Stages Of Microsporogenesis Research Articles

Exploring the role of 28-homobrassinolide in regulation of temperature induced clastogenic aberrations and sugar metabolism of Brassica juncea L.

The present research primarily focuses on Brassica juncea's physiological and cytological responses to low and high temperature stress at 4 °C and 44 °C respectively, along with elucidating the protective role of 28-Homobrassinolide (28-homoBL). Cytological investigations performed in floral buds of Brassica juncea L. under temperature (24, 4, 44 °C) stress conditions depict the presence of some abnormalities associated with cytomixis such as chromosome stickiness or agglutination, pycnotic nature of chromatin, irregularities in spindle formation, disoriented chromatins, and non-synchronous chromatin material condensation in Brassicaceae family that subsisted at diploid level (2n = 36). Spindle abnormalities produce various size pollen grains such as sporads micronuclei at some stages of microsporogenesis, polyads, triads, dyads that irrupted the productiveness of pollen grains. Furthermore, sugars play an imperative role in protecting plants under stress besides being energy sources. Therefore, the present study revealed accumulation of total soluble sugars (TSS), with 28-homoBL treatment which pinpoints protective role of 28-homoBL under temperature stress. Sugar profiling was done by using high-performance liquid chromatography (HPLC) which helped in analyzing different sugars both quantitatively and qualitatively under 28-homoBL and temperature stress conditions. The results indicate that the 28-homoBL treatment substantially enhances plant tolerance to heat stress, as evident by higher mitotic indices, fewer chromosomal abnormalities, and significantly more sugar accumulation. The findings of the study acknowledge the potential of 28-homoBL in inducing temperature stress tolerance in B. juncea along with improving the metabolic stability thereby implying application of 28-homoBL in crop strengthening under variable temperature conditions.

Correlation of stages of microsporogenesis with bud and anther morphology in pepper genotypes through DAPI staining with different levels of mordant in cytological fixative.

The haploid and doubled haploid plants serve as valuable tools for breeders due to their ability to expedite the mapping of genes of agronomic importance, as well as accelerate the breeding cycle for generation of novel hybrids and improved homogenous varieties. Successful anther/microspore culture largely depends on the use of microspores at appropriate developmental stages at the time of culture, which can be specific for each plant species and genotype. In the present study, we described the visible morphological characteristics of flower buds and anthers at different developmental stages to identify the optimal microspore stage within the anther/buds of two pepper hybrids, Indra and Lakshmi. This information enabled us to predict the suitable microspore stage for successful haploid production. To enhance the visualization of nuclei in the pepper microspores, different concentrations of FeCl3 were employed as a mordant to Carnoy's fixative I, followed by DAPI staining. A clear and distinct nucleus was observed using DAPI staining procedures in the pepper microspores when fixed in Carnoy's solution containing ferric chloride (40-90µl) as mordant. The use of mordant thus facilitated the efficient cytological analysis of the pepper microspores. Present results indicate that, to achieve efficient haploid production, flower buds with an average length of 4.4 to 5.02mm for the hybrid Indra and 5.15 to 5.40mm for the hybrid Lakshmi should be utilized. Additionally, these buds should have a calyx covering approximately 80-90% of the total bud length. We observed that in such buds, microspores are in the late-uninucleate and early binucleate stage which has been reported to be the most conducive stage for androgenesis induction in pepper.

Meiotic segregation and post-meiotic drive of the Festuca pratensis B chromosome

In many species, the transmission of B chromosomes (Bs) does not follow the Mendelian laws of equal segregation and independent assortment. This deviation results in transmission rates of Bs higher than 0.5, a process known as “chromosome drive”. Here, we studied the behavior of the 103 Mbp-large B chromosome of Festuca pratensis during all meiotic and mitotic stages of microsporogenesis. Mostly, the B chromosome of F. pratensis segregates during meiosis like standard A chromosomes (As). In some cases, the B passes through meiosis in a non-Mendelian segregation leading to their accumulation already in meiosis. However, a true drive of the B happens during the first pollen mitosis, by which the B preferentially migrates to the generative nucleus. During second pollen mitosis, B divides equally between the two sperms. Despite some differences in the frequency of drive between individuals with different numbers of Bs, at least 82% of drive was observed. Flow cytometry-based quantification of B-containing sperm nuclei agrees with the FISH data.

