Albinism is a common problem encountered by researchers in anther/microspore cultures of cereal crops. The present study investigates the effects of temperature variations on embryogenesis of wheat (Triticum aestivum ...Albinism is a common problem encountered by researchers in anther/microspore cultures of cereal crops. The present study investigates the effects of temperature variations on embryogenesis of wheat (Triticum aestivum L.) microspores. Following a cold (4°C - 13°C) vs. heat (33°C) shock to wheat tillers, microspores were isolated and cultured in a liquid medium to obtain embryoids. Data on embryogenic microspore%, embryoid yield, plant regeneration% and green plant% were collected and analyzed. Cold pretreatment of 4°C or 10°C for a period of 6 or 10 days were more effective than other cold temperature regimes in inducing microspore embryogenesis. The heat shock of 33°C yielded the highest numbers of embryogenic microspores and embryoids. The albino-prone genotypes produced significantly higher green plant% following optimal cold shock, as compared to the standard 33°C heat shock. Results from present study suggest that cold shock may be a desirable alternative for germplasm that produce lower green plant% using heat shock. Lowered incubation temperature during embryoid development did not result in higher green plant.展开更多
Populus bolleana is a variety of P. alba, commonly used in poplar breeding programs in China. Developmental biology that involves staminate flowers, microsporogenesis and microgametogenesis ofP. bolleana is essential ...Populus bolleana is a variety of P. alba, commonly used in poplar breeding programs in China. Developmental biology that involves staminate flowers, microsporogenesis and microgametogenesis ofP. bolleana is essential for Populus improvement in cross breeding for better characteristics in sexual reproduction. Flower morphology and pollen development were described and illustrated using anatomical, sectioning and stain-clearing techniques. The results show that microsporocytes undergo a regular meiotic process, but some multi-nucleate microspores occur at the microspore stage. It takes five days for microsporocytes to develop to mature pollen by forcing flower branches under greenhouse conditions. Additionally, an important relationship was found between stages of meiosis and anther colors. Microspore tetrads formed when the anther color turned yellow, whereas, when the pollen matured, the anther was red and the tapetum degenerated completely. When mature pollen grains are formed, flower buds develop into male catkins. In the end, filament elongated and pollen grains were released from dehisced anthers.展开更多
Most aspects of microspore culture protocol have the capacity to cause stress to microspores, hence, less stressful treatments might be required to avoid deleterious effects. In stressed plants, polyamines and trehalo...Most aspects of microspore culture protocol have the capacity to cause stress to microspores, hence, less stressful treatments might be required to avoid deleterious effects. In stressed plants, polyamines and trehalose can act as compatible solutes or osmoprotectants by stabilizing proteins and biological membranes. To improve green plant regeneration in wheat microspore culture, this study assessed the effects of polyamines (putrecine, spermidine, spermine) and trehalose on androgenic response namely embryogenesis, green plant regeneration and ploidy of green plants regenerated in three spring wheat genotypes. Microspores of the genotypes produced significant numbers of embryos and green plants among polyamine treatments but trehalose had no effect (P ≤ 0.05). Polyamine treatments for 30 min generally produced more green plants per 100 microspores than the 60 min treatments in all three genotypes. At least three out of twelve polyamine treatments in each genotype improved the production of double haploid plants and seed setting in regenerants. Wheat genotype, concentration and duration of polyamine treatment had significant impact on embryogenesis and regeneration of green plants in this study. The study also showed that polyamines could be used to accelerate cultivar development in wheat breeding.展开更多
Doubled haploid production via microspore culture is a technique known to accelerate crop breeding by shortening the breeding cycle through achieving homozygosity in one generation. Prior research observed that some e...Doubled haploid production via microspore culture is a technique known to accelerate crop breeding by shortening the breeding cycle through achieving homozygosity in one generation. Prior research observed that some embryogenic microspores aborted their development before reaching the embryoid stage. Such embryogenic abortion reduces embryoid yield, making microspore cultures less efficient. The present research aims at identifying stages during which microspore development is susceptible to embryogenic abortion. Information gained through delineation of the developmental dynamics of microspores in culture could be used to improve the efficiency of microspore culture. Embryogenic microspores were isolated from stress-treated wheat (Triticum aestivum L.) tillers and cultured in liquid medium. The development of embryogenic microspores was monitored over a 35 day period. At day 7, 10, 14, 21, 28, and 35, the developing microspores were counted and categorized into multicellular structures, pre-embryoids, immature embryoids and mature embryoids. The results showed that 44% - 62% of embryogenic microspores halted their development before the mature embryoid stage. Of these aborted embryogenic microspores, 21% - 33% terminated as multicellular structures, 16% - 19% arrested their development as pre-embryoids, and 7% - 10% halted development as immature embryoids. Identifying factors that are responsible for embryogenic abortion and finding remedy to the issue will help improve the efficiency of doubled haploid production.展开更多
