Influence of Temperature and Ageing of Ovules and Pollen on Reproductive Success in Trifolium repens L.

Four clones of Trifolium repens L., originating from Iceland,Denmark and Israel, were cultivated in controlled environmentchambers at 10, 14, 18 and 22°C. Seed set at 10°C was,on average, 45% lower than at 18°C. Generally, seed frequencieswere higher in the stylar ovule positions than in the basal.This polarization became more pronounced at low temperatures.At least 30% of the decrease in seed set was caused by inadequatepollen tube growth. This was further supported by in vitro studieswhich showed that pollen developed significantly shorter pollentubes at low temperatures and a strong positive correlationbetween the capacity of male parents to induce seed set in basalovule positions and the length of pollen tubes in vitro. Femaleage, determined by days relative to anthesis, was also foundto have a significant influence on seed set, i.e. when pollinationwas delayed until the fifth day of flowering, seed set decreasedby an average of 60% when compared to florets pollinated onthe first day of anthesis. Pollen age did not influence seedset. Ageing of pollen decreased in vitro germinability, tubeelongation and the percentage of pollen tubes with sperm cells.Seed set, pollen germinability, pollen tube lengths, and abilityto form sperm cells in vitro varied significantly among clones.Copyright1994, 1999 Academic Press Trifolium repens, white clover, temperature, pollen tube, sperm cells, ovule fertility, seed set     

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     

Viability and Germination of the Pollen of Sorghum [Sorghum bicolor (L.) Moench]

Previous studies have demonstrated that pollen of sorghum [Sorghumbicolor (L.) Moench] loses capacity to both germinate in vitroand to set seed in vivo soon after being shed. The current studyevaluates the capacity for dehydrated pollen to effect in vitrogermination, reduce tetrazolium chloride, and set seed on cytoplasmicmale sterile plants. Morphological changes during pollen germinationwere examined by scanning electron microscopy (SEM). Close to70% of the pollen germinated in 5 min, or less, when collectedat 80% relative humidity (RH) and stored in sealed glass vials.Pollen tubes elongated autotropically with atmospheric humidityapparently being a controlling factor in the process. Pollendehydrated at 50% RH and 25°C for 15-30 min neither germinatedin vitro, reduced tetrazolium chloride, nor set seed on malesterile plants. Rehydrating the pollen did not restore the capacityfor germination. SEM micrographs demonstrated that elongatingpollen tubes encircled the pollen grain and were contiguousto the surface. A fibrillar-like material existed on the exineof separated pollen grains at the point where the grains hadbeen previously attached.Copyright 1994, 1999 Academic Press Sorghum pollen, germination, seed-set, viability, scanning electron microscopy, Sorghum bicolor (L.) Moench     

Pollen Viability and Pollen-tube Growth Following Controlled Pollination and their Relation to Low Fruit Production in Teak (Tectona grandisLinn. f.)

Pollen released at 1100 h has the highest viability (92.2%)but is no longer viable 3 d (84 h) after anthesis.In vitropollen-tubegrowth is fast (140 µm h^-1) and increases significantlywithin the first 8 h.In vivopollen tubes also grow quickly andreach the base of the style within 2 h after pollination andenter the micropyle 8 h after pollination. There is no significantdifference between self- and cross-pollination in either therate and the number of pollen tubes in the pistil and the numberof ovules penetrated by a pollen tube. Teak has late-actinggametophytic self-incompatibility; the majority of pollen tubesgrow through the style but some do not continue to grow fromthe style towards the embryo sacs. Pollen-tube abnormalitiesinclude swollen, reversed, forked and tapered tips and irregularand spiralling tubes. These are most prevalent in self-pollination(20.4%). The index of self-incompatibility of 0.17 and low fruitset following self-pollination (2.49%) indicates that teak ismostly self-incompatible. Drastic fruit abortion occurs withinthe first week following controlled pollination. Within 14 d,fruit size and fruit set from cross-pollination is generallymuch greater than from self-pollination. Tectona grandis; pollen viability; pollen-tube growth; pollination; controlled pollinations; incompatibility.     

The significance of the methods of stigmatal and placental pollinationin vitro inAntirrhinum majus L.; seed and callus formation on placentae

A study was made on certain problems connected with the application of methods of stigmatal and placental pollinationin vitro in the snapdragon. Germinable pollen without microbial contamination could be obtained only from the flowers which were left, after surface sterilization, on the stem till the dehiscence of anthers. The germination of pollen, and especially the growth of pollen tubes was better in a 0.3 M lactose solution with 10^?3 per cent H₃BO₃ than in usual sucrose media. The seedsin vitro could be obtained only after normal pollination and the successive artificial cultivation of the entire pistil (the method of stigmatal pollinationin vitro). The seeds did not differ either in their morphology or in their size from those developed under natural conditions, they were viable and without dormancy. Though the pollinated pistils were cultivated in the medium without growth substances, callus formation from uncovered ovules and placenta was observed in some cases. A dependence was revealed of the proliferation on pollination and on the cultivar employed. When using the method of placental pollinationin vitro we failed to obtain seeds. The main difficulty in the use of this method seems to consist in the fact that pollen tubes are not capable of growing from the surface of ovules and placenta into the micropyle.     

