Cytogenetic studies in A4 cytoplasmic-nuclear male-sterility system of pigeonpea

Efforts were made to study microsporogenesis and genetics of fertility restoration of A(4) cytoplasmic-nuclear male-sterility (CMS) system in pigeonpea. The process of microsporogenesis in the male-sterile (ICPA 2039) and its maintainer (ICPB 2039) plants was normal up to the tetrad formation stage. The tapetal cells in the male-sterile anthers degenerated soon after tetrad formation, resulting in shriveled and degenerated microspores. In the maintainer plants, the tapetal cells were normal and microspores were functional. The breakdown of the tapetum before the completion of microsporogenesis was the major cause for the expression of male sterility in A(4) CMS system. The studies on the inheritance of fertility restoration showed that in 3 crosses, a single dominant gene; in 1 cross, 2 duplicate genes; and in another cross, 2 complimentary genes governed the fertility restoration.     

不同倍性水稻亚种间杂种小孢子发生的细胞学观察

利用塑料半薄切片对水稻同源四倍体亚种间杂种F1及其对应的二倍体杂种F1小孢子母细胞减数分裂过程的细胞学变化进行观察研究.结果表明,同源四倍体水稻亚种间杂种造孢细胞期和小孢子母细胞期已经表现出较高频率的异常;减数分裂过程中,小孢子母细胞出现异常更加复杂,主要包括小孢子母细胞液泡化和退化两大类,这些异常是导致杂种花粉败育和花粉低育性的重要原因之一;此外,绒毡层异常也是导致杂种的花粉育性降低的因素.二倍体杂种小孢子母细胞在减数分裂过程的异常类型与水稻同源四倍体亚种间杂种基本相似,其绒毡层异常频率较低,对其花粉低育性影响不大.     

水稻雄性不育系珍汕97A小孢子发育过程中的微管骨架

水稻（Oryza sativaL.)雄性不育系珍汕97A,保持系珍汕97B和恢复系测64三系小孢子发生过程的研究表明;恢复系测64小孢子母细胞细胞质浓,有明显的微管荧光围绕着细胞核。小孢子母细胞经两次减数分裂形成四分体。四分体和小孢子的微管从细胞核表面向胞质周缘延伸,形成放射性排列格局,花粉发育正常。细胞质中有少量点状微管荧光,保持系珍汕97B小孢子发生过程的细胞形态和微管结构与恢复系测64相似。但细胞质中的点状微管荧光多一些。雄性不育系珍汕97A小孢子发生早期,小孢子母细胞内出现液泡,核中染色质凝集,微管荧光很弱,没有清晰的微管丝结构。细胞质中有许多点状微管荧光等不正常现象。小孢子母细胞经过减数分裂形成的四分体也没有清晰的丝状微管结构。随后,所有的小孢子迅速败育,雄性不育系珍汕97A在小孢子母细胞发生的很早时期,微管结构就明显不正常。     

水稻非花粉型细胞质雄性不育系及其保持系花药发育过程中Ca2+的分布变化

钙在高等植物中被称为第二信使,与植物的有性生殖有关。为了研究水稻（Oryza sativa L.）花药中钙的定位与花粉败育的关系,利用焦锑酸钾沉淀法研究了非花粉型细胞质雄性不育系G37A及其保持系G37B花药的发育过程及其细胞中Ca^2＋ 的分布变化。研究发现,在2个材料间花药中钙的分布存在大量差异。G37B的可育花药在花粉母细胞时期及二分体时期,很少看到有Ca^2＋的沉积;而在单核花粉时期,Ca^2＋沉积急速地增加,主要定位在绒毡层细胞、花粉外壁外层及乌氏体的表面;随后花药壁上沉积的Ca^2＋减少而花粉的外壁外层仍然有很多Ca^2＋沉积物。相反,G37A的不育花药在花粉母细胞时期和二分体时期有大量的Ca^2＋沉积在小孢子母细胞和花药壁,中间层和绒毡层特别多。在二分体时期之后,不育花药的Ca^2＋沉积减少,特别是绒毡层内切向质膜附近的Ca^2＋几乎消失。但是同时期的可育花药中,有大量的Ca^2＋沉积在绒毡层。不育花药的Ca^2＋沉积在开花几天后消失。根据研究结果推测在不育花药发育早期中更多的钙离子与花粉败育有一定的关系。     

Tetrad pollen formation in quartet mutants of Arabidopsis thaliana is associated with persistence of pectic polysaccharides of the pollen mother cell wall

