The transcriptional landscape of the developmental switch from regular pollen maturation towards microspore-derived plant regeneration in barley

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.

Studies on Enhancing Yield of Embryos in Microspore Culture of Brassica napus L.

In ecological zone of Chengdu, Sichuan, microspore culture was carried out in Brassica napus L. to study the influencing factors on microspore culture. The results showed that the temperature on microspore formation stage, day and night temperature, disinfection solution of buds, cultivation concentration on microspore and strain-age were both important influencing factors on microspore culture. At a temperature below 5 ℃ or above 20 ℃, the material had a much lower embryo producing rate or even could not produce any embryo, but at the optimum temperature of 10 -15 ℃ the embryo yield was up to 300 pieces per bud; the best cultivation effect appeared when 0. 1% HgCl2 was used for disin- fection; the best density of microspore was 3 -4 buds per dish; In 2009, while strain-age was from 125 d to 150 d, the microspore embryo yield increased as strain-age increased. When stain-age was 150 days, the microspore embryo yield was up to the highest, but the yield declined after 150 days.

Embryogenesis and Development of Isolated Microspore in Chinese Cabbage

[Objective] The aim was to observe embryogenesis and development of isolated microspores in Chinese cabbage. [Method] Chinese cabbage F 1 hybrids were used as the experimental materials, and optical microspore was employed to observe the embryogenesis and development of isolated microspores. [Result] Cells swelled after heat shock treatment, which was the critical factor of embryoid induction. Three pathways equal division, unequal division and germination of microspores were discovered to lead to the embryogenesis from isolated microspores after swelling. Microspore could grow directly to embryoid through germination path way. Equally divided microspores formed the original embryos after successive multiple equal divisions. Original embryos could develop into cotyledon-shaped embryos via globular, heart-shaped and torpedo-shaped embryos. The large one of the two cells from unequally divided microspores continued to divide and finally formed a polar embryoid. [Conclusion] The study will provide cytological basis for high induction frequency and embryoid of Chinese cabbage.

Establishment on Isolated Microspore Culture Optimized System for Restorer of New Cytoplasmic Male Sterile (NER) of Brassica napus L.

[Objective]The aim was to establish optimized system for NER isolated microspore culture of Brassica napus L.. [Method]Twenty varieties of NER of Brassica napus were grown under uncontrolled temperature and light conditions,and their isolated microspore from anthers were used as explants in vitro culture. The influencing factors of microspore culture were preliminarily studied. [Result]The difference of genotypes was important influencing factors to embryoid yield. The embryoid yield increased by supplementing with 6-BA and NAA,culturing in solid-liquid double layer medium with activated charcoal; The difference was not significant of embryoid yield between culturing in medium supplemented with colchicines and the CK. The rates of cotyledonous embryoids directly developed into normal plantlets increased through enriching with 0.1-0.2 mg/L NAA and being treated on slim illumination two days before being inducted into normal plantlets. [Conclusion]The technical system of microspore culture of restorer of new cytoplasmic male sterile （NER） was established,which and lays a foundation for accelerating genotype purification of NER introgressive line.

Embryogenesis and Development of Isolated Microspores in Raphanus sativus

[Objective] The aim was to observe embryogenesis and development of isolated microspores of Raphanus sativus.[Method] By using Chinese radish variety Shandong Huaye Xinlimei as the experimental materials,the caryocinesia features and development of isolated microspores in radish were studied.[Result] Two types of cell division appeared in the isolated microspore：unequal division and equational division.The unequal divided microspores had two different size nucleuses,while the equational divided microspores had two same size nucleuses.The equational divided microspores developed to 4-cell structure,multi-cell structure,ball-shaped embryos,heart-shaped embryos,torpedo-stage embryos and cotyledon-stage embryos.[Conclusion] This result will provide cytological basis for the isolated microspore culture of radish.

Pollen development and multi-nucleate microspores of Populus bolleana Lauche

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.

Assessment of Polyamines and Trehalose in Wheat Microspores Culture for Embryogenesis and Green Regenerated Plants

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.

Effects of different high temperatures and incubation time periods on embryogenesis in isolated microspore culture of Chinese cabbage (Brassica campestris L. ssp pekinensis)

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.

Nuclear Fusion during Early Stage of Microspore Embryogenesis Indicates Chromosome Doubling in Wheat (Triticum aestivum)

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.

Isolated Microspore Culture and Plant Regeneration of Leaf Mustard(Brassica juncea(L.)Czern.et Coss.)

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

Morphological Characteristics of Floral Organ at Different Microspore Developmental Stage in Balsam Pear （Momordica Charantia L.）

The relationship between microspore developmental stages and morphological characteristics of floral organ in balsam pear was studied. The results showed that flower buds developed a series of morphological changes at different microspore developmental stages, and morphological characteristics of floral organ were significantly different when sampling from different part or cultivar of balsam pear. Anther at late-uninucleate stage was best for culture, in which stage flower buds swelled obviously, and white floss on the surface reduced. In addition, calyxes were obvious but did not spread, and the most of anthers were light green.

OsMAPK6 Affects Male Fertility by Reducing Microspore Number and Delaying Tapetum Degradation in Oryza sativa L.

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.

