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。     

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。     

iaaL基因在烟草绒毡层中的特异表达对花粉胚胎发生的影响

来源于丁香假单孢杆菌的吲哚乙酰胺赖氨酸酯合成酶(indoleacetic acid-ly-sine synthetase)基因(iaaL)与来自烟草的在花药绒毡层特异表达(tapetal-specific)的启动子TA29融合后导入烟草,在转基因烟草中研究了这种嵌合基因的表达特性,并测定了各器官内源生长素水平.结果表明:TA29启动子只能启动iaaL基因在转基因植株的花药中特异表达,并导致转基因植株花药内源IAA含量的下降,但在完整植株上并没有对花药的正常发育造成明显的影响.当转基因植株的花药在不附加任何激素的改良Nistch H(NH)培养基上培养时,花粉胚胎发生频率下降至11%(对照在50%以上);当在NH培养基上补加0.2mg/L IAA时,转基因植株的花粉胚胎发生频率恢复到与对照相当(达55.7%),由此说明花药绒毡层细胞中IAA的代谢对花药培养中花粉胚的发育具有重要的意义.     

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。     

葡萄半不育花粉粒形态和细胞学观察及相关基因表达分析

以葡萄品种‘魏可’(可育)及其自交后代单株‘魏可实生-3’和‘魏可实生-12’以及品种‘钟山红’(雄性不育)为材料,对花器官特征、花粉萌发率、花粉粒形态以及小孢子发育过程的细胞学特征进行观察,并对绒毡层发育相关基因(DYT1、TDF1和MYB4)的表达进行分析,以初步鉴定葡萄半不育单株的不育特征,揭示花粉败育与基因调控的关系。结果显示:(1)‘魏可实生-3’和‘魏可实生-12’的花粉萌发率均在10%左右,属于半不育花粉;其雄蕊主要特征为花丝卷曲,花丝短于花柱,花药内花粉量少,花药中既有正常长球形花粉粒也有异常近球形花粉粒。(2)‘魏可实生-3’、‘魏可实生-12’没有明显的药室壁结构,小孢子母细胞稀少、疏松;‘魏可实生-3’双核期绒毡层未完全降解,绒毡层细胞中的营养物质滞留没有及时供应小孢子的发育,致后期部分小孢子败育;‘魏可实生-12’绒毡层细胞在单核早期就大部分降解而导致营养供应异常,使部分小孢子败育。(3)在减数分裂期,‘魏可’的绒毡层发育相关基因DYT1和TDF1的表达均最高,MYB4最低,‘钟山红’的3个基因的表达与‘魏可’相反,‘魏可实生-3’和‘魏可实生-12’的表达水平均在‘魏可’与‘钟山红’之间;单核早期以后,3个基因在‘魏可’的表达一直均较低,钟山红’的DYT1基因表达一直最高,其余2个基因均在最低水平,而‘魏可实生-3’的TDF1、MYB4基因在单核早期表达量最高,‘魏可实生-12’的TDF1在双核期表达量最高。研究表明,‘魏可实生-3’的花粉败育发生在单核晚期至双核期,‘魏可实生-12’的花粉败育发生在单核早期;部分药室绒毡层的异常降解,小孢子发育营养供应不均衡可能是导致其花粉半不育发生的原因;‘魏可实生-3’的绒毡层延迟降解与减数分裂期DYT1基因的表达量不足、单核期TDF1基因的过量表达以及MYB4基因在单核期的异常表达有关,而‘魏可实生-12’的绒毡层提前降解与减数分裂期DYT1基因的表达量不足而MYB4基因过量表达,以及双核期TDF1基因过量表达有关。     

