In vitro pollen germination and pollen tube growth differences among Quercus robur L. clones in response to meteorological conditions

The impact of meteorological conditions on in vitro pollen germination and pollen tube growth during the initial phases of the development of male flowers in the Pedunculate Oak, Quercus robur, is studied. Phenological observations of male flowers and pollen sampling were performed on the field trial established with grafted Pedunculate Oak clones. During the investigation, weather conditions (absolute minimum and maximum daily air temperature, minimum absolute relative humidity of air and amount of precipitation) were recorded by an automatic meteorological station installed at the field trial. Influence of meteorological conditions on pollen germination and pollen tube growth was studied in the following stages of male flower: (I) during the last ten days of flower bud dormancy, (II) during swelling of the buds, (III) during bud burst and beginning of male catkins elongation, (IV) during the final stage of male flower catkins elongation. High temperatures and low relative air humidity during the bud burst and beginning of the male catkins elongation reduced pollen germination and pollen tube growth. Weather conditions did not significantly affect pollen germination and pollen tube growth during the swelling of flower buds, or in the final stage of male catkins elongation.     

Polyethylene glycol improves the in vitro growth of Brassica pollen tubes without loss in germination

The culture media for the in vitro pollen assay of Brassicaspecies have so far shown good percentage germination, but limitedpollen tube growth. It is reported here that by lowering sucroseconcentration from 20% to 5 or 10 % and by adding polyethyleneglycol 4000 in the culture medium, high percentage germinationand high tube growth (10 times that in the standard media) canbe achieved in three species of cultivated Brassica. This improvedmedium should be useful in fundamental and applied studies onthe pollen biology of Brassica species. Key words: Brassica, pollen, pollen germination, pollen tube growth, polyethylene glycol     

The influence of gelling agent purity and ion additions on in vitro tomato pollen germinability and pollen tube growth on semi-solid substrates

The physicochemical properties and inherent ion content of the gelling agents used for the preparation of semi-solid substrates significantly affect the germination of tomato pollen in vitro. The addition of Ca, K and Mg to semi-solid, agar-based, substrates improved the germination of tomato pollen when the inherent Ca content of the agar was low, but was without effect or even inhibitory when the Ca level was high. However, Κ and/or Mg addition was beneficial irrespective of the agar source. When agarose replaced agar and K, Mg and Ca were added individually or in combination, pollen germination and pollen tube growth were affected differently by each ion but were optimal only in the presence of all three ions, reflecting the absence of these ions in agarose. An involvement of Na was also implicated since reduction of the inherently high Na content of agar by washing improved germination, with or without the addition of Κ, Mg and Ca. Since >3 mM Ca in the semi-solid substrate impairs tomato pollen germination, the gelling agent must be of high purity, which in the case of agar may entail washing, followed by the addition of K, Mg and Ca. The adoption of such a medium would permit the standardization of semi-solid substrates for in vitro tomato pollen germination studies.     

Structural aspects of in vitro pollen tube growth and micropylar penetration inGasteria verrucosa (Mill.) H. Duval andLilium longiflorum Thunb.

Summary In vitro penetration of the micropyle of freshly isolatedGasteria verrucosa ovules by pollen tube was monitored on agar medium. 40–60% of the micropyles were penetrated, comparable with in vivo penetration percentages. When germinated on agar,Gasteria pollen tube elongation lasts for up to 8 h while plasma streaming continues for about 20–24 h. The generative cell divides between 7 and 20 h after germination, and after 20 h the pollen tube arrives at one of the synergids. The sperm cells arrive after 22 h. The whole process takes more time in vitro than in vivo. In fast growing pollen tubes, a pulsed telescope-like growth pattern of tube elongation is observed. The formation of pollen tube wall material precedes tube elongation and probably prevents regular enlargement of the pollen tube tip-zone. Rapid stretching of the new pollen tube wall material follows, probably due to gradually increased osmotic pressure and the use of lateral wall material below the tip. The stretching ceases when the supplies of plasma membrane and excretable wall material are exhausted. Multiple pollen tube penetration of the micropyle occurs in vitro as it does in vivo. Most pollen tube growth ceases within the micropyle but, if it continues, the pollen tubes curl. Inside the micropyle the pollen tube shows haustorial growth. At the ultrastructural level, the wall thickening of in vitro pollen tubes is quite similar to that in vivo. Before transfer of pollen tube cytoplasm a small tube penetrates one of the synergids. Sperm nuclei with condensed chromatin are observed in the pollen tube and the synergid. In vivo prometaphase nuclei are found in the most chalazal part of a synergid, against the egg cell nucleus and nucleus of the central cell at a later stage. Using media forLilium ovule culture,Gasteria ovules were kept alive for at least 6 weeks. Swelling of the ovule depends on pollen tube penetration. The conditions for fertilization to occur after in vitro ovular pollination seem to be present.     