Tomato POLLEN DEFICIENT 2 encodes a G-type lectin receptor kinase required for viable pollen grain formation.

Pollen development is a crucial biological process indispensable for seed set in flowering plants and for successful crop breeding. However, little is known about the molecular mechanisms regulating pollen development in crop species. This study reports a novel male-sterile tomato mutant, pollen deficient 2 (pod2), characterized by the production of non-viable pollen grains and resulting in the development of small parthenocarpic fruits. A combined strategy of mapping-by-sequencing and RNA interference-mediated gene silencing was used to prove that the pod2 phenotype is caused by the loss of Solanum lycopersicum G-type lectin receptor kinase II.9 (SlG-LecRK-II.9) activity. In situ hybridization of floral buds showed that POD2/SlG-LecRK-II.9 is specifically expressed in tapetal cells and microspores at the late tetrad stage. Accordingly, abnormalities in meiosis and tapetum programmed cell death in pod2 occurred during microsporogenesis, resulting in the formation of four dysfunctional microspores leading to an aberrant microgametogenesis process. RNA-seq analyses supported the existence of alterations at the final stage of microsporogenesis, since we found tomato deregulated genes whose counterparts in Arabidopsis are essential for the normal progression of male meiosis and cytokinesis. Collectively, our results revealed the essential role of POD2/SlG-LecRK-II.9 in regulating tomato pollen development.

Fossilized pollen malformations as indicators of past environmental stress and meiotic disruption: insights from modern conifers

AbstractPollen malformations have been proposed as a paleoenvironmental stress proxy. However, the frequency and variability of pollen malformations under near-optimal conditions and environmental stress, as well as their developmental origins, remain unclear. To bridge these gaps, we compared pollen malformation frequencies and assemblages of 14 extant conifer genera of Pinaceae and Podocarpaceae producing saccate (winged) grains grown under near-optimal conditions. These baseline pollen yields were compared with those produced by Pinus mugo ‘Columnaris’ cultured under an abiotic stress—three experimentally heightened ultraviolet-B radiation (UV-B) regimes proposed for the end-Permian crisis. We additionally reviewed previous cytological literature of abnormal microsporogenesis in conifers. Under near-optimal conditions, malformations comprise <3% of pollen yields in 12 out of 13 bisaccate genera and >10% of yields in the naturally trisaccate Dacrycarpus dacrydioides. We detected no phylogenetic pattern in malformation assemblages of the baseline comparisons. UV-B–irradiated P. mugo produced significantly higher malformation frequencies and different assemblage compositions when compared with baseline bisaccate lineages. We propose that pollen malformations originate during the meiotic and tetrad stages of microsporogenesis and present a framework for the ontogeny of different malformation types seen in the fossil record. Malformations comprising >3% of bisaccate pollen yields can be used as a paleoenvironmental stress proxy, but rare, naturally trisaccate lineages are not suitable for such assessments. Furthermore, heightened UV-B not only increases pollen malformation production, but also alters the types of abnormalities trees produce. Different environmental stresses may therefore leave behind distinct fingerprints in the fossil record.

Evaluation of mitotic activity in tapetal cells of grapevine (Vitis L.)

The knowledge with reference to the grapevine tapetum has been centered on its anatomy/morphology and hardly anything at all is known about its mitotic activity throughout the microsporogenesis. The aim of this study was to ascertain the mitotic activity in tapetal cells of some grapevines (Vitis L.) broadening knowledge about this tissue and simultaneously corroborating the viability of its use as an alternative tissue for further cytogenetic studies. Young buds of 12 grapevine varieties at different meiotic stages were squashed and tapetal cells a prometaphase/metaphase scored in each meiotic stage. Mitotic activity was observed since the beginning of microsporogenesis, where it was more intense, decreasing toward tetrad. Polyploid tapetal cells arose through endomitosis while the microsporogenesis advanced. Two types of polyploid cells were evidenced, those with two or more individualized diploid chromosome groups and those with only one polyploid group. The percentage of diploid cells and of polyploid cells with two or more individualized diploid groups was higher during the first stage of microsporogenesis, though decreasing and giving way to cells with one large polyploid group as microsporogenesis moved toward tetrad. The nucleolus number was scored at interphase at different stages. Two and four nucleoli prevailed in tapetal cells at all stages except at tetrad where one large nucleolus was seen. The results showed that despite of the squashing technique applied, grapevine tapetum has a substantial amount of cells with mitotic activity with a satisfactory chromosome spreading therefore establishing an interesting alternative and promising tissue for later cytomolecular studies.

