Showing papers on "Microspore published in 2009"
TL;DR: This work provides a comprehensive analysis of the molecular events underlying the HSR of maturing microspores of a crop plant, tomato.
Abstract: Above-optimal temperatures reduce yield in tomato largely because of the high heat stress (HS) sensitivity of the developing pollen grains. The high temperature response, especially at this most HS-sensitive stage of the plant, is poorly understood. To obtain an overview of molecular mechanisms underlying the HS response (HSR) of microspores, a detailed transcriptomic analysis of heat-stressed maturing tomato microspores was carried out using a combination of Affymetrix Tomato Genome Array and cDNA-amplified fragment length polymorphism (AFLP) techniques. The results were corroborated by reverse transcription-PCR (RT-PCR) and immunoblot analyses. The data obtained reveal the involvement of specific members of the small heat shock protein (HSP) gene family, HSP70 and HSP90, in addition to the HS transcription factors A2 (HSFA2) and HSFA3, as well as factors other than the classical HS-responsive genes. The results also indicate HS regulation of reactive oxygen species (ROS) scavengers, sugars, plant hormones, and regulatory genes that were previously implicated in other types of stress. The use of cDNA-AFLP enabled the detection of genes representing pollen-specific functions that are missing from the tomato Affymetrix chip, such as those involved in vesicle-mediated transport and a pollen-specific, calcium-dependent protein kinase (CDPK2). For several genes, including LeHSFA2, LeHSP17.4-CII, as well as homologues of LeHSP90 and AtVAMP725, higher basal expression levels were detected in microspores of cv. Hazera 3042 (a heat-tolerant cultivar) compared with microspores of cv. Hazera 3017 (a heat-sensitive cultivar), marking these genes as candidates for taking part in microspore thermotolerance. This work provides a comprehensive analysis of the molecular events underlying the HSR of maturing microspores of a crop plant, tomato.
318 citations
TL;DR: The results suggest that not all tapetal genes are inhibited by increased temperatures and the tapetum itself is not degraded in such an environment, however, high temperatures may disrupt some of theTapetum functions required for pollen adhesion and germination on the stigma.
Abstract: Plant male reproductive development is highly organized and sensitive to various environmental stressors, including high temperature. We have established an experimental procedure to evaluate high temperature injury in japonica rice plants. High temperature treatment (39 ° C/30 ° C) starting at the microspore stage repeatedly reduced spikelet fertility in our system. Morphological observations revealed that pollen viability in plants exposed to high temperatures was lower than that in control plants. Most pollen grains in high temperature-treated plants displayed a normal round shape and stained reddish purple with Alexander’s reagent; however, the pollen grains were very poorly attached and displayed limited germination on the stigma. To investigate gene regulatory mechanisms in the anther in high temperature environments, DNA microarray analysis was performed by comparing non-treated samples with samples treated with 2–4 d of high heat. Genes responsive to high temperatures were identifi ed from clustering of microarray data. Among these, at least 13 were designated as high temperature-repressed genes in the anther. Expression analyses revealed that these genes were expressed specifi cally in the immature anther mainly in the tapetum at the microspore stage and down-regulated after 1 d of high temperature. The expression levels of Osc6 , OsRAFTIN and TDR , which are tapetum-specifi c genes, were unaffected by high temperatures. These results suggest that not all tapetal genes are inhibited by increased temperatures and the tapetum itself is not degraded in such an environment. However, high temperatures may disrupt some of the tapetum functions required for pollen adhesion and germination on the stigma.
294 citations
TL;DR: The ability of CPPs to deliver macromolecules (protein as well as linear plasmid DNA) noncovalently has been demonstrated in triticale isolated microspores and it is shown that Tat2 can successfully deliver GUS gene in near to 2% triticalsemicrospores.