The quartet (qrt) mutants of Arabidopsis thaliana produce tetrad pollen in which microspores fail to separate during pollen development. Because the amount of callose deposition between microspores is correlated with tetrad pollen formation in other species, and because pectin is implicated as playing a role in cell adhesion, these cell-wall components in wild-type and mutant anthers were visualized by immunofluorescence microscopy at different stages of microsporogenesis. In wild-type, callose was detected around the pollen mother cell at the onset of meiosis and around the microspores during the tetrad stage. Microspores were released into the anther locule at the stage where callose was no longer detected. Deposition and degradation of callose during tetrad pollen formation in qrt1 and qrt2 mutants were indistinguishable from those in wild-type. Enzymatic removal of callose from wild-type microspores at the tetrad stage did not release the microspores, suggesting that callose removal is not sufficient to disperse the microspores in wild-type. Pectic components were detected in the primary wall of the pollen mother cell. This wall surrounded the callosic wall around the pollen mother cell and the microspores during the tetrad stage. In wild-type, pectic components of this wall were no longer detectable at the time of microspore release. However, in qrt1 and qrt2 mutants, pectic components of this wall persisted after callose degradation. This result suggests that failure of pectin degradation in the pollen mother cell wall is associated with tetrad pollen formation in qrt mutants, and indicates that QRT1 and QRT2 may be required for cell type-specific pectin degradation to separate microspores.     

Microsporogenesis of the feathers (fertile branches derived from annual buds) in Vitis vinifera,cv Picolit giallo

Abstract

Using light and electron microscopy, we have studied the microsporogenesis and tapetal development of the feathers in two different low producing clones of Picolit giallo (sp. Vitis vinifera). In these clones while the productivity of the main branches (fertile branches originated from buds, formed in the previous year, that remained silent during the winter) is very low, that of the feathers (fertile branches derived from annual buds) is always normal.

The microsporogenesis and tapetal development proceed normally in almost all the examined anthers; it is remarkable that at the tetrad stage the tapetal cells appear well structured without any degeneration symptom, unlike what observed for the main branches. Moreover in most of the mature anthers the pollen grains are numerous, pleinty of organelles and show sometimes thickenings in the callose layer under their wall. The tapetal cells of these anthers have disappeared. Only in few anthers we observed the presence of collapsed pollen grains and tapetal cells with anomalous development, that are still present when the pollen grains are mature. This rare situation for the feathers is on the contrary frequent for the main branches.     

Maturation and secretion of a serine proteinase is associated with events of late microsporogenesis

An antiserum against meiotic proteins which bind to DNA cellulose was generated as a tool to assist the identification and purification of microsporogenesis-specific proteins. In immunoblotting experiments, this antiserum identified three meiotic proteins which are differentially expressed in anthers during microsporogenesis. One of these proteins was purified and characterized by biochemical and immunological techniques. This 82 kDa protein is synthesized as a preproprotein, acquires glycans as it moves through the endoplasmic reticulum and Golgi body, and is secreted into the anther locule. Immunocytochemical experiments demonstrate that the protein is expressed primarily in tapetal cells, and reaches peak concentrations as the microsporocytes reach the tetrad stage. Zymogram analyses and protein sequence comparisons indicate that the protein is a member of the serine proteinase family. The possible roles of the proteinase in microsporogenesis and pollen development are discussed.     

Identification and molecular characterization of novel anther-specific genes in Oryza sativa L. by using cDNA microarray

The complicated genetic pathway regulates the developmental programs of male reproductive organ, anther tissues. To understand these molecular mechanisms, we performed cDNA microarray analyses and in situ hybridization to monitor gene expression patterns during anther development in rice. Microarray analysis of 4,304 cDNA clones revealed that the hybridization signal of 396 cDNA clones (271 non-redundant groups) increased more than six-fold in every stage of the anthers compared with that of leaves. Cluster analysis with the expression data showed that 259 cDNA clones (156 non redundant groups) were specifically or predominantly expressed in anther tissues and were regulated by developmental stage-specific manners in the anther tissues. These co-regulated genes would be important for development of functional anther tissues. Furthermore, we selected several clones for RNA in situ hybridization analysis. From these analyses, we found several novel genes that show temporal and spatial expression patterns during anther development in addition to anther-specific genes reported so far. These results indicate that the genes identified in this experiment are controlled by different programs and are specialized in their developmental and cell types.     

Microsporogenesis and tapetal development in fertile and cytoplasmic male-sterile sugar beet (Beta vulgaris L.)

