A comparative light and electron microscopic analysis of microspore and tapetum development in fertile and cytoplasmic male sterile radish

To gain further insight into the abortive stages and ultrastructural changes leading to pollen degeneration of a novel cytoplasmic male sterile radish 805A, we compared differences of cellular and subcellular structure of sterile anther with fertile anther by light and electron microscopy analysis. Two types of locule degeneration in sterile anther were detected, of which the time of degeneration occurred and completed was different. In type I, abnormality of pollen mother cells (PMCs) and tapetal cells, including condensation of cytoplasm and large vacuoles within tapetal cells, was shown at PMC stage. In type II, meiosis and early tetrad stage progressed normally except for large vacuoles that appeared in tapetal cells. Ultrastructural alterations of the cellular organization were observed in the type II locules, such as chromatin condensation at the periphery of the nucleus and degeneration of the karyotheca, compared with normal pollen development. The results suggested that the cytoplasmic male sterility anther degeneration was probably caused by dysfunctions of tapetum and vacuolation of tapetum, PMCs, and microspores. Thus, the identical factors, which induced CMS in the same cytoplasmic and nuclear genetic background, might affect development of tapetum and microspore at different stages during the cytoplasmic male sterile 805A anther development.     

Abnormal Vacuolization of the Tapetum During the Tetrad Stage is Associated with Male Sterility in the Recessive Genic Male Sterile <Emphasis Type="Italic">Brassica napus</Emphasis> L. Line 9012A

In the recessive genic male sterile line 9012A of Brassica napus, pollen development is affected during the tetrad stage. According to the light and electron microscopy analysis of tapetal cells and tetrads, the sterile tapetal cells swelled with expanded vacuoles at the early tetrad stage and finally filled the center of the locules where a majority of tetrads encased with the thick callose wall collapsed and degraded. We suggested that an absence of callase, which is a wall-degrading enzyme stored in the vacuoles of tapetal cells before secretion, resulted in the failure of tetrad separation. Moreover, transmission electron microscopy analysis showed that the secretory tapetal cells were not observed in sterile anthers, which indicated that the transition of the tapetum from the parietal type to the secretory type was probably aberrant. In plants, degeneration of the tapetum is thought to be the result of programmed cell death (PCD). PCD of tapetal cells was investigated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay and signals indicative of deoxyribonucleic acid fragmentation were detected much earlier in sterile anther than in fertile anther. This suggests that tapetal breakdown does not occur by the normal procession of PCD and might be following an alternative mechanism of unscheduled apoptosis in line 9012A. This research supports the hypothesis that premature PCD is associated with male sterility in B. napus.     

Cytological and ultra-structural study on microsporogenesis of cytoplasmic male sterility in <Emphasis Type="Italic">Raphanus sativus</Emphasis>

The cytological development of microspores and tapetum in cytoplasmic male sterile (CMS) line A₁₄ and its maintainer B₁₄ in radish were studied using light- and transmission electron microscopy (LM and TEM). The microspores of the CMS line began to abort soon after they were released from tetrads in pollen sacs with light microscopy investigation, while abnormal behavior of pollen mother cells (PMC) were observed during its meiotic stage in its ultra-structural study, including degeneration of organelles and irregularity of nuclear membrane. At the same time, development of tapetal cells was similar to that of the maintainer. With further development of the anther, the tapetal cells of CMS line showed an abnormal increase in size and other appearances, such as fewer organelles and indistinct cytoplasm. The microspores of the CMS line were always distinguishable from the maintainer line with irregular structure, more osphilic deposits and abnormal exine. It is inferred that abortion of microspores is attributed to mutation of genes controlling male sterility, which further leads to hypertrophy of tapetum and destruction of ultra-structure.     