Abstract: Microspore culture is contributing significantly in the field of plant breeding for crop improvement in general and cereals, in particular. In the present study, we investigated the uptake of fluorescently labeled cell-penetrating peptides (CPP; Tat, Tat2, M-Tat, peptide vascular endothelial-cadherin, transportan) in the freshly isolated triticale microspores (mid-late uninucleate stage). We demonstrated that Tat (RKKRRQRRR) and Tat2 (RKKRRQRRRRKKRRQRRR) are able to efficiently transduce GUS enzyme (272 kDa) in its functional form in 5 and 14% of the microspores, respectively, in a noncovalent manner. Pep-1, a synthetic CPP, was able to transduce GUS enzyme in its active form in 31% of the microspores. The effect of various endocytic and macropinocytic inhibitors on Tat2-mediated GUS enzyme delivery was studied and revealed a preferred micropinocytosis entry. DNase I protection assay and confocal laser microscopy was carried out to recommend a ratio of 4:1 Tat2-linear plasmid DNA (pActGUS) in complex preparation for microspore transfection. We further show that Tat2 can successfully deliver GUS gene in near to 2% triticale microspores. The negative control mutated Tat (M-Tat: AKKRRQRRR) failed to transducer the GUS protein and transfect the GUS gene in microspore nucleus. The ability of CPPs to deliver macromolecules (protein as well as linear plasmid DNA) noncovalently has been demonstrated in triticale isolated microspores. It further confirms potential applications of CPPs in developing simple, time saving, cost effective plant genetic engineering technologies.
95 citations
TL;DR: It is argued that the interplay between PCD and oxidative stress in anthers might be a cause of pollen sterility in drought-stressed rice.
Abstract: Male gametophyte development of eukaryotic plants in general and rice in particular is especially sensitive to drought. Water deficit during this stage inhibits microspore development resulting in male sterility. To elucidate the molecular mechanism of the phenomenon, a water deficit-induced experiment was conducted during anther development. Microscopic observations of anther cross-sections labelled with TdT-mediated dUTP nick-end labelling (TUNEL) indicated programmed cell death (PCD) signals after three consecutive days of water deficit. PCD is a biological process featured by the fragmentation of genomic DNA and plays an important role in plant reproduction. PCD is often concurrent with biochemical and physical changes in the cytoplasm, nucleus and plasma membrane of the cells. In this study, biochemical assays showed depletion of the adenosine triphosphate pool, increased concentration of hydrogen peroxide (ROS) and down-regulation of antioxidant transcripts in anthers. We argue that the interplay between PCD and oxidative stress in anthers might be a cause of pollen sterility in drought-stressed rice.
80 citations
TL;DR: It is concluded that native cross-talk from the plastid cannot compensate for complete blockage of the MVA pathway, at least during male gametophyte development, because either HMG1 or HMG2 is required for male gametic development.
Abstract: Plants have two isoprenoid biosynthetic pathways: the cytosolic mevalonate (MVA) pathway and the plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Since the discovery of the MEP pathway, possible metabolic cross-talk between these pathways has prompted intense research. Although many studies have shown the existence of such cross-talk using feeding experiments, it remains to be determined if native cross-talk, rather than exogenously applied metabolites, can compensate for complete blockage of the MVA pathway. Previously, Arabidopsis mutants for HMG1 and HMG2 encoding HMG-CoA reductase (HMGR) were isolated. Although it was shown that HMGR1 is a functional HMGR, the enzyme activity of HMGR2 has not been confirmed. It is demonstrated here that HMG2 encodes a functional reductase with similar activity to HMGR1, using enzyme assays and complementation experiments. To estimate the contribution of native cross-talk, an attempt was made to block the MVA pathway by making double mutants lacking both HMG1 and HMG2, but no double homozygotes were detected in the progeny of self-pollinated HMG1/hmg1 hmg2/hmg2 plants. hmg1 hmg2 male gametophytes appeared to be lethal based on crossing experiments, and microscopy indicated that approximately 50% of the microspores from the HMG1/hmg1 hmg2/hmg2 plant appeared shrunken and exhibited poorly defined endoplasmic reticulum membranes. In situ hybridization showed that HMG1 transcripts were expressed in both the tapetum and microspores, while HMG2 mRNA appeared only in microspores. It is concluded that native cross-talk from the plastid cannot compensate for complete blockage of the MVA pathway, at least during male gametophyte development, because either HMG1 or HMG2 is required for male gametophyte development.
78 citations
TL;DR: Received data indicate that in triticale, anther preculture conditions could generate oxidative stress and change the cell metabolic activity which could next be reflected in the cell viability and the efficiency of microspore embryogenesis.
