Evaluation of pollen tube growth ability in Petunia species having different style lengths

Pollen tube growth is essential for the fertilization process in angiosperms. When pollen grains arrive on the stigma, they germinate, and the pollen tubes elongate through the styles of the pistils to deliver sperm cells into the ovules to produce the seeds. The relationship between the growth rate and style length remains unclear. In previous studies, we developed a liquid pollen germination medium for observing pollen tube growth. In this study, using this medium, we examined the pollen tube growth ability in Petunia axillaris subsp. axillaris, P. axillaris subsp. parodii, P. integrifolia, and P. occidentalis, which have different style lengths. Petunia occidentalis had the longest pollen tubes after 6 h of culture but had a relatively shorter style. Conversely, the pollination experiments revealed that P. axillaris subsp. parodii, which had the longest style, produced the longest pollen tubes in vivo. The results revealed no clear relationship between the style lengths and the growth rate of pollen tubes in vitro. Interspecific pollinations indicated that the styles affected pollen tube growth. We concluded that, in vitro, the pollen tubes grow without being affected by the styles, whereas, in vivo, the styles significantly affected pollen tube growth. Furthermore, interspecific pollination experiments implied that the pollen tube growth tended to be suppressed in the styles of self-incompatibility species. Finally, we discussed the pollen tube growth ability in relation to style lengths.     

Effects of Previous Pollination and Stylar Ethylene on Pollen Tube Growth in Petunia hybrida Styles

The effect of ethylene on the growth rate of pollen tubes in styles of Petunia hybrida was examined. Apart from its strong inhibition of pollination-induced ethylene synthesis, aminoethoxyvinylglycine, placed on the stigma, did not impede tube growth. The inhibitors of the action of ethylene, silver thiosulfate and 2,5-norbornadiene, were similarly ineffective. Application of the ethylene precursor, 1-amino-cyclopropane-1-carboxylic acid, onto the stigma at different intervals prior to pollination evoked synthesis of ethylene, but was without effect on tube growth. However, prepollination (by 24 hours) with Nicotiana tabacum pollen, significantly enhanced tube growth of Petunia pollen. This enhancement was not counteracted by the pretreatment of stigmas with aminoethoxy-vinylglycine. It is concluded that the ethylene associated with pollination is without effect on pollen tube growth in the style, but that other pollination-induced factors may lead to an acceleration of growth.     

Anther response to high-temperature stress during development and pollen thermotolerance heterosis as revealed by pollen tube growth and in vitro pollen vigor analysis in upland cotton

Main conclusion

Pollen tube growth in styles was strongly inhibited by temperature above 35 °C, and the yield of cotton decreased because of the adverse effect of high temperatures during square development. High-temperature stress during flowering influences the square development of upland cotton (Gossypium hirsutum L.) and cotton yield. Although it is well known that square development is sensitive to high temperature, high-temperature sensitive stages of square development and the effects of high temperature on pollen tube growth in the styles are unknown. The effect of high temperature on anther development corresponding to pollen vigor is unknown during anther development. The objectives of this study were to identify the stages of square development that are sensitive to high temperatures (37/30 and 40/34 °C), to determine whether the abnormal development of squares influenced by high temperature is responsible for the variation in the in vitro germination percent of pollen grains at anthesis, to identify the effect of high temperature on pollen germination in the styles, and to determine pollen thermotolerance heterosis. Our results show that the stages from the sporogenous cell to tetrad stage (square length <6.0 mm) were the most sensitive to high temperature, and the corresponding pollen viability at anthesis was consistent with the changes in the square development stage. Pollen tube growth in the styles was strongly inhibited by temperature above 35 °C, and the yield of cotton decreased because of the effect of high temperature during square development. The thermotolerance of hybrid F₁ pollen showed heterosis, and pollen viability could be used as a criterion for screening for high-temperature tolerance cultivars. These results can be used in breeding to develop new cotton cultivars that can withstand high-temperature conditions, particularly in a future warmer climate.

     

Studies on growth and utilization of stylar carbohydrate by pollen tubes and callose development in self-incompatibleOenothera organesis

The addition of boric acid or calcium, or both boric acid and calcium to the warm water in which the styles were treated led to a further enhancement of pollen tube growth in otherwise incompatible styles. It was established that developing pollen tubes ofOenothera organesis obtain sugar from the stylar tissue through which they grow. The rate of this utilization of endogenous sugar was stimulated by the addition of boric acid. It was further established that with increased pollen tube development there was reduction in callose plugs as well as in content of endogenous stylar sugar.     