羽叶薰衣草雄性不育的细胞学研究

用光镜和电镜观察羽叶薰衣草(Lavandula pinnata L.)雄性不育小孢子发育过程的细胞形态学特征.结果表明:羽叶薰衣草花药4枚,每枚花药通常具4个小孢子囊.花药壁发育为双子叶型,从外向内分为表皮、药室内壁、中层和绒毡层4层细胞.减数分裂形成的四分体为四面体及十字交叉型.小孢子的发育过程可分为造孢细胞期、减数分裂时期、小孢子发育早期、小孢子发育晚期.未观察到二胞花粉期和成熟花粉期.羽叶薰衣草花粉败育主要发生在单核花粉时期,细胞内物质解体并逐渐消失变成空壳花粉或花粉皱缩变形成为各种畸形的败育花粉.在此之前小孢子的发育正常.羽叶薰衣草小孢子不育机制体现在绒毡层过早解体、四分体时期以后各细胞中线粒体结构不正常、胼胝质壁与小孢子母细胞脱离、花药壁细胞中淀粉出现时间异常等. 壁发育为双子叶型,从外向内分为表皮、药室内壁、中层和绒毡层4层细胞.减数分裂形成的四分体为四面体及十字交叉型.小孢子的发育过程可分为造孢细胞期、减数分裂时期、小孢子发育早期、小孢子发育晚期.未观察到二胞花粉期和成熟花粉期.羽叶薰衣草花粉败育主要发生在单核花粉时期,细胞内物质解体并逐渐消失变成空壳花粉或花粉皱缩变形成为各种畸形的败育花粉.在此 前小孢子的发育正常.羽叶薰衣草小孢子不育机制体现在绒毡层过早解体、四分体时期以后各细胞中线粒体结构不正常、胼胝质壁与小孢子母细胞脱离、花药壁细胞中淀粉出现时间异常等. 壁发育为双子叶型,从外向内分为表皮、药室内壁、中层和绒毡层4层细胞.减数分裂形成的四分体为四     

雄性不育与可育楸树花发育的细胞学比较研究

楸树（Catalpa bungei C.A.Meyer.）属紫葳科(Bignoniaceae)梓树属(Catalpa),落叶乔木,是我国特有的珍贵优质用材树种。本文用石蜡切片法对可育株和雄性不育株楸树的大、小孢子发生及雌、雄配子体发育过程进行了详细地比较观察。结果表明：可育株和不育株楸树雌蕊的发育基本相同,胚珠倒生,薄珠心,单珠被,胚囊发育为蓼型。可育株雄蕊花药四室,药隔薄壁组织发达;异型绒粘层,由药壁绒粘层和药隔绒粘层组成;花药壁表皮细胞在小孢子母细胞减数分裂前后开始径向伸长加厚,直到花药开裂并不降解,这可能与花药开裂有关;成熟花粉为四合花粉。雄性不育株花药的早期发育到次生造胞细胞时期与可育雄蕊的相同,小孢子母细胞减数分裂前绒毡层发育不充分;四分体时期,绒毡层细胞高度液泡化,细胞质稀薄,已提前降解,小孢子四分体因绒毡层结构和功能异常而不能正常发育,因此楸树雄性不育为结构型雄性不育。     

iaaM基因在烟草花粉中的表达及其在花粉发育中的作用

试验利用花粉特异表达的启动子(Lat52)和绒毡层特异表达的启动子(TA29)引导外源生长素合成代谢基因(iaaM)在烟草花粉中表达以研究生长素在花粉发育中的作用。转Lat52-iaaM基因或转TA29-iaaM基因烟草在形态上表现出变异,如从茎上形成不定根,叶呈卷曲状等典型的生长素过量表达的性状。另外,与对照相比,转基因烟草花药中IAA水平显著增加,且植株矮化,开花期推迟,有的转基因烟草未能开花。上述现象表明:Lat52和TA29启动子的表达并不仅限于花粉或绒毡层,或者说这两个启动子的表达有些泄漏。转基因烟草的花药形状有较大的变异,早期的每个花药中花粉数明显减少,但这些花粉可被醋酸一洋红染色。所有能开花的转基因烟草均可收到种子,但收自某些转基因株系的种子不能萌发。所有这些结果表明生长素在花粉发育过程中起重要作用,过量的生长素会导致花粉发育的异常。     