Abstract: Isolated microspore cultures of two spring triticale (x Triticosecale Wittm.) cultivars were used to examine the effect of various stress treatments (either high—32°C or low—5°C temperature with or without nitrogen/carbohydrate starvation) applied to excised anthers on the effectiveness of microspore embryogenesis induction. To quantify the effects of pretreatment conditions, the activity of antioxidative enzymes (catalase, peroxidase and superoxide dismutase) together with respiration rate and heat emission were measured. It was observed that heat shock treatment applied as the only one stress factor increased the activity of antioxidative enzymes which suggests intensive generation of reactive oxygen species. Such pretreatment effectively triggered microspore reprogramming but drastically decreased microspore viability. After low temperature treatment, the activity of antioxidative enzymes was similar to the control subjected only with the stress originated from the transfer to in vitro culture conditions. This pretreatment decreased the number of microspores entering embryogenesis but sustained cell viability and this effect prevailed in the final estimation of microspore embryogenesis effectiveness. For both, low- and high-temperature treatments, interaction with starvation stress was beneficial increasing microspore viability (at 5°C) or efficiency of embryogenesis induction (at 32°C). The latter treatment significantly reduced cell metabolic activity. Physiological background of these effects seems to be different and some hypothetical explanations have been discussed. Received data indicate that in triticale, anther preculture conditions could generate oxidative stress and change the cell metabolic activity which could next be reflected in the cell viability and the efficiency of microspore embryogenesis.
70 citations
TL;DR: Several lines of evidence suggest a sporophytic role in exine patterning, and a gametophytic mode of action of BcMF9 in intine formation, in pollen development and about this family in general.
67 citations
TL;DR: This protocol can be used instead of the labor-intensive inter-generic crossing with maize as an economically feasible method to obtain DHs for most crosses involving the durum wheat cultivars grown in Spain.
Abstract: The objective of this study was to produce durum wheat doubled haploid (DH) plants through the induction of microspore embryogenesis. The microspore culture technique was improved to maximize production of green plants per spike using three commercial cultivars. Studies on factors such as induction media composition, induction media support and the stage and growth of donor plants were carried out in order to develop an efficient protocol to regenerate green and fertile DH plants. Microspores were plated on a C17 induction culture medium with ovary co-culture and a supplement of glutathione plus glutamine; 300 g/l Ficoll Type-400 was incorporated to the induction medium support. Donor plants were fertilized with a combination of macro and microelements. With the cultivars ‘Ciccio’ and ‘Claudio’ an average of 36.5 and 148.5 fertile plants were produced, respectively, from 1,000 anthers inoculated. This technique was then used to produce fertile DH plants of potential agronomic interest from a collection of ten F1 crosses involving cultivars of high breeding value. From these crosses 849 green plants were obtained and seed was harvested from 702 plants indicating that 83% of green plants were fertile and therefore were spontaneously DHs. No aneuploid plant was obtained. The 702 plants yielded enough seeds to be field tested. One of the DH lines obtained by microspore embryogenesis, named ‘Lanuza’, has been sent to the Spanish Plant Variety Office for Registration by the Batlle Seed Company. This protocol can be used instead of the labor-intensive inter-generic crossing with maize as an economically feasible method to obtain DHs for most crosses involving the durum wheat cultivars grown in Spain.
51 citations
TL;DR: Comparison of events recorded in the recent pollen developmental mutants in Arabidopsis indicates that several failures during tetrad dissolution may convert to a common recurring phenotype that has evolved independently several times, whenever this grouping conferred advantages for pollen transfer.
Abstract: In most flowering plants, pollen is dispersed as monads. However, aggregated pollen shedding in groups of four or more pollen grains has arisen independently several times during angiosperm evolution. The reasons behind this phenomenon are largely unknown. In this study, we followed pollen development in Annona cherimola, a basal angiosperm species that releases pollen in groups of four, to investigate how pollen ontogeny may explain the rise and establishment of this character. We followed pollen development using immunolocalization and cytochemical characterization of changes occurring from anther differentiation to pollen dehiscence. Our results show that, following tetrad formation, a delay in the dissolution of the pollen mother cell wall and tapetal chamber is a key event that holds the four microspores together in a confined tapetal chamber, allowing them to rotate and then bind through the aperture sites through small pectin bridges, followed by joint sporopollenin deposition. Pollen grouping could be the result of relatively minor ontogenetic changes beneficial for pollen transfer or/and protection from desiccation. Comparison of these events with those recorded in the recent pollen developmental mutants in Arabidopsis indicates that several failures during tetrad dissolution may convert to a common recurring phenotype that has evolved independently several times, whenever this grouping conferred advantages for pollen transfer.
47 citations
TL;DR: Genes related to stress response, transcription and translation regulation, and degradation of pollen-specific proteins were associated with green plant production, while expression of genes related to plastid development was associated with albino plant regeneration.
