RNAi-mediated male sterility of tobacco by silencing TA29

The superior performance of F₁ hybrids has a significant impact on agricultural productivity. For commercial application, the availability of an efficient system for obtaining male-sterile lines of crops is an essential prerequisite. Here we have investigated the use of RNA interference (RNAi) technology to silence a male-specific gene in the model host tobacco. TA29 is expressed exclusively in anthers at the time of microspore development. About 10 out of 13 tobacco lines transformed with a hairpin RNAi construct containing TA29 sequences were male sterile. Transgenic plants were phenotypically indistinguishable from non-transgenic plants. At the anthesis stage, pollen grains from transgenic, male-sterile plants were aborted and lysed in comparison to the round and fully developed pollen in non-transgenic plants. Microscopic analysis of anthers showed selective degradation of tapetum in transgenic plants with no microspore development. One week after self-pollination, the ovules of non-transgenic plants were double the size of those in transgenic plants, due to successful self-fertilization. Male sterile transgenic plants set seed normally, when cross-pollinated with pollen from non-transgenic plants, confirming no adverse effect on the female parts of the flower. These results show that silencing of male-specific genes by RNAi is potentially a useful tool for generating male-sterile lines for producing hybrid seed.     

Anther developmental defects in Arabidopsis thaliana male-sterile mutants

 We identified Arabidopsis thaliana sterility mutants by screening T-DNA and EMS-mutagenized lines and characterized several male-sterile mutants with defects specific for different anther processes. Approximately 44 and 855 sterile mutants were uncovered from the T-DNA and EMS screens, respectively. Several mutants were studied in detail with defects that included the establishment of anther morphology, microspore production, pollen differentiation, and anther dehiscence. Both non-dehiscencing and late-dehiscencing mutants were identified. In addition, pollenless mutants were observed with either apparent meiotic defects and/or abnormalities in cell layers surrounding the locules. Two mutant alleles were identified for the POLLENLESS3 locus which have defects in functional microspore production that lead to the degeneration of cells within the anther locules. pollenless3–1 contains a T-DNA insertion that co-segregates with the mutant phenotype and pollenless3–2 has a large deletion in the POLLENLESS3 gene. The POLLENLESS3 gene has no known counterparts in the GenBank, but encodes a protein containing putative nuclear localization and protein-protein interaction motifs. The POLLENLESS3 gene was shown recently to be the same as MS5, a previously described Arabidopsis thaliana male-sterility mutant. Three genes were identified in the POLLENLESS3 genomic region: GENEY, POLLENLESS3, and β9-TUBULIN. The segment of the Arabidopsis thaliana genome containing the POLLENLESS3 and β9-TUBULIN genes is duplicated and present on a different chromosome. Analysis of the POLLENLESS3 expression pattern determined that the 1.3-kb POLLENLESS3 mRNA is localized specifically within meiotic cells in the anther locules and that POLLENLESS3 mRNA is present only during late meiosis. Received: 15 October 1998 / Revision accepted: 19 November 1998     

一个新的小麦核不育材料的发现和鉴定

从轮选可育株衍生的后代中发现一个雄性不育株,即P740不育材料。在P7 40不育材料第1、2、3次姊妹交后代中,可育株与不育株的分离比分别符合4:1、 7:5和15:13。在第2次姊妹交后代中可育株产生的40个株系中,7个株系的育性分离比是15:1,33个株系是3:1,二者约分别占1/7和6/7。以上结果表明,P740是一个双隐性基因控制的核雄性不育材料。本文提出了计算隐性核不育材料姊妹交后代育性分离比率的公式。 Abstract:A male sterile material was found in the progeny derived from a fertile plant in the population of recurrent selection.The ratios of fertile to sterile plants in three sib-mating of the male sterile material with fertile plants were to 4:1,7:5 and 15:13,respcctively.In the 40 plant lines derived from the fertile plants of second sib-mating population,33 lines approximated to the segregation ratio of 3(fertile):a double recessive nuclear male sterile material.A genetic pattern for calculation of fertile segregation in sib-mating progenies of recessive nuclear male sterile material was advanced in this paper.     