Induced mutations are important for genetic research and breeding.Mutations induced by physical or chemical mutagenesis are usually heterozygous during the early generations.However,mutations must be fixed prior to ph...Induced mutations are important for genetic research and breeding.Mutations induced by physical or chemical mutagenesis are usually heterozygous during the early generations.However,mutations must be fixed prior to phenotyping or field trials,which requires additional rounds of self-pollination.Microspore culture is an effective method to produce double-haploid(DH)plants that are fixed homozygotes.In this study,we conducted ethyl methanesulfonate(EMS)-induced mutagenesis of microspore cultures of barley(Hordeum vulgare)cultivar‘Hua30’and landrace‘HTX’.The EMS concentrations were negatively correlated with the efficiency of callus induction and the frequency of mutant plant regeneration.The two genotypes showed different regeneration efficiencies.The phenotypic variation of the regenerated M1 plants and the presence of genome-wide nucleotide mutations,revealed by whole-genome sequencing,highlight the utility of EMS-induced mutagenesis of isolated microspore cultures for developing DH mutants.Genome-wide analysis of the mutation frequency in the regenerated plants revealed that a considerable proportion of mutations resulted from microspore culture(somaclonal variation)rather than EMS-induced mutagenesis.In addition to producing a population of 1972 homozygous mutant lines that are available for future field trials,this study lays the foundation for optimizing the regeneration efficiency of DH plants and the richness of mutations(mainly by fine-tuning the mutagen dosage).展开更多
By using DNA-specific fluorescent dye and a confocal laser scanning microscope, the present study was designed to investigate the cytological characteristics of dedifferentiating initiation during pretreatment and em-...By using DNA-specific fluorescent dye and a confocal laser scanning microscope, the present study was designed to investigate the cytological characteristics of dedifferentiating initiation during pretreatment and em-bryogenesis during culture in freshly-isolated microspores of barley, and the difference in main developmental pathway between freshly-isolated and cold-treated microspores. The results revealed that ( i ) freshly-isolated microspores started the initiation within 12 h of mannitol pretreatment, whose main cytological characteristics were that: cell vol-ume was obviously extended; the volume of nuclei and nucleoli were also greatly increased; nucleoli were extremely clear and highly condensed; N/C ratio was very high; ( ii ) all the pretreatment methods led to the initiation of the mi-crospores, thus triggering the embryogenic process; ( iii ) pretreatment methods influenced the main developmental pathway of microspores by changing the pattern of the first mitosis. The cold-treated展开更多
Selaginella is the largest and most taxonomically complex genus in lycophytes.The fact that over 750 species are currently treated in a single genus makes Selaginellales/Selaginellaceae unique in pteridophytes.Here we...Selaginella is the largest and most taxonomically complex genus in lycophytes.The fact that over 750 species are currently treated in a single genus makes Selaginellales/Selaginellaceae unique in pteridophytes.Here we assembled a dataset of six existing and newly sampled plastid and nuclear loci with a total of 684 accessions(74%increase of the earlier largest sampling)representing ca.300 species to infer a new phylogeny.The evolution of 10 morphological characters is studied in the new phylogenetic context.Our major results include:(1)the nuclear and plastid phylogenies are congruent with each other and combined analysis well resolved and strongly supported the relationships of all but two major clades;(2)the Sinensis group is resolved as sister to S.subg.Pulviniella with strong support in two of the three analyses;(3)most morphological characters are highly homoplasious but some characters alone or combinations of characters well define the major clades in the family;and(4)an infrafamilial classification of Selaginellaceae is proposed and the currently defined Selaginella s.l.is split into seven subfamilies(corresponding to the current six subgenera t the Sinensis group)and 19 genera(the major diagnosable clades)with nine new species-poor genera.We support the conservation of Selaginella with a new type,S.flabellata,to minimize nomenclatural instability.We provide a key to subfamilies and genera,images illustrating their morphology,their morphological and geographical synopses,a list of constituent species,and necessary new combinations.This new classification will hopefully facilitate communication,promote further studies,and help conservation.展开更多
The formation of mature and fertile pollen grains, taking place inside the anther, depends on supply of assimilates, in the form of sucrose, provided mainly by the leaves. Data is limited, however, with respect to the...The formation of mature and fertile pollen grains, taking place inside the anther, depends on supply of assimilates, in the form of sucrose, provided mainly by the leaves. Data is limited, however, with respect to the understanding of sucrose metabolism in microspores and the supporting tissues. The aims of the present work were to 1) follow the changes in total and relative concentrations of sucrose, glucose, fructose and starch in the stamen parts and microspores up until anthesis, 2) follow the activities of sucrose-metabolism-related enzymes, in the anther walls fraction and microspores of the crop plant tomato. Sucrose was found to be partially cleaved in the filament, decreasing by more than twofold in the anther wall layers and the locular fluid, and to accumulate in the mature pollen grains, constituting 80% of total soluble sugars. Thus, sucrose was both the starting sugar, supporting microspore development, and the main carbohydrate accumulated at the end of the pollen-development program. The major invertase found to be active in both the anther wall layers and in maturing microspores was cell-wall-bound invertase. High fructokinase 2 and sucrose phosphate synthase activities during pollen maturation coincided with sucrose accumulation. The potential importance of sucrose accumulation during pollen dehydration phase and germination is discussed.展开更多
To facilate breeding process of Brassica napus,a microspore culture and molecular marker-assisted screening combined system were proposed in this research.At early flowering stage,F1 offspring of hybridized combinatio...To facilate breeding process of Brassica napus,a microspore culture and molecular marker-assisted screening combined system were proposed in this research.At early flowering stage,F1 offspring of hybridized combination HY15 A×HF06 was used as donor for microspore culture to analyze effects of colchicine concentration on embryogenic and diploid rates of microspore.Treatment with 50 mg/L colchicine resulted in embryogenic rate of3.56 embryos/bud,which was substantially higher than control(0.78 embryos/bud).A total of 1,387 embryos and 862 single plants were obtained after induction culture.Ploidy detection was performed for the regenerated plants by flow cytometry.Diploid rates of microspores treated with 50 mg/L and 70 mg/L colchicine were 17.2%and 21.0%respectively,which was significantly higher than control(10.5%).Totally 108 single plants that doubled successfully were randomly selected and screened using molecular marker BE10.Approximately 54 of108 plants generated a 305 bp amplification product,whereas the other 54 plants showed a 398 bp band,thereby satisfying 1:1 separation ratio(x0.052=0.0093).These coincided with field identification results.Findings of this study indicated that homozygous breeding material could be obtained by microspore culture in a short time,thereby remarkably accelerate breeding.展开更多
