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.     

Calcium distribution in fertile and sterile anthers of a photoperiod-sensitive genic male-sterile rice

Potassium antimonate was used to locate Ca²⁺ in fertile and sterile anthers of a photoperiod-sensitive genic male-sterile rice (Oryza sativa L. japonica). During the development of fertile anthers, abundant calcium precipitates accumulated in the anther walls and on the surface of pollen grains and Ubish bodies at the late developmental stage of the microspore, but not in the cytoplasm of pollen grains. Following the accumulation of starch grains in pollen, calcium precipitates on pollen walls diminished and increased in parenchymatous cells of the connective tissue. In sterile anthers, calcium precipitates were abundant in the middle layer and endothecium, but not in the tapetum, as was found in fertile anthers. A special cell wall was observed between the tapetum and middle layer of sterile anthers that appeared to relate to distinctive calcium accumulation patterns and poor pollen wall formation in the loculi. The formation of different patterns of antimonate-induced calcium precipitates in the anthers of photoperiod-sensitive genic male-sterile rice indicates that anomalies in the distribution of calcium accumulation correlate with the failure of pollen development and pollen abortion. Received: 30 May 1997 / Accepted: 5 July 1997     

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     

Acid phosphatase changes associated with development of male sterile and fertile maize (Zea mays L.)

Free and bound acid phosphatase were investigated biochemically and electrophoretically in male sterile and fertile plants. Scutella at the 72-hr seedling stage and anthers with enclosed reproductive tissue at the premeiosis, meiosis, and mature developmental stages were tested. Biochemical data show that while the total amount of free acid phosphatase behaved similarly in fertile and sterile plants, specific activities decreased in fertile plants and remained unchanged or increased in sterile plants. Total amounts of bound acid phosphatase increased significantly in fertile plants while specific activities decreased. In sterile anthers both specific activity and amount of bound acid phosphatase decreased significantly (except cms-S). Electrophoretic results indicated that the basic form of the enzyme was very similar in each location.Published with the approval of the Director of the West Virginia Agricultural Experiment Station as Scientific Paper No. 1573. Based in part on a dissertation by C. V. W. submitted to the Division of Plant Science, West Virginia University, Morgantown, West Virginia.     

ORGANELLE SIZE AND NUMBER IN FERTILE AND T-CYTOPLASMIC MALE-STERILE CORN

Anthers of inbred F₄₄ fertile (N) and cytoplasmic male-sterile (T) corn plants (Zea mays L.) were compared cytologically. No differences between fertile and sterile anthers were observed in size and number of mitochondria or plastids until after the start of anther degeneration. A rapid division of mitochondria was observed, however, in the tapetum and sporogenous cells of both fertile and sterile anthers during early growth stages. This rapid increase in mitochondrial numbers per cell (some 20-to 40-fold) preceded tapetal breakdown in sterile anthers and did not occur in other anther cells or in plastids. Limited observations on the megagametophyte and nucellus revealed that mitochondria in ovules remain relatively constant in size and number during gametogenesis and do not undergo degeneration.     

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。     

太谷核不育小麦可育花药内游离脯氨酸的来源、利用及与不育花药败育的关系

太谷核不育小麦营养体内的游离脯氨酸能迅速地运入并积累于花药中,花药有将谷氨酸转化成脯氨酸的能力。脯氨酸向花药内以主动运输为主,能被DNP所抑制,与旗叶的功能密切相关。在发育阶段Ⅰ(小孢子释放前后),脯氨酸已不能由营养体运入不育花药,运输的障碍在花丝或花药中,不育花药缺乏游离脯氨酸可能是主要原因。发育阶段Ⅰ的可育花药内,脯氨酸是合成蛋白质或其他大分子物质的原料。因而,脯氨酸的缺乏可能是加速不育花药小孢子败育和小孢子释放后急剧解体的重要原因。     

A case of early dissolution of the microsporocyte callose wall in male-sterile (CMS) sweet pepper (Capsicum annuum L.)

On squash preparations of anthers from pollen fertile and sterile plants of sweet pepper (Capsicum annuum L. cv. Severka) callose envelopes of microsporocytes, stained specifically with resorcin blue, were investigated microscopically. During normal course of microsporogenesis in fertile plants the envelopes remained intact up to the stage of microspore tetrads. Then callose begins to dissolve, and that from individual microspores towards the envelope periphery. In sterile analogues of the same cultivar the callose breakdown occurred precociously, usually in the course of the second, but sometimes as early as the first meiotic division of PMCs. Having completed meiosis sporadic microsporocytes formed microspore tetrads. Most PMCs contained an undivided four-nucleate protoplast rimmed with a narrow or wider unstained zone of dissolved callose. In certain cases more condensed callose septa pointing to the furrows on the surface of the PMC protoplast were well-observable in this lytic zone, as a residuum of normal mechanism of tetradogenesis.     

