Vanillin-hydrochloric acid as a histochemical test for tannin.

Condensed tannin (leucoanthocyanins and catechins) can be demonstrated in fresh plant sections with saturated alcoholic vanillin followed by addition of concentrated HCl. Bright red vanillin-tannin condensates are formed immediately. Preparations may then be made permanent by mounting in a 1:1 mixture of Hoyer's medium and concentrated HCl. Some fading and loss of tannin occurs. The phloroglucinol-HCl test for lignin can also be made permanent with this acidic mountant.     

Vanillin-Hydrochloric Acid as a Histochemical Test for Tannin

Condensed tannin (leucoanthocyanins and catechins) can be demonstrated in fresh plant sections with saturated alcoholic vanillin followed by addition of concentrated HC1. Bright red vanillin-tannin condensates are formed immediately. Preparations may then be made permanent by mounting in a 1:1 mixture of Hoyer's medium and concentrated HC1. Some fading and loss of tannin occurs. The phloroglucinol-HC1 test for lignin can also be made permanent with this acidic mountant.     

Expression of stylar incompatibility in the Andean clonal tuber crop oca (Oxalis tuberosa Mol., Oxalidaceae)

Long-, mid-, and short-styled clonal accessions of oca (Oxalis tuberosa) were intercrossed in a complete diallelic design. Pollen tube growth in styles was monitored in all diallelic crosses. Pollen fertility was estimated by two tests: staining of pollen grains with aceto-carmine and detection of β-galactosidase activity by the substrate X-Gal. The two methods of pollen fertility estimation were equally useful to detect fertility levels. Pollen originating from short stamens had the highest fertility (85%) and pollen from long stamens had the lowest fertility (70%). Pollen fertility was high throughout, but its degree varied with the stylar morph on which the pollen was formed. Long-styled accessions had the highest rates of fertile pollen. Differences in pollen fertility at different anther levels in the same style morph were also apparent. Pollen grain diameter of the six morph-anther level combinations was inversely correlated with pollen fertility. Pollen grains from long stamens were the largest and pollen grains from short stamens were the smallest. Neither pollen fertility nor pollen grain size had an influence on pollen tube growth in the style or on fruit and seed set. Pollen tubes growing within the styles were inhibited at a different level for each of the 18 cross combinations in the diallel. Although legitimate crosses had greatest pollen tube growth, some of the illegitimate inter- and intramorph crosses had equally high scores. Of all illegitimate crosses, mid-styled seed parents had the lowest level of stylar incompatibility. Fruit and seed set were highly correlated with the extent of pollen tube growth in the style. The number of pollen tubes entering ovules in a flower was in good agreement with the number of seeds produced per fruit. Therefore, it is concluded that stylar incompatibility is the major determinant of limited seed formation in oca even in the most successful legitimate cross combinations. Received: 1 September 1999 / Revision accepted: 4 April 2000     

A Method for Staining Pollen Tubes in Pistil

A quadruple staining procedure has been developed for staining pollen tubes in pistil. The staining mixture is made by adding the following in the order given: lactic acid, 80 ml; 1% aqueous malachite green, 4 ml; 1% aqueous acid fuchsia, 6 ml; 1% aqueous aniline blue, 4 ml; 1 % orange G in 50% alcohol, 2 ml; and chloral hydrate, 5 g. Pistils are fixed for 6 hr in modified Carnoy's fluid (absolute alcohol:chloroform:glacial acetic acid 6:4:1), hydrated in descending alcohols, transferred to stain and held there for 24 hr at 45±2 C They were then transferred to a clearing and softening fluid containing 78 ml lactic acid, 10 g phenol, 10 g chloral hydrate and 2 ml 1% orange G. The pistils were held there for 24 hr at 45±2 C, hydrolyzed in the clearing and softening fluid at 58±1 C for SO min, then stored in lactic acid for later use or immediately mounted in a drop of medium containing equal parts of lactic acid and glycerol for examination. Pollen tubes are stained dark blue to bluish red and stylar tissue light green to light greenish blue. This stain permits pollen tubes to be traced even up to their entry into the micropyle.     

