small SAS is disadvantageous due to the high load of self pollen, while very large SAS probably led to damage to the style during pollinator visits. We suggest that herkogamy may be a key character for determining the success of fruit production in self-incompatible hermaphroditic species.

Corolla wilting facilitates delayed autonomous self-pollination in Pedicularis dunniana (Orobanchaceae)

Structural changes associated with corolla wilting may serve as a mechanism for effecting self-pollination. Low pollinator visitation, high seed production and a corolla that persists after anthesis indicates that Pedicularis dunniana is autogamous. Delayed autonomous self-pollination is facilitated by corolla wilting. Wilting of the upper lip (galea) brought the pollen laden anthers into contact with the stigma resulting in the deposition of self pollen on the stigma. The seed set of flowers either emasculated, or with restrained galeae thus preventing anthers brushing against the stigma, was significantly lower than that of open-pollinated flowers. This demonstrates that autogamy occurs in this species through corolla wilting. Germination experiments indicated that outcross seedlings were more vigorous than selfed seedlings as a result of inbreeding depression. It is likely that autogamy provides reproductive assurance for P. dunniana under conditions of pollinator scarcity.

Minor quantitative trait loci underlie floral traits associated with mating system divergence in Mimulus.

The genetic basis of species differences provides insight into the mode and tempo of phenotypic divergence. We investigate the genetic basis of floral differences between two closely related plant taxa with highly divergent mating systems, Mimulus guttatus (large-flowered outcrosser) and M. nasutus (small-flowered selfer). We had previously constructed a framework genetic linkage map of the hybrid genome containing 174 markers spanning approximately 1800 cM on 14 linkage groups. In this study, we analyze the genetics of 16 floral, reproductive, and vegetative characters measured in a large segregating M. nasutus x M. guttatus F2 population (N = 526) and in replicates of the parental lines and F1 hybrids. Phenotypic analyses reveal strong genetic correlations among floral traits and epistatic breakdown of male and female fertility traits in the F2 hybrids. We use multitrait composite interval mapping to jointly locate and characterize quantitative trait loci (QTLs) underlying interspecific differences in seven floral traits. We identified 24 floral QTLs, most of which affected multiple traits. The large number of QTLs affecting each trait (mean = 13, range = 11-15) indicates a strikingly polygenic basis for floral divergence in this system. In general, QTL effects are small relative to both interspecific differences and environmental variation within genotypes, ruling out QTLs of major effect as contributors to floral divergence between M. guttatus and M. nasutus. QTLs show no pattern of directional dominance. Floral characters associated with pollinator attraction (corolla width) and self-pollen deposition (stigma-anther distance) share several pleiotropic or linked QTLs, but unshared QTLs may have allowed selfing to evolve independently from flower size. We discuss the polygenic nature of divergence between M. nasutus and M. guttatus in light of theoretical work on the evolution of selfing, genetics of adaptation, and maintenance of variation within populations.

MINOR QUANTITATIVE TRAIT LOCI UNDERLIE FLORAL TRAITS ASSOCIATED WITH MATING SYSTEM DIVERGENCE IN MIMULUS

The genetic basis of species differences provides insight into the mode and tempo of phenotypic divergence. We investigate the genetic basis of floral differences between two closely related plant taxa with highly divergent mating systems, Mimulus guttatus (large-flowered outcrosser) and M. nasutus (small-flowered selfer). We had previously constructed a framework genetic linkage map of the hybrid genome containing 174 markers spanning approximately 1800 cM on 14 linkage groups. In this study, we analyze the genetics of 16 floral, reproductive, and vegetative characters measured in a large segregating M. nasutus × M. guttatus F2 population (N = 526) and in replicates of the parental lines and F1 hybrids. Phenotypic analyses reveal strong genetic correlations among floral traits and epistatic breakdown of male and female fertility traits in the F2 hybrids. We use multitrait composite interval mapping to jointly locate and characterize quantitative trait loci (QTLs) underlying interspecific differences in seven floral traits. We identified 24 floral QTLs, most of which affected multiple traits. The large number of QTLs affecting each trait (mean = 13, range = 11–15) indicates a strikingly polygenic basis for floral divergence in this system. In general, QTL effects are small relative to both interspecific differences and environmental variation within genotypes, ruling out QTLs of major effect as contributors to floral divergence between M. guttatus and M. nasutus. QTLs show no pattern of directional dominance. Floral characters associated with pollinator attraction (corolla width) and self-pollen deposition (stigma-anther distance) share several pleiotropic or linked QTLs, but unshared QTLs may have allowed selfing to evolve independently from flower size. We discuss the polygenic nature of divergence between M. nasutus and M. guttatus in light of theoretical work on the evolution of selfing, genetics of adaptation, and maintenance of variation within populations.

