TA29—Barnase嵌合基因导入对烟草花药绒毡层及花粉发育的影响

比较研究了烟草(Nicotiana tabacum L.)TA29-Barnase转基因不育植株和正常植株的花药绒毡层及花粉发育的全过程。研究表明,外源基因在花药中特异表达导致绒毡层细胞的提前降解,这种降解一般在减数分裂早期开始,至四分体时期完成,而正常花药绒毡层的降解发生在二细胞雄配子体初期,至花粉发育的后期方才完成。转基因植株花药绒毡层的降解在细胞结构上表现为:最初发生细胞的液泡化,然后细胞核凝聚,最后整个细胞溃解。转基因植株的花粉母细胞则在减数分裂过程中逐渐降解、退化,只有少数花粉母细胞能够顺利完成减数分裂发育成小孢子。观察结果还表明外源基因在花药中的表达是不均一的。对转基因不育和自然败育在细胞结构上的不同表现进行了讨论。     

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.     

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.     

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.     

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     

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。     

凤仙花花药发育中多糖和脂滴组织化学研究

以不同发育时期的凤仙花花药为实验材料,采用组织化学方法,对花药发育中的结构变化及多糖和脂滴物质分布进行观察。结果表明:(1)凤仙花的花药壁由6层细胞组成,包括1层表皮细胞,2层药室内壁细胞,2层中层细胞和1层绒毡层细胞。其中绒毡层细胞的形态不明显,很难与造孢细胞区分,且在小孢子母细胞时期退化。(2)在小孢子母细胞中出现了一些淀粉粒,但减数分裂后,早期小孢子中的淀粉粒消失,又出现了一些小的脂滴;随着花粉的发育,小孢子形成大液泡,晚期小孢子中的脂滴也消失;小孢子分裂形成二胞花粉后,营养细胞中的大液泡降解、消失,二胞花粉中又开始积累淀粉;接近开花时,成熟花粉中充满细胞质,其中包含了较多的淀粉粒和脂滴。(3)在凤仙花的花药发育中,绒毡层细胞很早退化,为小孢子母细胞和四分体小孢子提供了营养物质;其后的中层细胞退化则为后期花粉发育提供了营养物质。     

PERSISTENT TAPETAL CELL1 encodes a PHD-finger protein that is required for tapetal cell death and pollen development in rice

In higher plants, timely degradation of tapetal cells, the innermost sporophytic cells of the anther wall layer, is a prerequisite for the development of viable pollen grains. However, relatively little is known about the mechanism underlying programmed tapetal cell development and degradation. Here, we report a key regulator in monocot rice (Oryza sativa), PERSISTANT TAPETAL CELL1 (PTC1), which controls programmed tapetal development and functional pollen formation. The evolutionary significance of PTC1 was revealed by partial genetic complementation of the homologous mutation MALE STERILITY1 (MS1) in the dicot Arabidopsis (Arabidopsis thaliana). PTC1 encodes a PHD-finger (for plant homeodomain) protein, which is expressed specifically in tapetal cells and microspores during anther development in stages 8 and 9, when the wild-type tapetal cells initiate a typical apoptosis-like cell death. Even though ptc1 mutants show phenotypic similarity to ms1 in a lack of tapetal DNA fragmentation, delayed tapetal degeneration, as well as abnormal pollen wall formation and aborted microspore development, the ptc1 mutant displays a previously unreported phenotype of uncontrolled tapetal proliferation and subsequent commencement of necrosis-like tapetal death. Microarray analysis indicated that 2,417 tapetum- and microspore-expressed genes, which are principally associated with tapetal development, degeneration, and pollen wall formation, had changed expression in ptc1 anthers. Moreover, the regulatory role of PTC1 in anther development was revealed by comparison with MS1 and other rice anther developmental regulators. These findings suggest a diversified and conserved switch of PTC1/MS1 in regulating programmed male reproductive development in both dicots and monocots, which provides new insights in plant anther development.     

Induction of Male Sterility in Oilseed Rape by TA29-Barnase Gene

Chimaeric TA29-Barnase gene was introduced into oilseed rape (Brassica napus) of good quality and high yield by Agrobacteriurn tumefaciens transformation. The transgenic plants were obtained and transformed genome was determined by Southern blot analysis. About 90~/40 TA29-Barnase transgenic plants were male sterile. However, about 80% transgenic plants turned to be male fertile at temperature higher than 25 ℃. It suggested that male sterility of these transgenic plants was probably temperature sensitive.     

