Regulation of seed number and female incitation of mate competition by a pH-dependent proteinaceous inhibitor of pollen grain germination in Leucaena leucocephala

Summary Leucaena leucocephala generally produces pods with more than 7–9 seeds. This is regulated by the stigmatic inhibition of pollen grain germination when the pollen grains are less than a critical number in the stigma. This number-dependent inhibition of pollen grain germination is effected by a pH-dependent proteinaceous inhibitor active at the stigmatic pH. Only when the pollen grains in the stigma exceed the critical number, they inactivate the inhibitor by collectively raising the stigmatic pH and thus overcoming the inhibition. The adaptive significance of such pre-fertilization mechanism for the female in inciting mate competition among the pollen grains is discussed. The evolution of en masse pollen grain dispersal units is explained as a sexual selection strategy by males in response to such stigmatic inhibition by females.     

Pollen morphology and germination inNarcissus

Pollen grains of several species and varieties ofNarcissus were examined with a light microscope and a scanning electron microscope. Normal pollen grains were kidney- or spindle-shaped with a germination furrow and a reticulate structure similar to that of the pollen grains ofAmaryllis. Pollen grains germinated within 2 to 3 hr. Percentage of germination was dependent upon temperature and treatment. Pollen tubes grew in length up to 1,000 μm and branched occasionally or behaved in strange fashion. Fresh pollen grains germinated more in distilled water at lower temperature than in sucrose-aqueous medium. Both in the presence and in the absence of stigmatic exudate calcium increased the percentage of germination. Gibberellic acid, abscisic acid and coumarin inhibited the pollen germination. Plasmoptysis occurred in all species and in all media tested except in a medium containing coumarin without stigmatic exudates. Plasmoptysis did not seem to be induced by hypotonic medium alone. Pollen of high germination capacity showed a high percentage of plasmoptysis. Based on the results obtained, evolution and sterility of theNarcissus plant was discussed.     

In vitro maturation and germination of <Emphasis Type="Italic">Orychophragmus violaceus</Emphasis> microspores

Microspores cultured in vitro can be regarded as a system to study gene regulation, cell fate determination and cell differentiation during pollen development as well as an alternative method of genetic transformation in plants. In our study, pollen development and viability in Orychophragmus violaceus in vivo were determined and then pollen from the late unicellular stage was cultured in vitro. MS liquid medium + White vitamins + 2% (V/V) coconut milk + 0.5 M maltose, pH = 7.0 was the most appropriate for in vitro culture of Orychophragmus violaceus microspores. With this medium, the rates of in maturation and germination were 19.3% and 4.7%, respectively. Liquid medium with 0.6 M maltose + 1.6 mM boric acid + 2.9 mM Ca(NO₃)₂ + 29.6 μM vitamin B1, pH = 7.0 was optimal for germination of pollen matured in vivo. The rate of germination was 70.7%. Pollen matured in vitro cultured in similar medium exhibited a rate of germination of 62.7%. Hence, the experimental study showed that in vitro maturation of microspores is feasible and this experimental system can be applied to further theoretical and practical research.     

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

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

Stigmatic exudate in the Annonaceae: Pollinator reward,pollen germination medium or extragynoecial compitum?

Although “dry‐type” stigmas are widely regarded as ancestral in angiosperms, the early‐divergent family Annonaceae has copious stigmatic exudate. We evaluate three putative functions for this exudate: as a nutritive reward for pollinators; as a pollen germination medium; and as an extragynoecial compitum that enables pollen tube growth between carpels. Stigmatic exudate is fructose dominated (72.2%), but with high levels of glucose and sucrose; the dominance of hexose sugars and the diversity of amino acids observed, including many that are essential for insects, support a nutritive role for pollinators. Sugar concentration in pre‐receptive flowers is high (28.2%), falling during the peak period of stigmatic receptivity (17.4%), and then rising again toward the end of the pistillate phase (32.9%). Pollen germination was highest in sugar concentrations <20%. Sugar concentrations during the peak pistillate phase therefore provide optimal osmolarity for pollen hydration and germination; subsequent changes in sugar concentration during anthesis reinforce protogyny (in which carpels mature before stamens), enabling the retention of concentrated exudate into the staminate phase as a pollinator food reward without the possibility of pollen germination. Intercarpellary growth of pollen tubes was confirmed: the exudate therefore also functions as a suprastylar extragynoecial compitum, overcoming the limitations of apocarpy.     