Summary The development of sporogenous and tapetal cells in the anthers of male-fertile and cytoplasmic male-sterile sugar beet (Beta vulgaris L.) plants was studied using light and transmission electron microscopy. In general, male-sterile anthers showed a much greater variability in developmental pattern than male-fertile anthers. The earliest deviation from normal anther development was observed to occur in sterile anthers at meiotic early prophase: there was a degeneration or irregular proliferation of the tapetal cells. Other early aberrant events were the occurrence of numerous small vesicles in the microspore mother cells (MMC) and a disorganized chromatin condensation. Deviations that occurred in sterile anthers at later developmental stages included: (1) less distinct inner structures in the mitochondria of both MMC and tapetal cells from middle prophase onwards. (2) dilated ER and nuclear membranes at MMC prophase, in some cases associated with the formation of protein bodies. (3) breakdown of cell walls in MMCs and tapetal cells at late meiotic prophase. (4) no massive increase in tapetal ER at the tetrad stage. (5) a general dissolution of membranes, first in the MMC, then in the tapetum. (6) abortion of microspores and the occurrence of a plasmodial tapetum in anthers reaching the microspore stage. (7) no distinct degeneration of tapetal cells after microspore formation. Thus, it seems that the factors that lead to abortive microsporogenesis are structurally expressed at widely different times during anther development. Aberrant patterns are not restricted to the tetrad stage but occur at early prophase.     

Pre-zygotic embryological characters ofPlatycrater arguta, a rare and endangered species endemic to East Asia

Platycrater arguta Sieb. et Zucc. is a rare and endangered species endemic to East Asia. It produces two floral morphs viz. bisexual and male flowers. For bisexual flowers, simultaneous cytokinesis in the microsporocyte meiosis leads to a tetrahedral tetrad. The mature pollen grain is shed at 2-cell stage. The young anther wall is composed of epidermis, endothecium that develops fibrous thickenings at maturity, 1–2 middle layers and tapetum. The tapetum with uninucleate to binucleate cells, disintegrates in situ (glandular tapetum), yet in a small percentage of the anthers (about 37.6%), the tapetum does not disintegrate, causing complete male sterility. The ovules are anatropous, unitegmic, tenuinucellar and the formation of the embryo sac follows the monosporic, Polygonum type. Antipodal cells are lacking in the mature embryo sacs. Before fertilization, two polar nuclei fuse into a secondary nucleus. The formation of microsporangial wall, microsporogenesis and male gametogenesis in male flowers are analogous to those in the bisexual. Prezygotic embryological characters ofP. arguta were reported for the first time, revealing that its endangerment is correlated with the abortion of pollen of a part but not to the female development that is normal.     

RUPTURED POLLEN GRAIN1, a member of the MtN3/saliva gene family, is crucial for exine pattern formation and cell integrity of microspores in Arabidopsis

During microsporogenesis, the microsporocyte (or microspore) plasma membrane plays multiple roles in pollen wall development, including callose secretion, primexine deposition, and exine pattern determination. However, plasma membrane proteins that participate in these processes are still not well known. Here, we report that a new gene, RUPTURED POLLEN GRAIN1 (RPG1), encodes a plasma membrane protein and is required for exine pattern formation of microspores in Arabidopsis (Arabidopsis thaliana). The rpg1 mutant exhibits severely reduced male fertility with an otherwise normal phenotype, which is largely due to the postmeiotic abortion of microspores. Scanning electron microscopy examination showed that exine pattern formation in the mutant is impaired, as sporopollenin is randomly deposited on the pollen surface. Transmission electron microscopy examination further revealed that the primexine formation of mutant microspores is aberrant at the tetrad stage, which leads to defective sporopollenin deposition on microspores and the locule wall. In addition, microspore rupture and cytoplasmic leakage were evident in the rpg1 mutant, which indicates impaired cell integrity of the mutant microspores. RPG1 encodes an MtN3/saliva family protein that is integral to the plasma membrane. In situ hybridization analysis revealed that RPG1 is strongly expressed in microsporocyte (or microspores) and tapetum during male meiosis. The possible role of RPG1 in microsporogenesis is discussed.     