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.     

Genetic Variation in Sorghum for the Inhibition of Maize Pollen Tube Growth

Aniline blue fluorescence was used to study the growth of maizepollen tubes in the stigmas of 13 diverse sorghum accessions.In 12, only short maize pollen tubes were formed, but in thesingle exception (Sorghum nervosum Nr481) maize pollen tubesgrew at least as far as the base of the style. The S. bicolorgenotypes S9B and CMS (a cytoplasmic male sterile line) werehybridized with Nr481, and analysis of maize pollen tube growthin F₁ plants, and BC₁ plants using Nr481 as the recurrent parent,suggested that differences in inhibition of pollen tube growthwere due to variation at a single locus, which we propose todesignate lap (Inhibition of alien pollen tubes). AccessionNr481 appears to be homozygous for a recessive allele permittingmaize pollen tube growth. Attempts were made to produce sorghumx maize hybrids using Nr481 and CMS derivatives which were knownto allow maize pollen tube growth to the base of the style.A putative hybrid endosperm was obtained in one Nr481 x Seneca60 maize cross, but this was not repeatable and no hybrid plantswere produced. A fundamental problem may be the large size ofthe maize pollen tube, which could have difficulty growing throughthe sorghum ovary and in entering the micropyle. Sorghum bicolor spp. bicolor (L.) Moench, Zea mays L, sorghum, maize, pollen tube growth, hybridization barriers     

Soya Bean Seed Growth and Maturation In vitro without Pods

Immature Glycine max (L.) Merrill seeds, initially between 50and 450 mg f. wt, were grown and matured successfully in vitro.Excised seeds were floated in a liquid medium containing 5 percent sucrose, minerals and glutamine in flasks incubated at25 °C under 300 to 350 µE m^–2 s^–1 fluorescentlight. During 16 to 21 d in culture, seeds grew to a matured. wt of 100 to 600 mg per seed at an average rate of 5 to 25mg d. wt per seed d^–1 depending on initial size. Growthrates were maximal during the first 8 to 10 d in vitro but declinedwith loss of green colour in the cotyledons. Seed coats rupturedwith rapid cotyledon expansion during the first 2 d in culture.Embryos were tolerant to desiccation and 80 to 90 per cent germinatedif removed from culture before complete loss of green colour.The growth of excised seeds in vitro exceeded the growth ofseeds in detached pods, but when windows were cut in pods topermit direct exposure of seeds to the medium, seed growth wascomparable. Glycine max (L.) Merrill, soya bean, seed culture, seed growth, seed maturation, germination     

Heterostyly and Pollen-tube Interactions in Luculia gratissima (Rubiaceae)

Observations on the floral biology of Luculia gratissima (Rubiaceae)showed that this species is distylous with complementary positioningof anthers and stigmas in the two floral forms. Unusual featuresof distyly in this species include the larger size of the corolla,the stigma surface and the stigmatic papillae in the thrum flowerscompared to the pin ones. Stigmatic surfaces have similar secretionsbut they appear more copious in thrum than pin. The floral dimorphismwas accompanied by a very effective self-incompatibility systemand no seed was set on selfing. Seed number per capsule on crossingwas significantly greater in thrum flowers compared to pin.Incompatible pollen tubes were inhibited within 24 h at thebase of the stigma/top of the style in both morphs. Amputationof this region of the gynoecium removed the self-incompatibilityreaction in thrum but not pin flowers. Pollination with a mixtureof compatible and incompatible pollen and sequential pollinationwith self followed by cross pollen showed that there were interactionsbetween the two types of pollen tube. The presence of compatibletubes was found to cause the excessive swelling of the pollen-tubetip of the incompatible ones. The incompatible tubes did notappear to have any effect on the growth of compatible ones. Luculia gratissima, distyly, floral biology, self-incompatibility, pollen-tube interactions     

Pollen Tube Growth and Fertilization Efficiency in Salpiglossis sinuata: Implications for the Involvement of Chemotropic Factors

The possible involvement of chemotropic factors influencingpollen tube growth in the style and ovary of Salpiglossis wasstudied. Pollen deposited at the base of excised styles germinatedreadily, with pollen tubes penetrating the transmitting tissueand growing towards the stigma, implying that no chemotropicrequirement exists for pollen tube growth in the style. However,the position and high efficiency of seed set following limited-pollinationof intact pistils would support the involvement of localizedchemotropism operating within the ovary. Chemotropism, limited pollination, fertilization efficiency, Salpiglossis sinuata     