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液泡的消失有关。而花粉外壁表面积累丰富的钙与以后花粉的萌发有关。     

太空诱变玉米核不育材料花粉败育的细胞学观察(简报)

玉米是最早利用雄性不育系生产杂交种的作物之一。在玉米T型细胞质雄性不育杂交种遭受毁灭性病害侵袭之后,科学家认识到利用细胞质雄性不育制种存在潜在的遗传脆弱性,从此试图通过多种途径来创造新的雄性不育．并对雄性不育材料的遗传多样性进行研究。空间诱变育种是80年代于我国发展起来的新技术,在农作物品种改良和种质创新上已初见成效。[第一段]     

新型甘蓝型油菜温敏核不育系SP2S的花药发育解剖学观察

SP2S是西北农林科技大学选育的甘蓝型油菜温敏核不育系,本文采用半薄树脂切片、扫描电镜对SP2S及其可育近等基因系SP2F的花药发育及花粉形态进行观察比较,发现SP2S花药发育在减数分裂时期出现异常,单核花粉时期彻底败育。其主要特征是：减数分裂时期绒毡层已经径向肥大且出现大液泡,胼胝质不能及时降解,使得单核小孢子相互粘连在一起,小孢子无花粉壁的形成且细胞质物质逐渐降解,最后小孢子仅剩下空壳残留物,聚集在一起。SP2S败育特征与现有的核不育材料不同,表明其有可能是一种新型温敏核不育材料。     

A Comparative Study of Anther and Pollen Development in Male Fertile and Male. Sterile Green Onion (Allium fistulosum L.)

Anther and pollen development in male-fertile and male-sterile green onions was studied. In the male-fertile line, both meiotic microspore mother ceils and tetrads have a callose wall. Mature pollen grains are 2-celled. The elongated generative cell with two bended ends displays a PAS positive cell wall. The tapetum has the character of both secretory and invasive types. From microspore stage onwards, many oil bodies or masses accumulate in the cytoplasm of the tapetal cells. The tapetum degenerates at middle 2-celled pollen stage. In male-sterile line, meiosis in microspore mother cells proceeds normally to form the tetrads. Pollen abortion occurs at microspore with vacuole stage. Two types of pollen abortion were observed. In type I, the protoplasts of the microspores contract and gradually disintegrate. At the same time the cytoplasm of microspores accumulates oil bodies which remain in the empty pollen. The tapetal cells behave normally up to the microspore stage and early stage of microspore abortion, but contain fewer oil bodies or masses than those in the male-fertilt line. At late stage of microspore abortion, three forms of the tapetal ceils can be observed: (1) the tapetal cells with degenerating protoplasts become flattened, (2) the tapetal cells enlarge but protoplasts retractor, (3) the cells break down and tile middle layer enlarges. In type Ⅱ, the cytoplasm degenerates earlier than the nucleus of the microspores and no protoplast is found in the anther locule. There are fibrous thickenings iii the endothecium of both types. It is difficult to verify whether the tapetum behavior and pollen abortion is the cause or the effect.     

Effects of specific expression of iaaL gene in tobacco tapetum on pollen embryogenesis

The indoleacetic-acid-lysine synthetase (iaaL) gene from Pseudomonas syringae subsp. savastanoi was fused to tobacco tapetum-specific expression promoter TA29, and introduced into tobacco. The expression pattern of this chimeric gene was studied, and the endogenous indoleacetic acid (IAA) levels in different organs were assayed. The results demonstrated that TA29 promoter was only able to direct the specific expression of iaaL gene in transgenic tobacco anther, and resulted in the decrease of endogenous IAA levels in transgenic tobacco anther. No significant phe-notype variation was observed among the transgenic plants at the whole plant level. However, the percentage of pollen embryogenesis was reduced to 11 % when anthers of the transgenic plants were cultured on the modified hormone-free Nistch H (NH) medium, while those of both CK1 and CK2 (see sec. 1.2.2) were more than 50% ; when the an-thers were cultured on NH medium supplemented with 0. 2 mg/L IAA, the percentage of pollen embryogenesis re-stor     

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。     

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。