Differential staining is a technique that helps to characterize the microorganisms depending on the difference in the physical and chemical nature of the microorganism. Gram stain the most commonly used differential stain. The shown results demonstrate that this method is applicable for pollen staining for a. N2 the ability to use chemical staining to discriminate aborted from non aborted pollen grains has wellknown practical applications in agriculture.
Although thousands of genes have been identified to be highly or specifically expressed in pollen grains, little is known about the functions and regulatory mechanisms of the genes in pollen formation. A simplified method for differential staining of aborted and non. Meiotic behavior was studied using the squashing technique. Pollination in pelargonium dolomiticum knuth geraniaceae. Images of representative seed populations are shown, with the top two rows in each image. Myb80 encodes a myb transcription factor that is essential for tapetal and pollen development. Della activity is required for successful pollen development. This tool splits rgb images of stained pollen grains into its primary channels. Dapi nuclear staining was performed as described previously tsutsui and higashiyama 2017. Staining is hastened by flaming the slide for loose thinwalled pollen or by immersing thickwalled pollen or anthers for 2448 hr at 50 c. The chromosomes are 1 3 flm in length, and comprise a larger submetacentric pair, four acrocentrics. Mutant analysis represents one of the most reliable approaches to identifying genes involved in plant development.
The shown results demonstrate that this method is applicable for pollen staining for a wide range of species from different genera. Glyphosate negatively affects pollen viability but not. However, the gene networks responsible for coordinate evolution of these complex. Differential staining, part i differential staining is a procedure that takes advantage of differences in the physical and chemical properties of different groups of bacteria. Production of triploid hydrangea macrophylla via unreduced gamete breeding. Arabidopsis galacturonosyltransferase gaut and gaut14.
The described method is based on differential staining of non viable and viable pollen. Negative staining electron microscope protocol for rash illness. Differential and cytological staining techniques theory. We compare the advantages and the disadvantages of each method and suggest some practical implications as revealed by the extensive data. Alexanders stain colors aborted pollen grains from most angiosperms. Among several mutated lines, five have been extensively studied at the genetic. Glyphosate negatively affects pollen viability but not pollination and seed set in glyphosateresistant corn volume 52 issue 5 walter e. Hierdie gedragspatroon bevorder kruisbestuiwing by p. Comparable, the alexander stain discriminates aborted pollen grains from. Pollen is a fine to coarse powdery substance comprising pollen grains which are male microgametophytes of seed plants, which produce male gametes sperm cells. The relationship between pollen staining and female fertility was weaker and non significant slope 0.
Differential staining of aborted and nonaborted pollen grains. The tapetal ahl family protein tek determines nexine. In flowering plants, pollen formation is a complex process and strictly controlled by genetic factors. Atnot1 is a novel regulator of gene expression during pollen. Pollen grains have a hard coat made of sporopollenin that protects the gametophytes during the process of their movement from the stamens to the pistil of flowering plants, or from the male cone to the female cone of coniferous plants. Ploidy level influences pollen tube growth and seed viability. An rnase step has been added to reduce cytoplasmic staining of propidium iodide. Differential staining is used to detect abnormalities in the proportion of different white blood cells in the blood. For all pollinations, fresh pollen was collected from flowers at anthesis.
Differential staining of aborted and nonaborted pollen. Aborted microspores acts as a master regulator of pollen wall formation in arabidopsisc w open jiexu,a zhiwending,a gemavizcaybarrena,b jianxinshi,a wanqiliang,a zhengyuan,a danielewerckreichhart,c lukas schreiber,d zoe a. A commonly used technique for assessing pollen vitality, alexanders stain, uses chloral hydrate, phenol and mercuric chloride, all of which are highly toxic. The transition from the syncytial to the cellularized state occurs at a defined time point during seed development. With this method, non aborted pollen grains are stained with a red color, whereas aborted pollen grains are stained blue. The author described in detail the procedure for staining free pollen or pollen from non dehiscent anthers. Alexander, differential staining of aborted and non aborted pollen. Pollen abortion was manually assessed for all genotypes using differential staining of aborted and non aborted pollen grains 53. Genetic male sterility in wheat triticum aestivum l. To study and gain expertise on differential and cytological staining techniques. In most angiosperms, the endosperm develops initially as a syncytium to facilitate rapid seed growth.
The latter is further divided into sexine and nexine. Male gametophyte defective 1, encoding the fad subunit of. A total of six anthers from three plants two anthers per plant was used for each experiment with three biological repeats. Reproductive studies on ipecac cephaelis ipecacuanha brot. In the typical stain, aborted pollen grains are green. A total of three plants and three flowers per plant of each cybrid. Cytokinesis at pmi displays specialized features, and it has been suggested that there might be distinct molecular pathways underpinning different modes of cytokinesis in plants.
The defects in pollen production in both col0 and ler rga gai mutants suggest that della activity in stamens does not merely inhibit development, but is also necessary for successful pollen production. A simplified method for differential staining of aborted and. Since flagella are too thin to be seen by compound light microscopy, staining methods employ the use of a mordant often tannic acid to make them thick enough to see using an oil immersion objective. A new imagebased tool for the high throughput phenotyping of pollen. Irregular chromosome segregation in meiosis i and ii, many sets of chromosomes in telophase ii, micronuclei, incorrect cytoplasm division, incomplete cytokinesis and anomalous postmeiotic products, mainly polyads, were. Potentially, della proteins might coordinate development between different anther tissues through inhibition at developmental checkpoints. To identify the stage of pollen development at which eif3 becomes essential it is desirable to examine viable pollen and distinguish mutant from wild type. Bacterial flagella are appendages used for motility. Although staining methods offer the possibility to distinguish aborted and nonaborted fresh pollen, they. It allows us to differentiate between different kinds of bacterial cells or different parts of a bacterial cell. Assessment of pollen viability for wheat plant science frontiers. Fluorescencetagged transgenic lines reveal genetic defects. J to m, enlargement of the areas enclosed by the dashed boxes in f to i. The ability to use chemical staining to discriminate aborted from non aborted pollen grains has wellknown practical applications in agriculture.