Morphological characterization of floral traits to predict ideal stage for haploid production in bell pepper

Double haploid (DH) breeding not only helps in shortening the breeding cycle, also in fastening the release of homozygous inbred lines with superior and desirable traits. In vitro microspore culture is the fastest approach for the production of haploid plantlets. The key parameter for success of the haploid production in any crop species is the stage of microspore or pollens at the time of culture as it decides the fate of cultured anthers/pollens. Therefore an easy, precise, fast, and reliable criterion to identify flower buds carrying microspores or pollen at particular stages are essential. In the present investigation, we developed easiest and accurate criterion to correlate visible, measurable traits of bud and anther development with individual stages of microsporogenesis in two sweet pepper F1 hybrids (Orobelle and Bomby). Anthers containing microspores at late uninucleate to early binucleate stage are optimal for induction of androgenesis in wide range of crop species. Anther containing uninucleate pollens were observed in flower buds with an average size of 5.39 mm in Orobelle and 4.8mm in Bomby, average anther length of 2.75 mm in Orobelle and 2.71mm in Bomby and corolla was slightly longer than that of the calyx in both the hybrids. Anthers were without any purple pigmentation in Orobelle and slight/no pigmentation at the top of the anther sac end in Bomby.

OsAPT1 a Pollen Preferentially Expressed Gene Is Essential for Pollen Tube Germination and Elongation in Rice

Pollen tube germination and elongation are important processes for double fertilization in flowering plants. However, little is known about the regulatory mechanism of pollen germination and tube growth in rice. Here, we reported that OsAPT1, a pollen-high expression gene and encoding a putative APT1 protein in Oryza sativa, regulating pollen tube germination and elongation in rice. Real-time quantitative PCR (qRT-PCR) and in situ hybridization analysis showed high expression level of OsAPT1 at late stage of microsporogenesis including late uninucleate stage, binucleate pollen stage, and mature pollen grains. OsAPT1 RNA interference (OsAPT1-RNAi) lines showed that the pollen tube germination and elongation rate were decreased. Compared with wild type (WT), the integrity and assembly of actin microfilaments were abnormal in hydrated pollen grains of OsAPT1-RNAi plants, and the fluorescence signals of actin microfilaments were altered in the pollen tube of OsAPT1-RNAi plants. GO analysis of the co-expressing genes of OsAPT1 showed that the GO terms such as pollination, cell growth, cellular cytoskeleton organization, and motor activity were enriched. In addition, the qRT-PCR results showed variations in the expression of some genes related to pollen tube growth. Overall, our results indicated that OsAPT1 is required for pollen tube germination and elongation in rice.

СКРЕЩИВАЕМОСТЬ СИНТЕТИЧЕСКИХ АМФИДИПЛОИДОВ ХЛОПЧАТНИКА (GOSSYPIUM L.) И ИХ ЦИТОЛОГИЧЕСКИЙ АНАЛИЗ

This paper discusses the results of the analysis of the different crossability of 4 species of synthetic cotton amphidiploids, with cultivated varieties of G. hirsutum L. and G. barbadense L., taking into account their cytological features and the creation of genetically enriched new polygenomic interspecific hybrids. The interrelation of cross-breeding in varying degrees with the fluctuation of chromosome numbers in somatic plant cells of parental forms and cotton hybrids, as well as the defects observed at the stages of microsporogenesis of maternal cells, is shown. The main reasons for the contrast difference between the degree of cross-breeding of synthetic cotton amphidiploids, with cultivated cotton varieties when producing an interspecific hybrid, as well as differences in the maturation of full seeds are established.