A light and electron microscopy analysis of the events leading to male sterility in Ogu-INRA CMS of rapeseed (Brassica napus)

Ogura cytoplasmic male sterility (CMS) occurs naturally in radishand has been introduced into rapeseed (Brassica napus) by protoplastfusion. As with all CMS systems, it involves a constitutivelyexpressed mitochondrial gene which induces male sterility tootherwise hermaphroditic plants (so they become females) anda nuclear gene named restorer of fertility that restores pollenproduction in plants carrying a sterility-inducing cytoplasm.A correlative approach using light and electron microscopy wasapplied to define what stages throughout development were affectedand the subcellular events leading to the abortion of the developingpollen grains upon the expression of the mitochondrial protein.Three central stages of development (tetrad, mid-microsporeand vacuolate microspore) were compared between fertile, restored,and sterile plants. At each stage observed, the pollen in fertileand restored plants had similar cellular structures and organization.The deleterious effect of the sterility protein expression startedas early as the tetrad stage. No typical mitochondria were identifiedin the tapetum at any developmental stage and in the vacuolatemicrospores of the sterile plants. In addition, some strikingultrastructural alterations of the cell's organization werealso observed compared with the normal pattern of development.The results showed that Ogu-INRA CMS was due to premature celldeath events of the tapetal cells, presumably by an autolysisprocess rather than a normal PCD, which impairs pollen developmentat the vacuolate microspore stage, in the absence of functionalmitochondria. Key words: Brassica napus, cell death, light and electron microscopy, mitochondria, plastids, pollen development, Ogu-INRA cytoplasmic male sterility, transgenic-restored plants, tapetum Received 30 September 2007; Revised 11 December 2007 Accepted 20 December 2007     

Reversible male sterility in transgenic tobacco carrying a dominant-negative mutated glutamine synthetase gene under the control of microspore-specific promoter

Metabolic engineering was used to disrupt glutamine metabolism in microspores in order to block pollen development. We used a dominant-negative mutant (DNM) approach of cytosolic glutamine synthetase (GS1) gene under the microspore-specific promoter NTM19 to block glutamine synthesis in developing pollen grains. We observed partial male sterility in primary transgenic plants by using light microscopy, FDA, DAPI and in vitro pollen germination test. Microspores started to die in the early unicellular microspore stage, pollen viability in all primary transgenic lines ranged from 40-50%. All primary transgenics produced seeds like control plants, hence the inserted gene did not affect the sporophyte and was inherited through the female germline. We regenerated plants by in vitro microspore embryogenesis from 4 individual lines, pollen viability of progeny ranged from 12 to 20%, but some of them also showed 100% male sterility. After foliage spray with glutamine, 100% male-sterile plants were produced viable pollen and seed set was also observed. These results suggested that mutated GS1 activity on microspores had a significant effect on normal pollen development. Back-cross progenies (T2) of DH 100% male-sterile plants showed normal seed set like primary transgenics and control plants.     

Effects of Introducing the Chimaeric Gene TA29 Barnase on the Tapetum and Pollen Development in Tobacco

The development of tapetum and pollen in transgenic tobacco (Nicotiana tabacum L. ) harboring a chimaeric gene TA29-Barnase was compared with that of the wild-type plant. The specific expression of the exogenous genes in anther led to premature tapetal degradation, which started at the early stage of meiosis and terminated at the tetrad stage. In the wild-type anthers, tapetal degradation started at the early stage of bicellular microgametophyte and ended at the later stage of pollen development. The cytological changes of tapetal degradation in the transgenic plants were characterized by vacuolization of the tapetal cells, then nuclear condensation, and consequent massive degradation of tapetal cells. Meanwhile, the pollen mother cells gradually degraded and became destroyed along with the progress of meiosis, leaving only a few which could successfully complete their meiosis to form microspores. This observation also indicated that the TA29-Barnase gene in anther was not uniformly expressed. In addition, the structural difference between the male sterility induced by exogenous gene and the natural sterile was also discussed.     