Microsporogenesis in a normal line and in the ogu cytoplasmic male-sterile line of Brassica napus

Summary Under an intermediate temperature regime (23° C/18° C; day/night), microsporogenesis in stamens of the ogu cytoplasmic male-sterile (CMS) line of Brassica napus terminated by the tetrad stage, although in some cases degeneration of the sporogenous tissue occurred prior to meiosis. In most cases the tetrads were collapsed and bounded by a sparse exine, but contained many organelles. Also, the tapetum in CMS anthers was abnormal and often highly vacuolated by the tetrad stage. Under low temperature conditions (18° C/15° C; day/night), neither microsporogenous nor tapetal tissues were observed. In the normal stamens, no differences were observed under different temperature regimes. In conjunction with the adjoining paper, this study demonstrates that temperature conditions strongly affect the cytological processes associated with microsporogenesis in the CMS anthers.     

Differences on the microsporogenesis and tapetal development of male fertile and cytoplasmic male sterile pepper (Capsicum annuum L.)

To clarify the time and cause of pollen abortion, differences on the microsporogenesis and tapetum development in the anthers of male fertile maintainer line and cytoplasmic male sterile (CMS) line pepper were studied using transmission electron microscopy. The results showed that CMS line anthers appeared to have much greater variability in developmental pattern than male fertile maintainer line ones. The earliest deviation from normal anther development occurred in CMS line anthers at prophase I was cytomixis in some microspore mother cells (MMCs), and vacuolisation in tapetal cells. Then, MMCs in CMS line anthers developed asynchronously and a small part of ones at the different stage degenerated in advance appearing to have typical morphological features of programmed cell death (PCD). Most MMCs could complete the meiosis, but formed non-tetrahedral tetrad microspores with irregular shape and different size and uncertain number of nuclei, and some degenerated ahead of time as well. Tapetal cells in CMS line anther degenerated during meiosis, and were crushed at the tetrad stage, which paralleled the collapse of pollens. Pollen abortion in CMS line anthers happened by PCD themselves, and the premature PCD of tapetal cells were closely associated with male sterility.     

Genetics and cytology of a new genic male-sterile soybean [Glycine max (L.) Merr.]

 Genetic and cytological studies were conducted with a new male-sterile, female-fertile soybean [Glycine max (L.) Merr.] mutant. This mutant was completely male sterile and was inherited as a single-recessive gene. No differences in female or male gamete transmission of the recessive allele were observed between reciprocal cross-pollinations in the F₁ or F₂ generations. This mutant was not allelic to any previously identified soybean genic male-sterile mutants: ms1, ms2, ms3, ms4, ms5, or ms6. No linkage was detected between sterility and flower color (W1 locus), or between sterility and pubescence color (T1 locus). Light microscopic and cytological observations of microsporogenesis in fertile and sterile anthers were conducted. The structure of microspore mother cells (MMC) in male-sterile plants was identical to the MMCs in male-fertile plants. Enzyme extraction analyses showed that there was no callase activity in male-sterile anthers, and this suggests that sterility was caused by retention of the callose walls, which normally are degraded around tetrads at the late tetrad stage. The tapetum from male-sterile anthers also showed abnormalities at the tetrad stage and later stages, which were expressed by an unusual formation of vacuoles, and by accumulation of densely staining material. At maturity, anthers from sterile plants were devoid of pollen grains. Received: 13 May 1996 / Revision accepted: 19 August 1996     

海桐大、小孢子发育及雌、雄配子体形成的研究

利用常规石蜡制片法研究了海桐大、小孢子发生及雌、雄配子体发育的过程。结果显示:(1)小孢子母细胞减数分裂过程中的胞质分裂为连续型,四分孢子为以四面体形为主,四分孢子后期部分小孢子壁皱缩;(2)花药壁由4层结构组成,由外到内为表皮、药室内壁、中层和绒毡层;(3)海桐具多个胚珠,单珠被,薄珠心,胚珠类型为倒生胚珠。大孢子母细胞减数分裂主要形成线形排列的4个大孢子,还具有少有的十字形排列,功能大孢子位于合点端;(4)胚囊发育属单孢型的蓼型,成熟的雌配子体为四细胞五核胚囊。     

Cold-induced repression of the rice anther-specific cell wall invertase gene OSINV4 is correlated with sucrose accumulation and pollen sterility

Low temperatures during rice (Oryza sativa L.) pollen development cause pollen sterility and decreased grain yield. We show that the time of highest sensitivity to cold coincides with the time of peak tapetal activity: the transition of the tetrad to early uni-nucleate stage (young microspore, YM stage). Low temperatures at this stage of pollen development result in an accumulation of sucrose in the anthers, accompanied by decreased activity of cell wall bound acid invertase and depletion of starch in mature pollen grains. Expression analysis of two cell wall (OSINV1, 4) and one vacuolar (OSINV2) acid invertase genes showed that OSINV4 is anther-specific and down-regulated by cold treatment. OSINV4 is transiently expressed in the tapetum cell layer at the YM stage, and later from the early binucleate stage in the maturing microspores. The down-regulation of OSINV4 expression in the tapetum at YM may cause a disruption in hexose production and starch formation in the pollen grains. In a cold-tolerant cultivar, OSINV4 expression was not reduced by cold; sucrose did not accumulate in the anthers and starch formation in the pollen grains was not affected.     