Abstract: Plant microspores can be reprogrammed from their normal pollen development to an embryogenic route in a process termed microspore embryogenesis or androgenesis. Stress treatment has a critical role in this process, inducing the dedifferentiation of microspores and conditioning the following androgenic response. In this study, we have used three barley doubled haploid lines with similar genetic background but different androgenic response. The Barley1 GeneChip was used for transcriptome comparison of these lines after mannitol stress treatment, allowing the identification of 213 differentially expressed genes. Most of these genes belong to the functional categories “cell rescue, defense, and virulence”; “metabolism”; “transcription”; and “transport”. These genes were grouped into clusters according to their expression profiles among lines. A principal component analysis allowed us to associate specific gene expression clusters to phenotypic variables. Genes associated with the ability of microspores to divide and form embryos were mainly involved in changes in the structure and function of membranes, efficient use of available energy sources, and cell fate. Genes related to stress response, transcription and translation regulation, and degradation of pollen-specific proteins were associated with green plant production, while expression of genes related to plastid development was associated with albino plant regeneration.
44 citations
TL;DR: Findings from experiments on rice plants exposed to water deficit for three consecutive days during early stages of anther development suggest a new underlying mechanism of water deficit-induced pollen abortion in rice.
Abstract: Pollen formation in rice (Oryza sativa L.) is highly vulnerable to environmental stresses such as heat, chilling and drought. In rice plants exposed to drought during male reproductive development, the most obvious damage often observed is a decline in the number of engorged pollen and grain set. This has been well characterized in rice under chilling and to a lesser extent under drought stress. Moreover, detailed literature on the immediate effects of drought on developing young microspores in rice is still limited. Here, we report findings from experiments on rice plants exposed to water deficit for three consecutive days during early stages of anther development. When the osmotic potential of the growing medium was equal to or less than −0.5 MPa, as induced by polyethylene glycol, the leaf water potential was significantly lowered and grain set was reduced. A strong correlation between grain set and viable young microspores (P < 0.001, r2 = 0.8223) indicates that water deficit immediately reduced fertility of rice plants at the time of exposure. This result suggests a new underlying mechanism of water deficit-induced pollen abortion in rice.
TL;DR: OsUgp2 complements OsUgp1 to fulfill the UGPase’s functions necessary for the full process of pollen development and plays a key role during pollen maturation, especially for starch accumulation.
Abstract: UDP-glucose pyrophosphorylase (UGPase) is predominantly present and plays significant role in carbohydrate metabolism in plants. Two homologous UGPase genes, OsUgp1 and OsUgp2, exist in rice genome. OsUgp1 has recently been reported to be essential for callose deposition during pollen mother cell and meiosis stages as well as for seed carbohydrate metabolism. In this study, a full-length cDNA of OsUgp2 was isolated from rice anther. Northern blot and RNA in situ hybridization indicated that the expression of OsUgp2 was preferentially in pollen and developmentally regulated. No transcripts were found in leaf, stem, lemma/palea, ripening grain and florets before the uninucleate microspore developmental stage, but a large quantity of OsUgp2 mRNA was found in pollen at the binucleate and mature stages. The immunolocalization of OsUgp2 showed a similar expression pattern to that by RNA in situ hybridization. The function of OsUgp2 was investigated by dsRNA-mediated transcriptional gene silencing. The pollen fertility of 16 independent transgenic rice plants was found between 25% and 90%, which was correlated with the amount of OsUgp2 mRNA. The results of morphological changes and starch variation during pollen development in transgenic rice showed that the abnormal feature of pollen development appeared after the uninucleate microspore stage. Starch failed to accumulate in pollen and thus led to sterile pollens. These results demonstrated that OsUgp2 is a pollen-preferential “late gene” and plays a key role during pollen maturation, especially for starch accumulation. OsUgp2 complements OsUgp1 to fulfill the UGPase’s functions necessary for the full process of pollen development.
TL;DR: This study provides the basis for understanding the biological mechanisms regulating floral development, thus expanding the prospects for Z. mauritiana breeding programs and for further molecular and genetic studies of this species.