A method for staining pollen tubes in pistil

A quadruple staining procedure has been developed for staining pollen tubes in pistil. The staining mixture is made by adding the following in the order given: lactic acid, 80 ml; 1% aqueous malachite green, 4 ml; 1% aqueous acid fuchsin, 6 ml; 1% aqueous aniline blue, 4 ml; 1% orange G in 50% alcohol, 2 ml; and chloral hydrate, 5 g. Pistils are fixed for 6 hr in modified Carnoy's fluid (absolute alcohol:chloroform:glacial acetic acid 6:4:1), hydrated in descending alcohols, transferred to stain and held there for 24 hr at 45 +/- 2 C. They were then transferred to a clearing and softening fluid containing 78 ml lactic acid, 10 g phenol, 10 g chloral hydrate and 2 ml 1% orange G. The pistils were held there for 24 hr at 45 +/- 2 C, hydrolyzed in the clearing and softening fluid at 58 +/- 1 C for 30 min, then stored in lactic acid for later use or immediately mounted in a drop of medium containing equal parts of lactic acid and glycerol for examination. Pollen tubes are stained dark blue to bluish red and stylar tissue light green to light greenish blue. This stain permits pollen tubes to be traced even up to their entry into the micropyle.     

Simplified Permanent Aceto-Carmine Smears with A Water-Miscible Mountant

By employing a water-miscible mountant with a good refractive index, permanent aceto-carmine smears can be made from any of a variety of killing, fixing, and staining schedules. Clear-col, a commercial preparation, orginally developed for mounting fungi from various water-containing media, is employed for anther smears by placing the medium directly on the final stage of aceto-carmine staining schedule. Thus, no dehydration and clearing are necessary. By controlling amounts of acetic acid either mixed with Clearcol or on smears, clear, well-stained preparations can be produced.     

Differential Staining of Aborted and Nonaborted Pollen

A single staining solution was made by compounding it in the following order (dyes were from British Drug Houses): ethanol, 10 ml; 1% malachite green in 95% ethanol, 1 ml; distilled water, 50 ml; glycerol 25 ml; phenol, 5 gm; chloral hydrate, 5 gm; acid fuchsin 1% in water, 5 ml; orange G, 1% in water 0.5 ml; and glacial acetic acid, 1-4 ml. For best results in differentiation to give green pollen walls and red protoplasm, the staining solution should be acidified with glacial acetic acid. The amount of acid to be added depends upon thickness of the pollen walls: for very thin-walled pollen, 1 ml; for moderately thin walls, 2 ml; and for thick-walled or spiny-walled pollen, 3 ml of acid. For pollen inside non-dehiscent anthers, 4 ml of acid should be used. Staining is hastened by flaming the slide (for loose thin-walled pollen) or by immersing thick-walled pollen or anthers for 24-48 hr at 50 C. In the typical stain, aborted pollen grains are green; nonaborted, red. The method is useful for pollen inside nondehiscent anthers if these are small and not too deeply coloured naturally. The stain is very durable, especially if the coverslips are sealed with param wax. The staining solution will keep well for about a month. It is useful both for angiosperms and gymnosperm microgametes.     

From the anther to the proctodeum: Pear (Pyrus communis) pollen digestion in Osmia cornuta larvae

Modifications of the pollen grains of Pyrus communis Linneaus that occur during the digestion by Osmia cornuta (Latreille) larvae were studied histochemically. We compared the features of the pollen grains found in the anthers, in the larval cell provisions and in the alimentary canal of the 5th instar larvae. Modifications were already evident in the provisions and consisted of protoplast protrusions through the apertures and a decrease in the number of starch-containing pollen grains. After pollen grains were ingested by the larvae, the protoplast appeared retracted from the pollen wall. Pollen digestion began in the anterior part of the midgut, where we observed: (1) disorganised intine at the apertures; (2) disappearance of DAPI staining of nuclear pollen DNA; (3) fewer pollen grains containing starch than in the anthers; (4) some empty pollen grains. Pollen grains in the proctodeum appeared extremely compressed and crushed. Some grains appeared to be unaffected by the digestive process. We hypothesise that the protrusion of the intine and of the protoplast from the apertures in bee provisions could be considered a kind of pre-treatment necessary to initiate the digestion process in the larval alimentary canal.     