Effects of five incubation temperatures (25℃ ,28℃ ,31℃ ,35℃ and 37℃ for 24hours) and four incubation time periods (0.4,16 and 24 hours at 35℃) on isolated microspore culture of Chinese cabbage were studied. The ...Effects of five incubation temperatures (25℃ ,28℃ ,31℃ ,35℃ and 37℃ for 24hours) and four incubation time periods (0.4,16 and 24 hours at 35℃) on isolated microspore culture of Chinese cabbage were studied. The results showed that cultured microspores of Chinese cabbage developed into embryos at all the incubation temperatues from 28℃ to 37℃ ,but the best response to high temperature occured at 35℃. Among the four kinds of time periods, the highest yield of embryos was obtained at the 24h treatment. Therefore, the isolated microspore culture of Chinese cabbege ran be efficiently carried out at 35℃ for 24 hours.展开更多
Studies of barley and maize indicate that chromosome doubling occurs via nuclear fusion during an early stage of microspore embryogenesis, but the time and mechanism by which chromosome doubling occurs in bread wheat ...Studies of barley and maize indicate that chromosome doubling occurs via nuclear fusion during an early stage of microspore embryogenesis, but the time and mechanism by which chromosome doubling occurs in bread wheat (Triticum aestivum) remains undetermined. The purpose of this study was to determine the relative time during induction culture when chromosome doubling may occur in wheat, and to identify early indicators for doubled haploid microspores. Microspore nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) and observed under a fluorescent microscope on the day of isolation, three days after isolation, and six days after isolation. The change in the percentage of microspores containing a single small nucleus, two small nuclei, a single enlarged nucleus, and three or more nuclei was then tracked throughout the six-day period. Ploidy levels were estimated by determining the cross-sectional area and number of nucleoli in microspores containing small and large nuclei then comparing the results of each respective cell-type. The percentage of microspores containing enlarged nuclei increased throughout the six-day test period, and the percentage of binucleated microspores containing small nuclei decreased. Comparison of the changes in average percentage of microspores containing a single small nucleus, binucleated microspores, microspores containing a single large nucleus, and multinucleate microspores on days 0, 3, and 6 indicates that nuclei classified as “small” are likely haploids and nuclei classified as “large” are doubled haploids. The percentage of microspores with enlarged nucleus (nuclei) during the first six days of induction culture could be used as an early indicator for the frequency of chromosome doubling in wheat microspore culture.展开更多
The mitogen-activated protein kinase(MAPK)cascade is important in stress signal transduction and plant development.In the present study,we identified a rice(Oryza sativa L.)mutant with reduced fertility,Oryza sativa m...The mitogen-activated protein kinase(MAPK)cascade is important in stress signal transduction and plant development.In the present study,we identified a rice(Oryza sativa L.)mutant with reduced fertility,Oryza sativa mitogen-activated protein kinase 6(osmapk6),which harbored a mutated MAPK gene.Scanning and transmission electron microscopy,quantitative RT-PCR analysis,TUNEL assays,RNA in situ hybridization,longitudinal and transverse histological sectioning,and map-based cloning were performed to characterize the osmapk6 mutant.The gene OsMAPK6 was expressed throughout the plant but predominantly in the microspore mother cells,tapetal cells,and microspores in the anther sac.Compared with the wild type,the total number of microspores was reduced in the osmapk6 mutant.The formation of microspore mother cells was reduced in the osmapk6 anther sac at an early stage of anther development,which was the primary reason for the decrease in the total number of microspores.Programmed cell death of some tapetal cells was delayed in osmapk6 anthers and affected exine formation in neighboring microspores.These results suggest that OsMAPK6 plays pivotal roles in microspore mother cell formation and tapetal cell degradation.展开更多
Isolated micmspore culture was carried out with Brassica juncea to study the effects of sterilization methods, culture period of embryos, hormone formula and the concentration of aetivatod carbon on embryogenesis, and...Isolated micmspore culture was carried out with Brassica juncea to study the effects of sterilization methods, culture period of embryos, hormone formula and the concentration of aetivatod carbon on embryogenesis, and the concentration of NAA on induction of roots. The results showed that the embryogenesis on medi- um subjected to filter sterilization is better than medium subjected to autoclaved sterilization. The embryos cultured for 15 d had the highest plantlat regenexafion rate (52%). NLN-13 liquid medium including 0. 1mg/L 6-BA and 0.2 g/L activated carbon significantly improved the planflet regeneration rate. 1/2 MS inclu- ding 0.3 mg/L NAA had the highest plantlet regeneration rate ( 100% ). Key words Brassica juncea ; Isolated micmspore culture ; Embryogenesis ; Planflet regeneration展开更多
During meiosis in flowering plants,degradation of the callose wall in tetrads releases newly produced microspores,which develop into mature pollen grains.In this study,we identified zbs1,a male-sterile mutant of naked...During meiosis in flowering plants,degradation of the callose wall in tetrads releases newly produced microspores,which develop into mature pollen grains.In this study,we identified zbs1,a male-sterile mutant of naked oat(Avena nuda L.)that displayed complete spikelet sterility due to inviable mature pollen.The abnormal pollen grains originated from microspores with a defective callose wall and cell plate during meiosis.The defective callose wall and cell plate of the zbs1 mutant were detected by the labeling of cell wall epitopes(β-1,3-glucan) with immunogold during meiosis,and an abnormal chromosome configuration was observed by propidium iodide staining.The mature pollen grains of the zbs1 mutant were irregular in shape,and abnormal germination was observed by scanning electron microscopy.Together,our results indicate that the cause of male sterility in zbs1 is abnormal meiosis.展开更多
