Marking live conifer pollen for long-distance dispersal experiments

Long-distance dispersal (LDD) theory requires a method for marking live LDD pollen. Such a method must complement more intensive sampling methods inclusive of molecular cytogenetics, proteomics and genomics. We developed a new method for marking live Pinus taeda pollen using two dyes, rhodamine 123 and aniline blue, dissolved in a sucrose solution. Marked and unmarked pollen were compared with respect to in vitro germination, storage, terminal velocity, and in vivo pollen tube penetration of ovules. We found that: (1) both types of marked pollen retained their capacity for germination, (2) both types of marked pollen had similar aerodynamic properties when compared to unmarked pollen controls, (3) marked pollen retained its germination capacity for 48 h, and (4) of the marked pollen, only the aniline-marked pollen penetrated ovules during pollination. Germination declined rapidly for both types of marked pollen after 48 h and before 37 days at -20°C storage, while unmarked pollen lots retained 93% germination at all stages. This method for marking live P. taeda pollen is feasible for tracing LDD pollen only if released and deposited within 48 h of dye treatment.     

Oxalis debilis in China: distribution of flower morphs, sterile pollen and polyploidy

BACKGROUND AND AIMS: Oxalis debilis is a South American tristylous species that is currently naturalizing in China. Based on field observations and herbarium data a report is made on its pollination, morph frequencies, pollen viability, seed set and chromosome number. In addition, a new chromosome count for the species in Africa is provided. METHODS: Field observations were conducted in six provinces in south-east China. Flower visitors were recorded and nectar sugar was measured with a refractometer. The species' compatibility system was determined by carrying out experimental self- and cross-pollinations on bagged inflorescences. Stigma receptivity and pollen viability was determined using the MTT test. KEY RESULTS AND CONCLUSIONS: Populations of O. debilis in China contain either the mid-style-length morph or the short morph, but not both. Pollination is by nectar- and pollen-foraging bees; pollen viability is low; and seed set in natural and experimentally self- or cross-pollinated flowers is extremely low. Chromosome counts indicate that O. debilis contains diploid and tetraploid forms in its native as well as introduced range, which does not support a previous hypothesis that the predominant vegetative reproduction in this species is an escape from pentaploidy.     

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。     

Stylar trimorphism in four functionally andromonoeciousProsopis species (Mimosaceae)

The reproductive system ofProsopis chilensis, P. pugionata, P. flexuosa, andP. torquata is described. Observations were made of floral morphology, stigmatic receptivity, pollen grain viability, and the nature of pollen reserves. Scanning electrone microscopy and statistical analysis were carried out. The results allow us to conclude that: 1) The studied species have three distinct floral morphs characterized by significantly different style lengths and, to a lesser extent, different stamen lengths; 2) style precocity found in all species does not indicate protogyny; 3) flowers with shortest styles are not receptive; 4) the pollen grains of all floral morphs showed high viability; 5) peroxidase is present in both the pollen and pistil; 6) viable pollen grains invariably have both starch and pollenkitt; 7) the studied species are andromonoecious.     

Limitations of MTT and MTS-Based Assays for Measurement of Antiproliferative Activity of Green Tea Polyphenols

Background

The chemopreventive effect of green tea polyphenols, such as (-)-epigallocatechin-3-gallate (EGCG), has been well demonstrated in cell culture studies. However, a wide range of IC₅₀ concentrations has been observed in published studies of the anti-proliferative activity of EGCG from different laboratories. Although the susceptibility to EGCG treatment is largely dependent on cancer cell type, the particular cell viability and proliferation assays utilized may significantly influence quantitative results reported in the literature.