iaaM基因在烟草花粉中的表达及其在花粉发育中的作用

试验利用花粉特异表达的启动子（Lat52)和绒毡层特异表达的启动子（TA29）引导外源生长素合成代谢基因（iaaM)在烟草花粉中表达以研究生长素在花粉发育中的作用。转Lat52-iaaM基因或转TA29－iaaM基因烟草在形态上表现出变异,如从茎上形成不定根,叶呈卷曲状等典型的生长素表达的性状。另外,与对照相比,转基因烟草花药中IAA水平显著增加,且植株矮化,开花期推迟,有的转基因烟草未能开花。上述现象表明：Lat52和TA29启动子的表达并不仅限于花粉或绒毡层,或者说这两个启动子的表达有些泄漏。转基因烟草的花药形状有较大的变异,早期的每个花药中花粉数明显减少,但这些花粉可被醋酸－洋红染色。所有能开花的转基因烟草均可收到种子,但收自某些转基因株系的种子不能萌发。所有这些结果表明生长素在花粉发育过程中起重要作用,过量的生长素会导致花粉发育的异常。     

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.     

Effects of Introducing the Chimaeric Gene TA29 Barnase on the Tapetum and Pollen Development in Tobacco

The development of tapetum and pollen in transgenic tobacco (Nicotiana tabacum L. ) harboring a chimaeric gene TA29-Barnase was compared with that of the wild-type plant. The specific expression of the exogenous genes in anther led to premature tapetal degradation, which started at the early stage of meiosis and terminated at the tetrad stage. In the wild-type anthers, tapetal degradation started at the early stage of bicellular microgametophyte and ended at the later stage of pollen development. The cytological changes of tapetal degradation in the transgenic plants were characterized by vacuolization of the tapetal cells, then nuclear condensation, and consequent massive degradation of tapetal cells. Meanwhile, the pollen mother cells gradually degraded and became destroyed along with the progress of meiosis, leaving only a few which could successfully complete their meiosis to form microspores. This observation also indicated that the TA29-Barnase gene in anther was not uniformly expressed. In addition, the structural difference between the male sterility induced by exogenous gene and the natural sterile was also discussed.     

洋葱花药发育过程中的细胞化学研究(简报)

高等植物花药结构复杂,其发育更是一个迅速、多变的过程,如小孢子母细胞减数分裂期间的细胞质改组、胼胝质壁的形成与降解、大液泡的形成与消失、花粉内外壁的形成、绒毡层细胞的降解、营养物质的积累与转化等。除了上述花药组成细胞的形态和结构发生明显变化外。花药发育的另一个显著特点是以花粉为中心的营养物质单向运输和转化,尤其是小孢子有丝分裂形成二胞花粉后开始积累大量的营养储存物以供成熟花粉萌发时利用。     

巴戟天花药发育过程中多糖和脂滴分布特征

巴戟天花药发育中多糖和脂滴类物质的分布呈现一定的规律：减数分裂之前,花药壁的绒毡层细胞中有少量脂滴,其他细胞中脂滴和淀粉粒都很少。四分体时期,四分体小孢子中开始出现脂滴,绒毡层细胞中的脂滴较以前增加,其他细胞中的脂滴和淀粉粒仍然很少。小孢子早期,游离小孢子在其表面形成了花粉外壁,靠外壁下方有一层周缘分布的多糖物质。绒毡层细胞中的脂滴明显减少。发育晚期的小孢子中形成一个大液泡,细胞质中出现淀粉粒;同时在药壁和药隔组织中也出现了淀粉粒。此时绒毡层退化。在二胞花粉早期,花粉中积累了大量淀粉粒和一些脂滴。但在成熟的花粉中（二胞花粉晚期）,淀粉粒消失,只有一定数量的脂滴保留。巴戟天成熟花粉中积累的营养物质主要为脂滴。     

The Post-meiotic Deficicent Anther1 （PDA1） gene is required for post-meiotic anther development in rice