Phytohormones play important roles in plant growth and development,and polyploids are thought to be an important method for plant breeding.However,the relationship between ploidy and phytohormone is still unclear.In t...Phytohormones play important roles in plant growth and development,and polyploids are thought to be an important method for plant breeding.However,the relationship between ploidy and phytohormone is still unclear.In this study,barley at three ploidy levels were produced by microspore culture.Therefore,we further analyzed the phytohormone content in the shoots and roots of the three kinds of barley materials to study the effect of ploidy on phytohormones accumulation and distribution.The results showed that Abscisic acid(ABA),gibberellin(GA),jasmonic acid(JA),auxin(IAA),salicylic acid(SA)and cytokinin(CTK)were successfully determined in shoots and roots using LC-MS(liquid chromatography mass spectrometry).By comparing the shoots of the haploid and diploid plants,it was found that the distribution trend of the six phytohormones was consistent,and another consistent trend was found in the roots of the diploid and tetraploid plants.In addition,we further analyzed the shoot/root ratio of the different phytohormones to identify the potential differences for haploid,diploid and tetraploid.Here,the relationship between ploidy and phytohormone we provided would provide new insights into understanding the new phenotypes that occur in polyploid species.展开更多
There were four microsporanges in the mature anther of B. rossica Fedtsch. et Flerov. The development of the anther wall belonged to dicotyledonous type. The tapetum cell was dichotypic. Simultaneous cytocinesis in th...There were four microsporanges in the mature anther of B. rossica Fedtsch. et Flerov. The development of the anther wall belonged to dicotyledonous type. The tapetum cell was dichotypic. Simultaneous cytocinesis in the microspore mother cells formed tetrad by reduction division and the array of microspore tetrads was different. There were tetrahedron type and bilateral symmetry type. The male gametophyte was 2-cell. The mature pollen was global and tricolporate. All those results were investigated under both light microscope (LM) and electron microscope (EM)展开更多
Plant formation from in vitro-cultivated microspores involves a complex network of internal and environmental factors.Haploids/doubled haploids(DHs)derived from in vitro-cultured microspores are widely used in plant b...Plant formation from in vitro-cultivated microspores involves a complex network of internal and environmental factors.Haploids/doubled haploids(DHs)derived from in vitro-cultured microspores are widely used in plant breeding and genetic engineering.However,the mechanism underlying the developmental switch from regular pollen maturation towards microspore-derived plant regeneration remains poorly defined.Here,RNA-sequencing was employed to elucidate the transcriptional landscapes of four early stages of microspore embryogenesis(ME)in barley cultivars Golden Promise and Igri,which exhibit contrasting responsiveness to microspore-derived plant formation.Our experiments revealed fundamental regulatory networks,specific groups of genes,and transcription factor(TF)families potentially regulating the developmental switch.We identified a set of candidate genes crucial for genotype-dependent responsiveness/recalcitrance to ME.Our high-resolution temporal transcriptome atlas provides an important resource for future functional studies on the genetic control of microspore developmental transition.展开更多
Lily was grown worldwide as a fresh cutting flower because of its colorful petals, but its anther contained a large number of pollen grains that cause serious pollen contamination, however, pollen abortion can effecti...Lily was grown worldwide as a fresh cutting flower because of its colorful petals, but its anther contained a large number of pollen grains that cause serious pollen contamination, however, pollen abortion can effectively reduce the level of pollen pollution. Our analysis aims to use cytological observation to detect the critical stage when pollen abortion occurs and to provide comprehensive gene expression information at the transcriptional level. The result showed that pollen abortion in ‘Little Kiss’ began at the mononuclear stage and the callose that covers the microspores failed to degenerate when young pollens were released from the tetrads. In addition, compared with the normally developed one,the tapetum of ‘Little Kiss’ degraded in advance while the degradation of callose was delayed. Furthermore, 103 differentially expressed genes(DEGs) related to the advance degeneration of tapetum cells and callose were found in the expression levels, including 22 transcription factors(TFs). In particular, two β-glucanase genes(endo-1,3(4)-β-glucanase, exo-β-glucanase) responsible for callose degeneration were significantly down-regulated. These results suggested that pollen abortion may occur at mononuclear stage and that early degeneration of tapetum cells resulted in a significant down-regulation of β-glucanase genes. As a result, the callose to cover microspores impedes the formation of pollen walls, which may possibly lead to pollen abortion.展开更多
文摘Albinism is a common problem encountered by researchers in anther/microspore cultures of cereal crops. The present study investigates the effects of temperature variations on embryogenesis of wheat (Triticum aestivum L.) microspores. Following a cold (4°C - 13°C) vs. heat (33°C) shock to wheat tillers, microspores were isolated and cultured in a liquid medium to obtain embryoids. Data on embryogenic microspore%, embryoid yield, plant regeneration% and green plant% were collected and analyzed. Cold pretreatment of 4°C or 10°C for a period of 6 or 10 days were more effective than other cold temperature regimes in inducing microspore embryogenesis. The heat shock of 33°C yielded the highest numbers of embryogenic microspores and embryoids. The albino-prone genotypes produced significantly higher green plant% following optimal cold shock, as compared to the standard 33°C heat shock. Results from present study suggest that cold shock may be a desirable alternative for germplasm that produce lower green plant% using heat shock. Lowered incubation temperature during embryoid development did not result in higher green plant.