Suhu Tinggi Berpengaruh Negatif pada Perkembangan Polen Cabai (Capsicum annum L.) cv. Tanjung-2

The development of male reproductive organ in plants is seriously affected by the adverse abiotic stresses. In this study, we investigated pollen development, particularly at microsporogenesis and micro gametogenesis stages of chili pepper (Capsicum annuum) cv. Tanjung-2 upon exposure with high temperature (day/night = 36/33°C). For this objective, anther of different flower sizes ranging from <2.5 mm; 3-4.5 mm; 4.5-7 mm; to 7-11 mm from treated and non-treated plants were collected. The results revealed that the early microsporogenesis stage was highly sensitive to high temperature indicated by a low progression of cells into subsequent process in meiotic division. This result was followed by the inhibition of tetrad formation up to 2%. Consequently, plant produced smaller microscpores and pollens with thin exin that resulted in the decreased pollen viability to 90%. In conclusion, high temperature has negative impact on the development of male reproductive program in chili pepper, including microsporogenesis and micro gametogenesis. Extending approach should be allocated to overcome this problem so that such environmental stress would not decrease the productivity of chili pepper. Keywords: Capsicum annuum, high temperature, microgametogenesis, microsporogenesis

LSSR1 facilitates seed setting rate by promoting fertilization in rice

Seed setting rate is one of the major components that determine rice (Oryza sativa L.) yield. Successful fertilization is necessary for normal seed setting. However, little is known about the molecular mechanisms governing this process. In this study, we report a novel rice gene, LOW SEED SETTING RATE1 (LSSR1), which regulates the seed setting rate by facilitating rice fertilization. LSSR1 encodes a putative GH5 cellulase, which is highly conserved in plants. LSSR1 is predominantly expressed in anthers during the microsporogenesis stage, and its encoded protein contains a signal peptide at the N-terminal, which may be a secretory protein that stores in pollen grains and functions during rice fertilization. To explore the physiological function of LSSR1 in rice, loss-of-function mutants of LSSR1 were created through the CRISPR-Cas9 system, which showed a significant decrease in rice seed setting rate. However, the morphology of the vegetative and reproductive organs appears normal in lssr1 mutant lines. In addition, lssr1 pollen grains could be normally stained by I2-KI solution. Cytological results demonstrate that the blockage of fertilization mostly accounted for the low seed setting rate in lssr1 mutant lines, which was most likely caused by abnormal pollen grain germination, failed pollen tube penetration, and retarded pollen tube elongation. Together, our results suggest that LSSR1 plays an important role in rice fertilization, which in turn is vital for maintaining rice seed setting rate.

Meiosis analysis in soft winter wheat varieties — carriers of wheat-rye translocations 1BL.1RS and 1AL.1RS

Aim. Investigation of the features of the meiosis flow in winter wheat varieties — carriers wheat-rye translocations 1BL.1RS and 1AL.1RS. Methods. A comparative analysis of the meiosis flow in winter wheat varieties, the carriers of various translocations, was carried out using the method of temporary pressure preparations. Results. The frequency and spectrum of meiosis anomalies in introgressive winter wheat varieties with wheat-rye translocations 1BL.1RS and 1AL.1RS are established. The variability of the frequency of violations at different stages of microsporogenesis in the carrier classes of a certain translocation is shown, which may be due to a different period of stabilization of their genomes. It was found that the frequency of violations in varieties with different translocations did not exceed 10 %, which is normal for stable introgressive varieties. Conclusions. It was found that in all stages of microsporogenesis in the number of cells with violations and their spectrum, varieties with translocation of 1BL.1RS are slightly different from those with translocation of 1AL.1RS. Keywords: bread winter wheat, translocations 1BL.1RS and 1AL.1RS, meiotic flow.

Cytological Features of Developing Anthers in Rose Balsam

Our goal was to determine the ultrastructure features and the dynamic changes in polysaccharides and neutral lipids in developing anthers of rose balsam (Impatiens balsamina), and to better understand the mechanisms controlling male reproductive development. Transmission electron microscope (TEM) techniques were used to study the ultrastructural characteristics of the anthers, and histochemical methods were used to determine levels of polysaccharides and lipids. The cytokinesis in the microsporocyte meiosis was simultaneous type, and microspore tetrads were mainly tetrahedral. The pollen exine began to form at the tetrad stage. The mature pollen grains were oval-shaped and bicellular. Accumulation of reserve substances began at the late microsporogenesis stage, and an abundance of starch grains and lipids were stored in pollen grains at anthesis. Polysaccharides and lipids changed in different stages and played an important role in anther development. Moreover, the calcium oxalate crystals may protect the pollen and suggest that calcium distribution is related to anther development.