The Effects of Modifying Sucrose Concentration on the Development of Maize Kernels Grown in vitro

Intact Zea mays L. kernels attached to cob tissue develop tomaturity when grown in vitro. This experiment was designed todetermine if it is possible to prolong kernel growth by refreshingthe culture medium. Blocks of maize kernels were grown in vitroon media containing several concentrations of sucrose. Kernels,at all concentrations of sucrose, developed to maturity at 30–35d post-pollination, indicating that it is not possible to extendthe kernel growth phase by supplying a carbohydrate source.Kernels grown on media containing 80 g 1^–1 or higher sucroseconcentration had a significantly greater percentage of kernelsthat developed to maturity, and had greater weight and starchcontent per seed. Zea mays, kernel culture, seed development, starch     

Evidence for Stigmatic Self-incompatibility, Pollination Induced Ovule Enlargement and Transmitting Tissue Exudates in the Paleoherb, Saururus cernuus L. (Saururaceae)

Saururus cernuus, a species belonging to the primitive herbaceousangiosperm family Saururaceae, exhibits high rates of self-sterility.We investigated the structural and functional aspects of pollen-carpelinteractions following cross and self pollination to assessthe tissue specific site and timing of self-sterility and factorsimportant for successful cross pollen tube growth. Self-sterilitywas due to inhibition of self pollen germination at a dry stigma.Self pollination was associated with anomalous foot formation,reduced cell wall expansion and secretory activity of stigmaticpapillae, and callose production in stigmatic papillae. Followinggermination, cross compatible pollen tubes entered a solid coreof transmitting tissue and grew to the base of a short style.Entry of cross pollen tubes into the ovary was coincident withovule enlargement which placed the micropyle in the proximityof cross pollen tube tips. Ovule enlargement also occurred followingself pollination. Cross pollen tubes either entered an exudate-filledmicropyle directly from the style, or growth in the ovary waslocalized to the epidermis of the locule and outer integumentprior to entry into the micropyle. Prior to pollination, thetransmitting tract was void of secretions except for exudatein the micropyle. Growth of pollen tubes on the locule and integumentwas associated with exudate apparently arising from transmittingcells adjacent to growing pollen tubes. The present study providesthe first evidence in a primitive herbaceous species of stigmaticself-incompatibility (SI) in association with a dry stigma,pollination-induced signalling events affecting developmentof carpellary tissues, and micropylar exudates. Copyright 1999Annals of Botany Company SI evolution, dry stigma, exudates, pollen-carpel signalling, Saururaceae.     

Pistil Exudate Production and Pollen Tube Growth in Lilium longiflorum Thunb.

Exudate production in the pistil of Lilium longiflorum was studiedin relation to pollen tube growth, using scanning electron microscopy(SEM), transmission electron microscopy and light microscopy.In contrast with conventional fixation for SEM, during whichthe exudate of L. longiflorum largely washes away, the exudateremains present through freezing in case of cryo-SEM. Usingthe latter method we observed that exudate production on thestigma and in the style started before anthesis. Just underneaththe stigma the exudate was first accumulated at the top of eachsecretory cell, followed by a merging of those accumulationsas exudate production proceeded. Exudate is also produced bythe placenta. It was however not possible to determine whetherany of this fluid originated from the micropyle. Apart fromthe cell shape and the cuticle present in between the secretorycells, the ultrastructure of the secretory cells covering theplacenta was comparable to those of the stylar canal. The transferwall of the secretory cells of the placenta originated fromfusing Golgi vesicles but the endoplasmic reticulum seemed tohave an important role as well. After pollination the pollen tubes grew across the stigma andentered the style through one of the slits in the three stigmalobes. The pollen tubes grew straight downward through the styleand were covered by exudate. As the pollen tubes approachedthe ovary their growth was restricted to the areas with secretorycells. In the cavity the pollen tubes formed a bundle and theybent from this bundle in between the ovules towards the micropylarside. There they bent again to stay close to the secretory cells.After bud pollination the pollen tube growth was retarded. Laterarriving pollen tubes had a tendency to grow close to the secretorycells of the style, which resulted in a growth between thesecells and preceding pollen tubes. If there was still a littleexudate produced, it resulted in a lifting up of the pollentubes, out of the exudate. The relationship between exudateproduction and pollen tube growth is discussed. Both the speedand the guidance of the pollen tube seemed determined by theproperties of the exudate.Copyright 1994, 1999 Academic Press Cryo-scanning electron microscopy, exudate, Lilium longiflorum, lily, ovary, pollination, pollen tube growth, secretory cell, stigma, style     

Pollen-pistil interactions in Hermodactylus tuberosus Mill. (Iridaceae)