Optimization of conditions for germination of cold-stored <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> pollen

One of the rare weak points of the model plant Arabidopsis is the technical problem associated with the germination of its male gametophyte and the generation of the pollen tube in vitro. Arabidopsis pollen being tricellular has a notoriously low in vitro germination compared to species with bicellular pollen. This drawback strongly affects the reproducibility of experiments based on this cellular system. Together with the fact that pollen collection from this species is tedious, these are obstacles for the standard use of Arabidopsis pollen for experiments that require high numbers of pollen tubes and for which the percentage of germination needs to be highly reproducible. The possibility of freeze-storing pollen after bulk collection is a potential way to solve these problems, but necessitates methods that ensure continued viability and reproducible capacity to germinate. Our objective was the optimization of germination conditions for Arabidopsis pollen that had been freeze-stored. We optimized the concentrations of various media components conventionally used for in vitro pollen germination. We found that in general 4 mM calcium, 1.62 mM boric acid, 1 mM potassium, 1 mM magnesium, 18% sucrose at pH 7 and a temperature of 22.5°C are required for optimal pollen germination. However, different experimental setups may deviate in their requirements from this general protocol. We suggest how to optimally use these optimized methods for different practical experiments ranging from morphological observations of pollen tubes in optical and electron microscopy to their bulk use for molecular and biochemical analyses or for experimental setups for which a specific medium stiffness is critical. F. Bou Daher and Y. Chebli contributed equally to this study.     

The significance of ant and plant traits for ant pollination in Leporella fimbriata

Summary Ant metapleural glands secrete surface antibiotics that affect pollen as well as bacteria and fungi. This may be one reason why ant pollination is rare. It is predicted that pollination by ants is possible only in the presence of certain ant and/or plant traits. Two traits are investigated; first, absence of the metapleural glands, and second, the presence of stigmatic secretions that insulate pollen from the ant integument. The pollinator of the orchid Leporella fimbriata is the ant Myrmecia urens. Only one caste is involved, the winged males, and they differ significantly from the queen and worker castes in that they do not possess metapleural glands. This paper reports experiments which test for differential effects on pollen between the males and other castes and evaluates the importance of stigmatic secretions. The results show that the absence of metapleural glands makes no difference as all three castes have strong disruptive effect on pollen artificially applied to the integument. However, during pollination the orchid secures the pollen mass to the ant surface by stigmatic secretions and normal pollen function, fruit production and seed set occur. It appears that both ant and plant traits are pre-adaptive having evolved for functions other than ant pollination.This is contribution number 98 to the Research Unit for Biodiversity and Bioresources, Macquarie University     

Fungicide sprays can injure the stigmatic surface during receptivity in almond flowers

Fungicides can be detrimental to flower development, pollen function and fruit set in a number of crops. Almond is a self-incompatible nut crop that has a fruit set of only approx. 30 % of the total number of flowers. Thus, interference of pollination and fertilization by fungicide sprays is of concern, and identification of chemicals having the least detrimental effects would be desirable. The objective of this study was to evaluate the effect of fungicide sprays on stigma morphology in almond using a laboratory spray apparatus that simulated field applications. Four fungicides (azoxystrobin, myclobutanil, iprodione and cyprodinil) were applied, and fresh, unfixed stigmatic surfaces were observed using a scanning electron microscope at 4 and 24 h after spraying. Increased exudate accumulation was induced by azoxystrobin at both time periods, and localized damage and collapse of stigmatic cells were observed after 24 h. Damaged stigmatic papillae exhibited wrinkling, surface distortion or collapse. Likewise, myclobutanil caused significant damage to and collapse of papillae; these were more extensive at later observations. Iprodione had no effect on exudate accumulation but caused marked and severe collapse of stigmatic papillae which was pronounced at 24 h. Cyprodinil promoted a copious increase in exudate secretion and caused the most severe collapse of stigmatic cells of all the fungicides evaluated. Damage was somewhat localized at 4 h but more global at 24 h. This study has verified that certain fungicide sprays have direct detrimental effects on stigma morphology and enhance exudate production in almond flowers.     