基金supported by the National Natural Science Foundation of China(Grant No.30471407)the Graduate Student Foundation of Beijing Forestry University(No.0@008).
文摘Populus bolleana is a variety of P. alba, commonly used in poplar breeding programs in China. Developmental biology that involves staminate flowers, microsporogenesis and microgametogenesis ofP. bolleana is essential for Populus improvement in cross breeding for better characteristics in sexual reproduction. Flower morphology and pollen development were described and illustrated using anatomical, sectioning and stain-clearing techniques. The results show that microsporocytes undergo a regular meiotic process, but some multi-nucleate microspores occur at the microspore stage. It takes five days for microsporocytes to develop to mature pollen by forcing flower branches under greenhouse conditions. Additionally, an important relationship was found between stages of meiosis and anther colors. Microspore tetrads formed when the anther color turned yellow, whereas, when the pollen matured, the anther was red and the tapetum degenerated completely. When mature pollen grains are formed, flower buds develop into male catkins. In the end, filament elongated and pollen grains were released from dehisced anthers.
文摘Most aspects of microspore culture protocol have the capacity to cause stress to microspores, hence, less stressful treatments might be required to avoid deleterious effects. In stressed plants, polyamines and trehalose can act as compatible solutes or osmoprotectants by stabilizing proteins and biological membranes. To improve green plant regeneration in wheat microspore culture, this study assessed the effects of polyamines (putrecine, spermidine, spermine) and trehalose on androgenic response namely embryogenesis, green plant regeneration and ploidy of green plants regenerated in three spring wheat genotypes. Microspores of the genotypes produced significant numbers of embryos and green plants among polyamine treatments but trehalose had no effect (P ≤ 0.05). Polyamine treatments for 30 min generally produced more green plants per 100 microspores than the 60 min treatments in all three genotypes. At least three out of twelve polyamine treatments in each genotype improved the production of double haploid plants and seed setting in regenerants. Wheat genotype, concentration and duration of polyamine treatment had significant impact on embryogenesis and regeneration of green plants in this study. The study also showed that polyamines could be used to accelerate cultivar development in wheat breeding.
文摘Doubled haploid production via microspore culture is a technique known to accelerate crop breeding by shortening the breeding cycle through achieving homozygosity in one generation. Prior research observed that some embryogenic microspores aborted their development before reaching the embryoid stage. Such embryogenic abortion reduces embryoid yield, making microspore cultures less efficient. The present research aims at identifying stages during which microspore development is susceptible to embryogenic abortion. Information gained through delineation of the developmental dynamics of microspores in culture could be used to improve the efficiency of microspore culture. Embryogenic microspores were isolated from stress-treated wheat (Triticum aestivum L.) tillers and cultured in liquid medium. The development of embryogenic microspores was monitored over a 35 day period. At day 7, 10, 14, 21, 28, and 35, the developing microspores were counted and categorized into multicellular structures, pre-embryoids, immature embryoids and mature embryoids. The results showed that 44% - 62% of embryogenic microspores halted their development before the mature embryoid stage. Of these aborted embryogenic microspores, 21% - 33% terminated as multicellular structures, 16% - 19% arrested their development as pre-embryoids, and 7% - 10% halted development as immature embryoids. Identifying factors that are responsible for embryogenic abortion and finding remedy to the issue will help improve the efficiency of doubled haploid production.
基金The whole genome re-sequencing clean reads datasets were deposited in NCBI database with the accession ID“SAMN32366957”to“SAMN32366969”under the project ID“PRJNA915006”.
文摘Induced mutations are important for genetic research and breeding.Mutations induced by physical or chemical mutagenesis are usually heterozygous during the early generations.However,mutations must be fixed prior to phenotyping or field trials,which requires additional rounds of self-pollination.Microspore culture is an effective method to produce double-haploid(DH)plants that are fixed homozygotes.In this study,we conducted ethyl methanesulfonate(EMS)-induced mutagenesis of microspore cultures of barley(Hordeum vulgare)cultivar‘Hua30’and landrace‘HTX’.The EMS concentrations were negatively correlated with the efficiency of callus induction and the frequency of mutant plant regeneration.The two genotypes showed different regeneration efficiencies.The phenotypic variation of the regenerated M1 plants and the presence of genome-wide nucleotide mutations,revealed by whole-genome sequencing,highlight the utility of EMS-induced mutagenesis of isolated microspore cultures for developing DH mutants.Genome-wide analysis of the mutation frequency in the regenerated plants revealed that a considerable proportion of mutations resulted from microspore culture(somaclonal variation)rather than EMS-induced mutagenesis.In addition to producing a population of 1972 homozygous mutant lines that are available for future field trials,this study lays the foundation for optimizing the regeneration efficiency of DH plants and the richness of mutations(mainly by fine-tuning the mutagen dosage).