Cellular energy levels and their effect on male cell abortion in cytoplasmically male sterile lines of Petunia hybrida

Summary Using an HPLC method it has proved possible to follow the levels of adenine and pyridine nucleotides in the anthers of normal and cytoplasmically male sterile (CMS) lines of Petunia hybrida. Well before the appearance of any structural differences, anthers of CMS plants begin to show lower ATP/ADP ratios. In anthers, as in other non-photosynthetic plant tissues, there is a strong correlation between the ATP/ADP ratio and levels of NADPH and, when NADPH was assayed in fertile and CMS anthers, very dramatic differences were discovered. In male fertile plants, the NADPH/NADP ratio differs strikingly between somatic and reproductive tissues and reaches a peak in anthers at the early prophase of meiosis. The ratio in male sterile anthers remains largely unchanged from that normally seen in somatic tissues over this period. A cytochemical localisation technique revealed that, at the stage of development in question, the major reserves of anther NADPH are held within the tapetal cells and that levels within CMS tapeta are strikingly lower than those of normal plants. These findings are discussed in the perspective of theories proposed to explain the operation of CMS, and also in terms of the now conclusive evidence that the genetical basis of CMS lies in changes in the organisation of the mitochondrial genome.     

Involvement of phenylalanine ammonia-lyase in the development of pollen in broccoli (Brassica oleracea L.)

Summary To determine whether phenylalanine ammonia-lyase (EC 4.3.1.5) is involved in the maturation of microspores to fertile pollen, anthers of a fertile strain of broccoli (Brassica oleracea L.) were studied in a comparison with anthers of a cytoplasmic male sterile strain. In the normal fertile strain, immature anthers of about 2 mm in length exhibited higher phenylalanine ammonia-lyase activity than mature anthers or those shorter than 2 mm. The 2-mm-long anthers corresponded to the mononucleate stage, just after release of the microspores during pollen development. Immunohistochemical localization of phenylalanine ammonia-lyase in the anthers indicated that the protein was present predominantly in the tapetal cells. The immature anthers of cytoplasmic male sterile broccoli had a lower phenylalanine ammonia-lyase activity than those of the normal fertile strain. The level of phenylalanine ammonia-lyase activity in the immature anthers was positively correlated with the number of fertile pollen grains at the flowering stage in both strains. It seems possible, therefore, that phenylpropanoid metabolism, which involves phenylalanine ammonia-lyase, may play an important role in the maturation of microspores in flowering plants.Abbreviations CHS chalcone synthase - CMS cytoplasmic male sterility - DAPI 4, 6-diamidmo-2-phenylindole dihydrochloride - PAL L-phenylalanine ammonia-lyase     

Mechanism of male sterility in Petunia

Summary The free amino acid contents in the anthers of male fertile, cytoplasmic male sterile (cms) and genic male sterile (gms) petunia lines were compared at different developmental stages of the male gametophyte. Quantitative differences in the amounts of free amino acids were found between the fertile and male sterile lines and between the cms and gms lines. The differences between the sterile lines were correlated with the different developmental stages at which the breakdown in microsporogenesis occurred. In the Rosy Morn (RM) cms line, where breakdown of microsporogenesis occurred at the end of prophase 1, there was an associated increase in asparagine and decrease in the other amino acids. In the RM gms line, in which breakdown occurred at the tetrad stage, an accumulation of asparagine in the anthers corresponded with an accumulation of glutamine beginning at prophase 1. Compared with fertile anthers, the sterile anthers accumulated much proline at the early meiotic stages, but no -aminobutyric acid. Comparison of the free amino acids of the fertile and the male sterile lines indicates that certain biochemical events leading to breakdown of microsporogenesis precede the observed cytological breakdown. The results from adding asparagine and glutamine to extracts of anthers at different developmental stages suggest that the amino acid balance may contribute to the changes in pH in the fertile and male sterile anthers which we observed previously.Contribution from the Volcani Center, Agricultural Research Organization Bet Dagan, Israel. 1972 Series, No. 2083 E.     

