Influence of temperature on the in vitro pollen germination and pollen tube growth of various native Iranian almonds (Prunus L. spp.) species

Pollen germination and pollen tube growth was quantified among various native Iranian wild almonds (P. dulcis (Mill.) D. A. Webb, P. eleaegnifolia Mill., P. orientalis Mill., P. lycioides Spach, P. reuteri Bioss. et Bushe, P. arabica Olivier, P. glauca Browick and P. scoparia Spach in order to identify differences in the tolerance of pollen to temperature variations. Pollen germination and pollen tube growth were observed after incubation in darkness in a germination medium for 24?h at 10?C50°C at 5°C intervals. Maximum pollen germination of the wild almond species and specify that 60% was obtained for P. orientalis pollen and 98% for P. scoparia. Pollen tube length ranged from 860???m was obtained in P. lycioides and 1490???m in P. scoparia. A modified bilinear model best described the response to temperature of pollen germination and pollen tube length. Almond species variation was found for cardinal temperatures (T _min, T _opt and T _max) of pollen germination percentage and pollen tube growth. Mean cardinal temperatures averaged over eight almond species were 14.7, 24.2, and 43.7°C for maximum percentage pollen germination and 14.48, 25.3, and 44.4 °C for maximum pollen tube length. The principal component analysis (PCA) identified maximum percentage pollen germination and pollen tube length of the species, and T _max for the two processes as the most important pollen parameters in describing a species tolerance to high temperature. PCA also classified Prunus L. spp. into four groups according to the tolerance of pollen to temperature variations. The T _min and T _opt for pollen germination and tube growth, rate of pollen tube growth were less predictive in discriminating species for high temperature tolerance.     

Pollen Tube Development and Characteristics of the Protein Emission in Conifers

When ripe, viable pollen of Pinus contorta, Pinus nigra, Piceasitchensis, Abies alba and Cedrus deodara is germinated on asuitable artificial substrate, the process of pollen tube growthbegins 12–36 h later, according to the species. The tubeemerges at the leptoma, a structurally specialized site in thepollen wall, and the early tube wall is continuous with thetwo microfibrillar intine strata within the grain. Later inpollen tube development, when cytoplasmic zonation has beenestablished, only the inner of these two wall layers is differentiated. Cytochemical methods show that, during hydration and throughoutthe period of tube growth in culture, proteins are releasedfrom the pollen grains; before germination most conspicuouslyfrom the leptoma, subsequently from the tube itself. The emissioncontains a number of hydrolytic enzymes and a non-catalyticmoiety. Gel-electrofocusing reveals that the hydrolases releasedfrom germinating Pinus contorta pollen include several acidphosphatase and esterase isozymes, and that there are differencesbetween the composition of the emission and the spectrum ofsoluble proteins extracted from the pollen grains before germination.Analysis by immunodiffusion demonstrates that two componentswith antigenic characteristics present in the quiescent pollengrains are represented in the emission. The possible utilityof the released components in the biology of conifer reproductionis discussed. Pinus contorta, Pinus nigra, Picea sitchensis, Abies alba, Cedrus deodara, lodgepole pine, Austrian pine, Sitka spruce, European silver fir, deodar, pollen tube, pollen germination, protein release, isoelectric focusing     

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.     

Effect of Spermidine and Methylglyoxal-bis (Guanyl-hydrazone) (MGBG) on In Vitro Pollen Germination and Tube Growth in Catharanthus roseus

Incorporation of polyamine-spermidine into the nutrient mediumat 10^–6 and 10^–5 M concentrations stimulates pollen-tubegrowth in vitro in Catharanthus roseus L. G. Don. MGBG, an inhibitorof spermidine biosynthesis, at 0.5 x 10^–3 and 1 x 10^–3M concentrations reduced the percentage of germination as wellas tube growth and at a concentration of 1.5 x 10^–3 Mgermination was totally inhibited. Pollen grains incubated inthe medium containing 1.5 x 10^–3 M MGBG, when transferredto a fresh medium with 10^–5 M spermidine, resulted in80% germination recovery, along with considerable tube growth.Experiments with actinomycin-D indicate that stimulation ofpollen-tube growth by spermidine may involve de novo synthesisof protein. Catharanthus roseus, pollen germination, tube growth, spermidine, MGBG, inhibition, actinomycin-D     