Resolvase OsGEN1 Mediates DNA Repair by Homologous Recombination.

Yen1/GEN1 are canonical Holliday junction resolvases that belong to the RAD2/XPG family. In eukaryotes, such as budding yeast, mice, worms, and humans, Yen1/GEN1 work together with Mus81-Mms4/MUS81-EME1 and Slx1-Slx4/SLX1-SLX4 in DNA repair by homologous recombination to maintain genome stability. In plants, the biological function of Yen1/GEN1 remains largely unclear. In this study, we characterized the loss of function mutants of OsGEN1 and OsSEND1, a pair of paralogs of Yen1/GEN1 in rice (Oryza sativa). We first investigated the role of OsGEN1 during meiosis and found a reduction in chiasma frequency by ∼6% in osgen1 mutants, compared to the wild type, suggesting a possible involvement of OsGEN1 in the formation of crossovers. Postmeiosis, OsGEN1 foci were detected in wild-type microspore nuclei, but not in the osgen1 mutant concomitant with an increase in double-strand breaks. Persistent double-strand breaks led to programmed cell death of the male gametes and complete male sterility. In contrast, depletion of OsSEND1 had no effects on plant development and did not enhance osgen1 defects. Our results indicate that OsGEN1 is essential for homologous recombinational DNA repair at two stages of microsporogenesis in rice.

Cytological and biophysical comparative analysis of cell structures at the microsporogenesis stage in sterile and fertile Allium species

Main conclusionUsing a live-cell-imaging approach and autofluorescence-spectral imaging, we showed quantitative/qualitative fluctuations of chemical compounds within the meiocyte callose wall, providing insight into the molecular basis of male sterility in plants from the genusAllium.Allium sativum (garlic) is one of the plant species exhibiting male sterility, and the molecular background of this phenomenon has never been thoroughly described. This study presents comparative analyses of meiotically dividing cells, which revealed inhibition at the different microsporogenesis stages in male-sterile A. sativum plants (cultivars Harnas and Arkus) and sterile A. ampeloprasum var. ampeloprasum (GHG-L), which is phylogenetically related to garlic. Fertile species A. ampeloprasum (leek) was used as the control material, because leek is closely related to both garlic and GHG-L. To shed more light on the molecular basis of these disturbances, autofluorescence-spectral imaging of live cells was used for the assessment of the biophysical/biochemical differences in the callose wall, pollen grain sporoderm, and the tapetum in the sterile species, in comparison with the fertile leek. The use of techniques for live-cell imaging (autofluorescence-spectral imaging) allowed the observation of quantitative/qualitative fluctuations of autofluorescent chemical compounds within the meiocyte callose wall. The biophysical characterisation of the metabolic disturbances in the callose wall provides insight into the molecular basis of male sterility in A. sativum. In addition, using this method, it was possible for the first time, to determine precisely (on the basis of fluctuations of autofluorescence compounds) the meiosis stage in which normal microsporogenesis is disturbed, which was not visible using light microscopy.

Comparative microsporogenesis and flower development in Eucalyptus urophylla × E. grandis

Microsporogenesis and flower development in Eucalyptus urophylla × E. grandis were examined using chromosome tableting to provide a method to predict the meiotic stages in this species. Although microsporogenesis was normal, cytokinesis during meiosis of pollen mother cells occurred simultaneously, with strong asynchronism observed in the two different lengths of stamens in a flower bud. In a single flower, the developmental period of microsporogenesis in anthers on the longer stamens was always ahead of those on the shorter stamens. Flower development was also asynchronous at different locations on a branch. Flower buds on the upper side of the branch were larger in diameter than those on the lower side. In addition, a correlation was observed between microsporogenesis development and flower bud diameter growth. The pachytene stage was first observed when the diameter of the flower buds increased to 3.0 mm, and the majority of the meiotic stages were observed when bud diameters ranged from 3.5 to 5.0 mm. This study showed that the developmental stages of microsporogenesis in Eucalyptus urophylla × E. grandis could be distinguished readily, which may be applicable to future breeding studies.