ABSTRACT

Hermodactylus tuberosus Mill. (Iridaceae) is a species with a very short style and a tricarpellar unilocular ovary. For ornamental purposes it is usually reproduced by means of tubers, although both vegetative and seed reproduction occur in nature. However, its capsules do not contain many seeds in comparison to the high number of ovules inside the ovary. To understand this conflicting process a study on H. tuberosus pollen-pistil interaction was carried out. Pollen viability was evaluated by different techniques. Germination rate was tested in vitro and in vivo after stigma, intrastylar and intraovarian pollinations. A high percentage of the pollen grains were found to be viable resulting in a high percentage of in vitro germination and a low rate of germination on the stigma. Indeed, many pollen tubes developed after intrastylar and intraovarian pollinations. Several anomalies characterized pollen germination and pollen tube growth in vitro and in vivo. Observations from field trials indicated that capsules were only produced from open or hand, cross- pollinated flowers, confirming that the plant is self- incompatible and not apomictic. In addition, all the mature capsules contained only a few mature seeds suggesting the existence of pre- and postzygotic seed set inhibition.     

Sensitivity to Low Temperature in Pollen Germination and Fruit-Set in Cicer arietinum L.

Cicer arietinum L. cv. G 62404 was seen to have a very low fruit-setpercentage at low temperatures and as temperatures increased,either in nature or in culture conditions, the fruit-set alsoincreased. This effect of temperature could also be seen inpercent pollen germination and pollen tube growth. M 450, amutant of G 62404, was seen to have a better fruit-set percentageat low temperature and better pollen germination. This may bedue to differences in malic acid concentration, which increasedpollen germination, and pollen tube growth under in vitro cultureconditions.     

Pollination in vitro: effects on the growth of pollen tubes,seed set and gametophytic self-incompatibility in Trifolium pratense L. and T. repens L.

Summary Growth of pollen tubes and seed set were compared after hand pollination in situ and in vitro in two self-incompatible species, Trifolium pratense and Trifolium repens. Adhesion of pollen grains to the stigma was greater in vitro for both species. After cross-pollination, in vitro culture gave a significant increase in the cumulative growth of pollen tubes in pistils of T. pratense compared to in situ conditions. After selfing in T. repens, pollen tube growth was significantly increased by in vitro culture of florets. Seed set after crossing in situ and in vitro was similar for both species. Seed set after selfing in vitro was not increased in T. pratense. Several genotypes of T. repens were classified as very good, good and poor selfers based on their capacity for seed set following selfing in situ. In vitro pollination increased self seed formation by 1.7-, 18.0- and 31.0-fold for each class, respectively. Ovules located nearest to the style were fertilized more often after selfing than after crossing.     

Overcoming Incompatibility in Brassica campestris L. by Carbon Dioxide, and Dark Fixation of the Gas by Self- and Cross-pollinated Pistils

Supplementing pollen suspension cultures with CO₂ (3–5per cent) caused a marked increase in germination and tube growthin vitro in Brassica campestris L. cv. toria. A weakening ofself-incompatibility by increased CO₂ levels from 3–5per cent was observed. The percentage of pollen tubes whichpenetrated the cuticle layer of stigmatic papilla cells in self-pollinatedpistils was high when CO₂ level was 5 per cent. Phosphoenolpyruvate (PEP) carboxylase activity was greater in the pollengerminated in 4 per cent CO₂ as compared to air (0.03 per cent).A possible role of CO₂ for self-recognition and control of pollentube growth is proposed, proposed. Brassica campestris L., carbon dioxide, self-incompatibility, phosphoenol pyruvate carboxylase     

Floral Biology, Pollination, Pistil Receptivity, and Pollen Tube Growth of Teak (Tectona grandis Linn f.)

Teak flowers are weakly protandrous and pollen is shed withina few hours of flower opening. Pollen is tricolpate and 29 µmin diameter. The papillate stigma is of the wet type and isreceptive from 1100–1300h. The style is hollow throughoutits length. Nectar and pollen are the major floral rewards forpollinators. The major pollinators areCeratina sp. which carryteak pollen on most parts of their bodies, especially the specializedhair structures (scopal brushes) on the tibia. The most effectivepollination period in terms of flowers pollinated and pollenper flower is between 0900 and 1300h. At 1300h the number ofpollen per flower is the highest, ranging from 1–36 (average7). Pollen tubes grow very fast. Within 2 h after pollination8% of the pollen tubes have reached the micropylar end of theovule and pollen tubes first enter the embryo sac at 8 h. Onlyone to two pollen tubes enter the micropyles of a flower. Although78% of flowers were pollinated in open-pollination, the lowfruit set (3.5%) suggests that there are factors other thanpollination limiting fruit set. The main factor appears to bea high amount of selfing, and self-incompatibility occurs whenpollen tubes are arrested at the lower portion of the ovary. Tectona grandis ; floral biology; pollen tube growth; pollination; receptivity; pollinators