Feb 19, 2020 differential staining of aborted and non aborted pollen. In 1884, hans christian gram, a danish doctor, developed a differential staining technique that is still the cornerstone of bacterial identification and taxonomic division. It may be that size uniformity indicates euploid gametes and an irregular shape indicates an aneuploid number. N, relative viable pollen per anther versus wild type. The endosperm is a terminal seed tissue that is destined to support embryo development. Preparation of the alexander staining solution add to a light protected bottle. Information about the openaccess article a simplified method for differential staining of aborted and non aborted pollen grains in doaj.
Download scientific diagram differential staining of aborted and nonaborted pollen grains. Arabidopsis thaliana myb80 formerly myb103 is expressed in the tapetum and microspores between anther developmental stages 6 and 10. Manipulating the timing of endosperm cellularization through interploidy crosses. A new imagebased tool for the high throughput phenotyping of. Nov 01, 20 the defects in pollen production in both col0 and ler rga gai mutants suggest that della activity in stamens does not merely inhibit development, but is also necessary for successful pollen production. Pollen abortion was evaluated under a light microscope throughout the flowering season using alexanders stain alexander, 1969. A simplified method for differential staining of aborted and non aborted pollen grains author. Ionizing radiation from chernobyl and the fraction of viable. A simplified method for differential staining of aborted and non aborted pollen grains. A major improvement in differential pollen staining was reported by m. A single staining solution was made by compounding it in the following order dyes were from british drug houses.
The myb80 transcription factor is required for pollen. Pollen grains have a hard coat made of sporopollenin that protects the gametophytes during the process of their movement from the stamens to the pistil of flowering plants, or from the male cone to the female cone of coniferous. A simplified method for differential staining of aborted and nonaborted pollen grains. A new basic chromosome number in pelargonium geraniaceae. Pdf a simplifed method for differential staining of aborted. Using microarray analysis of anther mrna, we identified 404 genes differentially expressed in the myb80 mutant.
A seeds were removed from ears, arranged according to size, and counted. We recognize eight main hypotheses concerning the ecology and the evolution of pollen. Keywords dichogamy geraniaceae mellitophily pelargonium selfcompatibility references acocks, 1988 j. Activation of the nackpqr map kinase signaling pathway, which is. Background mutations in several subunits of eukaryotic translation initiation factor 3 eif3 cause male transmission defects in arabidopsis thaliana. Five gametophytic mutations affecting pollen development and. The differential staining method of alexander 1969 for aborted and non aborted pollen was used to obtain an estimate of pollen viability. Differential staining of aborted and non aborted pollen. The method is useful for pollen inside nondehiscent anthers if these are small and not too deeply coloured naturally. Pdf a simplified method for differential staining of aborted and non. The differential and cytological staining techniques discussed in this chapter help to differentiate between acid fast and non. A simplifed method for differential staining of aborted. To accomplish this we have developed a broadly applicable method to track mutant alleles.
Using multiple stains can better differentiate between different microorganisms or structurescellular components of a single organism. The percentage of pollen abortion was scored from a minimum of 400 grains pooled from at least two flowers per month for each species or clone. Lateral organ boundaries domain lbd10 interacts with. T1 a simplified method for differential staining of aborted and non aborted pollen grains. Differential staining is a staining process which uses more than one chemical stain. Jul 12, 2009 staining is hastened by flaming the slide for loose thinwalled pollen or by immersing thickwalled pollen or anthers for 2448 hr at 50 c. Pollen grains have evolved mechanisms to avoid negative impacts of adverse environmental conditions on viability, including the ability to sustain ionizing radiation and repair dna. We recognize eight main hypotheses concerning the ecology and the evolution of pollen longevity and. Reproductive studies were carried out on brazilian accessions of ipecac, cephaelis ipecacuanha.
Flowers were dipped into 100 l of dapi staining buffer 1 gml dapi, 0. The pollen wall, an essential structure for pollen function, consists of two layers, an inner intine and an outer exine. Staining and fluorescent microscopy methods for pollen. This multistep, sequential staining protocol separates bacteria into four groups based on cell morphology and cell wall structure. A simplified method for differential staining of aborted. Pollen viability is an important component of reproductive success, with inviable pollen causing failure of reproduction.
Aug 01, 2001 mutant analysis represents one of the most reliable approaches to identifying genes involved in plant development. The screening of the versailles collection of arabidopsis thaliana tdna insertion transformants has allowed us to isolate different mutations affecting male gametophytic functions and viability. A commonly used technique for assessing pollen vitality, alexanders stain, uses chloral hydrate, phenol and. Negative staining electron microscope protocol for rash illness introduction electron microscopic em visualization of negatively stained poxvirus virions was a valuable technique for confirming poxvirus infections during the smallpox eradication campaign. High expression in maize pollen correlates with genetic.
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