An ms2 male-sterile, female-fertile soybean sharing phenotypic expression with other ms mutants

Genetic and cytological studies of microsporogenesis and microgametogenesis were conducted with a natural male‐sterile, female‐fertile soybean mutant (BR97‐17971) found in an F₄ breeding line of the Brazilian soybean breeding programme. Allele tests with ms Type Collection from USDA/ARS showed that the gene was allelic to ms2. Conventional analysis of microsporogenesis revealed a differential meiotic behaviour from the normal one reported in ms2 male‐sterile plants. Sterile plants have different levels of meiotic abnormalities related to chromosome segregation. Despite segregational abnormalities in the first and second divisions leading to micronuclei formation, the main meiotic cause of pollen sterility was absent or the defective cytokinesis following telophase II. Absent or defective cytokinesis has never been reported in ms2 mutants, but it was reported in the ms1 and ms4 mutants. After telophase II, one to four nucleate microspores underwent degeneration. Cross‐sections of sterile plant anthers showed that, at telophase II, the tapetal cells with large vacuoles degenerated. Despite callose deposition around meiocytes, sterile plants did not form tetrads. They degenerated and collapsed after callose dissolution, forming an amorphous mass. Anthers from male‐sterile plants were shrivelled.     

Pollen and coenocytic microspore germination in male-fertile and malesterilemsl soybean

Summary Light and fluorescence microscopy were used to study coenocytic microspore germination from male-sterile (ms1 ms1) soybean plants. Anther squashes from male-sterile plants revealed that a low frequency of natural coenocytic microspore germination occurred in male-sterile anthers of four independent lines; [ms1-North carolina (T260H),ms1-Urbana (T266H),ms1-Tonica (T267H), andms1-Ames (T268H)]. Abnormalities such as giant tubes, branched tubes, tubes with swollen areas, and multiple tubes were observed from coenocytic microspores from all four lines. The Urbana line, however, demonstrated a higher percentage of coenocytic microspore germination than did the other three lines. Flowers of the Urbana line from both malefertile and male-sterile plants, as well as gynoecia pollinated with coenocytic microspores from sterile plants, were used for in vivo studies. Pollen-tube growth appeared normal in male-fertile plants. In contrast, coenocytic microspore tubes rarely were observed in gynoecia from male-sterile plants or in gynoecia from malefertile plants that had been artificially cross-pollinated withms1 ms1 plants. Few tubes from coenocytic microspores were observed in the vicinity of the micropylar region. A low frequency of seed set was achieved in the greenhouse on Urbana male-sterile plants grown in the absence of male-fertile plants. Thus, we believe either that some gametes from coenocytic microspores are able to participate in fertilization at low frequency or that apomixis occurs inms1 ms1 plants.Joint contribution: Agricultural Research Service, U.S. Department of Agriculture, and Journal Paper No. J-12310 of the Iowa Agriculture and Home Economics Experiment Station, Ames IA, 50011 USA. Project 2471.     

Identification of AFLP markers linked to one recessive genic male sterility gene in oilseed rape, Brassica napus

The commercial utilization of heterosis in seed yield by means of hybrid varieties is of great importance for increasing oilseed rape production in China. This requires a functional system for the production of hybrid seed. The Brassica napus oilseed rape line 9012AB is a recessive epistatic genic male sterility (GMS) two‐type line, in which the sterility is controlled by two pairs of recessive duplicate sterile genes (ms1 and ms2) interacting with one pair of a recessive epistatic inhibitor gene (rf). Homozygosity at the rf locus (rfrf) inhibits the expression of the recessive male sterility trait in homozygous ms1ms1ms2ms2 plants. This study was conducted to identify molecular markers for one of the male fertility/sterility loci in the B. napus male sterility line 9012AB. Sterile bulk (BS) and fertile bulk (BF) DNA samples prepared from male sterile and male fertile plants of the homozygous two‐type line 9012AB were subjected to amplified fragment length polymorphic (AFLP) analysis. A total of 256 primer combinations were used and seven markers tightly linked to one recessive genic male sterile gene (ms) were identified. Among them, six fragments co‐segregated with the target gene in the tested population, and the other one had a genetic distance of 4.3 cM. The markers identified in this study will greatly enhance the utilization of recessive GMS for the production of hybrid seed in B. napus oilseed rape in China.     