Methodology/Principal Findings

We compared five widely used methods to measure cell proliferation and viability after EGCG treatment using LNCaP prostate cancer cells and MCF-7 breast cancer cells. Both methods using dyes to quantify adenosine triphosphate (ATP) and deoxynucleic acid (DNA) showed accuracy in the measurement of viable cells when compared to trypan blue assay and results showed good linear correlation (r = 0.95). However, the use of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) as indicators of metabolically active mitochondria overestimated the number of viable cells by comparison with the ATP, DNA, or trypan blue determinations. As a result, the observed IC₅₀ concentration of EGCG was 2-fold higher using MTT and MTS compared to dyes quantifying ATP and DNA. In contrast, when cells were treated with apigenin MTT and MTS assays showed consistent results with ATP, DNA, or trypan blue assays.

Conclusions/Significance

These results demonstrate that MTT and MTS -based assays will provide an underestimation of the anti-proliferative effect of EGCG, and suggest the importance of careful evaluation of the method for in vitro assessment of cell viability and proliferation depending on the chemical nature of botanical supplements.     

Effect of enhanced UV-B radiation on pollen quantity,quality, and seed yield in Brassica rapa (Brassicaceae)

We conducted three experiments to examine the influence of ultraviolet-B radiation (UV-B; 280–320 nm) exposure on reproduction in Brassica rapa (Brassicaceae). Plants were grown in a greenhouse under three biologically effective UV-B levels that simulated either an ambient stratospheric ozone level (control), 16% (“low enhanced”), or 32% (“high enhanced”) ozone depletion levels at Morgantown, WV, USA in mid-March. In the first experiment, we examined whether UV-B level during plant growth influenced in vivo pollen production and viability, and flower production. Pollen production and viability per flower were reduced by ≈50% under both enhanced UV-B levels relative to ambient controls. While plants under high-enhanced UV-B produced over 40% more flowers than plants under the two lower UV-B treatments, whole-plant production of viable pollen was reduced under high-enhanced UV-B to 17% of that of ambient controls. Whole-plant production of viable pollen was reduced under low-enhanced UV-B to 34% of ambient controls. In the second experiment, we collected pollen from plants under the three UV-B levels and examined whether source-plant UV-B exposure influenced in vitro pollen germination and viability. Pollen from plants under both enhanced-UV-B treatments had initially lower germination and viability than pollen from the ambient level. After in vitro exposure to the high-enhanced UV-B levels for 6 h, viability of the pollen from plants grown under ambient UV-B was reduced from 65 to 18%. In contrast, viability of the pollen from plants grown under both enhanced UV-B treatments was reduced to a much lesser extent: only from ≈43 to 22%. Thus, ambient source-plant pollen was more sensitive to enhanced UV-B exposure. In the third experiment, we used pollen collected from source plants under the three UV-B levels to fertilize plants growing under ambient-UV-B levels, and assessed subsequent seed production and germination. Seed abortion rates were higher in plants pollinated with pollen from the enhanced UV-B treatments, than from ambient UV-B. Despite this, seed yield (number and mass) per plant was similar, regardless of the UV-B exposure of their pollen source. Our findings demonstrate that enhanced UV-B levels associated with springtime ozone depletion events have the capacity to substantially reduce viable pollen production, and could ultimately reduce reproductive success of B. rapa.     

Fluorescent dye particles as pollen analogues for measuring pollen dispersal in an insect-pollinated forest herb

In flowering plants, pollen dispersal is often the major contributing component to gene flow, hence a key parameter in conservation genetics and population biology. A cost-effective method to assess pollen dispersal consists of monitoring the dispersal of fluorescent dyes used as pollen analogues. However, few comparisons between dye dispersal and realized pollen dispersal have been performed to validate the method. We investigated pollen dispersal in two small populations of the insect-pollinated herb Primula elatior from urban forest fragments using direct (paternity analyses based on microsatellite DNA markers) and indirect (fluorescent dyes) methods. We compared these methods using two approaches, testing for the difference between the distance distributions of observed dispersal events and estimating parameters of a dispersal model, and related these results to dye dispersal patterns in three large populations. Dye and realized (based on paternity inference) pollen dispersal showed exponential decay distributions, with 74.2?C94.8% of the depositions occurring at <50?m and a few longer distance dispersal events (up to 151?m). No significant difference in curve shape was found between dye and realized pollen dispersal distributions. The best-fitting parameters characterizing the dye dispersal model were consistent with those obtained for realized pollen dispersal. Hence, the fluorescent dye method may be considered as reliable to infer realized pollen dispersal for forest herbs such as P. elatior. However, our simulations reveal that large sample sizes are needed to detect moderate differences between dye and realized pollen dispersal patterns because the estimation of dispersal parameters suffers low precision.     