Abstract: Floral development of the synchronous dichogamous species Ziziphus mauritiana, as followed by light and scanning electron microscopy (SEM), was divided into 11 stages using a series of landmark events. Main cellular events happen synchronously in the female and the male structures, such as meiosis in micro- and macrosporocyte cells, tetrad microspore formation and appearance of the functional megaspore cell, and onset of embryo sac differentiation coinciding with mitosis in the microspores. The last stage was characterized by anthesis and continued development of the flower, beginning with anther dehiscence (male phase) and proceeding to the female phase, which was characterized by style elongation. Flowers exhibit synchronous protandrous dichogamy; anthesis takes place in the morning (group A, e.g., clone Q-29) and afternoon (group B, e.g., clone B5/4). Stigma receptivity started after the male phase and occurred synchronously and complementarily with pollen dispersal in the two clones. Pollen viability and production were similar in the two clones, but the pollen diameter of Q-29 was significantly larger than that of B5/4. This study provides the basis for understanding the biological mechanisms regulating floral development, thus expanding the prospects for Z. mauritiana breeding programs and for further molecular and genetic studies of this species.
TL;DR: Four phenomena were observed in a study of Populus tremula and P. tremula f.
Abstract: Four phenomena were observed in a study of Populus tremula and P. tremula f. gigas microspores from before microspore mitosis through mature pollen which may have general significance in the ontogeny of pollen grains: 1) The exine and orbicules (Ubisch bodies) were covered by membranes. 2) The exine and the tapetal surfaces where orbicules form were covered by a polysaccharide (PAS positive) coat until after microspore mitosis; subsequently the tapetum became plasmodial. 3) Material having the staining characteristics of the nexine 2 (endexine in the sense of Faegri) accumulated on membranes in microspores in the space between the exine and the plasma membrane. That material was almost completely gone from the wall in mature pollen. The membranes on which material had accumulated migrated through the exine. Following passage through the exine these membranes were seen as empty fusiform vesicles in micrographs of anthers prepared by commonly used methods. 4) At about microspore mitosis when the cel...
TL;DR: An efficient protocol for microspore culture is established and offers great potential for DH breeding in purple flowering stalk and regenerated plants were double haploid.
TL;DR: This study focused on the optimization of a protocol to allow for the exogenous DNA to enter the microspore in an efficient manner and provides an efficient means to integrate extraneous DNA into rapeseed microspores prior to androgenesis induction.
Abstract: The physical barrier imposed by the thick microspore wall constitutes an obstacle for an efficient Agrobacterium-mediated transformation of vacuolate microspores prior to androgenic induction and haploid embryogenic commitment. It is thus necessary to implement additional methods to overcome this drawback. In this study, we focused on the optimization of a protocol to allow for the exogenous DNA to enter the microspore in an efficient manner. We tested different options, based on microprojectile bombardment, to be applied prior to agroinfiltration. From them, the best results were obtained through co-transformation by microspore bombardment with DNA-coated microprojectile particles, followed by Agrobacterium tumefaciens infection. This method provides an efficient means to integrate extraneous DNA into rapeseed microspores prior to androgenesis induction.
1 Jan 2009
TL;DR: This review report on the progress made to develop doubled haploid methods in triticale finds isolated microspore culture is a method particularly attractive that will open new opportunities for gene transfer in trticale.
Abstract: This review report on the progress made to develop doubled haploid methods in triticale. Wide hybridization, anther culture and isolated microspores methods have been well established in triticale, and adopted in breeding and germplasm development programs for diverse genotypes. The development of these methods and culture media has co-evolved with those for its wheat counterpart. Essentially the critical factors for success remain the same: genotype, growing condition of mother plants, stress-like pretreatment and the culture medium composition. Today, C17, N6 and NPB99 induction medium for microspore culture are preferred. They carry maltose, sucrose or a combination of both, amino acids such as glutamine or proline, a low concentration of auxin and/or cytokinin. Spontaneous doubling has been reported at a low frequency in microspore derived plants making chromosome doubling by colchicine treatment an absolute need, as for wide hybridization derived plants. Isolated microspore culture is a method particularly attractive that will open new opportunities for gene transfer in triticale.
TL;DR: The data demonstrated that the induction of embryogenesis induced by heat stress can be enhanced by inhibitors of ethylene biosynthesis, and a special response to ethylene during the early stages of microspore development was shown to occur.