Localization and fine mapping of gaMS-1, a male gametophytic mutant of maize

Post-meiotic mutants affecting pollen development are fundamental tools for defining the genetic program controlling microsporogenesis and pollen function. An example of such mutants is gametophytic male sterile-1 (gaMS-1). Heterozygous plants gaMS-1/+ that have a normal phenotype and are female fertile, segregate 1:1 normal:sterile pollen grains and their selfed progeny segregates 1:1 normal:semi-sterile plants. With the final aim of isolating the gene, a positional cloning strategy was adopted. In this paper, we report the results of fine mapping GaMS-1 by different types of molecular markers. Two back crosses were used as mapping populations. They were obtained by crossing the line carrying the mutation with the inbred lines Mo17 and WF9, used as recurrent male parents. Linkage disequilibrium analysis allowed assigning GaMS-1 to the short arm of chromosome 2.By the combined use of SSR, AFLP, PCR markers and ESTs a region of 1 cM containing GaMS-1 was delimited. Received: 15 November 2000 / Revision accepted: 24 May 2001     

甘蓝型油菜几个雄性不育系花药发育的细胞形态学研究

选用甘蓝型油菜6个细胞质雄性不育系和1个细胞核雄性不育系为材料,与可育系比较,确定花药发育受阻的时期和方式。根据研究结果,将其雄性不育系分为三类:1.湘矮A,Po-aA,陕2A和7s-3A花药发育受阻于孢原细胞分化期,没有分化形成花粉囊。2.萝AⅠ和萝AⅡ花药发育受阻于四分体至单核花粉期。败育方式为小孢子难以从四分体中释放出来,或释放出来后细胞质液泡化,核不能分裂,花粉壁发育不良。此外,还见到绒毡层径向肥大、延迟消失和维管束分化不良等异常现象。3.宜3A为核不育系,花药发育受阻于花粉母细胞期。败育方式为花粉母细胞死亡,减数分裂异常,或不能进行减数分裂。绒毡层和维管束一般都能正常发育。     

不同温度条件下高粱温敏雄性不育系冀130A 育性变化规律及花粉败育研究

以回18A为母本、抗蚜保持系130B为父本,回交8个世代育成夏播高粱不育系冀130A。在选育过程中发现,冀130A的不育度受温度影响。为进一步了解温度对该不育系育性的影响,明确影响育性的临界温度,为杂交制种提供依据,对其进行了播期试验、异地种植和关键时期不同温度处理试验,结果表明:旗叶伸展至抽穗期间平均气温低于23℃,不育度为100%,株间无差异;平均气温高于23℃,部分花粉可育,随着温度升高,可育花粉增多,平均气温超过26℃可明显观察到可育花粉,自交结实率可达30%,株间有差异。通过石蜡制片及显微摄影观察,对不同温度处理条件下花药和花粉特征进行了研究。结果显示:低温导致绒毡层细胞出现明显异常,主要表现为液泡化、径向肥大和延迟退化;低温条件下早期败育的花药,造孢细胞或花粉母细胞较早出现解体,无单核花粉粒产生。旗叶伸展至抽穗期间温度高于26℃时,花粉母细胞减数分裂形成单核花粉粒,之后部分花粉粒出现畸形,但仍有约40%的正常花粉粒可以存活到花药开裂并散粉。低温导致绒毡层细胞出现异常,是花粉败育的主要原因。冀130A的育成,扩大了高粱不育系的差异,有利于商业化的高粱杂交种选育,同时为深入研究高粱不育系的败育机理提供了新种质资源。对冀130A育性转换特性研究为利用该不育系进行杂交种制种选择制种区域和播期奠定了基础。     

A rapid,single leaf assay for detecting the presence of ‘S’-male-sterile cytoplasm in maize

Summary A simple, rapid, and reproducible assay is described for determining unambiguously the presence of S-type cytoplasm in male-sterile and male-fertile (restored) maize lines. Because the assay requires only 0.5 g leaf segment per sample, it is a single plant assay and the plant is not destroyed. Plants at any developmental stage can be used. The assay involves a 30 sec homogenization, 20 min centrifugation, one hour lysis, overnight agarose electrophoresis, 30 min gel staining, and photography of the gel to produce a result in much less than 24 hr. Many samples can be assayed simultaneously. The various assay methods available for classifying maize cytoplasms are compared and discussed.     

