A Rapid Method for Mounting Pollen Grains: With Special Regard To Sterility Studies

A method is described for the simultaneous mounting and double staining of mature pollen grains. The medium consists of 50 ml. of glycerol jelly to which 2.5 ml. of methyl green and 2 ml. of phloxine, both in 50% alcoholic solutions, have been added. Prior to the application of the jelly-dye mixture, the pollen is washed with 70% ethanol to remove adhering oils and resins. The staining reaction is differential and permits the rapid classification of pollen grains. The “functional” pollen expands and stains with both dyes whereas the aborted grains remain shrunken and take only the methyl green wall stain. The same reaction functions with orchid seed.     

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.     

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.     

用花粉粒染色法标记虫媒植物的花粉流

花粉作为植物有性生殖过程中的雄性供体,其释放后的去向和散布范围直接影响植物的交配模式和居群遗传结构,但花粉的运动肉眼不易观察,因此标记花粉运动的方法一直是传粉生物学中的关键技术［１,２］。尽管已有多种分子标记的技术被用来研究花粉的运动,包括同工酶［３...     

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.     

To Repeat the Effects of Hormones on the Early Microspore Developments of Wheat in Vitro in Anther Culture

1. The normal development of pollen cells can be transformed by the exoision itself of anther culture: The second mitotic division of pollen grains has been prevented; The frequency of anomalous division of pollen grains was higher than that present in anthers in vive; The generative nuclei after the first mitosis were more or less globular in form and in their subsequent developments most of them do not become spindly-shape which is particular to the generative cells in vive. In the meantime, they show a weak staining reaction with Feulgen reagent. 2. The higher concentrations of hormones were found to enhance the frequency of abnormal division obviously. Of anthers cultured on the four N6 media added with various concentration ratios of IAA to Kinetin 2:10, 10:2, 2:12, and 12:2 mg/l. The mean percentages of abnormal pollen grains were 34.02%, 35.28%, 34.27% and 36.65% respectively. 3. The higher hormone level may promote the formation of multicellular pollen grains obviously. When the IAA concentration was raised up to 12 mg/l, the mean multieellular pollen grain yields per anther increased to 13.3 unit, while the control without hormone was only 4 unit.     

Pyronin Y in the Methyl-Green-Pyronin Histological Stain

Two samples of pyronin Y were found which, with the exception of eosinophilic granules and osteoid, stained only nucleic acids in animal tissues. Good differentiation was obtained. with n-butyl alcohol. It was therefore possible to prepare a differentially staining mixture of either of these pyronins combined with methyl green. This mixture stains polymerized desoxyribose nucleic acid (DNA) clear green, depolymerized DNA and ribonucleic acid red. The red staining of eosinophilic granules and osteoid is readily distinguished by its persistence after ribonuclease or warm-buffer extraction. The staining mixture consists of: (1) pyronin Y (Edward Gurr or G. T. Gurr), CHCl₃ extracted, 2% aq, 12.5 ml; (2) methyl green, CHCl₃ extracted, 2% aq, 7.5 ml; (3) distilled water, 30 ml. The staining procedure is as follows. (1) Immerse slides 6 min in the dye mixture. (2) Blot with filter paper. (3) Immerse in 2 changes of n-butyl alcohol, 5 min each. (4) Xylene, 5 min. (5) Cedar oil, 5 min. (6) Apply Permount and cover.     

太子参花药发育及精细胞分离

太子参花药壁发育为基本型,腺质绒毡层。小孢子母细胞减数分裂为同时型,小孢子四分体为四面体型,成熟花粉具两个精细胞,为3胞花粉。在花粉表面具散孔,孔数22—30个,均匀分布于花粉粒表面上。花粉在10％甘露醇或15％蔗糖溶液中可直接爆破,精细胞易被释放并散开,通过显微操作仪可收集到一定数目的精细胞。FDA染色荧光显示释放出来的精细胞活力可维持25—50min。花粉在舍O．03％CaCl2、0．01％H3803、0．01％KH2P04和20％PEG、pH5．8的培养液中2—5min即萌发花粉管．花粉管生长2h可达815μm。一般花粉管伸长500—600μm时,一对精细胞才进入花粉管。DAPI染色后荧光观察．可观察到精细胞和营养细胞核在花粉管中的移动状况。爆破花粉管后可释放出一对精细胞。     