Development of a cytoplasmic male‐sterile line NJCMS4A for hybrid soybean production

Cytoplasmic male‐sterile (CMS) lines are being used to produce hybrid seeds. Thus far, four CMS sources in soybean [Glycine max (L.) Merr.] have been reported in China. However, they are not sufficient or efficient in meeting the requirements of commercial soybean hybrid seed production. In this study, 33 varieties were tested for CMS using 45 crosses among 37 landraces and 17 annual wild soybean accessions (Glycine soja Sieb. et Zucc.). The cross of N23661 × N23658 showed partial to complete male sterility in backcross generations, while the corresponding reciprocal cross showed normal male fertility. Thus, the cytoplasm of N23661 is male‐sterile, the continuously backcrossed line is a male‐sterile line (designated NJCMS4A), and N23658 is its maintainer (designated NJCM4B). The male fertility of NJCMS4A was restored by another accession, Nansheng9403. Accordingly, NJCMS4A along with its maintainer and restorer composes a complete set of three lines for producing hybrid soybean. Using mitochondrial markers and sequence analyses, NJCMS4A is a CMS line with its cytoplasm not identical to the four previously reported CMS sources in soybean.     

大麦的小孢子发生和雄配子体发育的细胞学研究

本文研究了大麦(Hordeum vulgare L.)的小孢子发生和雄配子体发育的细胞学过程,主要结论是:小孢子母细胞减数分裂胞质分裂为连续型,四分体排列为左右对称型.小孢子第一次有丝分裂形成生殖和营养细胞.生殖细胞再分裂一次,形成一对精子.所以,大麦的成熟花粉粒为三细胞型.绒毡层为分泌型.母细胞减数分裂时,绒毡层细胞内多数具双核.小孢子释放前,绒毡层开始退化,二核花粉粒时仅留残痕.     

芝麻核雄性不育系ms86-1小孢子败育过程的超微结构

运用透射电子显微镜对芝麻核雄性不育系ms86-1的可育和不育花药进行了超微结构的比较观察。根据小孢子的细胞学形态特征,将芝麻花粉发育过程划分为小孢子母细胞形成期、减数分裂期、四分体期、单核小孢子早期、单核小孢子中期、单核小孢子晚期、花粉成熟期7个时期。对比观察表明芝麻核雄性不育的败育迹象起始于小孢子母细胞形成期,并伴随着进一步发育,败育现象逐渐明显,小孢子母细胞形成期小孢子母细胞壁形状不规则;减数分裂期小孢子母细胞壁严重扭曲变形,质膜外缺少早期外壁成分--原基粒棒;四分体期胼胝质壁外沉积物异常,呈绒毛状;四分体解体后形成畸形小孢子,孢子外壁不健全,绒毡层异常肥厚、降解延迟,释放极少量的畸形乌氏体;随后小孢子愈发皱缩,胞质凝集,内含物减少并逐渐凝聚成一团电子致密物质,最终走向完全败育。本研究揭示了不育小孢子的败育过程和败育特征,为深入研究芝麻核雄性不育败育机理奠定了基础。     

Effects of low temperature storage on the pollen of Brassica campestris,B. oleracea and B. napus

Summary Four indica cultivars viz. Kalinga-I, Ptb. 10, IR 27280-13-3-3-3 and Co. 41 were found to possess male sterile cytoplasm with fertility restoring genes while the cultivar Krishna was found to maintain the male sterility in all the cases. All the plants in the F₁ of Kalinga-I × Krishna were observed to be completely male sterile and continued to show complete pollen sterility in subsequent backcross generations when backcrossed with recurring pollen parent, Krishna. Thus, it was posible to develop a new cytoplasmic-genetic male sterile line in indica rice (Krishna A) with Kalinga-I male sterile cytoplasm and this male sterile cytoplasm was found to be genetically different from others. Further, the newly developed male sterile line (Krishna A) was observed to be tolerant for low temperature at seedling stage.     

大豆雄性不育突变体NJ89-1的细胞学研究

通过对大豆雄性不育突变体NJ89-1不育株与可育株小 孢子母细胞减数分裂的比较研究,发现NJ89-1雄性不育株小孢子母细胞减数分裂高度变异,联会异常,出现单价体、染色体落后、染色体桥、染色体不均等分离等;在形成四分体的早期阶段,小孢子母细胞里见到微核,以后产生大小不等的二分体、三分体、四分体及多分体;单核小孢子     

Development of the male gametophyte of an infertile plant Armoracia rusticana Gaertn.