Abstract: Procedures that induce microspore embryogenesis have been described for a range of Brassica species, but embryo yield remains low for a number of genotypes. We have carried out experiments with the microspores from a weakly responsive line of B. napus to determine the culture conditions that optimize their in vitro embryogenesis by treating them with effectors of ethylene synthesis and action. The results revealed that isolated microspores subjected to an initial heat stress in a medium supplemented with inhibitors of ethylene synthesis such as AVG and CoCl2 exhibited significantly increased embryo yields. This suggested that regulatory effects are exerted by the ethylene produced by the isolated microspores on the early processes of gametogenesis. As a consequence, treatment of microspores with SAM, an ethylene synthesis precursor, or with the ethylene-releasing agent ethephon, led to decreases in embryo yield. A special response to ethylene during the early stages of microspore development was finally shown to occur through experiments where isolated microspores were treated for increasing periods of time with CoCl2. Collectively, our data demonstrated that the induction of embryogenesis induced by heat stress can be enhanced by inhibitors of ethylene biosynthesis.
TL;DR: Results indicate that the Zm 401 gene is one of the major components of the molecular network regulating maize anther development and male fertility, and that Zm401p10 is expressed from the longest ORF of the gene.
Abstract: In flowering plants, the tapetum is proposed to play a vital role in the early stages of pollen development. Disruptions to tapetum development and degeneration typically result in male sterility. The present study characterised a maize (Zea mays L.) anther-specific gene, Zm401, which only contains short open reading frames (sORFs). The longest ORF of the Zm401 gene encodes a small protein designated Zm401p10 that accumulates in the nucleus. Overexpression of Zm401p10 in maize retarded tapetal degeneration and caused microspore abnormalities. A microarray analysis identified 278 downregulated and 150 upregulated genes in anthers overexpressing Zm401p10. These results indicate that the Zm401 gene is one of the major components of the molecular network regulating maize anther development and male fertility, and that Zm401p10 is expressed from the longest ORF of the gene.
TL;DR: The results of the real-time PCR suggest that Bnms1 might be involved in the metabolism of lipid/fatty acids, and the homologous mutation of BnMS1 may either block the biosynthesis of sporopollenin or block sporopollin from being deposited on the microspore surface, thus, preventing pollen exine formation.
Abstract: Male sterility in a near-isogenic line S45AB after 25 generations of subcrossing is controlled by two pairs of duplicate genes. The genotype of S45A is Bnms1Bnms1Bnms2Bnms2, and that of S45B is BnMs1Bnms1Bnms2Bnms2, respectively. Histological observations revealed that abnormal anther development appeared in the tapetum and pollen exine during the tetrad stage. This male sterility was characterized by hypertrophy of the tapetal cells at the tetrad stage and a complete lack of microspore exine after the release of microspores from the tetrads. To elucidate the mechanism of this recessive genic male sterility, the flower bud expression profiles of the S45A and S45B lines were analyzed using an Arabidopsis thaliana ATH1 oligonucleotide array. When compared with the S45B line, 69 genes were significantly downregulated, and 46 genes were significantly upregulated in the S45A line. Real-time polymerase chain reaction (PCR) was then used to verify the results of the microarray analysis, and the majority of the downregulated genes in the S45A line were abundantly and specifically expressed in the anther. The results of the real-time PCR suggest that Bnms1 might be involved in the metabolism of lipid/fatty acids, and the homologous mutation of Bnms1 may either block the biosynthesis of sporopollenin or block sporopollenin from being deposited on the microspore surface, thus, preventing pollen exine formation. The role of Bnms1 in the regulatory network of exine formation is also discussed as well.
TL;DR: It was confirmed that during the cold plus mannitol pretreatment, there was no apparent change in the cell cycle stage, with the majority of the microspores remaining at the G1 stage, and one might expect to find a higher frequency of homozygous doubled haploids by maintaining the temperature low during the 4 h osmotic adjustment period following the coldplus mannitor pretreatment than following the 21 day cold pretreatment.
Abstract: The objective of this study was to determine when DNA synthesis occurred during pretreatments of cultured barley (Hordeum vulgare L.) microspores and during their preparation for particle bombardme...
TL;DR: Microsatellite marker fingerprinting of 12 pairs of twinned embryos produced by microspore culture of heterozygous F1 lines revealed that pairs of twins were genetically identical.
Abstract: Microspore culture is used extensively in several crop species to generate diverse populations of homozygous, doubled haploid lines for breeding and genetic analyses. In our canola (Brassica napus L.) doubled haploid breeding programme we regularly observe conjoined microspore-derived embryos, most commonly twins, joined either at the base of the hypocotyl or along the length of the hypocotyl axis. The aim of this study was to determine if twinned embryos were genetically identical or non-identical in order to gauge their value for breeding and linkage analysis. Microsatellite marker fingerprinting of 12 pairs of twinned embryos produced by microspore culture of heterozygous F1 lines revealed that pairs of twins were genetically identical. Based on this finding, we recommend breeders and geneticists using microspore culture technology to retain only one embryo from each pair of twinned embryos.
