洋葱小孢子母细胞减数分裂过程中微管分布变化

应用间接免疫荧光标记技术和激光共聚焦扫描显微镜成像技术观察洋葱小孢子母细胞减数分裂过程中微管分布变化。减数分裂之前,小孢子母细胞中的微管较短,呈辐射状,由细胞核表面向四周扩散。减数分裂开始后,细胞质中的一部分微管蛋白聚集成纺锤体微管,控制染色体的分布。进入减数分裂I后期,纺锤体微管变为牵引染色体移向两极的着丝粒微管和连接纺锤体两极的极丝微管。之后,所有微管集中在两个核之间,构成成膜体。然后,微管解聚成微管蛋白弥散在细胞质中。减数分裂I完成后,二分体2个子细胞中的微管蛋白又聚集成2个纺锤体微管,开始减数分裂II过程。经过减数分裂II中期,2个二分体细胞中的微管再次集中在2个细胞核之间形成成膜体,隔离2个细胞核。此后,微管蛋白解聚,弥散分布在小孢子细胞质中。     

水稻小孢子发育过程中微管骨架的变化

对水稻（ＯｒｙｚａｓａｔｉｖａＬ．）小孢子发育过程中微管变化的研究表明,微管在小孢子不同发育阶段呈现多样性。在花粉母细胞内,微管形成许多粗束和分支,围绕着核分布形成一个网络。花粉母细胞经第一次减数分裂形成二分体。在每一个二分体细胞内,有许多微管束,从核周辐射至细胞质各部位;在细胞质存在一个疏松的微管束网络。二分体经第二次减数分裂形成四分体,在每一个四分体细胞内,微管束呈辐射状,从核膜辐射入细胞质内。四分体形成后不久,四分体的四个细胞便分开,每一个细胞变成一个独立的小孢子。在早期的小孢子细胞内,微管束呈疏松网状分布。其中有些微管束朝向胞质一个小突起聚集。当小孢子进入中期发育阶段,在胞质的小突起部位微管束密度增大。小突起最终形成为萌发孔。当小孢子发育至成熟期,细胞内的微管束变得纤细,而网络则变得紧密。之后的发育阶段（即花粉发育不同阶段）因荧光标记难以进入细胞,无法获得清晰的图像。     

水稻IR36及光温敏核雄性不育系培矮64S小孢子母细胞减数分裂期间微管骨架的变化

对水稻（Oryza sativa L.)IR36及光温敏核雄性不育系培矮64S小孢子母细胞减数分裂期间微管骨架的变化的研究显示,微管在小孢子母细胞正常发育的情况下,每一个发育阶段（即造孢细胞时期、细线期、偶线期、粗线期、双线期、终变期、中期Ⅰ、后期Ⅰ）都出现不同的组织结构形态和分布。在IR36中,还发现了一些新的和特殊的 管组织结构和分布。主要包括：（1）在细线期,从核膜向胞质射的微管呈螺旋状,显示核具旋转功能;（2）在偶线期,微管的分布呈极性分布现象;（3）在终变期,核被一组微管形成的宽带围绕着（perinuclear broad band）。而培矮64S小孢子母细胞分裂期间,各发育阶段的细胞内的;管管骨架结构呈现许多不正常现象。如：（1）在偶线期具极性分布的微管分布不存在;（2）在小孢子母细胞减数分裂期间的细胞内出现许多特别粗的微管束;（3）在终变期围绕核的微管宽带结构松散及内含微管密度低。由于微管骨架的分布变化可以使人们较早地在雄性不育细胞内看到败育现象,故此微管的分布和构型变化应可作为一个早期确立雄性不育现象的视觉标记(visual marker）或形态指标予以利用。     

兰州百合小孢子母细胞减数分裂异常现象的观察

对兰州百合(Lilium davidii var.unicolor)小孢子母细胞减数分裂异常进行了研究,发现存在不等二价体、同源染色体早分离、染色体桥、不均等分离、滞后染色体、核外染色体、微核等。分析了这些异常形成的可能机制及对正常小孢子形成的影响。人工花粉萌发实验表明：小孢子母细胞减数分裂异常是导致花粉败育的主要原因。认为兰州百合长期行无性繁殖引起染色体结构变异,导致减数分裂异常。     

矮牡丹小孢子母细胞减数分裂异常现象的观察

矮牡丹(Paeonia suffruticosa subsp. spontanea)(永济居群)存在多种结构杂合现象,减数分裂存在一些异常:如单价体、异形二价体、互锁四价体、六价体、后期I倒位桥、落后单价体、不均等分离、后期Ⅱ桥和微核等。统计了这些异常现象出现的频率,并对其形成的机制和对正常小孢子形成的影响进行了讨论。从细胞学水平上探讨了矮牡丹可能的濒危机制。同时结合前人的研究,对芍药属内3个组的结构杂合程度进行了比较。     