Effect of smoke derivatives on in vitro pollen germination and pollen tube elongation of species from different plant families

Plant‐derived smoke stimulates seed germination in numerous plant species. Smoke also has a positive stimulatory effect on pollen germination and pollen tube growth. The range of plant families affected my smoke still needs to be established since the initial study was restricted to only three species from the Amaryllidaceae. The effects of smoke‐water (SW) and the smoke‐derived compounds, karrikinolide (KAR₁) and trimethylbutenolide (TMB) on pollen growth characteristics were evaluated in seven different plant families. Smoke‐water (1:1000 and 1:2000 v:v) combined with either Brewbaker and Kwack's (BWK) medium or sucrose and boric acid (SB) medium significantly improved pollen germination and pollen tube growth in Aloe maculata All., Kniphofia uvaria Oken, Lachenalia aloides (L.f.) Engl. var. aloides and Tulbaghia simmleri P. Beauv. Karrikinolide (10^?6 and 10^?7 m ) treatment significantly improved pollen tube growth in A. maculata, K. uvaria, L. aloides and Nematanthus crassifolius (Schott) Wiehle compared to the controls. BWK or SB medium containing TMB (10^?3 m ) produced significantly longer pollen tubes in A. maculata, K. uvaria and N. crassifolius. These results indicate that plant‐derived smoke and the smoke‐isolated compounds may stimulate pollen growth in a wide range of plant species.     

Kanamycin resistance of germinating pollen of transgenic plants

The influence of kanamycin on the percentage of pollen germination and on tube growth of pollen from non-transformed and transformed plants of various species containing a chimaeric kanamycin resistance gene (NPTII) was investigated. Pollen grains isolated from kanamycin resistant plants expressed resistance when germinating in vitro, whereas kanamycin impaired tube growth of pollen from non-transformed plants. Pollen grains from transgenic plants were less sensitive and produced significantly longer tubes. mRNAs of the chimaeric gene are probably presynthesized concurrently with the other mRNAs during microsporogenesis, and kanamycin resistance is expressed by mRNA translation during pollen tube elongation. Received: 24 August 1999 / Revision accepted: 20 October 1999     

Stimulation of pollen tube growthin vitro by dicarboxylic acids

Summary Effect of eight dicarboxylic acids and three monocarboxylic acids on pollen growth ofCamellia japonica was tested. While monocarboxylic acids inhibited pollen germination and pollen tube elongation, dicarboxylic acids, namely oxalic, succinic, suberic, adipic, sebacic, traumatic cis-1,2-cyclohexane dicarboxylic, and 3,3-diethyl glutaric acids stimulated pollen tube elongation stronger than indoleacetic acid.     

类整合素在烟草花粉萌发和花粉管生长中的作用

The effects of antiserum against human VnR integrin and integrin antagonist GRGDSP peptides on Nicotiana tabacum pollen germination and tube growth both in in vitro and in semi-vivo conditions were studied. No obvious inhibitory effects on pollen germination and tube growth in vitro were observed when anti-VnR serum or GRGDSP peptides was added to BK culture medium, but the enhancement of pollen germination and tube growth in vitro promoted by calmodulin was depressed by adding anti-VnR serum or GRGDSP peptides to BK culture medium. In addition to that, pollen germination and tube growth on stigma, as well as tube growth in styles were also inhibited at some extent by treating stigma and microinjecting GRGDSP peptides or anti-VnR serum into styles. The role of integrin-like proteins in regulation of pollen germination and tube growth in situ was discussed.     