To understand the molecular mechanism of male reproductive development in the model crop rice,we isolated a complete male sterile mutant post-meiotic deficient anther1 (pda1) from a γ-ray-treated rice mutant library.Genetic analysis revealed that the pda1 mutant was controlled by a recessive nucleus gene.The pda1 mutant anther seemed smaller with white appearance.Histological analysis demonstrated that the pda1 mutant anther undergoes normal early tapetum development without obvious altered meiosis.However,the pda1 mutant displayed obvious defects in postmeiotic tapetal development,abnormal degeneration occurred in the tapetal cells at stage 9 of anther development.Also we observed abnormal lipidic Ubisch bodies from the tapetal layer of the pda1 mutant,causing no obvious pollen exine formation.RT-PCR analysis indicated that the expression of genes involved in anther development including GAMYB,OsC4 and Wax-deficient anther1 (WDA1) was greatly reduced in the pda1 mutant anther.Using map-based cloning approach,the PDA1 gene was finely mapped between two markers HLF610 and HLF627 on chromosome 6 using 3,883 individuals of F2 population.The physical distance between HLF610 and HLF627 was about 194 kb.This work suggests that PDA1 is required for post-meiotic tapetal development and pollen/microspore formation in rice.     

A comparative light and electron microscopic analysis of microspore and tapetum development in fertile and cytoplasmic male sterile radish

To gain further insight into the abortive stages and ultrastructural changes leading to pollen degeneration of a novel cytoplasmic male sterile radish 805A, we compared differences of cellular and subcellular structure of sterile anther with fertile anther by light and electron microscopy analysis. Two types of locule degeneration in sterile anther were detected, of which the time of degeneration occurred and completed was different. In type I, abnormality of pollen mother cells (PMCs) and tapetal cells, including condensation of cytoplasm and large vacuoles within tapetal cells, was shown at PMC stage. In type II, meiosis and early tetrad stage progressed normally except for large vacuoles that appeared in tapetal cells. Ultrastructural alterations of the cellular organization were observed in the type II locules, such as chromatin condensation at the periphery of the nucleus and degeneration of the karyotheca, compared with normal pollen development. The results suggested that the cytoplasmic male sterility anther degeneration was probably caused by dysfunctions of tapetum and vacuolation of tapetum, PMCs, and microspores. Thus, the identical factors, which induced CMS in the same cytoplasmic and nuclear genetic background, might affect development of tapetum and microspore at different stages during the cytoplasmic male sterile 805A anther development.     

Ethylene is Involved in the Control of Male Gametophyte Development and Germination in Petunia

The time courses of 1-aminocyclopropane-1-carboxylic acid (ACC) content and ethylene production in developing anthers of petunia fertile and sterile lines and the effects of exogenously applied ethylene and an inhibitor of ethylene action, 2,5-norbornadiene (NBD), on male gametophyte development and germination were investigated. Fertile male gametophyte development was accompanied by two peaks of ethylene production by anther tissues. The first peak occurred during microspore development simultaneously with degeneration of both tapetal tissues and middle layers of the anther wall. The second peak coincided with maturation and dispersal of pollen grains. The mature pollen is characterized by a high ACC content (up to 300 nmol/g). Exogenously applied ethylene (1–100 ppm) induced degradation of gametophytic generation at the meiosis stage. NBD completely inhibited anther development at the early stages of its development and delayed anther dehiscence. In anther tissues of the petunia sterile line, tenfold higher ethylene production was observed at the meiosis stage compared to that in fertile male gametophytes and this correlated with degeneration of both microsporocytes and tapetal tissues. In vitro male gametophyte germination was accompanied by an increase of ethylene production, whereas NBD completely blocked male gametophyte germination. These results suggest that ethylene is an important factor in male gametophyte development and germination.     