Extra-Large Seed Germplasms of Brassica Napus Created through Microspore Culture

Thousand Seed Weight （TSW） is one of the major yield components of rapeseed （Brassica napus L.）. Here reports an extra-large seed germplasm GM01 which was obtained through isolated microspore culture. Three-way cross was made： H8--a Yunnan spring early-maturing rapeseed variety, ＂Legeney＂--a Canadian canola variety and ＂020010＂--a semi-winter late-maturing rapeseed variety. One hundred and forty eight doubled hyploid lines were obtained from the F~ plants of three-way cross through isolated mierospore culture. Among them, the TSW of GM01 amounted to 8.68 g and the TSW of 53 lines were above 5.0 g. The TSWs of GM01 were relatively stable among the multi-location field trials from 2007 to 2014 with variation being only 10%-15% among the locations and years. Compared with H8, GM01 had larger flowers, stigmas, siliques and seed diameters, but less branches, siliques per plant and seeds per silique.

Differential Effects of Cold and Heat Shock on Embryogenic Induction and Green Plant Regeneration from Wheat (Triticum aestivum L.) Microspores

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.

Developmental Dynamics of Wheat (<i>Triticum aestivum</i>L.) Microspores under Culture

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.

Phytohormones Accumulation and Distribution in Shoots and Roots of Haploid,Diploid and Tetraploid Barley Seedlings Derived from Microspore Culture

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.

A Microspore-derived Rice Transformation System and Dissection of the Complex RCg2 Promoter

Meaningful evaluation of promoter activity following dissection of various promoter elements requires the production of many transgenic rice plants. For such purposes, we have developed highly effective Agrobacterium-

Transgene Homozygosity Following Particle Bombardment of Haploid Barley Microspores

Information from previously published studies that are basic to this study is: 1) Following isolated barley microspore culture, around 80% of the resulting barley plants are completely fertile and genetically

红树林内生拟盘多毛孢Pestalotiopsis microspora次级代谢产物及其抗肿瘤活性

【目的】以红树林内生拟盘多毛孢(Pestalotiopsis microspora)为对象挖掘结构特殊的甾体类化合物,分析其化学结构和抗肿瘤活性,为生物活性先导化合物提供模式结构。【方法】对课题组前期发现的活性显著的拟盘多毛孢(KX631718)添加海盐培养发酵,获得粗提物。采用柱层析结合高效液相,分离得到代谢产物;利用核磁共振波谱(NMR)、质谱(MS)等波谱解析方法以及与文献数据对照,鉴定化合物结构;对化合物进行抗肿瘤活性评价。【结果与结论】分离得到8个已知甾体类化合物,经鉴定化合物Ⅰ~Ⅷ分别为3-乙酰基-β-谷甾醇(3-O-acetyl-β-sitosterol)、7β-hydroxysitosterol、3β-hydroxy-5α-stigmastan-6-one、citreoanthrasteroid B、stigmast-4-en-6-ol-3-one、5,8-epidioxy-5α,8α-ergosta-6,22E-dien-3β-ol、β-谷甾醇(β-sitosterol)、7-ketositosterol,其中Ⅰ~Ⅳ为首次分离自拟盘多毛孢属真菌。生物合成途径推测拟盘多毛孢以常见的甾醇为前体,通过氧化、还原和重排反应,可代谢产生抗肿瘤先导物Sdy-1和结构特殊的甾醇化合物Ⅳ。生物活性评价发现,化合物Ⅰ、Ⅱ、Ⅴ和Ⅶ具有微弱的抗肿瘤活性。生物合成途径推测拟盘多毛孢菌可利用传统甾体类为前体代谢产生抗肿瘤先导化合物Sdy-1。

Non-separated microspores 1 controls male meiotic callose deposition at the cell plate in rice

In flowering plants,callose(b-1,3-glucan)plays a vital role in pollen development,especially in the separation and development of microspores.However,the molecular mechanism of callose deposition during rice pollen development remains unclear.In this study,we isolated and characterized a novel rice pollen defective mutant,non-separated microspores 1(nsm1),which produced“dyad”or“tetrad”pollen grains.Cytological analysis indicated disrupted interstitial callose deposition at the cell plate of dyads and tetrads in nsm1 pollens.This disruption caused sporopollenin to be massively deposited outside of the junction where the interstitial callose wall connected with the peripheral callose wall,or unevenly distributed on the interstitial pollen primexine at the late meiosis stage.Consequently,an excess tectum-like layer was formed outside of the junction,connecting with the tectum of two microspores during later developmental stages,which prevented the separation of microspores.Additionally,in the linkage area,the tectum of two microspores gradually fused or degenerated,resulting in a decreased contact area between microspores and the anther locule.Therefore,the defect in callose deposition resulted in unsuccessful separation of microspores,abnormal deposition of pollen exine,and also affected the accumulation of materials in microspores,resulting in pollen semi-sterility.NSM1,encoding a callose synthase located in the Golgi body,is ubiquitously expressed in anthers with its peak expression at the young microspore stage.The in vitro enzyme activity assay confirmed that NSM1 possesses callose synthase activity,and the enzyme activity in the nsm1 mutants was significantly reduced.Phylogenetic analysis indicated that NSM1 and its orthologs play a highly conserved role in callose biosynthesis among plant species.Taken together,we propose that NSM1 plays an essential role in male meiotic callose synthesis and later pollen wall development.