Genetic and cytological analyses of the male sterility mutation induced in a sorghum tissue culture with streptomycin

Treatment of sorghum callus cultures with 500–1000 mg/l streptomycin led to a high regeneration frequency of plants with complete or partial male sterility (MS), up to 100% of all green regenerants. The induced MS mutation (ms-str) was preserved in the F₁ and BC₁ progenies and was genetically unstable: many families produced semisterile and fertile revertants, whose progenies again contained semisterile and sterile mutants. The ms-str mutation was maintained through eight generations via selection and self-pollination of semisterile plants. The mutation was inherited as a recessive nuclear mutation in test crosses of sterile plants segregated in the progenies of fertile and semisterile revertants and was expressed only in single cases in a test cross for ms-str transfer through pollen of hybrids with restored male fertility. Recessive nuclear mutations determining a low plant height (dwarfness) and the lack of waxy bloom on the stem and leaves (bloomless) were found in male-sterile plants with the ms-str mutation. Cytological analysis of sterile plants reveal multiple abnormalities at various pollen development stages and in tapetal cells: cytomyxis, defects of chromosome conjugation, distorted cytokinesis in meiotic division II, a lack of tetrad separation, a defective formation of the microspore coat, generation of microspores with two to four nuclei, and the formation of micronuclei and large vacuoles in tapetal cells. A possible transfer of the induced cytoplasmic MS mutation into the nuclear genome and the causes of the high genetic instability are discussed.     

The Arabidopsis U–box/ARM repeat E3 ligase AtPUB4 influences growth and degeneration of tapetal cells,and its mutation leads to conditional male sterility

Pollen formation is a complex developmental process that has been extensively investigated to unravel underlying fundamental developmental mechanisms and for genetic manipulation of the male‐sterility trait for hybrid crop production. Here we describe identification of AtPUB4, a U–box/ARM repeat‐containing E3 ubiquitin ligase, as a novel player in male fertility in Arabidopsis. Loss of AtPUB4 function causes hypertrophic growth of the tapetum layer. The Atpub4 mutation also leads to incomplete degeneration of the tapetal cells and strikingly abnormal exine structures of pollen grains. As a result, although the Atpub4 mutant produces viable pollen, the pollen grains adhere to each other and to the remnants of incompletely degenerated tapetal cells, and do not properly disperse from dehisced anthers for successful pollination. We found that the male‐sterility phenotype caused by the Atpub4 mutation is temperature‐dependent: the mutant plants are sterile when grown at 22°C but are partially fertile at 16°C. Our study also indicates that the AtPUB4‐mediated pathway acts in parallel with the brassinosteroid pathway in controlling developmental fates of the tapetal cells to ensure male fertility.     

Microscopy of a cytoplasmic male-sterile soybean from an interspecific cross between Glycine max and G. soja (Leguminosae)

Cytoplasmic male sterility has been found independently in soybean three times since 1995, but no microscopic investigation has been published. The purpose of this microscopic study was to establish the developmental sequence leading to sterility in a cytoplasmic male-sterile soybean line that has been found to be stable under all environmental conditions tested and to demarcate the temporal and spatial parameters that result in degeneration of the microspores and pollen grains. Light microscopy showed an abnormal development and/or premature degeneration of the tapetum after meiosis II, but some pollen grains persisted until after microspore mitosis. The pollen grains never completely filled with reserves. Premature formation of the endothecium also was evident. Histochemical staining for water-insoluble carbohydrates revealed an abnormal pattern of starch deposition in anther walls that coincided with lack of pollen filling. Electron microscopy showed degeneration of the inner mitochondrial membrane in the tapetal cells as the first detectable change leading to cell degeneration. Subsequently, the tapetal endoplasmic reticulum exhibited atypical concentric rings. Pollen grains displayed mitochondria with unusually enlarged inner mitochondrial spaces, degraded plastids, a rudimentary intine, and no starch or lipid reserves. Results link mitochondrial degeneration, premature formation of the endothecium, and energy deprivation to male sterility.     