文摘By using DNA-specific fluorescent dye and a confocal laser scanning microscope, the present study was designed to investigate the cytological characteristics of dedifferentiating initiation during pretreatment and em-bryogenesis during culture in freshly-isolated microspores of barley, and the difference in main developmental pathway between freshly-isolated and cold-treated microspores. The results revealed that ( i ) freshly-isolated microspores started the initiation within 12 h of mannitol pretreatment, whose main cytological characteristics were that: cell vol-ume was obviously extended; the volume of nuclei and nucleoli were also greatly increased; nucleoli were extremely clear and highly condensed; N/C ratio was very high; ( ii ) all the pretreatment methods led to the initiation of the mi-crospores, thus triggering the embryogenic process; ( iii ) pretreatment methods influenced the main developmental pathway of microspores by changing the pattern of the first mitosis. The cold-treated
基金partially supported by the Natural Science Foundation of China (#31900186,#32260050)Yunnan Fundamental Research Projects (Grant NO.202301BF07001-016)the Glory Light International Fellowship for Chinese Botanists at Missouri Botanical Garden (MO) to X.M.Zhou
文摘Selaginella is the largest and most taxonomically complex genus in lycophytes.The fact that over 750 species are currently treated in a single genus makes Selaginellales/Selaginellaceae unique in pteridophytes.Here we assembled a dataset of six existing and newly sampled plastid and nuclear loci with a total of 684 accessions(74%increase of the earlier largest sampling)representing ca.300 species to infer a new phylogeny.The evolution of 10 morphological characters is studied in the new phylogenetic context.Our major results include:(1)the nuclear and plastid phylogenies are congruent with each other and combined analysis well resolved and strongly supported the relationships of all but two major clades;(2)the Sinensis group is resolved as sister to S.subg.Pulviniella with strong support in two of the three analyses;(3)most morphological characters are highly homoplasious but some characters alone or combinations of characters well define the major clades in the family;and(4)an infrafamilial classification of Selaginellaceae is proposed and the currently defined Selaginella s.l.is split into seven subfamilies(corresponding to the current six subgenera t the Sinensis group)and 19 genera(the major diagnosable clades)with nine new species-poor genera.We support the conservation of Selaginella with a new type,S.flabellata,to minimize nomenclatural instability.We provide a key to subfamilies and genera,images illustrating their morphology,their morphological and geographical synopses,a list of constituent species,and necessary new combinations.This new classification will hopefully facilitate communication,promote further studies,and help conservation.
文摘The formation of mature and fertile pollen grains, taking place inside the anther, depends on supply of assimilates, in the form of sucrose, provided mainly by the leaves. Data is limited, however, with respect to the understanding of sucrose metabolism in microspores and the supporting tissues. The aims of the present work were to 1) follow the changes in total and relative concentrations of sucrose, glucose, fructose and starch in the stamen parts and microspores up until anthesis, 2) follow the activities of sucrose-metabolism-related enzymes, in the anther walls fraction and microspores of the crop plant tomato. Sucrose was found to be partially cleaved in the filament, decreasing by more than twofold in the anther wall layers and the locular fluid, and to accumulate in the mature pollen grains, constituting 80% of total soluble sugars. Thus, sucrose was both the starting sugar, supporting microspore development, and the main carbohydrate accumulated at the end of the pollen-development program. The major invertase found to be active in both the anther wall layers and in maturing microspores was cell-wall-bound invertase. High fructokinase 2 and sucrose phosphate synthase activities during pollen maturation coincided with sucrose accumulation. The potential importance of sucrose accumulation during pollen dehydration phase and germination is discussed.
基金supported by the Youth Talent Development Plan of Shanghai Municipal Agricultural System,China(No.20180103)the National Key Research and Development Program of China(No.2018YFD0100602)the Youth Talent Running Plan of Shanghai Academy of Agricultural Sciences,China(No.2017020)。
文摘To facilate breeding process of Brassica napus,a microspore culture and molecular marker-assisted screening combined system were proposed in this research.At early flowering stage,F1 offspring of hybridized combination HY15 A×HF06 was used as donor for microspore culture to analyze effects of colchicine concentration on embryogenic and diploid rates of microspore.Treatment with 50 mg/L colchicine resulted in embryogenic rate of3.56 embryos/bud,which was substantially higher than control(0.78 embryos/bud).A total of 1,387 embryos and 862 single plants were obtained after induction culture.Ploidy detection was performed for the regenerated plants by flow cytometry.Diploid rates of microspores treated with 50 mg/L and 70 mg/L colchicine were 17.2%and 21.0%respectively,which was significantly higher than control(10.5%).Totally 108 single plants that doubled successfully were randomly selected and screened using molecular marker BE10.Approximately 54 of108 plants generated a 305 bp amplification product,whereas the other 54 plants showed a 398 bp band,thereby satisfying 1:1 separation ratio(x0.052=0.0093).These coincided with field identification results.Findings of this study indicated that homozygous breeding material could be obtained by microspore culture in a short time,thereby remarkably accelerate breeding.