Flowering phenology and wind-pollination efficacy of heterodichogamous Juglans mandshurica (Juglandaceae)

BACKGROUND AND AIMS: Heterodichogamy differs from normal dichogamy, in that it involves two mating types (protogyny and protandry) that occur at a 1 : 1 ratio in a population. Flowering phases of the two mating types are synchronized and reciprocal, which was considered to ensure between-type outcrossing. This study aims to quantify the flowering pattern and pollination efficacy in Juglans mandshurica, a wind-pollinated heterodichogamous tree. METHODS: The pattern of flowering phenology was monitored within individual trees and pollen traps were used to measure air-borne pollen loads during the spring in 2003 and 2004. Pollen longevity was determined by staining technique. Also a pollen supplementation experiment was performed in 2004 to assess pollen limitation of fruit production. KEY RESULTS: There was no overlap between sexual functions within individual trees. Flowering periods of the two mating types were reciprocal and synchronous in both 2003 and 2004. Air-borne pollen loads were large, and protogynous and protandrous individuals each produced a high pollination peak, consistent with the two blooming periods. Maximum pollen longevity was about 4 h for protandrous individuals, and 3 h for protogynous individuals. Pollen supplementation did not increase fruit production in either protogynous or protandrous individuals. CONCLUSIONS: Heterodichogamous flowering in Juglans mandshurica effectively avoids selfing, promotes between-type outcrossing, and leads to efficient pollination in a natural population.     

Collodion Membranes in Pollen Tube Technique

Membranes are formed by allowing a drop of collodion-acetone solution to come into contact with the surface of warm sugar solution in a petri dish. Pollen is germinated upon the smooth areas of the membrane when all traces of acetone have evaporated. Semipermanent preparations are made by isolating the pollinated area of the membrane, floating it onto a slide, and, after the removal of excess sugar solution, adding a drop of acetic-stain fixative, followed by an albumenized cover slip. The preparation can be made permanent by inverting a slide in a mixture of 1 part glacial acetic acid and 3 parts absolute alcohol, when the collodion membrane will dissolve and allow the cover slip and adhering grains to fall free. The cover slip is then passed through absolute alcohol (2 changes), xylene, and mounted in neutral mountant on a clean slide. By substituting a drop of the alcohol-acetic acid mixture in place of acetic-stain fixative, the grains adhering to the cover slip may be stained by the Feulgen method.     

Pollen germinates precociously in the anthers of raring-to-go, an Arabidopsis gametophytic mutant

Pollen hydration is usually tightly regulated and occurs in vivo only when desiccated pollen grains acquire water from the female, thus enabling pollen tube growth. Pollen tubes are easily visualized by staining with decolorized aniline blue, a stain specific for callose. We identified a mutant, raring-to-go, in which pollen grains stained for callose before anther dehiscence. When raring-to-go plants are transferred to high humidity, pollen tubes dramatically elongate within the anther. As early as the bicellular stage, affected pollen grains in raring-to-go plants acquire or retain water within the anther, and precociously germinate. Thus, the requirement for contact with the female is circumvented. We used pollen tetrad analysis to show that raring-to-go is a gametophytic mutation, to our knowledge the first gametophytic mutation in Arabidopsis that affects early events in the pollination pathway. To aid in identifying raring-to-go alleles, we devised a new technique for screening pollen in bulk with decolorized aniline blue. We screened a new M(1) mutagenized population and identified several additional mutants with a raring-to-go-like phenotype, demonstrating the usefulness of this technique. Further, we isolated other mutants (gift-wrapped pollen, polka dot pollen, and emotionally fragile pollen) with unexpected patterns of callose staining. We suggest that raring-to-go and these other mutants may help dissect components of the pathway that regulates pollen hydration and pollen tube growth.     

早发生胚水稻小孢子发生及花粉的萌发生长

实验结果表明,早发生胚水稻（PDER）品系小孢子的发生发育与常规水稻品种相同,常规I-KI镜检显示其成熟花粉育性达87．4％．开花后,少量花粉在柱头上能正常萌发,并以短管形式进入柱头．大部分花粉在柱头上发生了异常行为：不萌发花粉管,但排放出大量内容物;花粉管细小或畸形;在枝头内或柱头外花粉管前端破裂;花粉管在柱头上绕行不进入柱头;花粉管在柱头内逆行生长;花粉管进入柱头后又穿出柱头;花粉管壁上粘有颗粒或被有一层膜等．文中就PDER的来源讨论了这些现象,并提出“萌败”这一新概念．     

The effects of natural variation in pollinator visitation on rates of pollen removal in wild radish,Raphanus raphanistrum (Brassicaceae)

Pollen removal from flowers is an important component of male fitness, but the effects of natural variation in visitation rates on pollen removal are poorly understood. We measured pollen removal over 2 yr in experimental field populations of wild radish, Raphanus raphanistrum. Pollen removal and pollinator visitation over 1-hr periods were measured on previously unvisited flowers. The effects of pollen production and visitation by different insect taxa on pollen removal were determined using multiple regression. Pollen removal rates were extremely high; a median of 84% of pollen produced was removed in 1 hr. Pollen production was far more important than visitation in determining the number of pollen grains removed. Pollen removal increased with increasing numbers of visits by honey bees and small native bees, but increased numbers of syrphid fly visits had no effect. Average visit duration had no effect on pollen removal in 1991, and a marginally negative effect in 1992.     