Stigmatic inhibition of pollen grain germination-its implication for frequency distribution of seed number in pods of Leucaena leucocephala (Lam) de Wit

Summary Seed number per pod is negatively skewed in Leucaena leucocephala. Post-fertilisation abortion of pods does not completely explain the skewed distribution. It is shown that a pre-fertilisation regulation effectively restricts the formation of pods with less than a certain number of seeds. The regulation is rendered through the inhibition of pollen grain germination by the stigmatic fluid when the pollen grain is less than a certain critical load in the stigma. Such a control is shown to have implications in the evolution of pollen package units, generation of male gametophytic competition and the economy of packing seeds in pods.     

胀果甘草花粉生活力测定及甘草的杂交育种

杂交育种是最为传统的选育新品种的方式之一,花粉生活力能够影响其中遗传物质的传递。胀果甘草是3种药典收录的甘草之一,具有许多特殊活性成分。通过TTC法和离体萌发法对2个胀果甘草(Glycyrrhiza inflata)种质资源GJJ-7和GJJ-9花朵开放5个时期(a~e)的花粉生活力进行测定。结果表明,GJJ-7花粉生活力趋势在5个时期呈现波浪型,在a、d时期达到最高;GJJ-9花粉生活力会随着花瓣展开度的变大而升高,在e时期达到最高。离体萌发结果表明GJJ-7和GJJ-9花粉生活力随花朵开放程度增大呈先升高后降低的趋势,萌发力均在c时期最高。杂交结荚率则与离体萌发结果一致,说明离体萌发法测定花粉生活力比TTC法更可靠。以光果甘草(G.glabra) S-7和S-12为母本的杂交组合结荚率低,但能够获得发芽率高的种子;以乌拉尔甘草(G.uralensis) GDN-16为母本的杂交组合结荚率高,但种子发芽率低,说明在甘草杂交中父母本的选择对结荚率有重要影响。     

Effects of pollen competitive environment on pollen performance in Mirabilis jalapa (Nyctaginaceae)

 We examined the influence of pollen competitive environment on pollen performance in Mirabilis jalapa. We used the number of pollen grains and the number of pollen tubes per pistil as measures of pollen competition. Pollen germination, pollen tube penetration into the style, and pollen tube growth rates were used as measures of pollen performance. All three measures of pollen performance were affected by the competitive environment. Pollen germination was greatest at intermediate pollen load sizes. The percentage of germinated pollen grains that penetrated the stigma and grew into the style decreased with pollen load size. Pollen tube growth rate in the style was greater and more variable with larger numbers of pollen tubes in the style. Controlling for the degree of selection at the stigma indicated that pollen-pollen or pollen-style interactions were the likely causes of increased growth rates. Received: 28 October 1996 / Revision accepted: 24 January 1997     

Zinc is critically required for pollen function and fertilisation in lentil

The role of zinc (Zn) in reproduction of lentil (Lens culinaris Medik. cv. DPL 15) and the extent to which the Zn requirement for reproduction can be met through supplementation of Zn at the time of initiation of the reproductive phase have been investigated. Low supply (0.1micromol/L) of Zn reduced the size of anthers, the pollen producing capacity and the size and viability of the pollen grains. Scanning electron microscopy (SEM) of pollen grains of Zn deficient plants showed enhanced thickening of exine and wide and raised muri. In vitro germination of pollen grains was reduced by >50% and growth of pollen tubes was retarded. Unlike Zn sufficient plants, the cuticle around the stigmatic papillae of Zn deficient plants remained intact, preventing the interaction between pollen grains and stigmatic exudates that provides the polarity for the growth of pollen tubes through the stylar tract. Zn deficiency increased the activity of acid phosphatase and peroxidase in extracts of pollen grains. Histochemical localisation on the stigmatic surface and native PAGE of the enzyme extracts of pollen grain and stigma exudates showed enhanced expression of acid phosphatase and peroxidase and suppressed expression of esterase in response to Zn deficiency. Zn deficiency reduced the setting of seeds and also their viability. The effect on seed setting was more marked than on in vitro germination of pollen grains, suggesting that the latter was not the exclusive cause of inhibition of fertility. Possibly, loss of fertility was also caused by impairment in pollen-pistil interaction conducive to pollen tube growth and fertilisation. Impairment in pollen structure and function and seed setting was observed even when plants were deprived of Zn at the time of flowering, but to a lesser extent than in plants maintained with low Zn supply from the beginning. Increasing the Zn supply from deficient to sufficient at the initiation of flowering decreased the severity of Zn deficiency effects on pollen and stigma morphology, pollen fertility and seed yield. In conclusion, structural and functional changes induced in pollen grains and stigma of Zn deficient plants and associated decrease in seed setting of lentil indicate a critical requirement of Zn for pollen function and fertilisation that can be partially met by supplementing Zn at the onset of the reproductive phase.     