Autoradiographs of Pollen Tube Nuclei with Calcium-45

Autoradiography with Ca⁴⁵ has been used to obtain information about the relation between calcium and chromosomes. Labelled pollen from the Easter lily, Lilium longiflorum, was allowed to develop into pollen tubes between 5 and 6 cm. long in the styles of non-radioactive flowers. All of the nuclei, namely the tube nucleus and the two sperm nuclei, retain Ca⁴⁵ after this period of growth and development. Since the two sperm nuclei have formed during this interval by the mitotic division of the generative nucleus and growth of the tube has occurred under the influence of the tube nucleus, it is inferred that the calcium was bound in a stable nuclear component, the chromosomes.     

Semi in vivo pollen tube growth of Aechmea fasciata

With semi in vivo pollen tube growth assays, stigmas are pollinated in vivo and, after a fixed time interval, the styles are isolated from the ovary and placed on culture medium in vitro. Semi in vitro pollination includes isolation of the stigma and style complex, followed by pollination and placing the stylar end on nutrient medium. After semi in vivo pollination more and longer pollen tubes protruded from the cut end of the styles into medium, in comparison to semi in vitro pollination. Medium with 3 g l^–1 agar was better than that with 6 g l^–1 agar for pollen tube growth after the tubes emerged from the cut style. Semi in vitro pollination of the reversed style indicated that pollen tube growth was not influenced by the direction of the style. Fructose and glucose inhibited pollen tube growth compared to sucrose. Swollen tips characterized tube growth inhibition. After semi in vivo pollination all generative nuclei had divided to give two sperm nuclei. The average distance between the last sperm nucleus and the pollen tube tip as well as the distance between the two sperm nuclei diminished in growing pollen tubes between 24 and 48 h after pollination. The arrangements between the vegetative and the generative nuclei did not differ in semi in vivo and in vitro cultured pollen tubes of Aechmea fasciata. This information is important to explain why fertilization rate is low after placental pollination in comparison to placental grafted style pollination of Aechmea fasciata. The data may also contribute to the improvement of in vitro fertilization methods in Bromeliaceae and other higher plants.     

Why fast-growing pollen tubes give rise to vigorous progeny: the test of a new mechanism

A positive correlation between the speed of pollen tube growth and the quality of the resulting progeny in several species of flowering plants has traditionally been explained as being caused by an overlap in gene expression between gametophytes and sporophytes. We experimentally manipulated the pollen tube growth rates of pollen donors, such that the genotype controlling the rate was uncoupled from the phenotype, to test the alternative hypothesis that the correlation arises because ovules fertilized early are better provisioned by the maternal plant than later-fertilized ovules. Crosses using Silene vulgaris individuals bearing distinctive genetic markers revealed a correlation between the order of fertilization by pollen grains and vigour of the resulting sporophytes, which was independent of the speed of growth of the pollen tubes. Seeds sired by donors with relatively fast-growing pollen germinated earlier and grew larger than those sired by slow-growing pollen when pollen from the two donors was applied simultaneously. Reversing the order of arrival in the ovary by placing slow-growing pollen on the styles earlier and closer to the ovary led to reverse results. These results suggest that differential provisioning by the maternal plant can lead to differences in progeny vigour following pollen competition.     

Interspecific incompatibility due to stylar barriers in tuber-bearing and closely related non-tuber-bearing Solanums

Summary The phenomenon of interspecific incompatibility between various wild tuber-bearing and closely related non-tuber-bearing Solanum species was studied. One area of investigation included an examination of possible protein interactions in the incompatibility reaction using SDS electrophoresis. Pollen tube inhibition and morphology were examined in conjunction with biochemical analysis. Two sets of crosses were examined: interspecific tuber-bearing species crosses and interspecific tuber-bearing × non-tuber-bearing species crosses. These crosses had consistent pollen tube inhibition in the upper one-third of the style. The upper third of the styles of incompatibly pollinated, compatibly pollinated, and unpollinated styles was studied under fluorescence microscopy to observe pollen tube growth and morphology. Interspecific tuber-bearing × non-tuber-bearing species crosses demonstrated consistent pollen tube inhibition just below the stigma with frequent pollen tube swelling and bursting and extensive callose deposition along the pollen tube wall. Interspecific tuber-bearing species crosses had pollen tube inhibition further down the style with pollen tube tip tapering and extensive callose deposition. Stylar proteins of the lower two-thirds of the styles were analyzed with SDS electrophoresis. No unique protein differences were found to be specifically associated with the interspecific incompatibility reaction in this portion of the style.Cooperative investigation of the U.S. Department of Agriculture, Agricultural Research Service, and the Wisconsin Experiment Station. Supported in part by the USDA, Cooperative States Research Service Competitive grant no. 83-CRCR-1-1253     