文摘Effects of five incubation temperatures (25℃ ,28℃ ,31℃ ,35℃ and 37℃ for 24hours) and four incubation time periods (0.4,16 and 24 hours at 35℃) on isolated microspore culture of Chinese cabbage were studied. The results showed that cultured microspores of Chinese cabbage developed into embryos at all the incubation temperatues from 28℃ to 37℃ ,but the best response to high temperature occured at 35℃. Among the four kinds of time periods, the highest yield of embryos was obtained at the 24h treatment. Therefore, the isolated microspore culture of Chinese cabbege ran be efficiently carried out at 35℃ for 24 hours.
文摘Studies of barley and maize indicate that chromosome doubling occurs via nuclear fusion during an early stage of microspore embryogenesis, but the time and mechanism by which chromosome doubling occurs in bread wheat (Triticum aestivum) remains undetermined. The purpose of this study was to determine the relative time during induction culture when chromosome doubling may occur in wheat, and to identify early indicators for doubled haploid microspores. Microspore nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) and observed under a fluorescent microscope on the day of isolation, three days after isolation, and six days after isolation. The change in the percentage of microspores containing a single small nucleus, two small nuclei, a single enlarged nucleus, and three or more nuclei was then tracked throughout the six-day period. Ploidy levels were estimated by determining the cross-sectional area and number of nucleoli in microspores containing small and large nuclei then comparing the results of each respective cell-type. The percentage of microspores containing enlarged nuclei increased throughout the six-day test period, and the percentage of binucleated microspores containing small nuclei decreased. Comparison of the changes in average percentage of microspores containing a single small nucleus, binucleated microspores, microspores containing a single large nucleus, and multinucleate microspores on days 0, 3, and 6 indicates that nuclei classified as “small” are likely haploids and nuclei classified as “large” are doubled haploids. The percentage of microspores with enlarged nucleus (nuclei) during the first six days of induction culture could be used as an early indicator for the frequency of chromosome doubling in wheat microspore culture.
基金This work was supported by the National Natural Science Foundation of China(31771750,31730063)National Key Research and Development Project(2017YFD0100201,2017YFD0100202)Natural Science Foundation of Chongqing,China(cstc2018jcyjAX0424).
文摘The mitogen-activated protein kinase(MAPK)cascade is important in stress signal transduction and plant development.In the present study,we identified a rice(Oryza sativa L.)mutant with reduced fertility,Oryza sativa mitogen-activated protein kinase 6(osmapk6),which harbored a mutated MAPK gene.Scanning and transmission electron microscopy,quantitative RT-PCR analysis,TUNEL assays,RNA in situ hybridization,longitudinal and transverse histological sectioning,and map-based cloning were performed to characterize the osmapk6 mutant.The gene OsMAPK6 was expressed throughout the plant but predominantly in the microspore mother cells,tapetal cells,and microspores in the anther sac.Compared with the wild type,the total number of microspores was reduced in the osmapk6 mutant.The formation of microspore mother cells was reduced in the osmapk6 anther sac at an early stage of anther development,which was the primary reason for the decrease in the total number of microspores.Programmed cell death of some tapetal cells was delayed in osmapk6 anthers and affected exine formation in neighboring microspores.These results suggest that OsMAPK6 plays pivotal roles in microspore mother cell formation and tapetal cell degradation.
基金Supported by Key New Product Project of Science and Technology Department of Yunnan Province(2015BB007,2012BB017)International Cooperation Program of Science and Technology Department of Yunnan Province(2014IA016)+2 种基金Science and Technology Specific Project for Enriching People and Strengthening County Economy of Science and Technology Department of Yunnan Province(2014EB033)National Large Vegetable Industry Technology System(CARS-25-G-45)New Vegetable Variety Collaborative Project of Agriculture Department of Yunnan Province[YCN(2012)58]
文摘Isolated micmspore culture was carried out with Brassica juncea to study the effects of sterilization methods, culture period of embryos, hormone formula and the concentration of aetivatod carbon on embryogenesis, and the concentration of NAA on induction of roots. The results showed that the embryogenesis on medi- um subjected to filter sterilization is better than medium subjected to autoclaved sterilization. The embryos cultured for 15 d had the highest plantlat regenexafion rate (52%). NLN-13 liquid medium including 0. 1mg/L 6-BA and 0.2 g/L activated carbon significantly improved the planflet regeneration rate. 1/2 MS inclu- ding 0.3 mg/L NAA had the highest plantlet regeneration rate ( 100% ). Key words Brassica juncea ; Isolated micmspore culture ; Embryogenesis ; Planflet regeneration
基金supported by the Natural Science Foundation of Hebei Province,China(C2004000747)
文摘During meiosis in flowering plants,degradation of the callose wall in tetrads releases newly produced microspores,which develop into mature pollen grains.In this study,we identified zbs1,a male-sterile mutant of naked oat(Avena nuda L.)that displayed complete spikelet sterility due to inviable mature pollen.The abnormal pollen grains originated from microspores with a defective callose wall and cell plate during meiosis.The defective callose wall and cell plate of the zbs1 mutant were detected by the labeling of cell wall epitopes(β-1,3-glucan) with immunogold during meiosis,and an abnormal chromosome configuration was observed by propidium iodide staining.The mature pollen grains of the zbs1 mutant were irregular in shape,and abnormal germination was observed by scanning electron microscopy.Together,our results indicate that the cause of male sterility in zbs1 is abnormal meiosis.