Pollen Diffusates of Crotalaria retusa and their Role in pH Regulation

Details of the release of proteins and amino acids from culturedpollen grains and the role of the leached metabolites in pollengermination, pollen tube growth and regulation of pH of theculture medium in Crotalaria retusa have been investigated.In unbuffered media, satisfactory pollen germination and tubegrowth occurred over a wide range of pH values 4.0–9.0.This was related to the ability of pollen diffusates to shiftthe pH to 6.25 in all these media. Similar pollen germinationand pH shift was observed when the pollen was eluted twice beforeculturing. When the pH shift was reduced by using buffered media,optimal germination and tube growth occurred only at pH 6.0.Pollen diffusates had a strong buffering capacity. Proteinsand amino acids released from pollen do not seem to have a directrole in pH regulation. The components involved in pH regulationmay originate from the pollen wall as well as from the cytoplasm. Crotalaria retusa L, pH regulation, pollen diffusates, pollen germination     

Effect of Growth Regulators and Light on Pollen Germination and Pollen Tube Growth in Pinus roxburghii Sarg

Pine (Pinus roxburghii) pollen grown in suspension cultureswas used to study the effects of growth regulators and lightconditions on germination and pollen tube growth. Indol-3-ylacetic acid, gibberellic acid, ethylene, abscisic acid and cyclicAMP (cAMP) at low concentrations (1–10 mg 1^–1) promotedgermination and tube growth. Addition of 1 and 10 mg 1^–1cAMP to any of the growth regulators had a promotory effect.Pollen tube growth decreased in white light as compared to thedark, and was increased in red light. Far-red light counteractedthe effect of red light. The effect of growth regulators incausing the enhanced tube growth appears to be manifested throughsubstances such as cAMP, and phytochrome seems to be involved. Pinus roxburghii, pine, pollen germination, pollen tube growth, growth regulators, cyclic AMP, phytochrome     

RNA and Protein Synthesis in Germinating Pine Pollen

The results of autoradiographic experiments demonstrate that,as with the pollen of most other species, both the generativeand vegetative nuclei of Loblolly Pine (Pinus taeda) activelyengage in RNA synthesis from the very early stages of pollengermination. Unlike most other species, however, this newlysynthesized RNA includes rRNA. Evidence is provided for theimportance of this newly synthesized RNA in the process of continuedpollen tube growth. One and two-dimensional gel electrophoretic analysis revealsa number of both qualitative and quantitative differences amongthe proteins synthesized during the early stages of germinationand the later stages of pollen tube growth. One of the mostnotable of these is a 36 kD protein, the synthesis of whichpredominates during the later stages of pollen germination.A similar pattern of 36 kD protein synthesis is observed whenmRNA extracted from pollen at each of these stages is translatedin vitro. Key words: Pinus, pollen tube growth     

Sugars in Natural and Artificial Pollen Germination Substrates

The stigmatic exudates and pollen grains of five unrelated specieswere tested for sugars. Glucose, fructose, and sucrose werefound in the stigmatic fluid of Yucca aloifolia L. and glucoseand fructose in that of Oenothera adrummondii Hook. In in vitroexperiments with Y. aloifolia pollen, high germination percentageswere obtained in artificial media containing glucose or sucrose.Fructose, which is present in the stigmatic fluid of the Yuccasp. resulted in high in vitro pollen germination only when borateand calcium were added to the medium. Presence of bound sugarsis indicated in the stigmatic secretion of Citrus aurantiumL. and pollen of the single plant tested germinated at a lowpercentage in artificial sugar media. No sugars were detectedin the stigmatic fluids of Hemerocallis fulva L. and Zea maysL. and in these two species in vitro pollen germination in sugarymedia was negligible or absent. The pollen grains of all five species contain sucrose and thoseof Oenothera and Citrus also reducing sugars.     

In vitro Arabidopsis pollen germination and characterization of the inward potassium currents in Arabidopsis pollen grain protoplasts

The focus of this study is to investigate the regulatory role of K(+) influx in Arabidopsis pollen germination and pollen tube growth. Using agar-containing media, in vitro methods for Arabidopsis pollen germination have been successfully established for the first time. The pollen germination percentage was nearly 75% and the average pollen tube length reached 135 microm after a 6 h incubation. A decrease in external K(+) concentration from 1 mM to 35 microM resulted in 30% inhibition of pollen germination and 40% inhibition of pollen tube growth. An increase in external K(+) concentration from 1 mM to 30 mM stimulated pollen tube growth but inhibited pollen germination. To study how K(+) influx is associated with pollen germination and tube growth, regulation of the inward K(+) channels in the pollen plasma membrane was investigated by conducting patch-clamp whole-cell recording with pollen protoplasts. K(+) currents were first identified in Arabidopsis pollen protoplasts. The inward K(+) currents were insensitive to changes in cytoplasmic Ca(2+) but were inhibited by a high concentration of external Ca(2+). A decrease of external Ca(2+) concentration from 10 mM (control) to 1 mM had no significant effect on the inward K(+) currents, while an increase of external Ca(2+) concentration from 10 mM to 50 mM inhibited the inward K(+) currents by 46%. Changes in external pH significantly affected the magnitude, conductance, voltage-independent maximal conductance, and activation kinetics of the inward K(+) currents. The physiological importance of potassium influx mediated by the inward K(+)-channels during Arabidopsis pollen germination and tube growth is discussed.     