The Nutritional Role of Pistil Exudate in Pollen Tube Wall Formation in Lilium longiflorum: I. Utilization of Injected Stigmatic Exudate

A quantity of labeled stigmatic exudate, collected from detached Lilium longiflorum (cv. Ace) pistils labeled with d-glucose-1-¹⁴C, was fractionated on Sephadex G-100 and the polysaccharide component, G-100-I, was injected into the hollow styles of unlabeled detached pistils (cv. Ace) which had been removed on the day after anthesis from the plant. Injected pistils were immediately cross-pollinated with L. longiflorum (cv. No. 44) pollen. Eighty-four hours later, pistils were dissected to recover the pollen tubes, expended exudate, and labeled tissues of the stigma and style. Distribution of label revealed that at least 25% of the carbohydrate substance in excised pollen tubes was derived from G-100-I. The composition of expended exudate adhering to pollen tubes, of pollen tube cytoplasm, and of pollen tube walls suggests that utilization of exudate by growing pollen tubes involves uptake and incorporation into pollen tube cytoplasm of exudate polysaccharide fragments followed by extensive metabolism of at least a portion of the incorporated carbohydrate prior to its utilization for pollen tube wall biosynthesis. Results suggest the presence of at least two polysaccharide components in G-100-I, one which resists major degradation following injection into the style and another which undergoes measurable degradation both before and after entry into the pollen tube.     

Timing of invasive pollen deposition influences pollen tube growth and seed set in a native plant

Invasive plants may threaten the reproductive success of native sympatric plants by modifying the pollination process. One potential mechanism takes place through the deposition of invasive pollen onto native stigmas when pollinators are shared among species. We explore how pollen from the invasive plant Brassica nigra influences pre- and post-fertilization stages in the native plant Phacelia parryi, through a series of hand pollination experiments. These two species share pollinators to a high degree. P. parryi flowers were hand-pollinated with either pure conspecific pollen (the control) or with B. nigra pollen applied prior to, simultaneously with, or following conspecific pollen. Application of B. nigra pollen lowered seed set, with the simultaneous application resulting in the highest reduction. Pollen tube growth was also influenced by the presence of invasive pollen, with fewer conspecific pollen tubes reaching the base of P. parryi styles in treatments where B. nigra pollen was applied prior to or simultaneously with conspecific pollen. The deleterious effects of invasive pollen on native seed set in this study are likely not due to loss of stigmatic receptivity since seed set was less affected when heterospecific pollen was applied prior to conspecific pollen, but may instead involve interactions between interspecific pollen grains on the stigma or within the style. Our study highlights the importance of timing of foreign pollen deposition on native stigmas and suggests that interspecific pollen transfer between native and exotic plants may be an important mechanism of competition for pollination in invaded plant communities.     

Observations of pollen tube growth in Nicotiana alata and their implications for the mechanism of self-incompatibility

 Style squashes and stylar grafts were used to examine the growth of Nicotiana alata pollen tubes in self-compatible and self-incompatible styles. Compatible tubes typically showed a uniform layer of callose deposition in the walls and in small plugs spaced at regular intervals within the tube. Incompatible tubes were characterised by the variability of callose deposition in the walls and by larger, closer and more irregularly spaced plugs. There was no difference in the growth rate of compatible and incompatible tubes during growth through the stigma, but within the style most compatible tubes grew 20–25 mm day^-1 (maximum 30 mm day^–1), whereas incompatible tubes grew 1.0–1.5 mm day^-1 (maximum 5 mm day^–1). Many incompatible tubes continued to grow until flowers senesced, and only a small proportion died as a consequence of tip bursting. Grafting compatibly pollinated styles onto incompatible styles showed that the incompatible reaction could occur in pollen tubes between 2 and 50 mm long, and that inhibition of pollen tube growth occurred in both the upper and lower parts of the transmitting tract. Grafting incompatibly pollinated styles onto compatible styles showed that the incompatible reaction was fully reversible in at least a proportion of the pollen tubes. The findings are not consistent with the cytotoxic model of inhibition of self-pollen tubes in solanaceous plants, which assumes that the incompatible response results from the degradation of a finite amount of rRNA present in the pollen tube. However, if pollen tubes do in fact synthesise rRNA, the findings become consistent with this model. Received: 23 May 1996 / Revision accepted: 22 August 1996     

