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.     

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.     

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.     

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。     

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.     

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.     

THE CYTOLOGY OF POLLEN ABORTION IN C-CYTOPLASMIC MALE-STERILE CORN ANTHERS

Anther development of the C-cytoplasmic male-sterile (cms C) and the normal cytoplasm version (N) in the W182BN corn inbred was studied by light and electron microscopy. Deviation from normal pollen development was first observed in the tapetal cells at the tetrad stage of development. Two types of tapetal abnormalities were observed in plants with C cytoplasm. The first behaved like the N anther until the tetrad stage, when numerous small vacuoles appeared in the tapetal cells. Inner and radial tapetal cell walls broke down normally, but irregular Ubisch body deposition was observed, and exine development was inhibited and delayed. The tapetum and microspores disintegrated at the intermediate microspore stage. The second type of tapetum was highly vacuolated at the early tetrad stage, with dense inner and radial cell walls that remained intact and enlarged when the tetrads aborted. No organellar abnormalities, such as the mitochondrial changes observed in cms T, were observed in C anthers.     

新型小麦胞质不育系花粉败育的细胞学观察

观察了１种新型小麦细胞质雄性不育素（ＣＭＳ）－（野生二粒小麦）中国春ＣＭＳ花药和花粉败育的细胞学过程,结果表明;（１）不育系在小孢子发育至单核晚期以前,除了雄蕊心皮化发生率（３７．２％）较高外,其花药和花粉发育绝大多数与同核保持系相似,是正常的,仅少量表现异常而导致败育,异常现象主要有：雄蕊心皮化。药室合并,药壁组织喙状突起,绒毡层异常,小孢子母细胞粘连,减数分裂异常,小孢子异常等。（２）不育系花     

雄性可育与雄性不育籽粒苋小孢子发育研究

栽培种籽粒苋（ＡｍａｒａｎｔｈｕｓｈｙｐｏｃｈｏｎｄｒｉａｃｕｓＬ。）是一种很有潜力的新型作物。它营养价值高、蛋白质含量丰富、氨基酸平衡好、耐旱、耐盐碱和酸、抗逆性强、适应性广,被认为是极有潜力的、为全球提供粮食的替代作物之一。但是籽粒苋千粒重仅０．７～１．２ｇ,种子易散落、植株易倒伏。而且,籽粒苋花冠微小、花期无限,难于采用人工去雄授粉进行杂交育种。于是,和许多其它植物一样,籽粒苋中也找到了雄性不育株。但是它的小孢子发育过程及其败育时期和不育特征尚不清楚,为它的杂交育种研究带来不便。本文通过电镜对雄性可育和不育的两种籽粒苋小孢子分别进行了观察。发现不育小孢子败育起始于四分体释放以后的单核花粉期。在此之前小孢子的发育是一样的。花粉分化早期,孢原组织分化出初级造孢组织、绒毡层、中间层、药壁内层和表皮层（图１）;造孢组织继续分裂,细胞不断扩大,形成小孢子母细胞（图２）;小孢子母细胞不断增大,周围积累胼胝质并逐渐与绒毡层分离,出现大液泡（图３）,小孢子母细胞减数分裂,四分体形成,包埋于胼胝质中;绒毡层有丝分裂,有双核细胞;大液泡消失;细胞壁开始降解（图４）。胼胝质逐渐消失,小孢子从四分体中释放以后（单核花粉期）,     

棉花转基因恢复系杂种F1小孢子发生的细胞学观察

对转gst基因棉花恢复系浙大强恢"配制的杂种F1(三系杂交棉)的成熟花药和花粉育性进行了研究.结果表明,在花药的长、宽、鲜重和成熟花粉粒的育性方面,浙大强恢"所配的F1和保持系DES-HAB277"接近,无显著性差异,但比受体恢复系DES-HAF277"所配的F1分别提高47.7%、61.8%、28.5%和39.6%.以不育系DES-HAMS277"和保持系DES-HAB277"的花药为对照,对浙大强恢"和受体恢复系所配的F1的小孢子发生进行了细胞学观察发现,不育系小孢子败育主要发生在造孢细胞增殖期和小孢子母细胞形成期,且在减数分裂期彻底败育,不能形成四分体;受体恢复系所配的F1在小孢子发生和雄配子形成的各个发育时期都有部分败育,平均败育率约为20%,且主要发生在小孢子母细胞减数分裂期和小孢子单核期;而浙大强恢"所配的F1与保持系一样,花药的发育、小孢子的发生以及雄配子的形成均正常.研究结果从细胞形态学方面证明gst基因对三系杂交棉具有防止部分小孢子败育和提高花粉育性的功能.     

