Co-suppression of <Emphasis Type="Italic">Si401</Emphasis>, a maize pollen specific <Emphasis Type="Italic">Zm401</Emphasis> homologous gene,results in aberrant anther development in foxtail millet

We previously reported that the maize pollen-specific gene Zm401 functions in anther development. In this study, a Zm401’s ortholog Si401 was amplified and cloned from foxtail millet (Setaria italica). Zm401 and Si401 are highly conserved with 99% of their coding sequences identical to each other. Southern blot and RT-PCR analysis reveal that Si401 has only one copy in the genome and is expressed exclusively in the panicle. Co-suppression of Si401 in foxtail millet results in multiple abnormalities during the late stages of anther development, including premature degeneration of the tapetum, pre-deposition of fibrous bands in endothecium cells, and aborted pollen grains. Our results demonstrate that Si401 plays an essential role in anther development in foxtail millet and might be useful for generation of male-sterile plants in cereal crops.     

Characterization of transgenic male sterility in alfalfa

Dependable male sterility would help to make hybrid cultivar development a reality in alfalfa once higher levels of heterosis are attained. Alfalfa plants obtained by genetic transformation with a construct containing the Barnase gene under the control of a tobacco anther tapetum specific promoter were studied. Vacuolization and degeneration of the tapetal cell cytoplasm at a premeiotic stage of development were observed in all five transformed plants (T₀)examined, but the severity of the abnormalities varied greatly among pollen sacs of a genotype. During the meiotic stage, some pollen sacs showed reduction in size, and the tapetum generally appeared thinner when compared to those of the non transgenic plants; tapetal cells showed abnormal vacuolization and signs of cytoplasm degeneration. Despite this, some microspores were formed and some pollen grains were shed in all the T₀ plants, but these were highly variable in size and had very low in vitro germinability. Self-fertility was negligible. The T₀ plants were crossed with one or two unrelated non transgenic male-fertile plants. Mendelian segregation was observed with two exceptions. Instability of the trait in F₁ progenies was noticed, varying for different T₀ parents. F₁ plants exhibiting higher sterility than the primary transformants were observed, indicating that it should be possible to obtain good male sterile plants by backcrossing this trait into different genetic backgrounds. The possible use of this transgenic male sterility in alfalfa breeding is briefly discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

玉米CMS-S小孢子败育过程中的细胞程序性死亡

以玉米（Zea mays L.）S型细胞质雄性不育系(CMS-S)的一对近等基因系S-Mo17^Rf3Rf3 和S-Mo17^rf3rf3为材料，采用TdT介导的dUTP DNA末端标记（TUNEL）、细胞色素C免疫原位杂交和DNA寡聚核小体片段电泳等方法，分别在细胞学水平和DNA水平上研究了玉米CMS-S小孢子败育的细胞程序性死亡（PCD）过程。结果表明，在花粉母细胞减数分裂后的四分体解离时期，不育花药的绒粘层细胞较可育花药提前裂解；在不育系S-Mo17^rf3rf3花药和花粉S-rf3中均明显出现PCD过程的DNA片段化以及线粒体细胞色素C外渗的现象，证明了玉米CMS-S的花粉败育与花药绒粘层细胞的提前凋亡和小孢子细胞的程序性死亡有关。     

棉花雄性不育研究和应用进展

综述了棉花雄性不育类型、不育机理和雄性不育系杂种优势利用状况,着重讨论了棉花雄性不育的细胞学、生理生化和分子生物学研究进展。并对本领域研究和应用中存在的问题及发展前景提出了看法。     

小麦新型化学杂交剂的筛选

为拓展小麦化控两系途径的药剂种类,降低化学杂交剂(CHA)成本,利用西农1376检验15种化学药剂、4个浓度、3个施用时期(小麦不同发育时期)的诱导不育效果, 并对花药败育的机制进行了研究。结果表明,各处理诱导小麦花粉败育效果差异较大,Feek’s 7.5和9.5时期,15种药剂各浓度处理诱导植株营养生长发育异常或花粉败育不明显;Feek’s 8.5时期,T6药剂0.24 kg hm^-2诱导雄性不育率为93.33%,植株表型及雌蕊发育正常,对其饱和授粉,能获得杂交种子,且饱和授粉结实率较高。石蜡切片观察该处理诱导雄性不育花药的绒毡层细胞降解过程异常,自单核早期绒毡层细胞核明显降解,单核中后期花粉内核也开始降解,直至三核期,绒毡层细胞及花粉内核及营养物质基本消失,仅剩少量花粉壁残留,最终导致花粉败育。因此认为,T6诱导的小麦生理型雄性不育与绒毡层的异常降解直接相关。     