A simple method for staining nuclei of mature and germinated maize pollen

A sugar acetocarmine staining technique has been developed for staining the sperm and vegetative nucleus of mature and germinated maize pollen grains. This procedure is simple, stable and highly repeatable. The physiological properties of the mature maize pollen grains are first adjusted by using an in vitro germinating culture solution. This solution is 15% sucrose and contains 360 ppm calcium chloride dihydrate, and 120 ppm boric acid. One part fresh pollen grains is uniformly mixed with nine parts of the solution and left at room temperature for at least 5 hr. One part of this solution is then mixed with two parts of regular acetocarmine stain and left overnight. The color of this mixture is pinkish red or raspberry. The sugar in the mixture helps to increase color contrast between the pollen cytoplasm (light pink) and the nuclei (reddish purple), decreases the frequency of burst pollen, increases pollen expansion, stabilizes pollen figures and automatically seals the coverglass.     

A Simple Method for Staining Nuclei of Mature and Germinated Maize Pollen

A sugar acetocannine staining technique has been developed for staining the sperm and vegetative nucleus of mature and germinated maize pollen grains. This procedure is simple, stable and highly repeatable. The physiological properties of the mature maize pollen grains are first adjusted by using an in vitro germinating culture solution. This solution is 15% sucrose and contains 360 ppm calcium chloride dihydrate, and 120 ppm boric acid. One part fresh pollen grains is uniformly mixed with nine parts of the solution and left at room temperature for at least 5 hr. One part of this solution is then mixed with two parts of regular acetocannine stain and left overnight. The color of this mixture is pinkish red or raspberry. The sugar in the mixture helps to increase color contrast between the pollen cytoplasm (light pink) and the nuclei (reddish purple), decreases the frequency of burst pollen, increases pollen expansion, stabilizes pollen figures and automatically seals the coverglass.     

Pyronin Y in the Methyl-Green-Pyronin Histological Stain

Two samples of pyronin Y were found which, with the exception of eosinophilic granules and osteoid, stained only nucleic acids in animal tissues. Good differentiation was obtained. with n-butyl alcohol. It was therefore possible to prepare a differentially staining mixture of either of these pyronins combined with methyl green. This mixture stains polymerized desoxyribose nucleic acid (DNA) clear green, depolymerized DNA and ribonucleic acid red. The red staining of eosinophilic granules and osteoid is readily distinguished by its persistence after ribonuclease or warm-buffer extraction. The staining mixture consists of: (1) pyronin Y (Edward Gurr or G. T. Gurr), CHCl₃ extracted, 2% aq, 12.5 ml; (2) methyl green, CHCl₃ extracted, 2% aq, 7.5 ml; (3) distilled water, 30 ml. The staining procedure is as follows. (1) Immerse slides 6 min in the dye mixture. (2) Blot with filter paper. (3) Immerse in 2 changes of n-butyl alcohol, 5 min each. (4) Xylene, 5 min. (5) Cedar oil, 5 min. (6) Apply Permount and cover.     

Differential Staining of Mucin Granules from Epoxy Resin Sections by a Phosphotungstic Acid-Methyl Green Procedure

After treatment of epoxy resin semithin sections from glutaraldehyde fixed rat large intestine with 5% aqueous phosphotungstic acid (PTA), staining with unpurified 0.2% solutions of methyl green at 60 C for 5 min produces a color differentiation between mucin granules of goblet cells. Some mucin granules and the glycocalyx appear deep green while the remaining granules, luminal mucin and collagen fibers are pink. The known contamination of unpurified methyl green with crystal violet seems to be responsible for the pink staining reaction of the latter structures, which also present an orange-red fluorescence under green exciting light. Electron microscopic observations show selective contrast of mucin granules which appear with a different amount of PTA deposits. This procedure is useful to reveal the heterogeneity of mucin granules in light and electron microscopy.     

Production of total and stainable pollen grains in Olea europaea L

The production of the total and stainable number of pollen grains per flower and per inflorescence of 13 olive cultivars (Olea europaea L.) has been investigated. Grains stainability was evaluated by using the acetic carmine staining method whereas the production of pollen grains was calculated by using a Bürker haemocytometer. All pollen characteristics taken into account varied significantly among the cultivars. The number of flowers per inflorescence ranged from a maximum of 31.3 (‘Cipressino’) to a minimum of 13.0 (‘Leccino’), the number of pollen grains per anther ranged from 123?346 (‘Arbequina’) to 40?975 (‘Sant'Agostino’), and the pollen stainability from 97.6% (‘Cipressino’) to 48.0% (‘Cellina di Nardò’). The number of stainable pollen grains per inflorescence averaged 2?559?512, ranging from a maximum of 3?913?678 (‘Nociara’) to a minimum of 940?143 (‘Sant'Agostino’). All parameters were positively correlated, whereas a linear negative correlation was found between stainability and total pollen grains both per anther and per inflorescence.     

