灯盏花雄性不育种质鉴定与花药发育的细胞学研究

对云南泸西栽培灯盏花群体进行调查,发现了灯盏花雄性不育种质个体,其出现频率约为1.06×10-4.对所发现的灯盏花不育株形态特征及其花药发育过程进行了观察,并对花粉活力进行鉴定.结果显示:(1)灯盏花不育株根、茎、叶形态与正常可育植株基本相似,管状花小,花丝短,花药瘦小,无花粉粒散出或花粉无活力.(2)灯盏花在其花药发育的小孢子母细胞时期、四分体时期、小孢子时期和单核早期,由于绒毡层细胞液泡化、提前解体,不能为小孢子或花粉发育提供所需物质,导致小孢子母细胞和四分体解体,产生无花粉的花药;或小孢子和单核花粉胞内降解,形成不同形状和外壁纹饰的败育花粉.研究认为,灯盏花花药绒毡层异常是其花粉败育的主要原因.     

雄性不育和雄性能育小麦(Triticum aestivum L.)花药和花粉发育的细胞形态学观察

本工作是小麦雄性不育杂种优势利用研究项目的一部分。从细胞形态学的角度,研究小麦细胞质雄性不育系及其保持系花药和花粉的发育,为探索雄性不育性的机理提供资料。应用石蜡切片法,对小麦“早熟1号”和“北京8号”细胞质雄性不育系及其保持系花药的发育过程进行了观察,得到如下的结果:(1)不育系花粉的败育,在发育的各个时期都发生,但败育的关键时期是在小孢子发育后期,具大液泡的小孢子不能进入配子体发育阶段。(2)不育系花药和花粉的发育,在小孢子发育早期以前,90%以上与保持系相似,是正常的;少数表现异常而导致败育。异常现象有:药室合并;小孢子母细胞解体,绒毡层发育正常;小孢子母细胞互相粘连,形成多核的原生质团;解体的小孢子母细胞与绒毡层融合形成多核的原生质团;药室中除正常发育的小孢子母细胞或小孢子外,还出现异常的巨型细胞;绒毡层提早在小孢子发育早期解体,形成多核的原生质困;绒毡层肥大生长。     

温光敏核不育水稻N28S 无花粉败育的显微结构观察

采用石蜡切片和荧光显微技术观察了温光敏核不育水稻N28S 无花粉败育过程中的显微结构变化, 结果显示: N28S 的小孢子母细胞形成后细胞质变得稀薄, 一部分不能进行减数分裂, 一部分减数分裂阻滞在细线期或胞质分裂异常, 最终所有细胞液泡化解体消失。在此过程中, 还观察到小孢子母细胞在细线期胼胝质壁不产生或提早消失, 以及小孢子发育后期花药壁绒毡层的异常解体。认为N28S 的无花粉败育是由小孢子母细胞的细胞质异常引起的, 胼胝质壁和绒毡层的异常是结果而不是原因。     

温光敏核不育水稻N28S无花粉败育的显微结构观察

采用石蜡切片和荧光显微技术观察了温光敏核不育水稻N28S无花粉败育过程中的显微结构变化,结果显示：N28S的小孢子母细胞形成后细胞质变得稀薄,一部分不能进行减数分裂,一部分减数分裂阻滞在细线期或胞质分裂异常,最终所有细胞液泡化解体消失。在此过程中,还观察到小孢子母细胞在细线期胼胝质壁不产生或提早消失,以及小孢子发育后期花药壁绒毡层的异常解体。认为N28S的无花粉败育是由小孢子母细胞的细胞质异常引起的,胼胝质壁和绒毡层的异常是结果而不是原因。     

甘蓝型油菜隐性上位互作核不育材料1665花药败育的细胞学研究

采用石蜡切片方法,对甘蓝型油菜隐性上位互作核不育材料1665的可育株与不育株花药进行细胞学观察.结果显示:(1)不育株花药在花粉母细胞减数分裂时期出现异常,部分花粉母细胞细胞分裂相不均等分裂或分裂异常.导致部分四分体形状异常.(2)不育株绒毡层细胞在四分体时期开始生长膨大,单核花粉时期出现液泡化和巨型化,侵占药室,使得小孢子不能正常释放或无法继续发育;部分释放出的小孢子未及时形成花粉壁,阻碍花粉继续发育.不能发育形成二核期和三核期花粉,导致花药败育.     