A cytological study of anther and pollen development in Camellia oleifera.

The plant species Camellia oleifera is an important producer of edible oil in China. However, it suffers from a low fruit-setting rate. This study used high resolution scanning electron microscopy of semi-thin tissue sections to investigate anther development and pollen formation, and to determine whether problems in forming functional pollen cause low seed setting rates. During anther development, cell sizes within the epidermis and end othecium gradually increase, and at the beginning of the microsporocyte phase, the cells become highly vacuolated. The cell walls of the inner wall cells thicken in a ribbon pattern during the later part of the two-cell pollen stage. Middle layer cells become flattened during the later part of the secondary sporogenous cell stage. A tapetum also forms at this stage; the secondary sporogenous cells begin to degrade at the early microspore stage and completely disappear by the time the pollen is mature, thus forming a glandular tapetum. In microsporocyte, cytokinesis occurs simultaneously with microsporocyte meiosis, and a tetrahedral arrangement is present in tetrads. Reproductive cells begin to form at the later stage of microsporogenesis; the nuclei of the reproductive cells are enlarged as the reproductive cells begin to move away from the pollen wall. Two-cell pollen is first formed after reproductive cells have left the pollen wall, and the number of inclusions in the pollen subsequently increases. The pollen matures for 5-8 days before flowering occurs. No pollen abortion phenomena were observed, suggesting that anther development in C. oleifera is normal and that functional pollen are formed.

Variations of total protein content and protein synthesis during microsporogenesis in Larix europaea L.

The variations of the total protein content and protein synthesis were analyzed in successive stages of microsporogenesis in &lt;em&gt;Larix europaea&lt;/em&gt; L. Pure fractions of microsporocytes containing the cells in successive phases of meiosis and fractions of the anther wall cells were used to perform the assays. It was revealed, that the protein content and the intensity of protein synthesis undergo considerable changes in the course of meiosis. It was proved also, by means of electrophoretic separation of protein fractions, that no new proteins are synthesised during differentiation of microsporocytes of larch, and that the microsporogenesis process is related rather to disappearance of some protein fractions.

Polysaccharides and lipids in microsporocytes and tapetum of Rhoeo discolor Hance. Cytochemical study

The present report, which mainly presents cytochemical results, establishes the determination, localisation and evolution of the cytoplasmic and wall polysaccharides and lipids in the microsporocytes and the plasmodial tape turn of &lt;em&gt;Rhoeo discolor&lt;/em&gt; Hance. Enzymatic controls, use of autoradiographic methods, and electron microscopy, have proved the validity of our cytochemical results and permitted to precise these results. In the microsporocytes and pollen grains, the callosic special wall, the carotenoid exine, the pectocellulosic cellulasic in tine, and the callosic and pectic young curved wail. between the generative cell and the vegetative cell, have mainly held our attention. As for the tapetum which remains poor in insoluble polysacharides till the ultimate stages of microsporogenesis, it grows richer in choline - phospholipids during meiosis and, more lightly, in carotenoids beyond the tetrad stage; the periplasmodium does not seem to participate directly in the increase of exine lipids.

Embryogenesis and plant regeneration of the perennial pasture and medicinal legume Bituminaria bituminosa (L.) C.H. Stirton

Bituminaria bituminosa (common name tedera) is a drought-tolerant perennial pasture species of agronomic and pharmaceutical interest for Mediterranean climates. Considering the importance of this legume, in vitro experiments were conducted to develop protocols for plant regeneration from embryogenic calli of leaves, petioles and anthers to efficiently exploit and maintain selected important clones from the tedera breeding program. The type of explant was a key factor in the frequency of embryogenesis and the number of embryos per callus. For plant regeneration from cultured anthers, appropriate anther physiological state (uninucleate stage of microsporogenesis), stress treatments (electroporation, 25 Ω, 25 µF, 1500 V) and culture conditions were determined. A robust flow-cytometry method was developed to analyse the ploidy status of callus, in vitro shoots and in vivo acclimatised plants derived from anther and leaf explants.