Two new male-sterile mutants of Zea mays (Poaceae) with abnormal tapetal cell morphology

Two new recessive male-sterile mutants of Zea mays (Poaceae), or maize, were studied to identify the timing of pollen abortion and to examine the involvement of anther wall cell layers. The results of test crosses indicated that these mutants were not allelic with any known male-sterile mutants of maize. Light and transmission electron microscopy were used to compare pollen development in homozygous male-sterile mutants to that in fertile heterozygous siblings. In both mutants, microspores abort soon after release from the meiotic tetrad. However, the two mutations have strikingly different phenotypes. Large lipid bodies accumulate in the tapetal cells as the microspores vacuolate and die in the mutant ms25. Large vacuoles appear in both the tapetal cells and the young microspores as they begin to disintegrate in the mutant ms26. Because abnormal tapetal cell morphology is detected in both mutants, it is possible that both of these mutations affect the expression of genes in tapetal cells.     

Molecular mapping of the fertility restorer gene for ms-CW-type cytoplasmic male sterility of rice

Cytoplasmic male sterility (CMS) of rice (Oryza sativa L.) was first reported using the cytoplasm of a Chinese wild rice, Oryza rufipogon Griff. strain W1. However, it was not possible to characterize this ms-CW-type CMS in more detail until a restorer line had been developed due to the lack of restorer genes among cultivars thus far tested. The breeding of a restorer line (W1-R) was eventually achieved by transferring the restorer gene(s) of W1 to a cultivar. We report here the characterization of the ms-CW pollen grains and mapping of the restorer gene for ms-CW-type CMS. Pollen grains of the male-sterile plants appeared to be normal and viable based on the fluorochromatic reaction test, but they did not germinate on normal stigmas. The 1:1 segregation of fertile and sterile plants in a BC₁F₁ population from a cross between W1-R and a maintainer line demonstrated that fertility restoration is controlled by a single gene. The fertile seed set of all the F₂ plants examined indicated that the fertility restoration functions gametophytically. We designated the fertility restorer gene Rfcw. Using cleaved amplified polymorphic sequence (CAPS) and simple sequence repeat (SSR) markers, we localized Rfcw to chromosome 4 with a genetic distance of 0.6 cM from the nearest SSR marker.     

甜椒胞质雄性不育雄配子发育的解剖学和超微结构研究

运用石蜡切片和电子显微镜超薄切片方法观察了甜椒(Capsicum annuum L．)细胞质雄性不育系8A和保持系8B雄配子发育过程。结果表明：不育系和保持系都能正常进行减数分裂,绒毡层细胞无明显差异,形成了正常的四分孢子。在四分体单核居中期后,不育系的绒毡层细胞异常膨大并伸进药室,挤压花粉粒,同时绒毡层细胞提前降解,不育系单核晚期花粉粒开始崩出内含物。致使不育系的雄配子在双核花粉粒形成之前就完全裂解,不能发育成正常的花粉粒。此外,超薄切片还观察到不育系花粉粒在单核早期绒毡层细胞线粒体空泡化,这种变化表明雄性不育的遗传缺陷包括在花药发育早期发生的线粒体结构变化。     

MALE STERILITY IN SOYBEAN (GLYCINE MAX). I. PHENOTYPIC EXPRESSION OF THE ms2 MUTANT

The effects of a nuclear male-sterile mutant (ms2) of soybean, Glycine max (L.) Merr., on anther development were analyzed by means of light- and electron-microscopy. The structure of microspore mother cells (MMCs) in male-sterile plants was identical to that of male-fertile plants. Meiosis was completed, and tetrads of microspores formed. Microspores degenerated after the deposition of primexine and probacullae. The sheath of callose surrounding microspores did not dissolve. No structural abnormalities of the microspores were detected before the onset of degeneration. The tapetal and anther wall layers were characterized by aberrant development. Tapetal abnormalities included premature vacuolation, a persistent inner tangential cell wall, failure to differentiate normal concentrations of endoplasmic reticulum and dictyosomes, disruption of plastids, and premature degeneration. Malfunction of the tapetal layer preceded, and may have induced, microspore degeneration. Gross anther morphology was not influenced until advanced stages of development.     

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

Summary In the ogu cytoplasmic male-sterile (CMS) line of Brassica napus, stamen morphology was influenced by temperature conditions. Under a high temperature regime (27° C/23° C; day/ night) CMS stamens had a near-normal morphology, but microsporogenesis proceeded to a maximum of the microspore stage. However, compared to the normal stamens, the occurrence of sporopollenin-like deposits in the tapetum and deposition of exine on the microspores was sparse. Also, the tapetal cells of the CMS line were often highly vacuolate and failed to degenerate at the same stage as the normal. Ultrastructural changes in the mitochondrial matrix and cristae plus dilation of the endoplasmic reticulum, which occurred during development in sporogenous tissues of the normal line, were often lacking or mistimed in the mutant. Due to extensive variation, even between adjacent locules, the cytological differences between the normal and CMS anthers cannot be ascribed as the cause of male sterility in the ogu CMS line of B. napus, rather they may be the consequence of it.     