基因工程雄性不育烟草及其温度敏感性

将含有抗溴苯腈基因bxn和雄性不育基因的重组载体pTA29-Barnase/bxn导入农杆菌(Agrobacterium tumefaciens)后转化烟草(Nicotiana tabacum L.),得到33个转基因植株。在27℃/23℃培养的16株中,有7株表现部分不育,另9株全部可育。而在20℃/15℃培养的17株中,12株表现不育,5株表现部分不育。部分不育的植株上同时开放可育花朵和不育花朵,不过其中的不育花朵中的花粉萌发活力很低。将在20℃/15℃条件下表现不育的12个不育株从20℃/15℃温室移至27℃/23℃温室后30d左右,其中9株表现程度不同的育性恢复现象:5株表现部分可育,另4株表现完全可育;但仍有3株表现雄性不育。雄性不育花朵的花丝变短,花药皱瘪,不散粉。细胞学观察证明,转基因植株的花药绒毡层降解提早于四分体时期,至单核小孢子时期降解殆尽。     

枸杞花药发育过程中钙的分布

在枸杞花药发育过程中,用焦锑酸钾沉淀的钙颗粒显示出了一个与花药发育事件有关的分布特征：在孢原细胞时期的花药中钙颗粒很少。在造孢细胞到小孢子母细胞时期,花药中钙颗粒增加。当花粉母细胞进行减数分裂时,花药中的钙颗粒进一步增加,尤其是在小孢子母细胞的胼胝质壁中。在小孢子发育早期,花药药隔部位的绒毡层细胞质中钙颗粒也明显增加并特异性地分布在其内切向壁上。当小孢子被释放出后,钙颗粒开始特异性积累在正在形成的花粉外壁中,尤其在萌发孔的部位聚集了大量的钙颗粒。当小孢子形成大液泡时,其细胞质中的钙颗粒明显减少。在小孢子分裂形成二胞花粉后,在二胞花粉的大液泡中又特异性地出现许多细小钙颗粒。随着二胞花粉的大液泡完全消失,其细胞质中又出现了许多钙颗粒。接近开花时的成熟花粉粒细胞质中,细小的钙颗粒主要分布在营养细胞和生殖细胞中。枸杞花药发育过程中钙的分布特征反映了其参与调控花粉发育过程。     

MS32-regulated timing of callose degradation during microsporogenesisin Arabidopsis is associated with the accumulation of stacked rough ER in tapetal cells

  In the male sterile32(ms32)mutant in Arabidopsis thaliana, pollen development is affected during meiosis of pollen mother cells (PMCs). In normal wild-type (WT) anthers, callose is deposited around PMCs before and during meiosis, and after meiosis the tetrads have a complete callose wall. In ms32, PMCs showed initial signs of some callose deposition before meiosis, but it was degraded soon after, as was part of the cellulosic wall around the PMCs. The early dissolution of callose in ms32 was associated with the occurrence of extensive stacks of rough ER (RER) in tapetal cells. The stacks of RER were also observed in the WT tapetum, but at a later stage, i.e., after the tetrads were formed and when callose is normally broken down for release of microspores. Based on these observations it is suggested that: (1) callose degradation around developing microspores is linked to the formation of RER in tapetal cells, which presumably synthesize and/or secrete callase into the anther locule, and (2) mutation in MS32 disrupts the timing of these events. Received: 27 April 1999 / Revision accepted: 21 June 1999     

莴苣花药发育过程中钙的分布特征

减数分裂前,莴苣花药中的钙颗粒很少。减数分裂后,花药绒毡层细胞中的钙颗粒明显增加, 同时在花药药室基质中也出现许多细小的钙颗粒。刚从四分体中释放出的小孢子内钙颗粒很少,伴随着花粉外壁物质在小孢子表面的沉积,钙颗粒开始积累在花粉壁部位。随后,小孢子中开始出现钙颗粒。当小孢子开始形成液泡后,钙颗粒向其中聚集,伴随着小液泡融合成大液泡,体积较大的钙颗粒主要集中在液泡中,而细胞质基质中的钙颗粒很少。随着二胞花粉中的大液泡消失,花粉细胞质中的钙颗粒变得很少。在以后的发育中,只有花粉壁中积累较多的钙颗粒。在莴苣花药发育过程中,钙与绒毡层细胞的退化和小孢子液泡形成以及二胞花粉中大波泡的消失有关。而花粉外壁表面积累丰富的钙与以后花粉的萌发有关。