POLLINATION IN LUPINUS NANUS SUBSP. LATIFOLIUS (LEGUMINOSAE)

Floral morphology and phenology and the timing of stigmatic receptivity and pollen viability were studied to elucidate the mechanisms by which self-pollination in Lupinus nanus subsp. latifolius is minimized under natural conditions. Pollen germination and pollen tube growth suggest that a physiological self-incompatibility system does not exist. Instead, self-pollination is minimized by protandry and by a collar of peristigmatic hairs, which initially inhibit access of autologous pollen to the stigma. These hairs subsequently wilt, permitting self-pollination of unvisited flowers. Pollen germinates in vivo one day before substantial metabolic enzyme activity can be detected. Citric acid cycle enzymes are not detectable in pollen, but those of anaerobic metabolism are. Beginning on the second day postanthesis, stigmatic secretions exude from weak areas on lateral walls of the elongate epidermal papillae, welling up in the interstices between these cells. This contrasts with the stigmas of other papilionoid legumes, in which secretions accumulate beneath the cuticle covering the stigmatic cells, and the often-thin cuticle must be ruptured before pollen and exudate can come into contact.     

Distribution of calcium in the stigma and style of tobacco during pollen germination and tube elongation

Potassium antimonate was used to locate loosely bound calcium in the stigma and style of tobacco. The tobacco stigma is wet and covered by a thick layer of glycoprotein exudate at anthesis. The exudate contains abundant vesicles, which are densely labeled with calcium precipitates. When pollen grains arrive at the stigma, become hydrated, and as the pollen swells, Ca²⁺ precipitates accumulate at the aperture. Calcium precipitates that accumulate in pollen cytoplasm are initially concentrated within small vacuoles, but as germination proceeds these appear to fuse, forming prominent, densely labeled vesicles that preferentially accumulate near the proximal region of the growing tube. Although the stigma has abundant particles, few calcium precipitates are observed in the transmitting tissue from anthesis to 11 h after pollination. However, at 22 h after pollination, accumulation of calcium increases distally from the stigmatic interface with the transmitting tissue through the length of the style to the ovary. An examination of flowering plants with differing floral biology will be needed to understand the role of loosely bound calcium accumulation and its relationship to tissue-level changes in calcium uptake, maintenance of other calcium pools, including [Ca²⁺]_cyt, and in pollen and style maturation during the progamic phase.     

Papillar integrity as an indicator of stigmatic receptivity in kiwifruit (Actinidia deliciosa)

Stigmatic receptivity is a major factor limiting fruit set inkiwifruit (Actinidia deliciosa). The aim of this work was toknow what determines the cessation of stigmatic receptivityin this species. Stigmatic receptivity has been analysed inkiwifruit through the capacity of the stigma to sustain pollengermination and has been related to stigmatic development anddegeneration.The stigma of kiwifruit flowers has a papillatesurface which from anthesis is covered with an abundant exudate.Papillae are unicellular and contain a number of phenolic deposits.During the lifetime of the flower these papillae gradually loseturgidity, while stigmatic secretion increases until 5 d afteranthesis when papillae start rupturing and the papillar contentis liberated into the germination medium. Stigmatic receptivityis high at anthesis and lasts for the next 4 d. However, itdrastically decreases 5 d after anthesis and it is nil 2 d later.The pattern of stigmatic receptivity closely fits that of papillarintegrity, indicating that stigmatic receptivity relies on thisintegrity. Since papillar integrity can easily be evaluatedin stigmatic arms, this criterion can be used as a quick methodto estimate stigmatic receptivity. Key words: Actinidia deliciosa, kiwifruit, stigma, receptivity, pollen     

Influence of season on rose pollen quality

Summary In vitro tests of pollen germination were carried out at different periods during an annual cycle in order to study environmental influence on the quality of Rosa hybrida L. pollen during its maturation process. This quality was evaluated by taking into account the rate of germination as well as the average length of emitted pollen tubes. In addition, during an annual hybridization period, a few pollinations were carried out in vivo with pollen of the same origin in order to study the evolution of the fertilization results, as attested by number of achenes per resulting hips. During the period covered by the experiments, the evolution of the pollen quality detected in vitro can be related to that of seed setting success. Of the two criteria used in vitro to evaluate pollen quality, the factor most liable to influence in vivo fertilization success seems to be the average length of emitted pollen tubes.     

