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.     

Studies on heteromorphic self-incompatibility systems: Physiological aspects of the incompatibility system of Primula obconica

Summary In Primula obconica, a species with a heteromorphic self-incompatibility system, the distinction between compatible and incompatible pollen tubes takes place on the stigma surface in thrum flowers, self tubes growing randomly over the papillar cells. No differences were seen between self and cross tube behaviour on the pin stigma surface, but self tubes were inhibited within the stigmatic tissue with differences in tube length evident after 24 h. The stigma surface bears a proteinaceous pellicle and binds the lectin Concanavalin A. Removal of the stigma removes the incompatibility barrier in mature gynoecia. Bud pollination shows that pollen tubes cannot grow in a normal manner on immature stigmas; the random growth of tubes over the stigma surface resembles that of mature thrum selfs. Fewer compatible tubes reach the style base of young gynoecia and smaller numbers of seeds are set than in mature flowers. Pin and thrum pollen grains germinate and grow in aqueous media, thrum tubes growing longer than pin. The presence of H₃BO₄ and CaCl₂ in the growth medium promotes tube elongation and lengths equivalent to compatible styles can be obtained. The pollen grains have proteinaceous materials in their walls which diffuse out on moistening. Prolonged washing in aqueous media removes these materials but the incompatibility reaction remains unchanged. Thus the incompatibility reaction is between pollen tubes and stigmatic tissue and differs from the homomorphic, sporophytic system where pollen wall proteins elicit the incompatibility response.     

The genetics of self-incompatibility inOenothera rhombipetala

Pollen tubes were grown under controlled temperatures in stigmas and styles which had been cut from the flowers at the point of juncture of the style with the ovary. At constant temperatures the tubes compatible with the styles grew rapidly at uniform rates. They showed accelerated growth with increase in temperature within the range 10°–30°C. Growth of incompatible tubes was irregular and slow and was neither measurably accelerated nor depressed within this range of temperatures. Routine tests for compatibility were conducted at 27°C; at this temperature compatible tubes grew through the entire length of the styles (ca. 58 mm) in 9 hours. Much of the incompatible pollen failed to germinate; that which did, grew to a maximum length ofca. 2 mm inca. 4 hours. The incompatible tubes did not even enter the styles but remained confined to the stigmas. ThreeS alleles were involved and conformed in their behavior to the gametophytic system of incompatibility. Colchicine-induced tetraploid derivatives of these plants showed no increase in compatibility.     

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     

梨远缘花粉原位萌发及生长特性

应用荧光标记方法对梨远缘花粉在‘丰水’和‘噢嗄二十世纪’柱头上萌发及花粉管生长特性进行观察,结果表明:(1)梨远缘花粉均能在柱头上萌发,但其萌发率不同,授粉后24 h,在‘丰水’柱头上‘红叶桃’花粉萌发率最高,达62.8%,而‘盖县大李’花粉萌发率仅为12.0%,各种远缘花粉在‘丰水’柱头萌发率均高于‘噢嗄二十世纪’柱头。(2)各种远缘花粉管在梨柱头或花柱内生长情况也有差异,‘红叶桃’等核果类花粉管在梨柱头上均表现为扭曲、盘绕等现象,不能穿过柱头;‘红星’和‘红富士’花粉管虽然有少量穿过柱头,但不能进一步在花柱内生长,表现为扭曲变形、先端膨大等不亲和性现象。因此,梨与远缘果树杂交不亲和在柱头上就已发生,这与梨自交不亲和反应发生在花柱内的现象不同。     

STUDIES ON THE SELF-INCOMPATIBILITY RESPONSE OF LILIUM LONGIFLORUM

Removal of substances loosely associated with the wall of Lilium longiflorum pollen did not affect in vitro germination and tube growth, tube growth within compatible styles, or production of viable seeds. Nor was growth of pollen tubes within incompatible styles enhanced by prior removal of the loosely bound materials. Hence, these materials appear not to be recognition factors in the gametophytic self-incompatibility system of Lilium and to have no role in pollen germination. Heat treatment of selected portions of lily pistils prior to pollination indicated that the incompatibility factor(s) was located in the lower half of the style, and that the stigma plays no role in incompatibility.     