A Versatile Stain for Pollen Fungi, Yeast and Bacteria

A versatile stain has been developed for demonstrating pollen, fungal hyphae and spores, bacteria and yeasts. The mixture is made by compounding in the following order: ethanol, 20 ml; 1% malachite green in 95% ethanol, 2 ml; distilled water, 50 ml; glycerol, 40 ml; acid fuchsin 1% in distilled water, 10 ml; phenol, 5 g and lactic acid, 1-6 ml. A solution has also been formulated to destain overstained pollen mounts. Ideally, aborted pollen grains are stained green and nonaborted ones crimson red. Fungal hyphae and spores take a bluish purple color and host tissues green. Fungi, bacteria and yeasts are stained purple to red. The concentration of lactic acid in the stain mixture plays an important role in the differential staining of pollen. For staining fungi, bacteria and yeasts, the stain has to be acidic, but its concentration is not critical except for bacteria. In the case of pollen, staining can be done in a drop of stain on a slide or in a few drops of stain in a vial. Pollen stained in the vial can be used immediately or stored for later use. Staining is hastened by lightly flaming the slides or by storing at 55±2 C for 24 hr. Bacteria and yeasts are fixed on the slide in the usual manner and then stained. The stock solution is durable, the staining mixture is very stable and the color of the mounted specimens does not fade on prolonged storage. Slides are semipermanent and it is not necessary to ring the coverslip provided 1-2 drops of stain are added if air bubbles appear below the coverslip. The use of differentially stained pollen mounts in image analyzers for automatic counting and recording of aborted and nonaborted pollen is also discussed.     

Study on the Pollen Morphology of the Family Berberidaceae

The pollen grains of 33 species representing 11 genera of the family Berberidaceae, mostly from China, were examined with the light microscope and scanning electron microscope. Their characteritic details can be used for generic diagnosis. A pollen key to the genera based on these observations is presented. Based on the morphology, the pollen grains can be grouped into the following three types: 1. The tetrad pollen type found only in the genus Sinopodophyllum. 2. The spiraperturate pollen type found in the genera Berberis and Mahonia. 3. The tricolpate pollen type found in the genera Diphylleia, Jeffersonia, Nandina, Dysosma, Caulophyllum, Leontice and Epimedium. A diagnostic key to the pollen grains of genera in the Berberidaceae. 1. Pollen grains single 2. Pollen grains spiraperturate .................... Berberis L., Mahonia L. 2. Pollen grains tricolpate 3. Exine with spinose sculpture ........................ Diphylleia Michx. 3. Exine with non-spinose sculpture 4. Exine with striate or striate-reticulate sculpture ...... Jeffersonia Barton 4. Exine with reticulate sculpture 5. Exine around colpus with distinct thickening ........ Nandina Thunb. 5. Exine around colpus without thickening 6. Pollen grains larger (45—50)×(32.5—37.5)μ 7. Colpus with membrane ................ Dysosma R. E. Woodson 7. Colpus without membrane ................ Caulophyllum Michx. 6. Pollen grains smaller (25—550)×(20—527.5) μ 7. Pollen grains prolate-perprolate .................... Leontice L. 7. Pollen grains spheroidal-prolate...Epimedium L., Podophyllum L. 1. Pollen grains tetrad ............................ Sinopodophyllum Ying     