白菜核雄性不育两用系可育和不育花药ATPase的分布

对白菜核雄性不育两用系可育花药和不育花药的ATPase做了定位分析。可育花药的花粉母细胞核中积累了大量的ATPase反应颗粒,而细胞质中ATPase反应颗粒较少,但在线粒体中特异地聚集了一些大的ATPase反应颗粒。减数分裂后,小孢子细胞质中ATPase反应颗粒明显增加。随着小孢子发育,其细胞质中ATPase反应颗粒逐渐减少,但在线粒体中又特异性地聚集了较多的AT-Pase反应颗粒。当花药发育到二胞花粉时期,花粉和绒毡层细胞中的ATPase反应颗粒已很少了。不育花药的花粉母细胞中呈现较多的ATPase,然而在线粒体中很少。异常四分体小孢子细胞质中虽然有较多的ATPase反应颗粒,但还是通过细胞质收缩和质壁分离方式退化。对可育花药的花粉母细胞线粒体中特异出现的簇状ATPase分布现象进行了分析,讨论了不育花药中花粉母细胞线粒体ATPase的异常与花粉败育的可能关系。     

冬季低温条件下香石竹小孢子败育的细胞学研究

利用压片法及石蜡切片法观察冬季低温下香石竹小孢子发育过程,以明确低温导致香石竹小孢子败育的因素,为杂交育种奠定基础。结果表明:(1)冬季低温下香石竹只有部分小孢子发育正常,经过小孢子母细胞、减数分裂和四分体等时期,最后发育成花粉。(2)石蜡切片法观察到冬季低温下香石竹1.5~1.6cm长花蕾中有61%的花粉母细胞发生败育,1.7~1.8cm长花蕾中有71%的花粉母细胞发生败育。(3)部分已经进入四分体时期的小孢子胼胝质未能及时溶解,妨碍了小孢子释放而导致败育。研究认为,花粉母细胞和四分体的发育异常是冬季低温下香石竹小孢子败育的主要原因。     

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     

光敏雄性不育谷子683小孢子败育途径观察

对光敏雄性不育谷子（Setaria italica）683在长日（＞15h）和短日（＝10h）下小孢子的发育途径、花药中药粉总数及游离花粉总数作了观察和统计,发现长日条件下,小孢了败育主要发生在造孢细胞和花偻母细胞时期,此时,有60％－80％的药室的造胞细胞和花偻母细胞严重收缩,细胞结构破坏,演变成着色极深的“异常黑块”（埃氏苏木精染色）。每花药中花粉平均数,长日条件下为66.84,短日照下为111.86,差异显著,每视野中的游离花粉平均数,长日条件下为13.73,而在短日条件下为20.1.研究对“异常黑块”的出现与花粉总数大幅度降低的关系作了分析,并讨论了“异常黑块”发生的可能原因及其与小孢子败育的关系。     

云南紫稻细胞质雄性不育系花药发育过程中Ca2+的分布特点

运用焦锑酸钾沉淀法研究了云南紫稻细胞质雄性不育系和保持系花药在发育过程中Ca^2 的分布特点。结果表明,保持系的花粉母细胞和小孢子的胞质内部基本无Ca^2 的沉淀,后期花粉外壁出现Ca^2 的沉淀;保持系早期的绒毡层细胞形态正常,胞内有少量Ca^2 沉淀,后期绒毡层细胞开始凋亡,胞质凝集,胞内出现大量Ca^2 的颗粒。不育系花粉母细胞在减数分裂时期败育,胞质液泡化,内部出现大量Ca^2 的沉淀;不育系绒毡层细胞形态正常,胞内无Ca^2 的沉淀。绒毡层与花粉母细胞、小孢子之间出现大量Ca^2 颗粒。探讨了不育系花药花粉母细胞中以及与绒毡层细胞之间Ca^2 的异常积累与雄性不育的关系。     

大白菜新型细胞质雄性不育系6w-9605A的育性鉴定和花药败育的细胞学观察

采用石蜡切片技术,研究了大白菜(Brassica campestris L.ssp.pekinensis)细胞质雄性不育系6w-9605A及其保持系6w-9605B的花药发育过程的细胞形态学特征,确定不育系花药败育时期及方式,并对不育系6w-9605A进行花器官观察和育性鉴定.结果表明:保持系6w-9605B花药发育正常;不育系6w-9605A花药发育受阻于孢原分化时期,占总败育花药的66.7％,不形成花粉囊和花粉粒,属于无花粉囊型败育;另外33.3％的败育花药可形成花粉囊,小孢子均受阻于单核靠边期或者二胞期,败育特点为绒毡层细胞异常肥大,挤压小孢子,导致小孢子和绒毡层解体;6w-9605A的不育性稳定、彻底,不育株率和不育度均为100％.     