A Moricandia arvensis– based cytoplasmic male sterility and fertility restoration system in Brassica juncea

 A cytoplasmic male-sterility system has been developed in mustard (Brassica juncea) following repeated backcrossings of the somatic hybrid Moricandia arvensis (2n=28, MM)+B. juncea (2n=36, AABB), carrying mitochondria and chloroplasts from M. arvensis, to Brassica juncea. Cytoplasmic male-sterile (CMS) plants are similar to normal B. juncea; however, the leaves exhibit severe chlorosis resulting in delayed flowering. Flowers are normal with slender, non-dehiscent anthers and excellent nectaries. CMS plants show regular meiosis with pollen degeneration occurring during microsporogenesis. Female fertility was normal. Genetic information for fertility restoration was introgressed following the development of a M. arvensis monosomic addition line on CMS B. juncea. The additional chromosome paired allosyndetically with one of the B. juncea bivalents and allowed introgression. The putative restorer plant also exhibited severe chlorosis similar to CMS plants but possessed 89% and 73% pollen and seed fertility, respectively, which subsequently increased to 96% and 87% in the selfed progeny. The progeny of the cross of CMS line with the restorer line MJR-15, segregated into 1 fertile : 1 sterile. The CMS (Moricandia) B. juncea, the restorer (MJR-15), and fertility restored F₁ plants possess similar cytoplasmic organellar genomes as revealed by ‘Southern’ analysis. Received: 17 September 1997 / Accepted: 18 February 1998     

Genetics and cytology of a genic male-sterile, female-sterile mutant from a transposon-containing soybean population

A male-sterile, female-sterile soybean mutant (w4-m sterile) was identified among progeny of germinal revertants of a gene-tagging study. Our objectives were to determine the genetics (inheritance, allelism, and linkage) and the cytology (microsporogenesis and microgametogenesis) of the w4-m sterile. The mutant was inherited as a single recessive nuclear gene and was nonallelic to known male-sterile, female-sterile mutants st2 st2, st3 st3, st4 st4, st5 st5, and st6 st6 st7 st7. No linkage was detected between the w4-m sterile and the w4w4, y10 y10, y11 y11, y20 y20, fr1 fr1, and fr2 fr2 mutants. Homologous chromosome pairing was complete in fertile plants. Chromosome pairing, as observed in squash preparation, was almost completely absent in sterile plants. Developmentally microsporogenesis proceeded normally in both the fertile and the w4-m sterile through the early microspore stage. Then the tapetal cells of the w4-m sterile surrounding the young microspores developed different-size vacuoles. These tapetal cells became smaller in size and separated from each other. Some of the microspores of the w4-m sterile also became more vacuolate prematurely and sometimes they collapsed, usually by the late microspore stage. In the w4-m sterile the microspore walls remained thinner and structurally different from the microspore walls of fertile plants. No pollen was formed in the mutant plants, even though some of the male cells reached the pollen stage, although without normal filling. The w4-m sterile was designated st8st8 and assigned Soybean Genetic Type Collection number T352.     

Alloplasmic male-sterile <Emphasis Type="Italic">Brassica juncea</Emphasis> with <Emphasis Type="Italic">Enarthrocarpus lyratus</Emphasis> cytoplasm and the introgression of gene(s) for fertility restoration from cytoplasm donor species

A new cytoplasmic male sterility (CMS) source in Brassica juncea (2n = 36; AABB) was developed by substituting its nucleus into the cytoplasm of Enarthrocarpus lyratus (2n = 20; E(l)E(l)). Male sterility was complete, stable and manifested in either petaloid- or rudimentary-anthers which were devoid of fertile pollen grains. Male sterile plants resembled the euplasmic B. juncea except for slight leaf yellowing and delayed maturity. Leaf yellowing was due mainly to higher level of carotenoids rather than a reduction in chlorophyll pigments. Female fertility in male-sterile plants varied; it was normal in lines having rudimentary anthers but poor in those with petaloid anthers. Each of the 62 evaluated germplasm lines of B. juncea was a functional maintainer of male sterility. The gene(s) for male-fertility restoration ( Rf) were introgressed from the cytoplasm donor species through homoeologous pairing between A and E(l) chromosomes in monosomic addition plants (2n = 18II+1E(l)). The percent pollen fertility of restored F(1) ( lyr CMS x putative restorer) plants ranged from 60 to 80%. This, however, was sufficient to ensure complete seed set upon by bag selfing. The CMS ( lyr) B. juncea compared favourably with the existing CMS systems for various productivity related characteristics. However, the reduced transmission frequency of the Rf gene(s) through pollen grains, which was evident from the sporadic occurrence of male-sterile plants in restored F(1) hybrids, remains a limitation.