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     

Response of in vitro pollen germination and pollen tube growth of almond (Prunus dulcis Mill.) to temperature,polyamines and polyamine synthesis inhibitor

Prunus dulcis L. ‘Mamaei’ is grown widely in souhtwest of Iran. It blooms in early spring when temperatures are still low. Based on our knowledge there are no reports in the literature regarding pollen behavior of this cultivar under specified condition. Thus, the possible factors for low germination percentage in this cultivar have not been reported. The effect of three different temperatures (10, 25, or 35 °C), polyamines (putrescine, spermidine, and spermine) and polyamine synthesis inhibitor, methylglyoxals-bis (guanyl-hydrazone) (MGBG) on in vitro pollen germination and pollen tube growth were investigated in P. dulcis L. ‘Mamaei’. All temperatures and chemicals significantly affected both pollen germination percentage and pollen tube growth. In general, different polyamines stimulated the pollen germination percentage compared to the control at all temperatures, but increasing the temperature, particularly to 35 °C, had demonstrated inhibitory effects on pollen germination. At a concentration of 0.05 mM putrescine and spermidine and 0.005 and 0.025 mM spermine revealed longer pollen tube growth than that of the control at 10 °C, while higher concentrations tended to inhibit pollen tube growth. At 25 °C, most of the treatments had an inhibitory effect on pollen tube growth except for 0.25 mM putrescine and 0.005 mM spermine, which slightly stimulated pollen tube growth. Pollen germination and pollen tube growth were inhibited by MGBG at all temperatures and in all concentrations.     

Towards an in vitro bioassay for the self-incompatibility response in Brassica oleracea

Summary Eluates of stigmas of Brassica oleracea that were known to contain S locus-specific glycoproteins (SLSG) discriminated between self and cross pollen in vitro in three different media. Discrimination was equally evident in experiments that were the in vitro equivalents of reciprocal pollinations. In a TAPS-buffered medium, self eluates depressed pollen germination in a dose-dependent manner. TAPS medium allowed a bioassay of the effects of SLSG in eluates because it optimized germination in a way that eliminated the complicating features of the stimulatory substances in the eluates. Stigma eluates affected percentage pollen germination and optimum calcium concentrations in vitro whether or not SLSG were present in the eluates, but differently in different media, and depending on whether the eluates were cross or self with respect to the pollen tested. Thus, the effect of stigma eluates on the in vitro germination of pollen in Brassica depends on the balance of stimulatory versus inhibitory substances in the eluates.     

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.     

Sodium thioglycollate enhances pollen germination and pollen tube elongation in cruciferous species

Sodium thioglycollate is a reducing agent used in microbiological growth media to enhance the growth of anerobic, microaerophilic, and facultative organisms, and in eukaryotic tissue extraction buffers to inhibit damaging oxidative reactions. Sodium thioglycollate was added to a semi-solid pollen germination medium to evaluate its effects on in vitro pollen germination and pollen tube elongation, based on the assumption that conditions within stylar tissues are less aerobic than in ambient conditions. We observed significant increases in the percent germination and pollen tube elongation of both crop and weedy mustard family species, on a medium containing 2.2 mM sodium thioglycollate. This suggests that sodium thioglycollate may be a useful amendment to semi-solid media and to solutions that are used to study pollen vigor, physiology, or gene expression, and to bioassay sensitivities of different species or genotypes to diverse physical and chemical factors.     

The Importance of Light Intensity for Pollen Tube Growth and Embryo Survival in Wheat x Maize Crosses

The success of Triticum aestivumxZea mays crosses, used to producewheat doubled haploids, is influenced by light intensity. Toexamine the basis for this response, pollen tube growth, embryosurvival and indicators of photosynthetic rate were measuredin two wheat cultivars (‘Karamu’ and ‘Kotuku’)crossed with maize at two irradiance levels (250 or 750 µmolm^-2s^-1, PAR). Pollen tube growth was significantly affectedby light intensity in ‘Karamu’ plants but not in‘Kotuku’ plants, despite both cultivars being pollinatedby the same maize source. The percentage of pollen tubes reachingthe cavity between the ovarian wall and integuments, or in themicropyle of ‘Karamu’ plants at high light intensity(65%) was nearly three-times greater than that at low lightintensity (22%). Thus, either low light intensity can affectthe maternal wheat plant in a way that inhibits pollen tubegrowth and/or high light intensity may promote pollen tube growthin ‘Karamu’ plants. Significant differences in ratesof electron transport in plants grown at the two light intensitiesindicated that the rate of photosynthesis may also have an effecton pollen tube growth. These results have importance for improvingthe efficiency of wheat x maize crosses and other wide cerealcrosses. Copyright 2001 Annals of Botany Company Intergeneric hybridization, light intensity, pollen tube growth, embryo survival, Triticum aestivum, wheat,Zea mays , maize     