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。     

湖北光敏感核不育水稻发育过程中花粉及花药壁超微结构的研究

本工作应用透射电子显微镜，对不同光周期处理下的湖北光敏感核不育水稻农垦５８Ｓ花粉及花药壁发育情况进行了研究，得到如下的结果：１、农垦５８Ｓ经长日照处理，小孢的败育主要发生在单核晚期，花粉内细胞器解体严重，花粉壁异常发育，花粉的外形不规则。２、可育花的绒层在单核时期已严重解体，而败育花药的绒毡层不能及时解体，直到与成熟花粉相当的时期仍有清晰的细胞结构。３、败育花药的中间怪解体也延迟。在以上基础上讨论     

烟草绒毡层特异启动子pTA29在棉花中的表达特性

烟草绒毡层特异启动子pTA29 (TA29 promoter)已用于多种植物雄性不育和花粉发育的研究。作为外源特异表达启动子pTA29在棉花中的表达特性尚不清楚,为了研究该启动子在棉花中的表达特异性,本文构建了pTA29:gus融合基因,并将其转入棉花。研究发现在GUS染液中添加10%的乙醇可以抑制棉花花药内源GUS活性。用改良的GUS染液进行组织化学染色表明,pTA29:gus转化植株的GUS活性主要存在于花药中,并在花蕾长为6 mm和15 mm的花药中有2个活性高峰,而在根、茎、胚珠、花瓣、苞叶中未被检测到。与烟草不同,在pTA29:gus棉花转化植株的叶片表皮毛和花粉中也能检测到GUS活性。定量RT-PCR分析表明转化子中gus基因的转录水平与GUS活性一致。上述结果表明,烟草绒毡层特异启动子pTA29可以控制下游基因在棉花花药中优势表达;在利用该启动子进行棉花基因功能以及雄性不育研究时,应注意该启动子在棉花中的表达范围和组织特异性。     

Sc2053诱导小麦雄性不育形态学和细胞学观察

应用化学杂交剂Ｓｃ２０５３诱导小麦雄性不育的形态学和细胞学特征观察结果显示，经处理的小麦植株变矮，植株及麦穗呈现浅绿色，穗子呈爆裂状，花药不开裂，呈现雄性不育株的形态特征，这些特征可做为估算小麦群体化学杀雄效果即绝对不育率的依据。经Ｓｃ２０５３处理的小麦雄性不育有无花粉和花粉败育两种类型。无花粉型是在小孢子刚形成时发生败育，从而是产生无花粉的空壳型花药。花粉败育型是小孢子只发育到单核花粉，二胞花粉     

热胁迫下对玉米自交系育性的鉴定

在山西运城市7-8月份气温偏高的特殊气候环境下,对120份玉米自交系材料进行了雄花育性鉴定。结果表明,高温条件下,材料间的雄花育性存在着明显的差异;不育性表现方式有:无花粉粒、花药吐不出、花药不散粉。模拟玉米螟为害雄花节打孔试验表明,即使玉米植株能得到充足的水分供应,雄花内部的水分胁迫是影响花粉正常散出的主要因素之一。     