Variation in pollen viability among Picea abies genotypes – potential for unequal paternal success

An in vitro germination method was used to study variation in pollen viability, that is pollen-tube growth rate together with germination percentage, among the Picea abies genotypes in a seed orchard. The method permits easy, rapid screening of large numbers of genotypes. Significant variation in pollen viability among the genotypes was evident, the differences among the pollen-lot means being 7–10–fold in different years. No correlation was found between the average pollen viability and the phenology, growth or growing-site characteristics of the pollen donors. However, there appeared to be pollen lots that either benefit from a higher germination temperature or else germinate faster at lower temperatures. The significant variation in pollen viability among the pollen donors indicates a potential for male gametophyte competition. This, together with the observed genotype-environment interactions in pollen performance, may contribute to the variable genetic composition of seed produced in the seed orchard. Received: 9 December 1999 / Accepted: 22 December 1999     

Assessment of Cell Viability in Intact Glandular Tissue in <Emphasis Type="Italic">Chironomus ramosus</Emphasis> using Dye-exclusion and Colorimetric Assays

Conventionally, dye-exclusion test for determining cell viability has been restricted only for cells in suspension in tissue culture. In this paper, salivary gland of Chironomus has been proposed as a simple tissue model system where dye-exclusion test can be reliably employed for the intact gland. We have compared suitability of commonly used vital dyes and nigrosin was found suitable for the salivary gland cells. Biochemical tests using tetrazolium salts are also commonly used for determining quantitative indices of cell viability in metabolically active cells. Ours is the first attempt to extend the same technique for the whole tissue. We standardized the conditions and prepared a protocol for MTT-based colorimetric assay suitable for the salivary gland of Chironomus. A strong correlation (r² = 0.9893) was obtained where increasing O.D. correlated linearly with the number of live glands. We concluded that nigrosin dye-exclusion and MTT metabolic inclusion assays are suitable methods for the viability test of metabolically active intact salivary gland of Chironomus which can serve as a potential model for the assessment of cytotoxicity in future.     

Collection and storage of pollen from Salix (Salicaceae)

Genetic improvement of willows through traditional breeding can be facilitated by pollen collection and storage so that female flower receptivity need not be synchronized with pollen shed for breeding. Two experiments were completed to test the effectiveness of various organic solvents for willow pollen collection. In the first experiment, seven pollen collection treatments and an untreated control were tested with two willow clones. The other experiment tested three treatments that showed promise in the initial experiment and an untreated control with eight willow clones. Toluene and carbon tetrachloride were effective for pollen extraction, with average pollen germination percentages that were >15%, but both chemicals reduced pollen viability by 10-20% compared with an untreated control based on in vitro germination tests. Pollen extracted with carbon tetrachloride or toluene was successfully used in controlled pollination, and >100 new families were produced with this technique. Pollen viability remained high after 18 mo of storage at -20°C. Based on our results, toluene is the preferred solvent for future willow pollen extractions because it is as effective as carbon tetrachloride, is not a known carcinogen, and is less expensive.     