Nocturnal Pollination by the Carpenter Bee Xylocopa tenuiscapa (Apidae) and the Effect of Floral Display on Fruit Set of Heterophragma quadriloculare (Bignoniaceae) in India1

ABSTRACTWe provide the first data on nocturnal pollination by bees. Heterophragma quadriloculare is a self–incompatible hermaphroditic tree solely pollinated at night by the carpenter bee Xylocopa (Mesotrichia) tenuiscapa Westwood, whose pollinating flights were lunar insensitive and unaffected by low nighttime temperatures (2–l4°C). The number of carpenter bees visiting a tree per minute and the number of flowers visited per foraging bout were positively related to the size of the floral display. We found that floral display size was negatively related to fruit set but positively related to absolute fruit numbers. In pollen carryover experiments, cross–pollen did not travel far in sequences of non–emasculated flowers (up to the 4th flower), but traveled farther in emasculated flower sequences (up to the 15th flower) as evidenced by fruit set in these sequences. The relatively poor fruit set on trees with many open flowers may have resulted from deposition of self–pollen on touch–sensitive stigmas, which close on first contact. Because large–girth trees tend to have large floral displays, trees may increasingly realize a higher percentage of fitness through male rather than female function as they age. The steady–state flowering phenology of H. quadriloculare may limit reduction in the female function compared to the cornucopia strategy of many other members of the Bignoniaceae and may also provide a reliable resource for X. tenuiscapa.RESUMESInvestigou‐se a relação entre salinidade intersticial, esclerofilia da folha, vigor da planta e densidade de galhas provocadas pelo inseto galhador foliar, Cecidomyia avicenniae (Diptera: Cecidomyiidae), em sua planta hospedeira Avicennia germinans (Avicenniaceae). As coletas foram realizadas em seis mangues e em uma mata de várzea, no Maranhão, nordeste do Brasil. Em cada local, dez ramos foram coletados aleatoriamente em cinco árvores de A. germinans. De cada ramo foram obtidos o número total de galhas e o comprimento (cm). Médias do comprimento, largura, biomassa e área das folhas por ramo foram também registradas. A esclerofilia foliar foi quantificada atraves da biomassa por unidade de área foliar (g/cm2). Amostras da água intersticial foram obtidas através de um tubo de PVC de 1,3 cm de diàmetro á uma profundidade de 80 cm, e a salinidade (ppm) medida com um refratômetro. A esclerofilia das folhas apresentou uma forte corrclação positiva com a salinidade intersticial (R2=0,77; P < 0,05). Observou‐se também correlações entre a densidade de galhas por unidade de área foliar (cm2) e a salinidade intersticial (r=0,36; P < 0,05), e entre densidade de galhas e a esclerofilia das folhas (r=0,40; P < 0,05). Salinidade e esclerofilia juntas explicaram 22 por cento da variação na densidade de galhas de C. avicenniae. Observou‐se, ainda, uma relação negativa entre a densidade de galhas por centímetre e o comprimento do ramo (R2=0,50; P < 0,05). Portanto, ramos mais longos de A. germinans apresentaram menor densidade de galhas. Nossos resultados sugerem que a densidade de galhas de C. avicenniae em A. germinansé afetada tanto pela salinidade do habitat da planta hospedeira quanto pela esclerofilia ao longo do gradiente de salinidade intersticial.

Nocturnal Pollination by the Carpenter Bee Xylocopa tenuiscapa (Apidae) and the Effect of Floral Display on Fruit Set of Heterophragma quadriloculare (Bignoniaceae) in India1

We provide the first data on nocturnal pollination by bees. Heterophragma quadriloculare is a self-incompatible hermaphroditic tree solely pollinated at night by the carpenter bee Xylocopa (Mesotrichia) tenuiscapa Westwood, whose pollinating flights were lunar insensitive and unaffected by low nighttime temperatures (2‐148C). The number of carpenter bees visiting a tree per minute and the number of flowers visited per foraging bout were positively related to the size of the floral display. We found that floral display size was negatively related to fruit set but positively related to absolute fruit numbers. In pollen carryover experiments, cross-pollen did not travel far in sequences of nonemasculated flowers (up to the 4th flower), but traveled farther in emasculated flower sequences (up to the 15th flower) as evidenced by fruit set in these sequences. The relatively poor fruit set on trees with many open flowers may have resulted from deposition of self-pollen on touch-sensitive stigmas, which close on first contact. Because largegirth trees tend to have large floral displays, trees may increasingly realize a higher percentage of fitness through male rather than female function as they age. The steady-state flowering phenology of H. quadriloculare may limit reduction in the female function compared to the cornucopia strategy of many other members of the Bignoniaceae and may also provide a reliable resource for X. tenuiscapa.