TA29—Barnase嵌合基因导入对烟草花药绒毡层及花粉发育的影响

比较研究了烟草(Nicotiana tabacum L.)TA29-Barnase转基因不育植株和正常植株的花药绒毡层及花粉发育的全过程。研究表明,外源基因在花药中特异表达导致绒毡层细胞的提前降解,这种降解一般在减数分裂早期开始,至四分体时期完成,而正常花药绒毡层的降解发生在二细胞雄配子体初期,至花粉发育的后期方才完成。转基因植株花药绒毡层的降解在细胞结构上表现为:最初发生细胞的液泡化,然后细胞核凝聚,最后整个细胞溃解。转基因植株的花粉母细胞则在减数分裂过程中逐渐降解、退化,只有少数花粉母细胞能够顺利完成减数分裂发育成小孢子。观察结果还表明外源基因在花药中的表达是不均一的。对转基因不育和自然败育在细胞结构上的不同表现进行了讨论。     

Abnormal Development of Tapetum and Microspores Induced by Chemical Hybridization Agent SQ-1 in Wheat

Chemical hybridization agent (CHA)-induced male sterility is an important tool in crop heterosis. To demonstrate that CHA-SQ-1-induced male sterility is associated with abnormal tapetal and microspore development, the cytology of CHA-SQ-1-treated plant anthers at various developmental stages was studied by light microscopy, scanning and transmission electron microscopy, in situ terminal deoxynucleotidyl transferasemediated dUTP nick end-labelling (TUNEL) assay and DAPI staining. The results indicated that the SQ-1-treated plants underwent premature tapetal programmed cell death (PCD), which was initiated at the early-uninucleate stage of microspore development and continued until the tapetal cells were completely degraded; the process of microspore development was then blocked. Microspores with low-viability (fluorescein diacetate staining) were aborted. The study suggests that premature tapetal PCD is the main cause of pollen abortion. Furthermore, it determines the starting period and a key factor in CHA-SQ-1-induced male sterility at the cell level, and provides cytological evidence to further study the mechanism between PCD and male sterility.     

芝麻核雄性不育系ms86-1微粉发生的细胞学观察

芝麻(Sesamum indicum)核雄性不育系ms86-1姊妹交后代表现为可育、部分不育(即微粉)及完全不育(简称不育)3种类型。不同育性类型的花药及花粉粒形态差异明显。Alexander染色实验显示微粉植株花粉粒外壁为蓝绿色, 内部为不均一洋红色, 与可育株及不育株花粉粒的染色特征均不相同。为探明芝麻微粉发生机理, 在电子显微镜下比较观察了可育、微粉、不育类型的小孢子发育过程。结果表明, 可育株小孢子母细胞减数分裂时期代谢旺盛, 胞质中出现大量脂质小球; 四分体时期绒毡层细胞开始降解, 单核小孢子时期开始出现乌氏体, 成熟花粉时期花粉囊腔内及花粉粒周围分布着大量乌氏体, 花粉粒外壁有11–13个棱状凸起, 表面存在大量基粒棒, 形成紧密的覆盖层。不育株小孢子发育异常显现于减数分裂时期, 此时胞质中无脂质小球出现, 细胞壁开始积累胼胝质; 四分体时期绒毡层细胞未见降解; 单核小孢子时期无乌氏体出现; 成熟花粉时期花粉囊腔中未发现正常的乌氏体, 存在大量空瘪的败育小孢子, 外壁积累胼胝质, 缺乏基粒棒。微粉株小孢子在减数分裂时期可见胞质内有大量脂质小球, 四分体时期部分绒毡层发生变形, 单核小孢子时期有部分绒毡层开始降解; 绒毡层细胞降解滞后为少量发育进程迟缓的小孢子提供了营养物质, 部分小孢子发育为正常花粉粒; 这些花粉粒比较饱满, 表面有少量颗粒状突起, 但未能形成覆盖层, 花粉囊腔中及小孢子周围存在少量的乌氏体。小孢子形成的育性类型与绒毡层降解是否正常有关。     