An efficient and rapid transgenic pollen screening and detection method using flow cytometry

Assaying for transgenic pollen, a major vector of transgene flow, provides valuable information and essential data for the study of gene flow and assessing the effectiveness of transgene containment. Most studies have employed microscopic screening methods or progeny analyses to estimate the frequency of transgenic pollen. However, these methods are time-consuming and laborious when large numbers of pollen grains must be analyzed to look for rare transgenic pollen grains. Thus, there is an urgent need for the development of a simple, rapid, and high throughput analysis method for transgenic pollen analysis. In this study, our objective was to determine the accuracy of using flow cytometry technology for transgenic pollen quantification in practical application where transgenic pollen is not frequent. A suspension of non-transgenic tobacco pollen was spiked with a known amount of verified transgenic tobacco pollen synthesizing low or high amounts of green fluorescent protein (GFP). The flow cytometric method detected approximately 75% and 100% of pollen grains synthesizing low and high amounts of GFP, respectively. The method is rapid, as it is able to count 5000 pollen grains per minute-long run. Our data indicate that this flow cytometric method is useful to study gene flow and assessment of transgene containment.     

A new rapid method for selective extraction of RNA from fixed mammalian tissues.

Treatment of formalin-fixed mammalian tissues with concentrated or 50% phosphoric acid at 5 degrees C for 20 and 50 min. respectively reveals complete extraction of RNA as judged by methyl green followed by staining with pyronin. This procedure also causes depolymerisation of DNA as indicated by the red staining of the nuclei. Sections treated with concentrated phosphoric acid at 5 degrees C for 30 min. causes disruption of the double helical structure of DNA what results in the depression of the pyronin staining. Similarly treated sections show Feulgen positive nuclei. Treatment of sections in 25 % phosphoric acid at 60 degrees C for 15 min. followed by staining with methyl green and pyronin show red nuclei, nucleoli and the cytoplasm. This indicates that extraction of RNA is only possible in cold and not at elevated temperature.     

The association between pollen size and Renner complex in Oenothera villaricae and O. picensis ssp. picensis and their hybrids: evidence for preanthesis pollen competition

In both Oenothera villaricae Dietrich and O. picensis ssp. picensis, chromosomes are transmitted as two Renner complexes. Reciprocal combinations of the Renner complexes produce eight different F₁ hybrids, but only seven are viable. Each species, and each F₁ hybrid, produces three sizes of pollen, approximately 50% small sterile grains, 15% medium-sized viable grains and 35% large viable grains. Medium- and large-sized grains were separated manually and subjected to random amplified polymorphic DNAs (RAPDs) analysis. A pattern of RAPD amplifications was obtained which indicates that, for each species and F₁ hybrid, one specific Renner complex characterizes the medium- and another the large-sized viable pollen. The results indicate that pollen size is determined in part by the pollen genotype and in part by the genotype of the other pollen grains developing within the same anther.     

A New Stain for Pollen Fertility Studies

The juice from the berries of Cocculus hirsutum was extracted and used for pollen fertility studies in various crops. Two stains were prepared: P. H. Ramanjini (PHR) stain and modified PHR stain. The modified PHR stain contains lactic acid and produces the best staining differentiation. The intensity of the staining was dependent on the thickness of the pollen cell walls, hence PHR stain is recommended for thick walled pollen grains and the modified PHR stain for pollen with relatively thin walls. The preparation of both the stains are very simple, quick and inexpensive.     

The Germination of Vinca rosea L. Pollen Grains and the Growth of the Pollen Tubes in vitro

The effect of medium concentration, pollen grain concentration, pH of the media, light and temperature on the germination of Vin ca rosea pollen grains, and the growth of their pollen tubes in vitro have been studied. The pollen grains germinate best at a sucrose concentration between 14.2% and 30%; when the pollen grain concentration exceeds 800 per 0.0234 ml; at near neutral pH (6.5); in darkness and at a temperature close to 30°. Moreover buffering ions affect the growth of the pollen tubes. Pollen grains remain viable in a wide range of temperatures, and the wall of the pollen grain is capable of withstanding severe osmotic imbalance. Low temperature induces spherical swellings at the tips of the pollen tubes, followed by accumulation of a hyaline plug.