TL;DR: It is demonstrated that WISH is a suitable technique for studying gene expression in embryogenic microspore populations and, because different structures can be examined individually, is an appropriate complement to transcriptomic profile analyses in the study of early microspores embryogenesis.
Abstract: Microspores genetically programmed to produce male gametes can be switched to the embryogenic pathway to give rise to haploid embryos. Microspore embryogenesis is usually induced in barley by stress pre-treatment applied to vacuolated microspores. We studied the expression of two genes during the early stages of microspore embryogenesis to gain further insight into the microspore transition from the gametophytic to the embryogenic pathway. RT-PCR together with in situ hybridization on sections (ISH) and whole-mount in situ hybridization (WISH) were used to analyse the expression of the early-culture abundant gene (ECA1), which is expressed in barley during microspore embryogenesis, and a polygalacturonase gene (HvPG1), a late pollen gene expressed during gametogenesis only after microspore division. Both ECA1 and HvPG1 genes were transcriptionally active after stress pre-treatment in the same populations of microspore-derived structures, representing the sporophytically induced ones. ECA1 transcripts were also detected after 3 days' culture. Our results point to the possibility of using ECA1 gene expression as a marker for the induction of microspore embryogenesis and the earliest stages of this process. Finally, we demonstrate that WISH is a suitable technique for studying gene expression in embryogenic microspore populations and, because different structures can be examined individually, is an appropriate complement to transcriptomic profile analyses in the study of early microspore embryogenesis.
TL;DR: The results of this study indicate that the mechanism causing male sterility in Shindai3 differs from that previously reported for other male-sterile trees of C. japonica.
Abstract: To determine the mechanism of male-sterility Cryptomeria japonica tree Shindai3, the process of microspore development was observed under light and fluorescence microscopy. Microspore development in the Shindai3 was normal until the tetrad stage, but separation of the microspores from the tetrads was not observed even after callose had been degraded. In contrast to the microspore stage in a male-fertile tree, amorphous substances were observed around tetrads in the Shindai3, and the amount of the amorphous substance increased from the middle of November to the middle of December. The substances are hypothesized to prevent the separation of individual microspores from tetrads by inducing adhesion between microspore cells within the tetrad or enclosing the tetrads. The form of the tetrads in the Shindai3 was maintained until just before the pollen dispersal season, but they ultimately degenerated. The results of this study indicate that the mechanism causing male sterility in Shindai3 differs from that previously reported for other male-sterile trees of C. japonica.
TL;DR: The best procedure for obtaining transgenic barley plants from this study was pretreatment C, leaving the cultures at either 4 or 25 degrees C during the 4 h prebombardment high-osmotic period, using the actin promoter and having arabinogalactan protein in the microspore culture medium.
Abstract: Based on paper I in this series, our goals in this paper were to determine the relationship between prebombardment pretreatments and temperatures, microspore cell cycle when bombarded, and the freq...
1 Jan 2009
TL;DR: The stress of pre-treatment may influence the triggering of PCD in both the anther tissues and the microspores and is likely involved in the competence of micro spore to the process of microspore embryogenesis.
Abstract: Programmed cell death (PCD) occurs in the anther during in vivo micro-spore/pollen development. The first hallmarks are detected in the tapetum at meiosis and consist in progressive degeneration of organelles such as mitochondria, as well as DNA degradation into multiple of 180 kpb fragments. This phenomenon radially extends in the anther sporophytic tissues and finally affects microspore/pollen at various stages of development depending upon species. PCD may infer with the process of microspore embryogenesis. Indeed, the inducing pre-treatment is per formed using the whole anther during the process of microspore vacuolation. Thus the stress of pre-treatment may influence the triggering of PCD in both the anther tissues and the microspores and is likely involved in the competence of microspore to the process of microspore embryogenesis. These data are also discussed consid ering the regeneration of albino microspore derived plants in cereals.
TL;DR: The effects of cold pretreatment with or without either mannitol or chemical + heat and also the effects of 5 embryo induction media on embryo production, percentage of total and green plant regeneration in isolated microspore culture of an Iranian spring bread wheat, cultivar ‘Falat’ were reported.