Unilateral incompatibility within the brassicaceae: further evidence for the involvement of the self-incompatibility (S)-locus

Unilateral pollen-pistil incompatibility within the Brassicaceae has been re-examined in a series of interspecific and intergeneric crosses using 13 self-compatible (SC, Sc) species and 12 self-incompatible (SI) species from ten tribes. SC x SC crosses were usually compatible, SI x SC crosses showed unilateral incompatibility, while SI x SI crosses were often incompatible or unilaterally incompatible. Unilateral incompatibility (UI) is shown to be overcome by bud pollination or treating stigmas with cycloheximide — features in common with self-incompatibility. Treating stigmas with pronase prevents pollen tubes from penetrating the stigma in normally compatible intra-and interspecific pollinations. The results presented show that the presence of an incompatibility system is important in predicting the outcome of interspecific and intergeneric crosses and, combined with the physiological similarities between UI and SI, would suggest an involvement of the S-locus in UI.     

Staining Germinating Pollen and Pollen Tubes

Germinating pollen on stigmas and pollen tubes in styles of Antirrhinum, Brassica, Oenothera, Raphanus, Rosa, solatium and Tagetes spp. were prepared for examination as follows: The styles were fixed in ethyl alcohol-acetic acid 3:1 for 1 hr, and hydrolyzed at 60°C for 5 to 60 min (depending on the species) in 45% acetic acid. The stigma with its attached strand(s) of stigmatoid tissue was then dissected out under a stereoscopic microscope, placed in a few drops of a staining solution made by dissolving 150 mg of safranin O and 20 mg of aniline blue in 25 ml of hot 45% acetic acid. After 5-15 min in this stain, the tissue was placed in a fresh drop of stain on a microscope slide and gently squashed under a cover glass. Because of a gradual precipitation of the aniline blue component, the stain had to be filtered regularly before use. However, a staining solution could be kept at room temperature for several weeks.     

INCOMPATIBILITY AND THE POLLEN ECONOMY OF JEPSONIA PARRYI

Jepsonia parryi (Saxifragaceae) has heterostylous flowers and is strongly self-incompatible. Pin flowers have long styles, large stigmas, short stamens, and numerous, small pollen grains with finely sculptured walls. Thrum flowers have short styles, small stigmas, long stamens, and fewer, larger pollen grains with coarsely sculptured walls. Pin plants and thrum plants occur in a 1:1 ratio in field populations. Although the insect pollinators of J. parryi transfer ample compatible pollen to pin and thrum stigmas to account for full seed production, much of the pollen deposited on stigmas is incompatible. Analysis of the pollen deposits on stigmas collected from field populations indicates that compatible “legitimate” pollination of pin and thrum flowers is essentially random and is not obviously aided by floral dimorphism. It is suggested that although heterostyly had a positive adaptive value in the past evolutionary history of Jepsonia it is no longer adaptive under the present pollination regime, although it is maintained because of its strong genetic fixity.     

POLLEN TUBE GROWTH AND INCOMPATIBILITY FOLLOWING INTRA- AND INTER-SPECIFIC POLLINATIONS IN LILIUM LONGIFLORUM

The growth of pollen tubes of a sampling of Lilium species in styles of L. longiflorum incubated at 24 C for 48 hr after pollination indicated two types of interspecific incompatibility. Pollen tubes of two self-compatible species of section Leucolirion, the section including L. longiflorum, stopped growth abruptly upon reaching the stylar canal, were of abnormal morphology, and were incapable of continued growth with longer incubation. Pollen tubes of self-compatible or self-incompatible species of sections Sinomartagon, Pseudolirium, Liriotypus, and Daurolirion approached but did not exceed the length of intraspecific incompatible pollen tubes in styles of L. longiflorum. Pollen tube morphology was normal and tubes were capable of continued growth with additional incubation. Unilateral interspecific incompatibility occurred in reciprocal crosses between self-incompatible L. longiflorum and self-compatible L. regale and L. formosanum, but exceptions occurred in Aurelian hybrids. Incubation of interspecifically pollinated L. longiflorum styles at 39 C, which removes the self-incompatibility reaction, had no effect on interspecific incompatibility.     