VARIATION IN MALE AND FEMALE REPRODUCTIVE SUCCESS AMONG FLORAL MORPHS IN THE TRISTYLOUS PLANT LYTHRUM SALICARIA (LYTHRACEAE)

The purpose of this study was to assess variation in male and female reproductive success among the three morphs of the tristylous plant, Lythrum salicaria. Fluorescent dyes were used as pollen analogs to determine whether morphs differ in their abilities to donate and receive pollen, and actual and potential seed set was measured with a hand pollination experiment. Dye transfer among morphs was highly asymmetric, with more frequent transfer from the short-styled morph to the long- and mid-styled morphs. This suggests that shorts are performing better at pollen donation and longs and mids are performing better at pollen receipt. All flowers on 95 plants were hand pollinated to test whether female reproductive success is more pollen-limited in the short-styled morph than in other morphs. Hand-pollinated short-styled plants had significantly higher total seed mass and more seeds per capsule than short controls, whereas hand pollination failed to increase seed set in long and mid morphs. As predicted, short-styled morphs showed significant pollen limitation, whereas seed set in long- and midstyled morphs was not pollen-limited. Thus, in Lythrum salicaria asymmetrical pollen flow generates morph-specific differences in male and female fitness.     

Autogamy of an endangered species: Loropetalum subcordatum (Hamamelidaceae)

The reproductive biology of Loropetalum subcordatum was studied. The floral phenology, pollen histochemistry, pollen-ovule ratio (P/O), pollen viability and floral visitors were investigated and determined. Assisted pollination experiments were carried out to examine the breeding system of L. subcordatum. Scanning electron microscopy (SEM) and fluorescence microscopy (FM) were employed to check the pollen germination and the growth of pollen tubes. Results were obtained as follows: (1) The flowering period of L. subcordatum was from September to the next February with a peak at September-October; the longevity of a single flower was 4-6 days. (2) L. subcordatum was protogynous, and pollen grains could be found on self-stigmata when the anthers opened 12-24 h after petals unrolling. (3)The pollen viability (MTT test) maintained for ca. 26 hours; the pollen was starchless; the P/O was 8420±720.86 (n=10); no nectar secretion was observed. (4) Thrips (Thrips sp.) were the only floral visitors observed but which seldom moved among inflorescences, thus played limited role in pollination. (5) Fruit sets of untreated bagged (5.30±1.83%) and hand assisted cross pollinated flowers (6.67±1.91%) were not significantly different from that of open flowers (4.79±1.45%). (6) SEM and FM observations proved that pollen germinated on self-stigmata and pollen tubes growed in self-styles. The results indicated that L. subcordatum was facultatively autogamous and without apomixes. The possibility of outcrossing and the protogyny might indicate that the species was originally a crosser. Flowers pollinated in September-October usually start ovule growth in next summer, and set mature fruits in next October (after the next flowering peak), suggesting the occurrence of retard embryo development. The possibility that autogamy in Hamamelidaceae could have been developed from fly pollination was also discussed.     

The estimation of pollen viability in rice

Methods for testing pollen viability in rice were evaluatedby comparing staining with aniline blue in lactophenol, twotetrazolium salts and fluorescein diacetate with germinationin an in vitro culture medium. Staining with thiazolyl blue(MTT) was nicely correlated with germination. Under non-saline conditions, rice pollen was only viable fora short period of time, with an approximately 50% loss of viabilitywithin 20 min of shedding. When plants were salinized at eitherpanicle initiation or the booting stage, pollen viability wasreduced. Key words: Rice, Oryza sativa, pollen viability, salinity     

Optimisation of pollen viability tests for Acacia podalyriifolia and two ploidys of Acacia mearnsii

Acacia mearnsii (black wattle) is a commercially important forestry species in South Africa, grown for its timber as well as its bark. It is, however, also considered to be an alien invader of indigenous vegetation and for this reason the production of a sterile variety would be highly desirable for commercial forestry in South Africa. Previous research on crosses between diploid and tetraploid parent plants to produce triploid progeny has resulted in poor seed set. One possible barrier preventing seed set could be the viability of the pollen used in the cross pollination operations. Thus a study was conducted to test the pollen viability. In vitro agar media germination tests (ACIAR and Brewbaker and Kwack media) were optimised on Acacia podalyriifolia pollen and then used together with vital stain tests (Sigma® DAB peroxidase and p-phenylendiamine) to test pollen germination and viability of A. mearnsii pollen. These were then compared to in vivo pollen germination on the stigma, and were conducted on both diploid and tetraploid pollen mixes. Results showed that the vital stain tests gave significantly (p < 0.05) higher pollen viability than the agar germination tests and were more in agreement with the results from the pollen germination rate on the stigma. For both the diploid and tetraploid pollen mixes tested, there were no significant differences (p > 0.05) between the two agar media germination tests and between the two vital stain tests.     