Pollen dispersion, pollen viability and pistil receptivity in Leymus chinensis

BACKGROUND AND AIMS: Leymus chinensis is an economically and ecologically important grass that is widely distributed across eastern areas of the Eurasian steppe. A major problem facing its propagation by man is its low sexual reproductivity. The causes of low fecundity are uncertain, largely because many aspects of the reproductive biology of this species remained unknown or incomplete. This study aims to address some of these issues. METHODS: Pollen dispersion, pollen viability, pollen longevity and pistil receptivity were studied in a representative, natural population of L. chinensis growing in Inner Mongolia. KEY RESULTS: Flowering of L. chinensis occurred at the end of June and lasted for 5 d. Pollination peaked between 1600 h and 1700 h, and about 56.1 % of the total pollen grains were released at this time. Pollen density was highest towards the middle of flowering spikes and lowest at the bottom over the 5 d measurement period. Pollen viability (62.4 %) assessed using TTC was more accurate than using IKI (85.6 %); 50 % of pollen arriving on stigmas germinated. Pollen remained viable for only 3 h and the pollen : ovule ratio was 79 333 : 1. Pistil receptivity lasted for only 3 h and, overall, 86.7 % of pistils were pollinated. Within the spike, the relative fecundity of different positions was middle > lower > upper throughout the period of pollination; daily variation of fecundity was similar to that of the pollen flow. The spikes that opened on the day of highest pollen density exhibited the highest fecundity (36.0 %). No seeds were produced by self-pollination. CONCLUSIONS: The data suggest that low pollen viability, short pollen longevity and short pistil receptivity all appear to contribute to the low seed production typical of this important forage crop.     

Taxonomy of Selaginella: a study of characters, techniques, and classification in the Hong Kong species

This study of 11 Hung Kong species of Selaginella indicates that sufficient characters exist by which they may be conveniently, consistently, and conclusively distinguished, and implies that the same is true for the rest of the genus, which suffers from chronic taxonomic confusion. Characters assessed are: leaf size, shape, epidermal cell patterns and mesophyll structure; ligule shape; patterns seen in stem transections; sporophyll shape; microsporangium shape; sporangial distribution; and spore surface ornamentation. Of these, the shape of median leaves, the maximum size of lateral leaves and epidermal cell patterns are the most significant and distinctive foliar features. These, together with spore surface ornamentation, are the most useful for distinguishing species. Correlations between the shapes of ligules, sporophylls, microsporangia, and vascular bundles indicate natural subgeneric groups.
These studies suggest that there are two ways by which taxonomic investigations of Selaginella can be improved. First leaves and spores should be prepared in permanent mountant (e.g. Hoyer's solution) for accurate observation of size, shape, and epidermal patterns. Secondly, data should be collected on a wide range of features.     

A Trichrome Stain for Insect Material

Deparaffinized insect sections are brought down to water and overstained in a 0.1% solution of azocarmine G in 1% acetic acid. They are then destained in a saturated solution of orange G until the azocarmine G is removed from the endocuticle and the latter is colored pale yellow. After washing, the sections are transferred to a 5.0 % solution of phosphotungstic acid in water for 3 min. They are then rinsed in distilled water and stained in a 0.1% solution of methyl green in 1% acetic acid until the endocuticle is green. Differentiation is done in 2 changes of 95% alcohol. The sections are then dehydrated either in absolute alcohol or dioxane, cleared in a mixture of “camsal”, eucalyptol, dioxane, and paraldehyde (1:2:2:1), and mounted in Mohr and Wehrle's medium, a mountant of the Euparal type.     

Pollen–vegetation relationships along steep climatic gradients in western Amazonia

Question: How accurately do Amazonian montane forest pollen spectra reflect the vegetation? Can compositional changes observed in the vegetation along environmental gradients be identified in the pollen spectra? How well do herbarium collection data and bioclimatic envelopes represent abundance changes along elevation gradients? Location: Amazonian montane forests, Peru. Methods: Moss polsters collected along five altitudinal transects spanning over 3000 m a.s.l. were used to characterize pollen spectra. Vegetation plot data from a network of 15 1‐ha permanent plots were used to correlate pollen spectra with present‐day vegetation. Probability density functions (PDFs) fitted to pollen and plot data allowed comparisons using Spearman correlation coefficients. Ordination analyses were used to summarize changes in pollen spectra. Correlations between pollen‐based PDFs and previously‐published herbarium collection PDFs were also evaluated. Results: Pollen spectra closely reflected changes in species composition along elevation gradients. A mid‐elevation shift in pollen spectra was identified using ordination analyses. Pollen spectra from the driest forest in our data set were statistically different from those of wet forests. Pollen abundance PDFs along the altitudinal gradient were significantly correlated (P<0.01) with PDFs fitted to plot abundance, basal area and herbarium collection data for ten out of 11 taxa analysed. Conclusions: Pollen spectra closely reflected the vegetation composition of Amazonian montane forests. The differentiation of pollen spectra from dry localities showed the potential of genus‐level pollen data to reflect precipitation gradients. Pollen spectra also reflected mid‐elevation compositional changes well along the lower elevation limit of ground cloud formation. Despite collection biases, herbarium‐based bioclimatic envelope PDFs also represented well forest compositional changes along elevation gradients.