Ultrastructural aspects of cytoplasmic male sterility inPetunia hybrida

Summary Anther development of isogenic male fertile and cytoplasmic male sterile types ofPetunia hybrida cv. Blue Bedder is studied by electron microscopy. First deviation in sporogenesis of the sterile type, is observed during leptotene stage of the meiocytes. Initial aberration is represented by the presence of large vacuoles in the cytoplasm of the tapetal cells. These vacuoles reveal the first aspects of degeneration; no other ultrastructural differences are observed. Vacuolation is accompanied by the condensation of cytoplasmic organelles. The tapetal cells become distorted and ultrastructural aberrations in mitochondria do occur. The mitochondria elongate and contain several tubular cristae.Substantial evidence suggests, that cytoplasmic male sterility in petunia is encoded by the mitochondrial genome (Boeshore el al. 1983). However, before degeneration becomes manifest, no consistent ultrastructural differences in mitochondrial organization are observed.Abortion of the tapetum and the sporogenous tissue in cytoplasmic male sterile plants, generally follows a corresponding pattern. Ultimately, the cells are highly distorted, the nucleus is disrupted and the cytoplasm disorganized. Mitochondria and plastids degenerate and many lipid droplets are present.     

Effects of Petunia cytoplasmic male sterile (CMS) cytoplasm on the development of sterile and fertility-restored P. parodii anthers

The developmental defects causing cytoplasmic male sterility in Petunia parodii are described in isonuclear fertile, sterile, and fertility-restored plants using both light- and scanning electron microscopy. The aberrant development of the sporogenous tissue and tapetal layer caused by the cytoplasmic male sterile cytoplasm in both Petunia hybrida and P. parodii nuclear backgrounds is similar in onset and progression. The degeneration of the sporogenous tissue and tapetal layer of sterile anthers is first apparent late in meiosis and results in highly abnormal sterile sporogenous tissue by tetrad stage of fertile anthers. The stomium and endothecium do not show major developmental differences between fertile and sterile anthers, but the inner connective tissue of sterile anthers contained calcium crystals not found at high abundance in fertile anthers. Ovoid bodies containing magnesium and phosphorus were seen only in the vascular bundles of fertile anthers. Material prepared for the scanning electron microscope by freeze drying showed better retention of fragile morphological features, while critical-point drying permitted examination of nonvolatile structures, such as cell walls.     

太空诱变玉米核不育材料花粉败育的细胞学观察(简报)

玉米是最早利用雄性不育系生产杂交种的作物之一。在玉米T型细胞质雄性不育杂交种遭受毁灭性病害侵袭之后,科学家认识到利用细胞质雄性不育制种存在潜在的遗传脆弱性,从此试图通过多种途径来创造新的雄性不育．并对雄性不育材料的遗传多样性进行研究。空间诱变育种是80年代于我国发展起来的新技术,在农作物品种改良和种质创新上已初见成效。[第一段]     

Abnormal anther development and high sporopollenin synthesis in benzotriazole treated male sterile Helianthus annuus L

Foliar application of 1.5% benzotriazole induced 100% pollen sterility in H. annuus. Pollen abortion in treated plants was mainly associated with abnormal behaviour of tapetum. A limited number of anther locule showed early degeneration of tapetum followed by disintegration of sporogenous tissues. On the other hand, some locules showed normal development of tapetum at initial stages. However, this tapetum exhibited degenerated and non-functional cell organelles. In both these situations tapetum failed to provide proper nourishment to developing microspores. The ultrastructure of both tapetum and microspores is different from that of control material with irregularities of exine deposition, endopolyploidy of tapetal nuclei and an alteration of organelle composition being correlated with sterility. Pollen grains thus developed were devoid of nucleus and cell organelles and were complete sterile.     