Freezing injury to ovules, pollen and seeds in winter rape

Winter rapeseed (Brassica napus, cv. Samouraï) flowersearly in spring and, under field conditions, short freezingperiods can occur. Unacclimatized plants were freeze-stressed(–3°C for 4 h) at different developmental stages ofbuds, open flowers and seeds. The dissection of pistils from stressed plants showed that freezingresults in shrivelled ovules. We assessed freezing injury onthe basis of per cent of shrivelled ovules: ovule sensitivitybegins early (8 d before anthesis) but increases up to anthesis.Crosspollination of stressed pistils with non-stressed pollenshowed that recording of freeze-injured ovules is a good methodfor early estimation of the effect of stress on seed yields. On the other hand, stress does not reduce the viability of pollen,except when it was applied at the binucleate pollen stage. Useof frozen pollen x nonstressed pistils has little effect onseed yields. Freeze injury on seeds was assessed by seed filling:seeds are very susceptible just after fertilization until 20d after fertilization (DAF). Freezing seems to inhibit seedfilling. A germination test of stressed seeds during their developmentindicated that embryo viability is not affected if the stressoccurs after 35 DAF. As the embryos develop, resistance to stressincreases. Key words: Brassica napus, rapeseed, freeze injury, pollen and ovule, seed filling     

Conservation of the germination capacity of pollen grains in three varieties of maize (Zea mays L.)

Maize pollen longevity is short under natural conditions, and the seed yield is hampered by poor synchronisation between pollen shed and ovule maturity. Hybridization during stress conditions could be improved if pollen is collected and stored for use at appropriate times. This study was initiated to determine the optimum conditions for in vitro maize pollen germination. The method of pollen conservation and its viability over time was also part of the study. The pollen from three Zea mays cultivars (Exp₁ 24 and 5057, two inbred lines, and Tuxpeño sequía which is an open pollinated cultivar) were used in this study. The conservation was evaluated using three different methods: room temperature, the refrigerator (+10°C), and deep‐freezing at ?20°C. Two base culture media (Brewbaker &; Kwack, Heslop‐Harrison) were used for in vitro pollen germination. The effects of saccharose concentrations, incubation and desiccation times, were also evaluated. Results from this study showed that, Heslop‐Harrison medium was better than Brewbaker and Kwack or and with pollen germination. Pollen stored in a refrigerator (+10°C) varying desiccation times, yielded the following findings: for a short conservation time (10 days maximum), there is no need for a prior desiccation. For a medium conservation time (between 16 days and 20 days), five to six hours desiccation time are needed to obtain maximum germination for inbred line Exp₁ 24 and for the composite variety Tuxpeño sequía respectively. It was also determined that, at room temperature, maize pollen could germination is longer than 24?hours. The use of deep‐freezer at ?20°C did not yield any consistent result. In vivo germination test in a maize field will be necessary to confirm the above in vitro germination.     

Responses of tobacco pollen to high humidity and heat stress: viability and germinability in vitro and in vivo