Cre/loxP定位重组系统在植物雄不育和杂种优势中的利用研究

Cre—loxP是来源于噬菌体P1的一种位点特异性重组系统。目前，它已广泛应用于基因功能鉴定，动植物及微生物基因组的修饰以及基因的表达和调控。雄性不育系在杂种优势的利用中具有重要意义。本实验是以Cre—loxP系统调控细胞毒素基因barnase在植物雄性器官发育的特异时期在特异组织表达，从而达到控制植物育性的目的。1．在本实验中，通过PCR方法克隆了解淀粉芽孢杆菌Bacillus amyloliquefaciens的barnase基因，及其抑制因子barstar的DNA序列，同时，构建成功双元表达载体及基因枪转化载体，并已获得经分子险测呈阳性的小麦植株。2．osg6B是来源于水稻花药绒毡层的特异性的启动子，它在小孢子发育的四分体时期至单核花粉期在花药绒毡层中特异启动基因的表达，它具有较为严紧的时空特异性。以osg6B为启动子，构建了一组受控于Cre／loxP位点特异性重组系统的适用于基因枪法转化小麦的载体系统。其中，转不育基因(og6B—barnase)小麦植株生长正常，但开花后，花粉量明显减少，自交不能得到正常种子。进行花粉活力检测发现，转基因小麦大部分花粉表现为败育。花粉离体萌发试验表明，转不育基因小麦的花粉离体萌发率极低，几近败育。说明以osg6B驱动barnase在小麦花药中的表达可以导致雄性不育。3．以osg6B为启动子，构建了一组受控于Cre／loxP的用于控制双子叶植物育性的双元表达载体系统。以转cre基因的烟草作为受体，以含阻断序列的质粒农杆菌进行二次转化。二次转化株营养生长正常，但开花期花器官表现异常。具体表现为花瓣异常开裂或不开裂，花丝变短或粘连，花药提早萎蔫或异常开裂。推测这种现象的出现与启动子的组织特异性或barnase的渗露表达有关。4．二次转化株花粉染色因不同株系出现全部败育、部分不育和多数可育的情况。花粉离体萌发实验也得到了相似的结果。这说明通过筛选，利用Cre／loxP得到完全不育的植物株系是完全可行的。5．获得了9个barnase的突变体，初步推测第十四位氨基酸及第八十位氨基酸与核糖核酸酶活性有关。同时，发现barnase的第316位核苷酸序列为易突变位点，自然发生的突变多插入一个C，从而产生终止位点的改变。这或许会为今后barnase的克隆与利用提供一些参考。     

Influence of cytoplasmic-nuclear male sterility systems on microsporogenesis in pearl millet (Pennisetum glaucum (L.) R. Br.)

Influence of a range of cytoplasms on microsporogenesis and anther development in pearl millet was studied using six isonuclear A-lines having five cytoplasms (A₁, A₂, A₃, A₄ and A_v) and the nuclear genome of 81B. 81B was used as a male-fertile control. Microsporogenesis and anther development were normal in 81B. However, pollen mother cell (PMC)/microspore/pollen degeneration in the six A-lines occurred at different stages of anther development. Each cytoplasm had its unique influence on microsporogenesis and anther development as evidenced by different developmental paths followed by them leading to pollen abortion. The cause of pollen abortion differed from line to line, from floret to floret within a spikelet, from anther to anther within a floret, and in some cases even from locule to locule within an anther. Events that led to male sterility included anomalies in tapetum and callose behaviour, persistence of tapetum, endothecium thickness, and other unknown causes. The present study also indicated that anther/pollen development was more irregular in Pb 406A₃. In 81A₄ and 81A₁ > 95% of anther locules followed a definite developmental path to pollen abortion. In the other A-lines many developmental paths were observed within the line and pollen degeneration occurred at various stages. This could be one of the reasons for greater instability of male sterility in the A₂ and A₃ systems and greater stability of male sterility in the A₁ and A₄ systems. This revised version was published online in July 2006 with corrections to the Cover Date.     

Particle-Bombardment-Mediated Co-Transformation of Maize with a Lysine Rich Protein Gene (sb401) from potato

Summary Little work has been reported on genetic transformation with maize inbred lines, especially elite inbred lines used in breeding. In this work, 7 self-pollinated inbred lines and 4 hybrid lines have been screened. The results revealed that calli derived from immature embryos from two inbred lines X333 and X301 were compact, hyperhydric and unsuitable for transformation, but the calli induced from other inbred lines and all the hybrid lines were friable and yellow and could be used for genetic transformation. The sb401 gene isolated from potato (Solanum berthaultii) encodes a protein with a high lysine content. Maize calli from 5 self-pollinated inbred lines and 4 hybrid lines were transformed using particle-bombardment with different plasmids to simultaneously introduce the sb401 lysine rich gene and the selectable gene hpt respectively. Two hundred and sixty-eight regenerated plants were obtained from these genotypes. Co-insertion was confirmed in 29 regenerated plants by PCR and Southern blot analysis. Transgene segregation of the R1 plants was observed and one marker-free transgenic maize line was recovered. Analysis of the crude protein content in mature seeds of R1 transgenic plants also showed an increase from 36.8% to 48.2%. This study thus provides a workable system for generating transgenic maize free from selectable marker genes and generates valuable resources for obtaining marker free transgenic maize with a high-lysine protein content.     