桃花粉离体萌发和花粉管生长特性研究

采用花粉离体萌发法研究不同培养基组分和培养条件对桃花粉萌发和花粉管生长的影响,同时对不同贮藏温度下的桃花粉寿命进行研究.结果表明:固体培养基与液体培养基对桃的花粉萌发率和花粉管长度影响差异不显著;10%蔗糖是大多数桃品种花粉的最适萌发条件;硼能提高桃花粉的萌发率,但对花粉管的生长没有促进作用;桃花粉在20℃～25℃的培养温度下萌发率最高,花粉管最长;桃花粉萌发率和花粉管长度在培养前3 h内上升最快,3～5 h上升趋势减弱,5 h后基本停止;随着贮藏温度的升高和贮藏时间的延长,花粉生活力呈降低的趋势.     

Computer-assisted Video Image Analysis of Spatial Variations in Membrane-associated Ca2+ and Calmodulin during Pollen Hydration, Germination and Tip Growth in Nicotiana tabacum L.

Video images of the distributional pattern of membrane-associatedcalcium (Ca²⁺) and calmodulin (CaM) have been documented andanalysed during pollen hydration, germination and tip growthin Nicotiana tabacum. Digitization of fluorescence microscopeimages of chlorotetracycline (CTC) and fluphenazine (FPZ)-fluorescenceemissions reveal that there is a maximum concentration of membrane-associatedCa²⁺ and also CaM in the vicinity of germination apertures ofhydrated pollen. With the onset of germination relatively higheramounts of Ca²⁺ and CaM were found to regionalize towards theaperture through which the pollen tube would emerge Both shortand long growing pollen tubes manifest tip-to-base Ca²⁺ andCaM gradients which are disturbed in non-growing tubes. Tubegrowth and the Ca²⁺-gradient were significantly affected byvanadate and verapamil suggesting that both a vanadate-sensitiveCa²⁺-transport system and verapamil-sensitive Ca²⁺ channelsare involved in maintaining Ca²⁺ homeostasis during pollen germinationand tube growth. The possible interactions of Ca²⁺ and CaM withdifferent cytoskeletal proteins modulating organelle movementare also briefly discussed. Image analysis, calcium, calmodulin, Nicotiana tabacum L., pollen germination, pollen tube, tip growth, Ca²⁺-channels, Ca²⁺ transport ATPase     

The Growth Activity and Fertilizing Capacity of Submersely Cultivated Pollen Tubes of Nicotiana tabacum L.

Tobacco pollen was cultivated in shaken suspension culturesand the growth estimated by weighing the mass of germinatedpollen separated from the nutrient solution. A formula for calculatingmean pollen tube length from the weight of the culture has beenderived for this pollen species. The growth of pollen tubes in vitro is shown to have a rhythmiccharacter. The rapid growth comparable to the mean growth ratein styles is limited to short time intervals alternating withprogressively extending periods of very depressed growth, whichceased entirely after 10–12 h of cultivation. When tested by placental pollination in vitro, the fertilizingcapacity of the pollen culture was found to increase duringthe first hour of cultivation but to decrease steadily thereafter.On the other hand, with the application of pollen tubes fromculture on stigma, a short precultivation period and even themere wetting of pollen had a negative effect on the seed set.With pollen cultivated longer than 4 h, no seed formation wasobserved.     

Intergeneric hybridization between Diplotaxis siifolia,a wild species and crop brassicas

Summary Attempts were made to obtain intergeneric hybrids between Diplotaxis siifolia, a wild species, and cultivars of Brassica (B. campestris, B. juncea, and B. napus). The crosses showed unilateral incompatibility. When the wild species was used as female parent, pollen germination and pollen tube growth were normal, but hybrid seeds aborted due to post-fertilization barriers. Reciprocal crosses (cultivars as female parent) showed strong pre-fertilization barriers; although pollen grains showed germination, pollen tubes failed to enter the stigma. Hybrids were realized in two of the crosses, D. siifolia x B. juncea and D. siifolia x B. napus, through ovary culture. The hybrids were multiplied in vitro by multiplication of axillary shoots, or somatic embryogenesis. Detailed studies were carried out on the hybrid D. siifolia x B. juncea. F₁ hybrids had shrivelled anthers and were pollen sterile. Amphiploids of this hybrid showed 60% pollen fertility and produced seeds upon self-pollination as well as backcross pollination with the pollen of B. juncea.     

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