基金Key Technology R&D Project of Shanghai Agriculture-developed with Science&Technology Program,China(Grant No.2018(1-2))the Natural Science Foundation of Shanghai,China(Grant No.19ZR1417000)+2 种基金the National Key Research and Development Program of China(2018YFD1000702-5)the Climbing Plan of Shanghai Academy of Agricultural Sciences(2022)the earmarked fund for China Agriculture Research System of MOF and MARA(CARS-05-01A-02).
文摘Phytohormones play important roles in plant growth and development,and polyploids are thought to be an important method for plant breeding.However,the relationship between ploidy and phytohormone is still unclear.In this study,barley at three ploidy levels were produced by microspore culture.Therefore,we further analyzed the phytohormone content in the shoots and roots of the three kinds of barley materials to study the effect of ploidy on phytohormones accumulation and distribution.The results showed that Abscisic acid(ABA),gibberellin(GA),jasmonic acid(JA),auxin(IAA),salicylic acid(SA)and cytokinin(CTK)were successfully determined in shoots and roots using LC-MS(liquid chromatography mass spectrometry).By comparing the shoots of the haploid and diploid plants,it was found that the distribution trend of the six phytohormones was consistent,and another consistent trend was found in the roots of the diploid and tetraploid plants.In addition,we further analyzed the shoot/root ratio of the different phytohormones to identify the potential differences for haploid,diploid and tetraploid.Here,the relationship between ploidy and phytohormone we provided would provide new insights into understanding the new phenotypes that occur in polyploid species.
文摘There were four microsporanges in the mature anther of B. rossica Fedtsch. et Flerov. The development of the anther wall belonged to dicotyledonous type. The tapetum cell was dichotypic. Simultaneous cytocinesis in the microspore mother cells formed tetrad by reduction division and the array of microspore tetrads was different. There were tetrahedron type and bilateral symmetry type. The male gametophyte was 2-cell. The mature pollen was global and tricolporate. All those results were investigated under both light microscope (LM) and electron microscope (EM)
基金funded by National Science Center in Poland Grant (2015/18/M/NZ3/00348) to Iwona·Zursupported by Czech Science Foundation Grant (21-02929S) to Ales Pecinka+2 种基金European Regional Development Fund project TANGENC (CZ.02.01.01/00/ 22_008/0004581)funded by Ad Agri F (CZ.02.01.01/00/22_008/0004635)supplied by the project “e-Infrastruktura CZ” (e-INFRA CZ LM2018140) supported by the Ministry of Education, Youth and Sports of the Czech Republic
文摘Plant formation from in vitro-cultivated microspores involves a complex network of internal and environmental factors.Haploids/doubled haploids(DHs)derived from in vitro-cultured microspores are widely used in plant breeding and genetic engineering.However,the mechanism underlying the developmental switch from regular pollen maturation towards microspore-derived plant regeneration remains poorly defined.Here,RNA-sequencing was employed to elucidate the transcriptional landscapes of four early stages of microspore embryogenesis(ME)in barley cultivars Golden Promise and Igri,which exhibit contrasting responsiveness to microspore-derived plant formation.Our experiments revealed fundamental regulatory networks,specific groups of genes,and transcription factor(TF)families potentially regulating the developmental switch.We identified a set of candidate genes crucial for genotype-dependent responsiveness/recalcitrance to ME.Our high-resolution temporal transcriptome atlas provides an important resource for future functional studies on the genetic control of microspore developmental transition.
基金supported by the Priority Academic Program Development of Jiangsu Higher Education Institutionsthe Fundamental Research Funds for the Central Universities(Grant No.KYTZ201602)+1 种基金the High Level Talent Project of the Top Six Talents in Jiangsu(Grant No.NY-077)the earmarked fund for germplasm resources of Nanjing Agricultural University(Grant No.KYZZ201920)。
文摘Lily was grown worldwide as a fresh cutting flower because of its colorful petals, but its anther contained a large number of pollen grains that cause serious pollen contamination, however, pollen abortion can effectively reduce the level of pollen pollution. Our analysis aims to use cytological observation to detect the critical stage when pollen abortion occurs and to provide comprehensive gene expression information at the transcriptional level. The result showed that pollen abortion in ‘Little Kiss’ began at the mononuclear stage and the callose that covers the microspores failed to degenerate when young pollens were released from the tetrads. In addition, compared with the normally developed one,the tapetum of ‘Little Kiss’ degraded in advance while the degradation of callose was delayed. Furthermore, 103 differentially expressed genes(DEGs) related to the advance degeneration of tapetum cells and callose were found in the expression levels, including 22 transcription factors(TFs). In particular, two β-glucanase genes(endo-1,3(4)-β-glucanase, exo-β-glucanase) responsible for callose degeneration were significantly down-regulated. These results suggested that pollen abortion may occur at mononuclear stage and that early degeneration of tapetum cells resulted in a significant down-regulation of β-glucanase genes. As a result, the callose to cover microspores impedes the formation of pollen walls, which may possibly lead to pollen abortion.