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。     

大白菜雄性败育的显微结构观察

通过对核质互作型雄性不育系169A和核雄性不育两用系88_3的细胞形态解剖学观察表明,两个不育系在开花时雄性细胞均表现100%的败育,花药的表皮细胞均具有生活力,但败育形式、时期、特点各异。169A败育发生于孢原细胞前后,以孢原细胞退化,或转变成薄壁细胞为主要特点。88_3败育从小孢子母细胞至二核花粉粒皆有发生,高峰期在四分体前后（约占80%）,小孢子母细胞不能进入减数分裂和不能完成减数分裂及小孢子不能正常发育是败育的主要形式,但败育特点均是败育一旦发生便是急剧而彻底的解体或凝集成一团。     

大白菜雄性败育的显微结构观察

通过对核质互作型雄性不育系169A和核雄性不育两用系88-3的细胞形态解剖学观 察表明,两个不育系在开花时雄性细胞均表现100%的败育,花药的表皮细胞均具有生活力,但败育形式、时期、特点各异。169A败育发生于孢原细胞前后,以孢原细胞退化,或转变成薄壁细胞为主要特点。88-3败育从小孢子母细胞至二核花粉粒皆有发生,高峰期在四分体前后（约占80%）,小孢子母细胞不能进入减数分裂和不能完成减数分裂及小孢子不能正常发育是败育的主要形式,但败育特点均是败育一旦发生便是急剧而彻底的解体或凝集成一团。     

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.     

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。     

Cytological analysis of early microspore divisions leading to gametic embryo formation in <Emphasis Type="Italic">Quercus suber</Emphasis> L. anther cultures

The correlation between the phenologic stage of the inflorescence and the microspore development stage was studied. Cytological examinations of the development of microspores during in vitro anther culture of cork oak (Quercus suber L.), were carried out during the first four weeks of culture. To observe the division occurring in the microspores, anthers were taken randomly from the cultures after heat shock treatment and were stained with DAPI. Most of the anthers responding to a heat stress treatment contained 91 % vacuolated microspores, indicating that this developmental stage is responsive to embryogenesis induction in cork-oak microspores. After the heat shock treatment some cork-oak microspores were induced and initiated the embryogenic pathway with the occurrence of numerous symmetric mitosis, producing structures with two to ten or more nuclei. These lead to the formation of high numbers of multicellular cork-oak microspores (pro-embryos). Twenty-forty days after induction, small white globular and cotyledonal embryos were observed, which further developed root and shoot, regenerating plantlets.     

Two recessive genes controlling thermophotoperiod-sensitive male sterility in wheat

Male sterility of wheat-breeding line 337S (Triticum aestivum L.) is sensitive to both short day-length/low temperature and long day-length/high temperature. 337S was crossed with the common wheat variety, Huamai No. 8 and the F₁ was highly fertile. The F₂ population segregated in a 15:1 ratio for fertility/sterility in 243 individuals under long day-length/high-temperature. The two thermophotoperiod-responsive male sterile genes were mapped to chromosomes 5B and 2B using Simple Sequence Repeat (SSR) markers and bulked segregant analysis. Partial linkage maps around the sterility loci of chromosomes 2B and 5B were constructed using the 243 individuals in the F₂ population. One gene (wptms1) for male sterility was flanked by the SSR markers Xgwm335 and Xgwm371 at a genetic distance in chromosome 5B of 4.1 and 24.4 cM, respectively. The second gene (wptms2) was mapped between markers Xgwm374 and Xgwm120 at a genetic distance of 6.6 and 20.9 cM, respectively. The closest linked markers Xgwm335 (wptms1) and Xgwm374 (wptms2) explained 53 and 38% of phenotypic variation for the fertility. The SSR markers provide a useful tool to transfer the male sterile genes into elite wheat germplasm.     

Apoptosis in developing anthers and the role of ABA in this process during androgenesis in Hordeum vulgare L.

Intra-nucleosomal cleavage of DNA into fragments of about 200 bp was demonstrated to occur in developing anthers, in which microspores had developed into the mid-late to late uni-nucleate stage in situ, i.e. at the verge of mitosis. The same was observed, but to a much larger extent, if these anthers were pre- treated by a hyper-osmotic shock. Pretreatment of anthers before the actual culture of microspores was required for optimal androgenesis of microspores. The use of the TUNEL reaction, which specifically labels 3 ends of DNA breaks, after intra-nucleosomal cleavage of DNA, revealed that DNA fragmentation mainly occurred in the loculus wall cells, tapetum cells and filament cells. TUNEL staining was absent or infrequently observed in the microspores of developing anthers in situ. Electron microscopy studies showed condensed chromatin in nuclei of loculus wall cells in the developing anthers. These observations at the chromatin and DNA level are known characteristics of programmed cell death, also known as apoptosis. Features of apoptosis were infrequently found in microspores from freshly isolated mature anthers. However, most tapetum cells had disappeared in these anthers and the remaining cell structures showed loss of cellular content. The viability of microspores in pre-treated anthers was comparable to those in freshly isolated anthers and almost four times higher than in anthers from control experiments. This observation was correlated with three to four times less microspores showing TUNEL staining and a two times higher level of ABA in the anther plus medium samples than in controls. Addition of ABA to the controls enhanced the viability and lowered the occurrence of apoptosis linked characteristics in the microspores. These data suggest that pre-treatment is effective in stimulating androgenesis because it leads to an increase in ABA levels which protects microspores from dying by apoptosis.     

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。