Structural analysis of stigma development in relation with pollen–stigma interaction in sunflower

Structural analysis of stigma development in sunflower highlights the secretory role of papillae due to its semi-dry nature. Production of lipid-rich secretions is initiated at the staminate stage of the flowers in stigma development and increases at the receptive stage, coinciding with an extensive development of elaioplasts and endoplasmic reticulum network in the basal region of the papillae. Transfer cells, earlier identified only in the wet type of stigma, are also present in the transmitting tissue of the sunflower stigma. Attainment of physiological maturity by the stigmatic tissue, accompanying development from bud to pistillate stage, appears to affect the initial steps of pollen–stigma interaction. The nature of self-incompatibility in Helianthus has also been investigated in relation with pollen adhesion, hydration and germination. Pollen adhesion to the stigma is a rapid process in sunflower and stigma papillae exhibit greater affinity for pollen during cross pollination as compared to self-pollination. Components of the pollen coat and the pellicle on the surface of stigmatic papillae are critical for the initial phase of pollen–stigma interaction (adhesion and hydration). The lipidic components of pollen coat and the proteinaceous and lipidic components from the surface of the papillae coalesce during adhesion, leading to the movement of water from stigma to the pollen, thereby causing pollen hydration and its subsequent germination. Pollen germination (both in self-and cross-pollen) on the stigma surface and the growth of the pollen tube characterize the flexibility of self-incompatibility in sunflower. Compatible pollen grains germinate and the pollen tube penetrates the stigma surface to enter the nutrient-rich transmitting tissue. The pollen tube from incompatible pollen germination, however, fails to penetrate the stigmatic tissue and it grows parallel to the papillae. Present findings provide new insights into structural and functional relationships during stigma development and pollen–stigma interaction.     

转基因插入对水稻花粉活力和杂交结实的影响

以花粉活力和杂交结实率评价转基因插入对水稻品种异交潜力的影响.选用含bar、crylAb、BADH和Xa21等4种基因的5份转基因水稻品系研究转基因插入对转基因受体品种花粉离体萌发率的影响.结果表明,不同受体品种花粉离体萌发率差异显著;转基因水稻花粉离体萌发率在0.416～0.584间,与受体品种(0.004～0.574)相近;转基因品种与相应受体品种间花粉离体萌发率差异不显著.对所选配的26个人工杂交组合杂种结实调查表明,bar、crylAb基因的插入对受体品种杂交结实率影响显著,而Xa21基因的插入对受体品种杂交结实率影响较小;转基因水稻品种(花粉供体)与非转基因水稻品种的杂交结实率变幅为0.056～0.13,在受体品种(花粉供体)与非转基因水稻品种的杂交结实率范围(0.052～0.417)之内.本研究花粉离体萌发和杂交结实结果表明转基因的插入对水稻品种异交潜力的影响甚微.     

Stigma-surface Esterase Activity and Stigma Receptivity in Some Taxa Characterized by Wet Stigmas

Stigma-surface esterase activity and stigma receptivity througha sequence of developmental stages of the pistil have been studiedin four taxa characterized by having wet stigmas — Petuniahybrida, Nicotiana tabacum, Crinum defixum and Amaryllis vittata.The style is solid in the first two and hollow in the lattertwo taxa. In all the taxa, stigma—surface esterase couldbe detected in a thin surface layer (pellicle) from a very earlystage of pistil development, irrespective of the presence orabsence of the exudate. However, the taxa showed variation instigma receptivity. In Petunia and Nicotiana, stigmas from pistilsof all the stages supported pollen germination and tube growth.In Amaryllis and Crinum, stigmas of only the mature pistils,when the exudate is present on the stigma, supported normalpollen germination and tube growth. It is inferred that in taxacharacterized by a wet stigma and solid style, the factors requiredfor pollen germination are present from an early stage of pistildevelopment and the exudate per se is not involved in pollengermination. In taxa characterized by a wet stigma and hollowstyle, however, the pellicle does not carry the factors requiredfor pollen germination and tube growth; they appear to be presentin the exudate. Petunia hybrida Vilm, Nicotiana tabacum L., Crinum defixum, Ker-Gawl, Amaryllis vittata Ait., tobacco, pollination, pollen germination, stigmatic exudate, stigma receptivity, stigma-surface esterase, esterase activity