Temperature as a determinant factor for increased and reproducible in vitro pollen germination in Arabidopsis thaliana

Despite much effort, a robust protocol for in vitro germination of Arabidopsis thaliana pollen has been elusive. Here we show that controlled temperatures, a largely disregarded factor in previous studies, and a simple optimized medium, solidified or liquid, yielded pollen germination rates above 80% and pollen tube lengths of hundreds of microns, with both Columbia and Landsberg erecta (Ler) ecotypes. We found that pollen germination and tube growth were dependent on pollen density in both liquid and solid medium. Pollen germination rates were not substantially affected by flower or plant age. The quartet1 mutation negatively affected pollen germination, especially in the Ler ecotype. This protocol will facilitate functional analyses of insertional mutants affecting male gametophyte function, and should allow detailed gene expression analyses during pollen tube growth. Arabidopsis thaliana can now be included on the list of plant species that are suitable models for physiological studies of pollen tube elongation and tip growth.     

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     

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.     

In vitro pollen germination in Hypericum perforatum L. and Hypericum rumeliacum Boiss

In vitro pollen germination and pollen tube growth investigations are valuable tools used in identification of the effects of environmental factors and genotypic differences on pollen viability, pollen germination and tube elongation. In this study pollen viability, in vitro pollen germination capacity, abnormality ratios and tube length in germinated pollens of Hypericum perforatum L. and H. rumeliacum Boiss. were investigated. Both of these species has spheroid-shaped and tricolporate pollen grains. The diameters of Hypericum perforatum and H. rumeliacum pollens were found as 24 +/- 3 microm and 19 +/- 2 microm, respectively. Pollen viability of H. perforatum and H. rumeliacum was found as 83% and 72%, respectively. The germination percentages were found as 12.85% for H. perforatum and 64.42% for H. rumeliacurm. Tube lengths in germinated pollens of both taxa were measured approximately as 95.25 +/- 38 microm in H. perforatum and 165.92 +/- 53 microm in H. rumeliacium 4 h after inoculation. In germinated pollen grains of H. perlbratum and H. rumeliacumn abnormality percentages were determined as 13.23% and 43.97%, respectively. In germinated pollens of these two species, highly significant (P < 0.00001) differences in in vitro germination percents and abnormality percents were observed. Abnormalities such as swollen tube tip, branched tube, spiralled tube and excessive tube formation were observed in pollen tubes. The results of this study showed that there were obvious differences in pollen germinability between these two species growing under the same environmental conditions.     

山玉兰花粉离体萌发和花粉管生长特性的研究

山玉兰(Magnolia delavayi)是木兰科木兰属的常绿乔木或大型灌木,是重要的园林造景、庭院绿化素材,也是重要的育种资源。山玉兰花粉的研究对其杂交育种的成败具有重要影响,但目前尚未见其花粉活力的相关报道。该研究以新鲜的山玉兰花粉为对象,采用悬滴培养法分析了温度、光照以及培养液的蔗糖和硼酸浓度对山玉兰花粉萌发的影响。结果表明:(1)山玉兰花粉萌发时,最适宜的温度为27℃。(2)光暗条件下,山玉兰花粉以浓度为5%的蔗糖培养效果最佳,其萌发率在16%以上;而硼酸浓度则以0.001%的培养效果最佳。(3)蔗糖与硼酸共同作用可有效促进花粉萌发和花粉管生长。其中,在光照条件下,以5.0%蔗糖+0.001%硼酸为最适宜的培养液,花粉萌发率达41.27%,花粉管长达281.49μm;而在黑暗条件下,则以5.0%蔗糖+0.01%硼酸为最适宜的培养液,花粉萌发率达45.71%,花粉管长达254.00μm。该研究结果为进一步开展人工辅助授粉、发掘山玉兰的种质资源工作奠定了基础。     

Microtubules and microfilaments are both responsible for pollen tube elongation in the coniferPicea abies (Norway spruce)

Summary InPicea abies (Norway spruce), microtubules and actin microfllaments both form a dense matrix throughout the tube mainly parallel to the direction of elongation. In these conifer pollen tubes the organization of this matrix is different from that in angiosperms. This study tests our hypothesis that differences in cytoskeletal organization are responsible for differences in tube growth and physiology. Pollen grains were germinated in media containing cytoskeletal disrupters and analyzed for germination, tube length, tube branching, and tip swelling. Disruption of microtubules significantly inhibits tube elongation and induces tube branching and tip swelling. Tip swelling is probably caused by disruption of the microtubules in the tip that are perpendicular to the direction of elongation. Confocal microscopy indicates that colchicine and propyzamide cause fragmentation of microtubules throughout the tube. Oryzalin and amiprophosmethyl cause a complete loss of microtubules from the tip back toward the tube midpoint but leave microtubules intact from the midpoint back to the grain. Disruption of microfilaments by cytochalasins B and D and inhibition of myosin by N-ethylmaleimide or 2,3-butanedione monoxime stops tube growth and inhibits germination. Microfilament disruption induces short branches in tubes, probably originating from defective microfilament organization behind the tip. In addition, confocal microscopy coupled with microinjection of fluorescein-labeled phalloidin into actively growing pollen tubes indicates that microfllament bundles extend into the plastid-free zone at the tip but are specifically excluded from the growing tip. We conclude that microtubules and microfilaments coordinate to drive tip extension in conifer pollen tubes in a model that differs from angiosperms.     