Horseradish, Armoracia rusticana , is propagated vegetatively through root cuttings. It is described as a male-sterile plant as it does not form seeds or only develops them in small quantities. This study discusses the anatomical structure of anthers and the events throughout meiosis up to the formation of a male gametophyte. Microscopic observations have shown that the anatomical structure of the anther wall of A. rusticana is typical of the whole family of Brassicaceae , and all its layers are properly developed and fully functional. Meiosis in the sporogenous tissue proceeds correctly and leads to the formation of microspores. Also the configurations of the cytoplasmic skeleton are typical of plants with simultaneous cytokinesis. Further development, however, exhibits many abnormalities. A majority of the cells formed in the sample studied were dysfunctional uninucleate microspores with apparent symptoms of degeneration of the cytoplasm and organelles. Binucleate pollen grains, in which mitosis did occur, constituted only 10% of the cells analysed. Anatomical and cytological observations of male sterility in A. rusticana indicated inhibition of development at the microspore, stage which was manifested by cytoplasm degeneration with a low number of membranes and a scarce amount of starch in proplastids. Usually, in the prolonged microspore stage and in the young pollen no mitosis occurs at the time of anthesis. Cytological disorders may, to a great extent, limit the number of functional bicellular male gametophytes. Both in microspores and in pollen grains, there is exceptionally little starch in plastids.     

红麻GMS与CMS小孢子败育过程的细胞学及组织化学比较

以红麻细胞质雄性不育系(GMS)L23A、保持系L23B及从该保持系中发现的细胞核雄性不育系(CMS)L23BS为材料,采用石蜡显微制片技术,在光学显微镜下比较观察了三者花药发育过程中小孢子的发育过程及组织化学变化。结果表明,L23A的花药发育过程中,小孢子发育的不同阶段均出现败育现象,最早的败育表现为花粉母细胞在减数分裂之前的退化解体,最终形成空的花粉囊; 有的因花粉母细胞减数分裂过程异常而导致不能形成小孢子四分体;有的因小孢子在四分体中不能正常释放而败育,同时绒毡层细胞过度液泡化,并提早解体死亡; 花药发育早期含有少量蛋白质和淀粉,随着花药的发育逐渐变少。而L23BS小孢子败育的时期集中于四分体至单核花粉期间,表现为小孢子发育异常,有些小孢子不能从四分体里释放出来而影响其正常发育。在四分体以前与可育系类似,花药含丰富的蛋白质和淀粉。在败育的过程中,花药中的蛋白质和淀粉含量渐渐减少,但药隔组织中的在颗粒淀粉含量几乎不变。     

甘蓝型油菜细胞质雄性不育系105A花药败育的细胞学观察

旨在了解并揭示不育系105A的花粉败育时期和细胞学特征,为进一步正确认识细胞质雄性不育系分子机制提供必要参考依据。采用石蜡制片法,对甘蓝型油菜杂交种‘青杂5号’的雄性不育系105A及其恢复系1831R的小孢子发生和花药发育过程进行观察,以确定其花粉败育的时期和细胞学特点。研究结果表明,在不育系105A花药败育过程中,一部分发生于造孢细胞时期,属于无花粉囊败育型;另一部分发生于单核晚期,属于单核败育型,其特点为单核晚期绒毡层细胞膨大向小孢子靠近,并逐步降解,其破裂胞质退化残余物侵入药室,与小孢子混合粘连在一起,甚至有些绒毡层细胞整块脱落,成为染色很深的团块状物质,占据药室一部分空间,最终小孢子降解,花药败育。     

New insights into genotypic thermodependency of cytoplasmic male sterility for hybrid barley breeding

The cytoplasmic male sterility (CMS) system msm1 in barley is known to be thermosensitive, sometimes resulting in spontaneous fertility restoration in the absence of the corresponding restorer gene Rfm1. Here, we investigated genotypic differences concerning temperature sensitivity and the plant developmental stage at which elevated temperature induces spontaneous fertility restoration in three CMS mother lines. While one line stayed completely male sterile, a significantly higher fertility was observed in two lines after treatment from growth stage DC 41 until maturation. Microscopic analysis revealed that sterile anthers contained neither intact pollen, nor remains of aborted pollen grains, whereas pollen was visible in anthers of potentially fertile plants. We conclude that the barley CMS system affects anther and pollen development prior to meiosis. Elevated temperature during heading and flowering can lead to a spontaneous fertility restoration by reactivating pollen growth. Nevertheless, genotypic variation exists enabling the selection for stable CMS mother lines and the development of F₁ hybrids with high hybridity. As spontaneous fertility restoration due to environmental effects is difficult to phenotype, further investigations will focus on the development of molecular markers for marker‐assisted selection.     