Pollen tube growth is affected by exogenous hormones and correlated with hormone changes in styles in <Emphasis Type="Italic">Torenia fournieri</Emphasis> L.

In the present report, we described the effects of indole-3-acetic acid (IAA), zeatin (ZT), gibberellin (GA₃), and abscisic acid (ABA) on in vitro pollen germination and pollen tube growth in Torenia fournieri L. The results showed that IAA and GA₃ stimulated in vitro pollen tube growth, ABA inhibited pollen tube growth, and ZT had no significant effect on the process. The stimulating effect of exogenous IAA was particularly distinct, and led to synchronous growth of straighter and more slender pollen tubes compared with the controls. However, no significant changes were found in the germination of the treated pollen. The auxin efflux inhibitor, 10 μM 1-N-naphthylphthalamic acid (NPA), was also found to stimulate pollen tube growth. We measured the content of hormones (free IAA, ZT, GA₃, and ABA) in the stigmas and styles before and after pollination. The hormone contents of stigmas measured 0.5 h after pollination (0.5 HAP) showed that ABA content decreased, whereas the content of IAA, ZT, or GA₃ did not change significantly. The hormone level in pollinated styles (4 HAP) when pollen tubes had grown into the middle part of style was characterized by an increase in free IAA and GA₃ and a decrease in ABA, which was in agreement with the results that IAA and GA₃ promoted but ABA inhibited pollen tube growth in vitro. Furthermore, the change of IAA level in styles was most notable, which was accordant to the fact that auxin stimulated significantly pollen tube growth in vitro. Using immunoenzyme and immunogold labeling techniques and an anti-IAA monoclonal antibody, we confirmed that free IAA was present throughout style tissues, and distributed in the nucleus and cytoplasm of style cells. All these results suggested that hormones, especially IAA, play important roles in pollen tube growth of T. fournieri. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> calmodulin-like protein CML24 regulates pollen tube growth by modulating the actin cytoskeleton and controlling the cytosolic Ca<Superscript>2+</Superscript> concentration

Cytosolic free calcium ([Ca²⁺]_cyt), which is essential during pollen germination and pollen tube growth, can be sensed by calmodulin-like proteins (CMLs). The Arabidopsis thaliana genome encodes over 50 CMLs, the physiological role(s) of most of which are unknown. Here we show that the gene AtCML24 acts as a regulator of pollen germination and pollen tube extension, since the pollen produced by loss-of-function mutants germinated less rapidly than that of wild-type (WT) plants, the rate of pollen tube extension was slower, and the final length of the pollen tube was shorter. The [Ca²⁺]_cyt within germinated pollen and extending pollen tubes produced by the cml24 mutant were higher than their equivalents in WT plants, and pollen tube extension was less sensitive to changes in external [K⁺] and [Ca²⁺]. The pollen and pollen tubes produced by cml24 mutants were characterized by a disorganized actin cytoskeleton and lowered sensitivity to the action of latrunculin B. The observations support an interaction between CML24 and [Ca²⁺]_cyt and an involvement of CML24 in actin organization, thereby affecting pollen germination and pollen tube elongation.     

AMONG- AND WITHIN-FLOWER COMPARISONS OF POLLEN TUBE GROWTH FOLLOWING SELF- AND CROSS-POLLINATIONS IN DIANTHUS CHINENSIS (CARYOPHYLLACEAE)

We compared the rate of pollen tube growth following self- and cross-pollinations both among and within flowers of two clones of Dianthus chinensis L. For among-flower comparisons, both styles of a flower were pollinated with either self- or cross-pollen. Within-flower comparisons were made between the two styles of the same flower, one of which was self-pollinated and the other cross-pollinated. Comparisons between flowers indicated that self-pollen grew slower than cross-pollen in both clones. However, differences in the growth rate of pollen tubes from self- and cross-pollinations were greater when comparisons were made between the two styles of the same flower than when pollinations were made in different flowers. These results suggest the existence of interstyle interactions in pollen tube growth.     