The effects of ethyl methanesulfonate treatment on <Emphasis Type="Italic">Eucalyptus</Emphasis> pollen behaviour in vitro

Treating pollen with mutagens prior to controlled pollination may facilitate the production of mutant trees for developmental studies and eventual plantation improvement. To establish a suitable dose of the chemical mutagen ethyl methanesulfonate (EMS) for the testing of this hypothesis, pollen of Eucalyptus globulus ssp. globulus and E. grandis was studied in vitro. Pollen germination, pollen tube elongation and generative cell division were examined after 48 h of culture, following exposure to between 0 and 1,000 ppm EMS. Doses of 600 to 1,000 ppm EMS reduced pollen germination in vitro in both species. Doses of up to 1,000 ppm EMS were not observed to significantly impact on either pollen tube length, or generative cell division in vitro of either species. A dose of 600 ppm EMS in paraffin oil is predicted to induce mutation in Eucalyptus species whilst impacting minimally on seed production based on the effect on pollen germination.     

Effects of air pollutants on pollen

In vivo and in vitro pollen germination and pollen tube growth are affected by air pollutants. The stimulation and inhibition of these pollen characteristics depend on the pollen species as well as on the pollutant and its concentration, the exposure time, the relative humidity during exposure and some other factors. Mitosis, gas exchange, size and chemistry of pollen can also be influenced by air contaminants. Pollen germination and tube elongation differ in their response to air pollution stress, and both are more susceptible in vitro compared to the in vivo situation. Some possible mechanisms of action are reported in this review study. The effects of air contaminants on pollen are discussed in relation to sexual reproduction, gametophytic selection and adaptation to stress. Pollen may be used as sensitive biological indicators of pollution     

The involvement of hydrogen peroxide in UV-B-inhibited pollen germination and tube growth of Paeonia suffruticosa and Paulownia tomentosa in vitro

Previous studies have shown that UV-B could affect pollen germination and tube growth. However, the mechanism of response of pollen to UV-B has not been clear. The purpose of this study was to investigate the role of hydrogen peroxide (H₂O₂) in the UV-B-induced reduction of in vitro pollen germination and tube growth of Paeonia suffruticosa Andr. and Paulownia tomentosa Steud. Exposure of pollen of the two species to 0.4 and 0.8 W m⁻² UV-B radiation for 3 h resulted in not only the reduction of pollen germination and tube growth, but also the H₂O₂ production in pollen grain and tube. Also, exogenous H₂O₂ inhibited pollen germination and tube growth of the two species in a dose-dependence manner. Two scavengers of H₂O₂, ascorbic acid and catalase, largely prevented not only the H₂O₂ generation, but also the reduction of pollen germination and tube growth induced by UV-B radiation in the two species. These results indicate that H₂O₂ is involved in the UV-B-inhibited pollen germination and tube growth.     

The effects of a triazole fungicide,propiconazole, on pollen germination,tube growth and cytoskeletal distribution in Tradescantia virginiana

The effects of propiconazole on germination and tube growth of Tradescantia virginiana pollen when incorporated in germination media at 0, 102, 136, or 170 l l^–1 were evaluated using light microscopy and immunocytochemistry. Propiconazole inhibited pollen germination, cytoplasmic streaming, and tube elongation. Treatments also induced abnormal tube morphology and cytoskeletal distribution. Tubes treated with propiconazole displayed weaker microfilament (Mf) signals along the pollen tubes, with amorphous staining. Microtubule (Mt) distribution was also severely affected. In treated tubes, the proximal portions had characteristically fragmented Mts. Fewer Mt bundles were seen in the subapical region, and these were located further from the apex. Propiconazole effects were generally concentration dependent. The results indicate that propiconazole affects both Mfs and Mts; however, the effects may be an indirect result of the drug's influence on membranes.