Characterization for agronomic use of cytoplasmic male-sterility in sunflower (Helianthus annuus L.) introduced from H. resinosus Small

A backcrossing programme was carried out both to assess the stability of a cytoplasmic male‐sterility (CMS) source from Helianthus resinosus, designated RES1, and to incorporate it into inbred sunflower lines (HA89, RHA271, RHA801). All the progenies, grown in different environments, were completely male‐sterile. This suggests that the expression of this cytoplasm is stable. Female‐fertility of lines HA89, RHA271 and RHA801 carrying CMS RES1 were compared with those of the corresponding fertile inbred lines. There were no differences in the number of seeds per head. This indicates that female‐fertility is not affected by RES1 cytoplasm. Cytological studies showed that meiosis proceeds normally until the tetrad stage; consequently, the absence of pollen is caused by alterations that take place during postmeiotic stages. With the aim of identifying male‐fertility restorer genotypes, crosses were made between HA89 (CMS RES1) plants and different annual diploid and perennial hexaploid Helianthus species. All the diploid germplasm evaluated behaved as a CMS RES1 maintainer. However, the hexaploid species, H. resinosus, H. x laetiflorus, H. pauciflorus and H. tuberosus, restored pollen fertility in CMS RES1 plants.     

Development of new cytoplasmic genetic male sterile lines through Indica × Japonica hybridization in rice

Summary From 28 Indica-Japonica crosses, two Indica cultivars, V.20B and Sattari were identified to possess male sterile cytoplasm with fertility restoring genes. It was possible to develop a new Japonica cytoplasmic genetic male sterile line (Zhunghua-1) on Indica male sterile cytoplasm (V 20B) by repeated backcrossing the complete pollen sterile plants of V 20B x Zhunghua-1 to the recurring male parent, Zhunghua-1. The study indicated that it would be possible to develop male sterile lines rom indica-japonica crosses only when there is sufficient amount of reciprocal differences with respect to pollen sterility. Further, it was inferred that it would be easier to develop Japonica male sterile lines on Indica cytoplasm than developing Indica male sterile line with japonica cytoplasm.     

The wi gene causes genic male sterility in Allium choenoprasum

Segregation studies following the transfer of the gene wi to different cytoplasm types, which have been distinguished by means of restriction fragment length polymorphism analyses using mitochondrial gene probes, revealed the formation of the wi‐sterility in each of the four cytoplasms examined. The male sterility is therefore only caused by the nuclear wi gene, i.e. an additional factor of a specific cytoplasm can be excluded. Hence, the wi‐sterility proved to be a genic male sterility (GMS) and not a cytoplasmic male sterility (CMS). The expression of the wi‐sterility appears to be stable, since it is not affected by high temperatures or tetracycline. Accordingly, a temporary pollen production, which would allow self‐fertilization for the maintenance of sterile lines, cannot be induced by controlling these environmental factors. In terms of hybrid breeding, this GMS therefore has no advantage over the previously described CMS system.     

甘蓝型油菜光、温敏雄性不育系Huiyou50S花粉败育的细胞学观察

Huiyou50S是从甘蓝型油菜品种汇油50中发现的半不育株选育而成的光、温敏雄性不育系,育性受隐性核基因控制,在高温、长光周期下不育而在低温、短光周期条件下可育。本文通过半薄树脂切片、扫描电镜、花粉压片染色、花药整体透明方法对Huiyou50S及其近等基因系Huiyou50F的花药发育过程及花粉形态观察比较。结果表明, Huiyou50S的花药造孢细胞、花粉母细胞、减数分裂、四分体阶段均正常,在单核期出现明显异常,虽然能形成花粉壁,但小孢子细胞质收缩、解体,最终只剩余空瘪的花粉壳; Huiyou50S的花药绒毡层在单核后期提前降解,绒毡层解体速度快于可育株Huiyou50F。Huiyou50S的小孢子的完全败育主要发生在单核时期,绒毡层发育与小孢子异常有某种关联,该结论为油菜光、温敏雄性不育类型划分及育种应用提供了依据。     

A unique introgression from Moricandia arvensis confers male fertility upon two different cytoplasmic male-sterile lines of Brassica juncea