烟草绒毡层特异启动子pTA29在棉花中的表达特性

烟草绒毡层特异启动子pTA29 (TA29 promoter)已用于多种植物雄性不育和花粉发育的研究。作为外源特异表达启动子pTA29在棉花中的表达特性尚不清楚,为了研究该启动子在棉花中的表达特异性,本文构建了pTA29:gus融合基因,并将其转入棉花。研究发现在GUS染液中添加10%的乙醇可以抑制棉花花药内源GUS活性。用改良的GUS染液进行组织化学染色表明,pTA29:gus转化植株的GUS活性主要存在于花药中,并在花蕾长为6 mm和15 mm的花药中有2个活性高峰,而在根、茎、胚珠、花瓣、苞叶中未被检测到。与烟草不同,在pTA29:gus棉花转化植株的叶片表皮毛和花粉中也能检测到GUS活性。定量RT-PCR分析表明转化子中gus基因的转录水平与GUS活性一致。上述结果表明,烟草绒毡层特异启动子pTA29可以控制下游基因在棉花花药中优势表达;在利用该启动子进行棉花基因功能以及雄性不育研究时,应注意该启动子在棉花中的表达范围和组织特异性。     

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。     

A comparative study of microsporogenesis and anther wall development in different types of genic and cytoplasmic male sterilities in chives

A new cytoplasmic and two genic male sterilities in chives are compared with male‐fertile counterparts for anther development and microsporogenesis. The genic male sterilities (wi and st1) fail to produce viable pollen, since microsporogenesis is stopped around the stage of tetrads. Compared with a previously described cytoplasmic male sterility, CMS₁, which also shows alterations during this stage, wi‐ and st1‐sterile plants fail to produce pollen walls. The last two male sterilities are very similar in development but can be distinguished by the missing breakdown of callose in st1‐sterile plants. In contrast to the sterilities described above, the second cytoplasmic male sterility, CMS₂, is caused by functional damages to sporophytic tissues, such as no degeneration of mesothecium cells and no thickening of the endothecial cell walls. These alterations prevent the release of pollen, although they are normally developed.     

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.     

花粉致死基因ZmAA1对玉米遗传转化的研究

为了研究花粉致死基因ZmAA1的功能,进而创制转ZmAA1基因玉米不育新材料。在Gen Bank中找到花粉致死基因ZmAA1及启动子Pg47,并在目的片段的上下游设计増加酶切位点,基因合成构建到克隆载体puc57上,采用传统酶切连接的方法构建了植物表达载体pCAMBIA3300-Pg47-ZmAA1-35S-bar,并利用农杆菌介导法转化优良玉米自交系郑58萌动胚。成功构建植物表达载体pCAMBIA3300-Pg47-ZmAA1-35S-bar;共获得94株除草剂抗性植株,其中47株目的片段PCR呈阳性反应,对温室中表现为花粉败育的PCR阳性植株进行目的片段及筛选标记bar基因RT-PCR与蛋白免疫学试纸条检测,结果表明,花粉致死基因ZmAA1及启动子Pg47已经整合到玉米基因组并表达。     

Bud pollination and hybrid seed production in detergent-induced male sterile plants of Brassica juncea

The efficacy of a synthetic detergent (Surf Excel) as a potential chemical hybridizing agent in Brassica juncea was studied. Foliar sprays with various concentrations of the detergent caused reductions in plant height, number of branches and leaves per plant, size of leaves, anther size, pollen per flower, ovules per flower, pollen fertility, fruits per plant, fruit size, seeds per fruit, total yield per plant and 100 seed weight as compared with those of untreated plants. The style in the floral buds of plants sprayed with different concentrations of Surf Excel elongated and so the receptive stigma protruded from the buds which facilitated cross‐pollination by honey bees. The plants sprayed once with 2% Surf Excel exhibited an elongated style with a raised receptive stigma and 100% pollen sterility without causing a significant reduction in total yield.