Cytochemical investigation of genic male-sterility in Chinese cabbage

A genic male sterile Chinese cabbage, Brassica campestris L. ssp. chinensis Makino, was examined using cytological and cytochemical methods to characterize the process of pollen abortion in this plant. Thick sections of both fertile and sterile anthers at different developmental stages were stained using Toluidine Blue O, Periodic Acid-Schiff’s (PAS) reaction and Sudan Black B to detect cytochemical changes that may occur in the distribution of insoluble polysaccharide and lipid storage bodies. Pollen abortion in sterile anthers occurs at an early stage of microspore development. During early microspore development, reductions in the number of starch grains in the connective tissue of fertile anthers coincide with the accumulation of starch grains in cells of the anther wall. In the late microspore stage, a large vacuole forms in the microspore, and tapetal cells synthesize and accumulate lipid droplets. The cellular organization of tapetal cells in sterile anthers appears similar to that in fertile anthers, except for the absence of lipid droplets in cells of sterile anthers and diffusely labeled tapetal polysaccharides, suggesting defects in nutrient storage. Supported by National Natural Science Foundation of CHINA (30170060)     

白菜细胞核雄性不育花药的细胞化学观察

对一种由一对隐性基因控制的白菜细胞核雄性不育和可育株的花药进行了细胞学和组织化学研究。种子播种后,有1／4植株为不育株,其余的为可育株。通过对不育株和可育株花药发育的细胞学观察,确认不育花粉的败育发生在小孢子发育时期。用组织化学的方法研究了可育株和不育株花药发育过程中的多糖和脂类的分布动态,发现在减数分裂前,可育花药和不育花药的药隔细胞中都储藏了大量的淀粉粒。二者的差异仅是不育花药的绒毡层细胞液泡化明显。在减数分裂后的小孢子发育时期,可育花药的绒毡层细胞具有将药隔细胞中的淀粉粒多糖吸收并转化成脂类的功能,小孢子及以后的二胞花粉中也积累了大量的脂类储藏物质。在不育花药中,虽然减数分裂后药隔细胞中的淀粉粒也都消失,但绒毡层细胞中的脂类物质相比很少,同时绒毡层细胞显示了明显的多糖反应,表明不育花药的绒毡层细胞将糖类转化为脂类的功能受阻。在小孢子的表面有些脂类物质,但在细胞质中却没有脂类积累。这一结果暗示在该种白菜细胞核雄性不育株中,由于花药绒毡层细胞转换多糖为脂类的功能失常,导致了小孢子的败育。     

THE ANTHER TAPETUM IN CYTOPLASMIC-GENETIC MALE STERILE SORGHUM

Measurements of anthers of five fertile lines of Sorghum vulgare Pers. indicate that the tapetum decreases in radial extent following the two-celled dyad stage of meiosis. At a mature pollen stage 78% of the anthers of fertile lines and 88% of those of a fertility restorer line have a narrow tapetum measuring between 4 and 16 μ radially, with the remainder having a degenerate tapetum less than 4 μ wide. In isogenic sterile lines, approximately 68% of the anthers have a narrow tapetum less than 16 μ wide at a “prepollen” stage, which represents the terminal stage in the sterile lines, whereas the remaining 32% have a well-developed or enlarged tapetum measuring over 28 μ in radial width. Tapeta in the sterile lines have a variation in width and in morphology not encountered in fertile lines and presumably display the variable manifestations of nuclear-cytoplasmic interaction characteristic of cytoplasmic-genetic male sterility.     

Hydroxycinnamic acid amides in fertile and cytoplasmic male sterile lines of maize

Hydroxycinnamic acid (HCA) amides in fertile and cytoplasmic male sterile lines of maize were determined in reproductive organs, developing grains and cobs. HCA amides occurred in large amounts in the anthers of fertile plants (line F7N) and were absent from the anthers of cytoplasmic male sterile lines (lines F7T and F7C). Restoration of fertility was associated with the production of these compounds (line FC31). Considerable variations were observed in the concentrations of HCA amides at different stages of growth and grain maturation. Changes of HCA amides in the grains which were to produce sterile plants followed a pattern similar to that obtained with the grains which were to produce fertile plants. Accumulation of HCA amides was substantially higher in fertile lines whatever their genotype (F7N, FC31 and F7T x FC31) than in sterile lines. Marked changes occurred in the HCA amide content of embryo and endosperm during grain development. Many changes in HCA amides were observed in cobs during development and maturation, but no substantial differences could be observed between fertile and sterile lines.     

Transfer of the CMS trait in Daucus carota L. by donor-recipient protoplast fusion

Summary X-irradiated protoplasts of Daucus carota L., 28A1, carrying cytoplasmic male sterile (CMS) cytoplasm and iodoacetamide-treated protoplasts of a fertile carrot cultivar, K5, were fused with polyethylene glycol (PEG), and 73 plants were regenerated. Twenty-six randomly chosen regenerated plants had non-parental mitochondrial DNA (mtDNA) as revealed by XbaI restriction fragment patterns, and all of the plants investigated had diploid chromosome numbers. Of the 11 cybrid plants that showed mtDNA fragment patterns clearly different from those of the parents, 10 plants showed male sterility with brown or red anthers, and one plant possessed partially sterile yellow anthers. The mtDNA fragment patterns of the ten cybrid plants with male sterile flowers resembled that of a CMS parent, 28A1; and four fragments were identified that were common between the sterile cybrid plants and 28A1, but absent from the partially sterile cybrid plants and a fertile cultivar, K5. The results indicated that the CMS trait of the donor was efficiently transferred into the cybrid plants by donor-recipient protoplast fusion.     