Summary Responses of pollen grains of Nicotiana tabacum to high humidity (95% RH, 4 h) and temperature (38°/45° C, 4 h) stresses were investigated. Pollen grains were subjected to only RH or only temperature, or to both of these stresses. Their viability was assessed on the basis of the fluorochromatic reaction (FCR) test, and vigour was assessed on the basis of the time taken for in vitro germination as well as on the emergence of pollen tubes through the cut end of semi-vivo implanted styles. None of the stress conditions affected pollen viability and high RH or high temperature stress did not individually affect pollen vigour. However, pollen vigour was markedly affected when both the stresses were given together. Pollen grains subjected to high RH at 38° C took a longer time to germinate in vitro and the pollen tubes emerged later from the cut end of the semi-vivo styles; division of the generative cell was also delayed. Pollen grains subjected to high RH at 45° C failed to germinate in vitro, but did germinate on the stigma. Many pollen tubes subjected to this treatment showed abnormalities, and the growth of pollen tubes in the pistil was much slower than that observed in other treatments. Pollen samples subjected to all of the stress conditions were able to induce fruit and seed set. The implications of these results on the relationship between the FCR test and viability, and between viability and vigour, especially in stressed pollen, are discussed.     

The effects of time,temperature and plant variety on pollen viability and its implications for gene flow risk

• Pollen viability affects the probability that a pollen grain deposited on a plant's stigma will produce a viable seed. Because a mature seed is needed before a gene flow event can occur, pollen viability will influence the risk of escape for genetically engineered (GE) crops.
• Pollen viability was measured at intervals for up to 2 h following removal of the pollen from the anthers. It was quantified at three temperatures and for different alfalfa varieties, including both conventional and Roundup Ready (RR) varieties. Pollen viability was assessed using in vitro germination.
• Time since removal from the anthers was the most prevalent factor affecting pollen viability in alfalfa. Pollen viability declined with increasing time at all three temperatures and for all varieties tested. Pollen viability was not affected by temperatures ranging between 25 and 37 °C and did not vary among plant varieties, including conventional and RR varieties.
• Bee foraging behaviour suggested pollen viability within the first 10 min following pollen removal from a flower to most affect seed production. Pollen longevity was predicted to have little impact on seed set and gene flow. Linking pollinator behaviour to pollen viability improved our understanding of its impact on gene flow risk.

     

Pollination, Pollen Viability and Pistil Receptivity in Cucurbita pepo

Cucurbita pepo carries male and female flowers on the same plant,and is pollinated by nectar-collecting bees. The nectaries aredimorphic in the two sexes and pollen is loaded and unloadedas the bees gain access to the nectar. Both types of flowerare open for only 6 h (from 0600 h to 1200 h); male flowersopen and close half an hour earlier than female flowers. Thelatter produce more nectar and are visited more often by thebees than the male flowers. Pollen viability determined by fluoresceindiacetate (fluorochromatic reaction) decreases by 20% duringanthesis and more rapidly after the flower closes. This decreaseis due to dehydration of the grain, especially around the porewhere the intine is exposed. An unusual feature of this speciesis that the grains do not dehydrate before anther dehiscence.Female receptivity has two aspects, that of the stigma lasting4 d, and that of the ovules lasting 2 d. The receptivity ofthe two sexes and the short period of anthesis are discussedin the light of the reproductive ecology of the species.Copyright1993, 1999 Academic Press Entomophilous pollination, anthesis, nectars, pollen viability, female receptivity, bees, pollinator efficiency, courgette Cucurbita pepo, Cucurbitaceae     

钝裂银莲花花粉活力测定方法的研究

以3种花色（黄色、浅黄色和白色）钝裂银莲花为研究对象,分别采用TTC染色、I2-I染色、无机酸测定和蔗糖萌发4种方法,测定了钝裂银莲花（Anemone obtusiloba）的花粉活力,同时比较了不种方法对花粉活力的测定效果,并了解了花粉活力在一天内的动态变化情况。结果表明：（1）钝裂银莲花花粉可育率很高,达80%左右。花粉直径为28.75~33.75 m 。（2）TTC浓度影响花粉活力的测定,当浓度为0.5%时,所测花粉活力的效果较好。（3）I2-I法的染色效果不理想,染色率仅为8.33%~22.2%,且3种花色花粉活力测定数据间差异不显著。（4）3种花色钝裂银莲花花粉活力有差异,黄色最高,浅黄色次之,白色最低。TTC染色法是简单快速测定钝裂银莲花花粉活力的最适方法。一天内花粉活力在12:00~14:00最大。