Nonrandom Mating in Clarkia gracilis (Onagraceae): A Case of Cryptic Self-Incompatibility

In plants capable of both self-fertilization and outcrossing, the selfing rate depends on the proportion of self pollen in pollen loads and on the relative postpollination success of self pollen in siring offspring. While the composition of pollen loads is subject to unpredictable variation, paternity success of self vs. outcross pollen following pollen deposition may be controlled by maternal plants. This study examined postpollination paternity success in Clarkia gracilis ssp. sonomensis, in which deposition of self pollen is common. Pure loads of self and outcross pollen produced similar numbers of mature seeds, but equal mixtures of self and outcross pollen yielded more than three times as many outcrossed offspring as selfed offspring. The finding that the paternity success of self pollen depends on whether it is in competition with outcross pollen helps to explain an earlier finding that the selfing rate in experimental populations was highest when pollinator activity was lowest. Cryptic self-incompatibility allows paternity by self pollen when outcross pollen is unavailable.

Nonrandom mating in Clarkia gracilis (Onagraceae): a case of cryptic self‐incompatibility

In plants capable of both self‐fertilization and outcrossing, the selfing rate depends on the proportion of self pollen in pollen loads and on the relative postpollination success of self pollen in siring offspring. While the composition of pollen loads is subject to unpredictable variation, paternity success of self vs. outcross pollen following pollen deposition may be controlled by maternal plants. This study examined postpollination paternity success in Clarkia gracilis ssp. sonomensis, in which deposition of self pollen is common. Pure loads of self and outcross pollen produced similar numbers of mature seeds, but equal mixtures of self and outcross pollen yielded more than three times as many outcrossed offspring as selfed offspring. The finding that the paternity success of self pollen depends on whether it is in competition with outcross pollen helps to explain an earlier finding that the selfing rate in experimental populations was highest when pollinator activity was lowest. Cryptic self‐incompatibility allows paternity by self pollen when outcross pollen is unavailable.

Gynodioecy and Male Sterility in Eucalyptus leucoxylon F. Muell. (Myrtaceae)

The breeding system of Eucalyptus leucoxylon was investigated using crosses, microscopy, and isozyme analysis. In the study population, 57% of trees were male sterile because microspores were produced by meiosis and formed exines; however, they contained little starch, lacked intines, and had collapsed by anthesis, surrounded by incompletely degenerated tapetum. Hermaphrodite trees produced bisexual flowers, which were protandrous. Anthers dehisced in the bud and 93% of pollen was deposited on the upper style and lower stigma as the filaments extended; only 7% remained on the anthers. Hermaphrodite flowers set low levels of autogamous and assisted selfed seed, compared with open pollinations. Counts of pollen tubes in open-pollinated pistils of each morph showed that female flowers received less pollen than hermaphrodite flowers, but despite this, there was no difference in open-pollinated seed set between the two morphs. Multilocus estimates of outcrossing rates (t̂) from male-sterile trees showed values approaching complete outcrossing (0.96), while hermaphrodite trees showed significant levels of inbred seed in open-pollinated seed crops (0.83) but still maintained a higher outcrossing rate than reported for most other eucalypts. This study has shown that E. leucoxylon maintains high levels of outbreeding, despite the deposition of self pollen on the stigma in the secondary pollen presentation mechanism; it may achieve this through protandry, pollen removal, male sterility, and late-acting self-incompatibility.

Pollen Storage and Breeding System in Relation to Controlled Pollination of Four Species of Acacia (Leguminosae: Mimosoideae)

Pollen of A. auriculiformis, A. iteaphylla, A. karroo and A. mangium was stored at 25, 5, -18 or -196°C for up to 3 years, and its viability tested by pollen staining, in vivo pollen tube growth or pod set 1 month after hand pollination. The effectiveness of staining methods using 2,3,5-triphenyltetrazolium chloride (TTC), 5-bromo-4-chloro-3-indole- β -galactoside (X-Gal) and fluorescein diacetate (FDA) to predict pollen viability was investigated. All of the staining methods gave variable results, but the TTC and X-Gal tests were particularly unreliable. FDA staining of pollen gave the best indication of its ability to germinate on the stigma and penetrate ovules. Pollen stored for up to three days at 25°C retained the ability to penetrate ovules following hand pollination, and of that stored for three years at 5°C, 19% of the grains fluoresced with FDA. Pollen stored at -18°C for 1 year retained the ability to penetrate ovules and produce pod set, and of that stored for 3 years, 23% of the grains fluoresced with FDA. Pollen stored at -196°C for one year retained the ability to penetrate ovules and produce pod set, but thawing and refreezing of the pollen reduced viability to zero. It was concluded that the most successful and convenient method of pollen storage was vaccuum drying followed by storage at -18°C. The Australian species of Acacia investigated (A. iteaphylla, A. auriculiformis and A. mangium) had protogynous flowers, with stigma receptivity preceding anther dehiscence, such that flowers could be reliably hand pollinated. The African species A. karroo had protandrous flowers, with stigmas unreceptive at anthesis, but receptive at 5 days after anther dehiscence. Deposition of self pollen on the stigma prior to attainment of receptivity rendered hand pollination of this species unreliable.