A Brassica juncea line carrying an introgression from Moricandia arvensis restored male fertility to two cytoplasmic male‐sterile (CMS) B. juncea lines carrying either M. arvensis or Diplotaxis catholica cytoplasm. Genetics of fertility restoration was studied in the F₁, F₂, F₃ and backcross generations of the cross between CMS and fertility‐restorer lines. No male‐sterile plants were found in F1‐F₃ generations of the cross between CMS [M. arvensis] B. juncea and the restorer. However, a 1: 1 segregation for male sterility and fertility was observed when the F1 was pollinated with non‐restorer pollen from a euplasmic line. These results clearly show that restoration is mono‐genic and gametophytic. In CMS lines carrying D. catholica cytoplasm, the restorer conferred male fertility to the F1 and showed 3: 1 and 1: 1 segregations for male fertility and sterility in F₂ and BC1 generations, respectively, indicating a monogenic, sporophytic mode of fertility restoration. The results were also supported by pollen stainability in the F1 which was about 65% in M. arvensis‐based CMS and >90% in D. catholica‐based CMS. The above results are discussed in the light of previous molecular studies which showed association between CMS and atpA in both systems.     

高粱A3细胞质雄性不育发生的细胞学观察及分析

以不育系A₃晋粱5号和相应保持系B₃晋粱5号为材料，对A₃不育系花药发育及雄配子形成过程进行了细胞学观察和分析。结果表明，与A₁、A₂型CMS不同，A₃ CMS孢母细胞减数分裂正常，从四分体形成到幼龄花粉粒发育阶段都未观察到不同于B₃晋粱5号的异常现象。A₃晋粱5号花药经石蜡包埋切片观察，在造孢细胞期、减数分裂前间期及减数分裂期，均未观察到异常现象，不育系花粉母细胞能完成正常的减数分裂过程，四分体正常游离，绒毡层发育正常。但在成熟的花药中，观察到所有的花粉粒皱缩、凹陷、变形。在开花前约1周内的花粉粒成熟期，不育系花粉粒蔗糖—淀粉代谢途径受阻，花粉粒壁上无淀粉粒沉积。A₃ CMS小孢子败育发生在小孢子形成晚期的花粉粒成熟期。     

Identification of AFLP markers linked to the epistatic suppressor gene of a recessive genic male sterility in rapeseed and conversion to SCAR markers

A recessive epistatic genic male sterility (REGMS) two‐type line, 9012AB, has been used for rapeseed hybrid seed production in China. The male sterility of 9012AB is controlled by two recessive duplicate sterile genes (ms1 and ms2) interacting with one recessive epistatic suppressor gene (esp). Homozygosity at the esp locus (espesp) suppresses the expression of the recessive male sterility trait in homozygous ms1ms1ms2 ms2 plants. In this study, we used a combination of bulked segregant analyses and amplified fragment length polymorphism (AFLP) to identify markers linked to the suppressor gene in a BC₁ population. From the survey of 1024 AFLP primer combinations, eight markers tightly linked to the target gene were identified. The two closest markers flanking both sides of Esp, P9M5₃₇₀ and S16M14₇₈₀, had a genetic distance of 1.4 cM and 2.1 cM, respectively. The AFLP fragment from P4M8₁₉₀, which co‐segregated with the target gene was converted into a sequence characterized amplified region marker. The availability of linked molecular markers will facilitate the utilization of REGMS in hybrid breeding in Brassica napus.     

高粱A3细胞质雄性不育发生的细胞学观察及分析

以不育系A₃晋粱5号和相应保持系B₃晋粱5号为材料,对A₃不育系花药发育及雄配子形成过程进行了细胞学观察和分析。结果表明,与A₁、A₂型CMS不同,A₃ CMS孢母细胞减数分裂正常,从四分体形成到幼龄花粉粒发育阶段都未观察到不同于B₃晋粱5号的异常现象。A₃晋粱5号花药经石蜡包埋切片观察,在造孢细胞期、减数分裂前间期及减数分裂期,均未观察到异常现象,不育系花粉母细胞能完成正常的减数分裂过程,四分体正常游离,绒毡层发育正常。但在成熟的花药中,观察到所有的花粉粒皱缩、凹陷、变形。在开花前约1周内的花粉粒成熟期,不育系花粉粒蔗糖—淀粉代谢途径受阻,花粉粒壁上无淀粉粒沉积。A₃ CMS小孢子败育发生在小孢子形成晚期的花粉粒成熟期。