Abstract: Isolated microspores of many plants can be induced in vitro to switch their developmental process from the gametophytic to a sporophytic pathway under appropriate conditions and produce haploid plants. This research reports the effects of cold pretreatment with or without either mannitol or chemical + heat and also the effects of 5 embryo induction media (NPB-99, C17, W14, CHB-2 and P2) on embryo production, percentage of total and green plant regeneration in isolated microspore culture of an Iranian spring bread wheat, cultivar ‘Falat’. The results showed that combination of 21 days cold (4°C) with mannitol (0.3 M) produced the highest number of embryos/spike while the combination of cold with chemical + heat produced the lowest number. In the case of total and green plant regeneration, "7 days cold + mannitol" was more superior than other pretreatments. NPB-99, W14 and C17 media produced the highest number of embryos/spike, while CHB-2 medium appeared to be a better medium for green plant regeneration.
TL;DR: The sexual progeny of C48 seems most suitable as female parents to be used in intra-and interspecific hybridization of Brachiaria brizantha, and the importance of these findings to theBrachiaria breeding program is discussed.
Abstract: Meiotic behavior was analyzed in 6 progenies from 3 artificially induced tetraploid (2n = 4x = 36) sexual genotypes (C31, C41, and C48) of the normally apomictic Brachiaria brizantha (Hochst. ex A. Rich.) Stapf., syn. Urochloa brizantha (Hochst. ex A. Rich.) R. Webster. These are key plants to allow intraspecific hybridization of this important forage species, widely used for pastures in the tropics. The percentage of abnormal cells among the plants ranged from 39.8% to 63.2%. In the single plant derived from C48, only the common meiotic abnormalities typical of polyploids were observed, while in plants derived from C31 and C41, a distinct behavior was found. In the majority of cells of those plants, the chromosomes remained scattered in the cytoplasm in the first division, without forming a metaphase plate. This abnormality blocked chromosome movements at anaphase I. Several micronuclei of various sizes were formed and, after the occurrence of an irregular first cytokinesis, the meiocytes progressed normally to the second division, generating polyads with unbalanced microspores. Pollen viability was not correlated with meiotic abnormalities. The importance of these findings to the Brachiaria breeding program is discussed. The sexual progeny of C48 seems most suitable as female parents to be used in intra- and interspecific hybridization.
TL;DR: This study elucidates that the microspore sacs, instead of having normal four microspores, possessed 1 (monad), 2 (dyads), 3 (triads) and even up to 5 (polyads) microsp spores, which may produce unreduced gametes at later stages.
Abstract: The present research work was undertaken to assess the effect of gibberellic acid (GA3) applied exogenously to the Safflower plant for the study of pollen behavior. A cytological characterization in safflower from meiosis to pollen formation revealed that whereas meiosis was recorded to be normal in control plants with normal microsporogenesis, GA3-treated plants possessed disturbed tetrad configurations of pollen. GA3 caused deductions in pollen yield and increased the formation of abnormal meiotic products. Our study elucidates that the microspore sacs, instead of having normal four microspores, possessed 1 (monad), 2 (dyads), 3 (triads) and even up to 5 (polyads) microspores, which may produce unreduced gametes at later stages. A significant variation in the degree of pollen mortality was also displayed amongst these pollens. Another interesting feature noticed during the study was the fusion among tetrads due to wall dissolution.
1 Jan 2009
TL;DR: This paper used sequence tags (ESTs) from cDNA libraries representing freshly-isolated microspores (0 hours) and microsores cultured for 3, 5 or 7 days under embryogenesisinducing conditions were prepared.
Abstract: Isolated microspores from selected cultivars of Brassica napus readily form embryos in culture after mild heat stress treatments (32°C for 1–3 days). Transcript profiling methods were used to identify differentially-expressed genes as well as shifts in metabolism during the early stages of microspore embryogen-esis. Approximately 20,000 expressed sequence tags (ESTs) from cDNA libraries representing freshly-isolated microspores (0 hours) and microspores cultured for 3, 5 or 7 days under embryogenesis-inducing conditions were prepared. In silico analyses of ESTs and semi-quantitative and real time reverse transcription-polymerase chain reaction (RT-PCR) based profiling identified differentially-regulated gene clusters and 16 genes that could be used as specific markers for microspore embryogenesis. These molecular marker genes also were expressed during zygotic embryogenesis, underscoring the common developmental path ways that function during zygotic and gametic embryogenesis. Future studies will focus on characterization of embryogenesis-related genes and development of fluorescently-labeled gene/protein probes to precisely mark and isolate early stages of microspore embryogenesis.