Experimental insights on the function of ancillary pollen and stigma polymorphisms in plants with heteromorphic incompatibility

Most heterostylous plants possess a reciprocal arrangement of stigmas and anthers (reciprocal herkogamy), heteromorphic self‐incompatibility, and ancillary polymorphisms of pollen and stigmas. The topographical complementarity hypothesis proposes that ancillary polymorphisms function in the rejection of incompatible pollen thus promoting disassortative pollination. Here, we test this hypothesis by investigating patterns of pollen transfer and capture in populations of dimorphic Armeria maritima and A. pubigera and distylous Limonium vulgare (Plumbaginaceae), and by studying pollen adherence and germination patterns in A. maritima following controlled hand‐pollinations. Armeria lacks reciprocal herkogamy allowing the evaluation of the extent to which ancillary polymorphisms affect the composition of pollen loads. We compared the amounts of compatible and incompatible pollen on stigmas in natural populations and calculated the proficiencies of pollen transfer for each mating type. We detected disassortative pollination in each species, and mating types did not differ in compatible pollen capture, although cob stigmas captured more incompatible pollen. Controlled hand‐pollinations revealed the failure of incompatible pollen to adhere and germinate on stigmas. Our results provided evidence that, while structural in nature, pollen‐stigma dimorphisms are tightly associated with heteromorphic incompatibility and likely function to promote disassortative pollination, especially in the absence of reciprocal herkogamy.     

烟草柱头和花柱中阿拉伯半乳糖蛋白的定位

通过Western印迹法、免疫组织化学和超微细胞化学等技术,研究了烟草柱头和花柱中阿拉伯半乳糖蛋白（arabinogalactan-proteins,AGPs）的分布。结果表明烟草柱头和花柱组织中含有大量的AGPs,主要分布于柱头表皮细胞的细胞质和分泌层细胞的胞外基质中,且授粉前后AGPs的分布情况差异不明显;而花柱中的AGPs主要分布于表皮细胞的外层细胞壁、维管组织周围细胞的细胞质及引导组织的胞外基质中;花粉管通过后,引导组织胞外基质中AGPs减少,而花粉管细胞质和花粉管壁中检测到大量AGPs。     

荞麦亲和与不亲和授粉后柱头和花粉管中ATP酶的超微细胞化学定位

利用磷酸铅沉淀技术对荞麦(Fagopyrum esculentum Month.)pin型植株分别进行亲和授粉和不亲和授粉后的柱头、花粉粒、花粉管进行了ATPase的超微细胞化学定位。结果表明(1)亲和授粉和不亲和授粉后0.5h,柱头细胞的ATPase活性反应水平较低或基本无酶活性;柱头表面、柱头上附着的花粉粒内ATPase活性在不亲和授粉时较低,亲和授粉时较高,花粉粒内ATPase主要定位于线粒体和精子细胞;(2)授粉后1.5h,不亲和授粉的柱头细胞及花粉管的ATPase活性均较低,花粉管停止生长,细胞质开始解体;而亲和授粉的柱头细胞及花粉管的ATPase活性均较高,ATPase主要定位于柱头细胞的质膜、胞基质以及花粉管的壁、质体的膜、高尔基体、线粒体上。根据不同时期不同部位ATPase活性的差异,我们认为荞麦发生自交不亲和时,花粉管在花柱中停止生长不仅是因为花粉管得不到花柱中的营养物质而引起的,可能也与花粉管自身物质代谢发生障碍有关。     

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.     

Disentangling the components of pollen limitation in a widespread herb with gametophytic self-incompatibility

Premise

Seed production is frequently limited by the receipt of insufficient or low-quality pollen, collectively termed “pollen limitation” (PL). In taxa with gametophytic self-incompatibility (GSI), incompatible pollen can germinate on stigmas but pollen tubes are arrested in styles. This allows for estimates of pollen performance before, during, and after self-recognition, as well as insight into the factors underlying pollen quality limitation in GSI taxa.