The PsEND1 promoter: a novel tool to produce genetically engineered male-sterile plants by early anther ablation

PsEND1 is a pea anther-specific gene that displays very early expression in the anther primordium cells. Later on, PsEND1 expression becomes restricted to the epidermis, connective, endothecium and middle layer, but it is never observed in tapetal cells or microsporocytes. We fused the PsEND1 promoter region to the cytotoxic barnase gene to induce specific ablation of the cell layers where the PsEND1 is expressed and consequently to produce male-sterile plants. Expression of the chimaeric PsEND1::barnase gene in two Solanaceae (Nicotiana tabacum and Solanum lycopersicon) and two Brassicaceae (Arabidopsis thaliana and Brassica napus) species, impairs anther development from very early stages and produces complete male-sterile plants. The PsEND1::barnase gene is quite different to other chimaeric genes previously used in similar approaches to obtain male-sterile plants. The novelty resides in the use of the PsEND1 promoter, instead of a tapetum-specific promoter, to produce the ablation of specific cell lines during the first steps of the anther development. This chimaeric construct arrests the microsporogenesis before differentiation of the microspore mother cells and no viable pollen grains are produced. This strategy represents an excellent alternative to generate genetically engineered male-sterile plants, which have proved useful in breeding programmes for the production of hybrid seeds. The PsEND1 promoter also has high potential to prevent undesirable horizontal gene flow in many plant species.     

Energy and Activated Oxygen Metabolisms in Anthers of Hubei Photoperiod-Sensitive Genic Male-Sterile Rice

The energy and activated oxygen metabolisms in male-sterile and fertile anthers of Hubei photoperiod-sensitive genic male-sterile rice (HPGMR)have been comparatively studied. Among the male-sterile anthers, lower total activities of cytochrome oxidase(COD), ATPase, peroxidase (POD), catalase (CAT) and superoxide dismutase(SOD) in six anthers of a spikelet were seen, which were equivalent to about 2–64%, 20–76%, 26–60%, 6–77%, 29–55%, respectively, of those of their fertile counterparts during different development stages of pollens. The sterile anthers lacked 1–5, 1, 1–2 isozyme bands of COD, POD and SOD, respectively, and also displayed lower content of ATP, higher contents of H202 and malondiald ehyde (MDA) and higher efficiency of O2- which were equivalent to about 14–77%, 152–424%, 153–238%, 230–340%, respectively, of those of their corresponding fertile ones. It is concluded that the physiological features of male-sterile anthers, viz. lower efficiency of oxidative phosphorylation, higher efficiency of O2 and H202 generation, weaker activity of scavenger system of activated oxygen and higher level of lipid peroxidation, are indicative of some relationship between abnormal energy, activated oxygen metabolisms in male-sterile anthers and male-sterility in HPGMR.     

利用粘果山羊草细胞质1B/1R易位系诱导小麦单倍体频率的研究

用具有粘果山羊草(Ae. kotschyi)细胞质的1B/1R易位系转育成的K型小麦雄性不育系为基本材料,通过随体数目和C—显带技术鉴定了不育系、恢复系及其亲本的染色体组成,以中国春重双端体2t″1B和缺—四体1B°1D~Ⅳ与其杂交,观察了F_1减数分裂中期Ⅰ的染色体构型和C—显带特征。调查了有代表性的10个k型不育系及其中三个不育系与中国春重双端体2t″1B或缺—四体1B°1D~Ⅳ杂交F_1的单倍体诱导频率。结果表明,多数k型1B/1R不育系均可产生较高频率的单倍体,但不育系间差异很大,个别k型1B/1R不育系不产生单倍体,说明单倍体的产生除受1R短臂上的诱导单倍体基因作用外,还受其他基因的影响,同时也受父本基因型和染色体结构的影响,试验证明,1B染色体缺失或端体等非整倍体作为父本可以大大提高诱导单倍体的频率(接近70%),这种方法诱导产生的是单倍体种子,一般发芽正常,发育良好,在异质小麦育种方面具有重要实践意义。但由于这种单倍体均为孤雌生殖所致,应用受到一定限制,因此本文提出了如何应用的参考方案。