Relationships of pollen size,pistil length and pollen tube growth rates in Rhododendron and their influence on hybridization

Summary Pollen size and pistil length data have been collected for 93 species of Rhododendron (Ericaceae) belonging to a number of different subgeneric taxa. For a sample of eight species in section Vireya, pollen tube growth in the style after selfor interspecific pollination has been quantified. Pollen volume and the time taken for pollen tubes to reach the ovary were both related to pistil length. Pollen-tube growth rates were generally greater for species with longer pistils and larger pollen. Increasing temperature increased the rate of pollen-tube growth. There was no detectable effect of pollen tube density on tube growth rate in the style. After interspecific pollinations tube growth rates in foreign styles could be faster or slower than in self styles. A semisterile individual with two viable pollen grains per tetrad and a plant grafted as scion to a longer-styled stock both showed more rapid pollen-tube growth than expected on the basis of pistil size. Data collected for 26 species in section Vireya showed that where extreme disparity of pollen/pistil size causes failure of interspecific crosses, one or more bridging species with intermediate pollen/pistil size can generally be selected.     

Pollen germination and tube growth: dependent on carbon dioxide and independent of ethylene

The influence of ethylene and CO₂ on pollen germination and tube growth was investigated employing ventilated culture systems. Ethylene had no effect on pollen germinability or tube growth. Germinating pollen did not produce a detectable amount of ethylene (less than 0.1 nl/g·hr). Supplementing the cultures with CO₂ caused a marked increase in germination and tube growth. The half-maximal response for germination was less than 0.5%. CO₂ levels ranging from 1.08 to 2.22% were found in the internal cavity of lily styles. CO₂ derived from stylar metabolism may, therefore, modulate pollen tube growth thus integrating the events leading to fertilization.     

Pollen tube competition as a mechanism of prezygotic reproductive isolation between Mimulus nasutus and its presumed progenitor M. guttatus

This paper considers the extent to which differences in pollen tube growth rates can provide prezygotic reproductive isolation between Mimulus nasutus and its presumed progenitor, Mimulus guttatus . Mimulus nasutus is partially cleistogamous, but its larger chasmogamous flowers offer appreciable opportunity for outcrossing. Mimulus nasutus was found to have smaller pollen grains and shorter styles than M. guttatus . No differences were observed in pollen grain germination on conspecific and heterospecific stigmas. However, pollen tube growth rates of M. nasutus were found to be much slower than those of M. guttatus in the styles of that species. Consequently, any M. nasutus pollen transferred to an M. guttatus stigma was found to be competitively disadvantaged in an M. gutattus style. By contrast, no difference in pollen tube growth rate was detected between the species when growing in M. nasutus styles, possibly because M. nasutus styles are unable to support fast pollen tube growth. We tested the prediction from the pollen tube studies that a 50:50 mix of M. guttatus and M. nasutus pollen would produce 50% hybrid seeds when M. nasutus was the maternal parent, and near to 0% hybrid seed when M. guttatus was the maternal parent. The results were found to support this prediction. We conclude that pollen–pistil interactions can effect strong reproductive isolation between these species, as M. guttatus pollen tubes have a competitive advantage over those of M. nasutus in an M. guttatus style, but not in an M. nasutus style.     

Sugar composition of pollen grain and pollen tube cell walls

The sugar composition of pollen grain and pollen tube cell walls was studied for Camellia japonica, C. sasanqua, C. sinensis, Tulipa gesneriana and Lilium longiflorum. In all species, the main components of pollen grain walls were arabinose, galactose, glucose and uronic acid. On the other hand, the pollen tube walls consisted mostly of glucose. The pollen tube wall of C. japonica was fractionated into hemicellulose, α-cellulose and pectic substance fractions in yields of 61, 19 and 3 %, respectively. The hemicellulose fraction was composed essentially of glucose. The sugar composition of the pollen tube wall was not influenced by the nature of exogenously supplied sugars. Rapid growth of the pollen tube seemed to correlate with the synthesis of hemicellulosic glucan.