基因工程雄性不育烟草及其温度敏感性

将含有抗溴苯腈基因bxn和雄性不育基因的重组载体pTA29-Barnase/bxn导入农杆菌(Agrobacterium tumefaciens)后转化烟草(Nicotiana tabacum L.),得到33个转基因植株。在27℃/23℃培养的16株中,有7株表现部分不育,另9株全部可育。而在20℃/15℃培养的17株中,12株表现不育,5株表现部分不育。部分不育的植株上同时开放可育花朵和不育花朵,不过其中的不育花朵中的花粉萌发活力很低。将在20℃/15℃条件下表现不育的12个不育株从20℃/15℃温室移至27℃/23℃温室后30d左右,其中9株表现程度不同的育性恢复现象:5株表现部分可育,另4株表现完全可育;但仍有3株表现雄性不育。雄性不育花朵的花丝变短,花药皱瘪,不散粉。细胞学观察证明,转基因植株的花药绒毡层降解提早于四分体时期,至单核小孢子时期降解殆尽。     

Expression of sunflower cytoplasmic male sterility-associated open reading frame, orf H522 induces male sterility in transgenic tobacco plants

Sterility in the universally exploited PET1-CMS system of sunflower is associated with the expression of orfH522, a novel mitochondrial gene. Definitive evidence that ORFH522 is directly responsible for male sterility is lacking. To test the hypothesis that ORFH522 is sufficient to induce male sterility, a set of chimeric constructs were developed. The cDNA of orfH522 was cloned in-frame with yeast coxIV pre-sequence, and was expressed under tapetum-specific promoter TA29 (construct designated as TCON). For developing control vectors, orfH522 was cloned without the transit peptide under TA29 promoter (TON) or orfH522 was cloned with or without transit peptide under the constitutive CaMV35S promoter (SCOP and SOP). Among several independent transformants obtained with each of the gene cassettes, one third of the transgenics (6/17) with TCON were completely male sterile while more than 10 independent transformants obtained with each of the control vectors were fertile. The male sterile plants were morphologically similar to fertile plants, but had anthers that remained below the stigmatic surface at anthesis. RT-PCR analysis of the sterile plants confirmed the anther-specific expression of orfH522 and bright-field microscopy demonstrated ablation of the tapetal cell layer. Premature DNA fragmentation and programmed cell death was observed at meiosis stage in the anthers of sterile plants. Stable transmission of induced male sterility trait was confirmed in test cross progeny. This constitutes the first report at demonstrating the induction of male sterility by introducing orfH522 gene that could be useful for genetic engineering of male sterility. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Inducing male sterility of tomato using two component system

Production of hybrid seeds and pursuing heterosis breeding of many crops have been accomplished using male sterile lines. However, not all crops have valuable male sterile lines due to instability of male sterility and absence of a restorer system. In this study, male sterile lines have been induced using a two-component system. The extracellular ribonuclease Barnase was cleaves into two inactive yet complementary fragments, designated as ??Bn-5?? and ??Bn-3??. Both components were controlled by a TA29 promoter. They were transferred into the tomato inbred line ??Yellow tomato?? by Agrobacterium method. Southern blotting identified that 11 transgenic Bn-5 plants (T₀) and 10 transgenic Bn-3 plants (T₀) were obtained. The vegetative phenotypes of all T₀ plants were similar to wild-type, and they were capable of producing viable pollen grains and normal fruit with seeds, indicating that Barnase had lost its function after it being split two partial fragments. After self-pollination, homozygous progenies (T₁) of transgenic Bn-5 and Bn-3 plants were chosen to cross each other, Barnase could be reconstituted and co-expressed in the same cell, which caused the hybrid plants to produce collapsed pollen grains with no viability and thus100?% male sterile plants were obtained. Stamens of male sterile plants were shorter than those of the wild type plants. PCR detection demonstrated that all male sterile plants contained Barnase, but male fertile plants did not. The male sterile plants were crossed with the male fertile inbred lines, and the result showed that hybrid (F₁) plants were capable of producing normal fruit with seeds, and their pollen grain fertility was restored. The co-segregation ratio of Bn-5 and Bn-3 fragments showed 1:1 among hybrid plants. In conclusion, the results verified that the male sterility could be generated by two component system and be used in hybrid seed production. The F₁ between the male sterile plant and the inbred line showed heterotic comparing to both parents. This system needs not breed restoration line.