Methods

We scored pollen performance following self and outcross pollinations in Argentina anserina to identify the location of self-recognition and establish the relationship between pollen tubes and seed production. We then estimated quantity and quality components of PL from >3300 field-collected styles. We combined our results with other studies to test the prediction that low pollen quality, but not quantity, drives higher PL in self-incompatible (SI) taxa than in self-compatible taxa (SC).

Results

Self and outcross pollen germinated readily on stigmas, but 96% of germinated self-pollen was arrested during early tube elongation. Reproduction in the field was more limited by pollen quality than by quantity, and pollen failure near the location of self-recognition was a stronger barrier to fertilization than pollen germination. Across 26 taxa, SI species experienced stronger pollen quality, but not quantity, limitation than SC species.

Conclusions

Evaluating pollen performance at multiple points within pistils can elucidate potential causes of pollen quality limitation. The receipt of incompatible pollen inhibits fertilization success more than insufficient pollen receipt or poor pollen germination in A. anserina. Likewise, pollen quality limitation drives high overall PL in other SI taxa.     

Analysis of stylar self-incompatibility competence by use of heat induced inactivation

Summary Immersion of Lilium longiflorum pistils in 49 °C water for increasing durations of 1,2,3, or 4 minutes immediately prior to incompatible pollination resulted in a correspondingly progressive decrease in the stylar self-incompatibility competence, as determined from the lengths attained by pollen tubes during 48 hours growth in the styles at 24 °C. Neither pistils remaining on the plant nor those detached from the plant which were immersed after anthesis in 49 °C water for 5 minutes regained self-incompatibility competence during a 48 hour incubation at 24 °C prior to incompatible pollination. Heat treatment of detached pistils as early as 39 hours prior to bud anthesis also resulted in an inactivation of stylar self-incompatibility competence when incompatible pollination was made at 24 hours after anthesis. Experiments utilizing heat treatment of partial lengths of detached whole styles revealed that pollen tubes which have grown through as much at 45 millimeters of either a physiologically incompatible or compatible portion of the style are still capable of shifting to either a higher growth rate or lower growth rate upon entry into respectively either a physiologically compatible or incompatible portion of the style.     

Pollen tube growth following compatible and incompatible intraspecific pollinations in Petunia hybrida

The observation that both compatible and incompatible pollen tubes grow at identical speeds on the stigma in many plants with gametophytically controlled self-incompatibility (SI) systems has, in Petunia, been extended to cover all other facets of pollen behaviour on this tissue. On entry into the stylar transmitting tissue both types of tubes accelerate, but the compatible achieve a higher terminal velocity than do the incompatible, which eventually slow and stop. Grafting experiments show that the top 1 mm of the stylar tissue can play an important rôle in determining the future development of the pollen tube. Following mixed pollinations, proportionally too many compatible pollen tubes reach the ovary than would be expected from the results of pure compatible and incompatible pollinations indicating that incompatible pollen in some way helps prime the style for growth of compatible pollen tubes. This data is considered in terms of recent structural studies of these tissues, and related to the pollination conditions pertaining to Petunia populations in the field.Abbreviation SI self-incompatibility     

Self-incompatibility inCrocus vernus subsp.vernus (Iridaceae)

Outcross, self- and mixed pollinations were performed inCrocus vernus subsp.vernus, a species with bicellular pollen, dry stigmas and hollow style. No differences were noted among the above pollinations concerning the germination of pollen and the growth of pollen tubes until the top of ovary. Within 45 min after pollinations 62% of pollen grains germinated. Pollen tubes penetrated the papilla cuticle extending along the papilla wall; on entry into stigmatic lobes they continued growth in the stylar secretion to ovarian locules. Here, however, self-pollen tubes failed to reach or to enter the ovule micropyle; while pollen tubes from either outcross- or mixed pollinations grew until fertilizing ovules. These observations gave evidence of a self-incompatibility system inCrocus, which appeared to be neutralized by mentor effect. The ovary as site of incompatibility response is discussed.