水稻不育系安农S-1育性转换及相关基因的表达分析

通过在自然环境和高温温室内对安农S-1的不同部位进行高温、低温诱导处理,对水稻温敏核不育系安农S-1的温度敏感时期和诱导部位进行了研究。总共进行了8种处理,结果表明：安农S-1的育性转换时期是从花粉母细胞形成到减数分裂的四分体时期之前。在育性转换时期,处于高温的条件下,根部低温处理不能诱导安农S-1可育,穗部低温处理可以使安农S-1保持可育,可见安农S-1的温度敏感部位在幼穗。aprz基因和育性相关,用RT-PCR方法研究了aprt基因在安农S-1不同部位和不同温度环境的表达变化,结果显示,在幼穗中aprt基因的表达在高温环境中被大幅度下调,而在叶中和根中的变化比较小,这说明幼穗对温度最敏感,从侧面验证了引起安农S-1育性转换的温度敏感部位是在幼穗。     

Ultrastructural expression of cytoplasmic male sterility in sugar beet (Beta vulgaris L.)

Summary The development of microspore mother cells (MMC) and tapetum in male-fertile and male-sterile anthers of Beta vulgaris L. was compared at the electron microscope level. These studies were complemented by morphometric analyses of mitochondria in both tissues through successive stages of microsporogenesis. The earliest irregularities in the ultrastructure of male-sterile anthers were noted within the tapetum at the tetrad stage. These disturbances were initially expressed by a slight reduction in mitochondrial size and the appearance of concentric configurations of endoplasmic reticulum. As development proceeded, a further decrease in mitochondrial size become more conspicuous and was accompanied by a reduction in ribosome population and a failure of the tapetum to produce Ubisch bodies. This failure to produce Ubisch bodies is reflected in the underdevelopment of sterile microspore exine.     

Changes in the Pattern of Organization of Microtubules During Microspore Formation in Rice (Oryza sativa L.)

Changes in the pattern of organization of microtubules in the developing microspore of rice ( Oryza sativa L. ) have been followed using immunofluorescence confocal microscopy. At the microsporocyte stage of development the cell possessed a network of highly branched and thickened microtubule bundles. In the central cytoplasm numerous bundles mn circumferentially around the nucleus. From the circumferentially distributed microtubule bundle network some microtubule bundles radiated towards the conical region of the cell. The microsporocyte after Meiosis Ⅰ became a dyad. In the dyad cell microtubule bundles emanated radially from the nucleus. In the cortex of the dyad cell some of the microtubule bundles became randomly oriented. The dyad then underwent Meiosis Ⅱ to become tetrad. Microtubule bundles in the tetrad cell radiated from the nucleus. No randomly oriented microtubule bundles were present in the cortical region of the tetrad cell. Mter- wards the four cells that made up the tetrad dissociated from each other and each became a microspore. At the early stage of the microspore development most of the microtubule bundles were randomly distributed. Later, some of the microtubules converged towards a bud-like cytoplasmic protrusion. This bud-like protrusion later developed into a germ pore (or pollen pore). At the late stage of microspore formation, microtubule bundles became thinner and reticulately oriented to form a tightly knitted network.     

萝卜CMS不育系与保持系小孢子发生的细胞学研究

研究了萝卜胞质雄性不育系A2、A4及其相应保持系B2、B4的小孢子发生与花药壁发育的细胞学特征.结果表明,不育系A2的绒毡层细胞在四分体时期出现异常,小液泡增多,至单核期汇合形成大液泡,绒毡层细胞异常膨大;小孢子外壁染色浅,细胞壁受到破坏,最后与绒毡层一同降解.不育系A4在减数分裂期即表现出异常,绒毡层异常肥大;花药发育后期,小孢子外壁亦染色较浅;绒毡层细胞融合形成细胞团块侵入药室挤压小孢子,两者一同降解.