Developmental and cytogenetic analyses of pollen sterility in Valeriana scandens L.

Valeriana scandens presents perfect and pistillate flowers, the latter with sterile anthers. The species is composed of two varieties with different ploidy; V. scandens var. scandens (2n = 28) and V. scandens var. candolleana (2n = 56), both of which occur in RS, Brazil. Crosses between these varieties may give rise to hybrids with pollen sterility. In this study, we analyzed the microsporogenesis and microgametogenesis of sterile and fertile anthers, and also investigate whether pollen sterility is caused by an irregular meiotic process. Developmental analysis using light microscopy and scanning electron microscopy showed that sterile anthers develop similarly to fertile anthers until the end of meiosis. After this stage, sterile tetrads do not separate as a consequence of exine fusion between adjacent microspores, which is similar to sterile pollen of Brassica ms-cdl1 mutants. In addition, vacuolated immature pollen grains degenerate after separation. The cytogenetic analysis of the microspore mother cell (MMC) showed that the diploid population of V. scandens var. scandens (2n = 28) has pollen sterility that is not caused by a cytogenetic disturbance. The MMCs analyzed from prophase I to tetrad stage showed a regular meiotic process, indicating the phenotype of V. scandens sterile pollen is a postmeiotic process formed by fusion of exine between opposite microspores.     

Microsporogenesis in male sterile Rosmarinus officinalis L. (Lamiaceae), an ultrastructural study

Within an extensive reproductive biology program on Rosmarinus officinalis a study of microsporogenesis in male sterile plants has been carried out. Two events, related to the two types of male sterile flowers found in this species (MS; Male sterile and INT; Intermediate male sterile flowers), are described. The first event is characterized by the early appearance of necrotic areas in the anther tissues during the final differentiation phases of the MS flowers, before meiosis takes place. Initially, these necrotic areas are small, later on they enlarge affecting the sporogenous tissue and tapetum. All the anthers in which necrosis has begun finally become aborted and lack pollen grains. The second event is manifested in the anthers of the INT flowers. Previous to the release of the microspores from the callosic envelope, vacuolisation of the tapetal cells takes place. The tapetum does not properly carry out its secretory function. It accumulates some substances in vacuoles and starch granules in plastids. The microspores degenerate in the vacuolate stage. In the epidermal and endothecial cells modifications are observed which may be related to the carbohydrate metabolism. Indehiscent, whitish anthers containing inviable microspores result. We have not found significant differences between the mitochondria in the anthers of the fertile flowers and MS or INT flowers. But we suggest, for both events, a metabolic failure, possibly controlled by cytoplasmic genes as the origin of male sterility. Paracrystalline material has been detected in microspore nuclei of developing INT anthers, though it does not seem to be related to the expression of male sterility.     

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.     

白菜细胞核雄性不育花药的细胞化学观察

对一种由一对隐性基因控制的白菜细胞核雄性不育和可育株的花药进行了细胞学和组织化学研究。种子播种后,有1/4植株为不育株,其余的为可育株。通过对不育株和可育株花药发育的细胞学观察,确认不育花粉的败育发生在小孢子发育时期。用组织化学的方法研究了可育株和不育株花药发育过程中的多糖和脂类的分布动态,发现在减数分裂前,可育花药和不育花药的药隔细胞中都储藏了大量的淀粉粒。二者的差异仅是不育花药的绒毡层细胞液泡化明显。在减数分裂后的小孢子发育时期,可育花药的绒毡层细胞具有将药隔细胞中的淀粉粒多糖吸收并转化成脂类的功能,小孢子及以后的二胞花粉中也积累了大量的脂类储藏物质在不育花药中,虽然减数分裂后药隔细胞中的淀粉粒也都消失,但绒毡层细胞中的脂类物质相比很少,同时绒毡层细胞显示了明显的多糖反应,表明不育花药的绒毡层细胞将糖类转化为脂类的功能受阻。在小孢子的表面有些脂类物质,但在细胞质中却没有脂类积累。这一结果暗示在该种白菜细胞核雄性不育株中,由于花药绒毡层细胞转换多糖为脂类的功能失常,导致了小孢子的败育。