卷丹小孢子母细胞减数分裂染色体行为的研究

对引种于陕西汉中地区的三倍体卷丹的小孢子母细胞减数分裂进程、花粉生活力和可育性进行了分析。结果显示:减数分裂时期与花蕾、花药长度及花药壁颜色呈现出一定的相关性,处于减数分裂时期的卷丹花蕾长度在2.5～3.2 cm之间,花药长度在1.4～1.8 cm之间;从细线期到成熟花粉粒,花药壁的颜色呈现出淡绿色-淡黄绿色-黄绿色-橘黄色的变化趋势,花药内容物颜色也从透明逐渐变为粘稠的橘红色。减数分裂终变期染色体构型以三价体为主,平均构型为:11.6Ⅲ+2Ⅱ+1.1Ⅰ,可判断该种源地卷丹为同源三倍体。减数分裂中出现染色体不均等分裂、染色体落后、染色体桥及微核现象,同时观察到成熟花粉粒大小差异较大,极轴与赤道轴乘积在839.465～3327.907μm2范围内,趋于正态分布,为三倍体卷丹花粉的高度不育提供了细胞学证据。以卷丹为母本与大花卷丹进行杂交,得到一定数量的种子,其中有胚率达到了43.0%,成苗率为67.4%,说明三倍体卷丹在百合育种中具有一定的价值。     

航天诱变凤仙花小孢子母细胞减数分裂的研究

对卫星搭载凤仙花与对照组的小孢子母细胞减数分裂以及四分孢子期内的小孢子数目、形状进行了对比研究,发现经过航天搭载的凤仙花种子在第一代(SP1)植物的小孢子母细胞减数分裂中出现了染色体桥、落后染色体和分散染色体;四分孢子时期易出现多分孢子及四分孢子不分离等现象。而对照组则很难发现染色体畸变和小孢子的异常现象。     

以单细胞免疫荧光技术观察烟草细胞融合过程中微管骨架的变化

本文建立了单细胞免疫荧光标记技术并以此结合单对细胞融合技术对细胞融合过程中微管骨架组织形式的动态变化进行了追踪观察。发现在聚乙二醇（PEG）诱导条件下,一旦细胞开始粘连,细胞内微管骨架便开始解聚。在细胞融合的整个过程中一直维持着这种解聚的状态,直到融合完成,在后续的培养中微管骨架才重新出现。在微管骨架呈解聚状态时融合产物不能完成与另外的细胞融合。实验揭示了细胞的再融合能力可能受细胞本身微管骨架状态的影响。该结果为解释高等植物如何避免多精入卵提供了新的可能性。     

胡杨小孢子发生及微管骨架变化与异常研究

利用压片法和间接免疫荧光结合DAPI(4′,6-diamidino-2-phenylindole)染色法,对胡杨小孢子母细胞减数分裂过程中微管骨架变化和染色体行为进行观察研究。结果表明:（1）胡杨小胞子母细胞减数分裂进程中染色体行为正常,其中:偶线期可观察到单价体,中期Ⅰ会出现落后染色体,末期Ⅰ和末期Ⅱ的核仁呈现动态变化。（2）胡杨小孢子发生过程中细胞内微管骨架呈动态变化过程,其中:中期Ⅱ形成平行纺锤体以及三极纺锤体;末期Ⅱ未观察到典型的成膜体结构,同时型胞质分裂受子核间辐射微管系统调节,通过胞质向心收缩而发生,胞质分裂后形成四边形和四面体型四分体。（3）胡杨小孢子母细胞减数分裂过程中还存在各种异常细胞学现象,其中:中期Ⅱ平行纺锤体发生融合;中期Ⅱ 和后期Ⅱ孢母细胞两个纺锤体间的胞质会出现裂沟;四分体时期存在三分体和二分体,并产生天然2n花粉和连体花粉。     

烟草花发育过程中花药和花粉IAA分布规律的研究

以烟草（Nicotiana tabacumL.）花药为材料,通过4’,6-二脒基-2-苯基吲哚（DAPI）染色详细观察花粉发育过程,获得了花药发育时期与花蕾大小的对应关系;通过吲哚乙酸（IAA）单克隆抗体、免疫组织化学技术以及DR5∶∶GUS转基因植株的GUS活性对花药和花粉发育过程中生长素的分布规律进行了研究。免疫酶标记结果表明,在不同的花药发育时期IAA水平呈现出明显的差别。小孢子母细胞时期,IAA在整个花药中均有分布,并且在小孢子母细胞发育晚期,IAA信号集中在小孢子母细胞的细胞核中;随着小孢子母细胞减数分裂后形成四分体,IAA信号逐渐减弱,四分体中几乎没有信号;单核花粉期的花药中IAA信号进一步减弱,仅存在于花药壁中;待小孢子继续发育为成熟二核期时,花粉和整个花药组织中均出现较强的IAA信号。GUS活性检测结果表明,烟草DR5∶∶GUS转基因植株中花药和花粉粒的GUS信号与IAA免疫酶定位结果基本一致。总的来说,IAA在烟草花药和花粉中的积累呈现出由强到弱、再由弱到强的分布规律,暗示IAA在被子植物花药和花粉发育过程中可能起着较为重要的作用。     

烟草柱头和花柱中阿拉伯半乳糖蛋白的定位

通过Western印迹法、免疫组织化学和超微细胞化学等技术,研究了烟草柱头和花柱中阿拉伯半乳糖蛋白（arabinogalactan-proteins,AGPs）的分布。结果表明烟草柱头和花柱组织中含有大量的AGPs,主要分布于柱头表皮细胞的细胞质和分泌层细胞的胞外基质中,且授粉前后AGPs的分布情况差异不明显;而花柱中的AGPs主要分布于表皮细胞的外层细胞壁、维管组织周围细胞的细胞质及引导组织的胞外基质中;花粉管通过后,引导组织胞外基质中AGPs减少,而花粉管细胞质和花粉管壁中检测到大量AGPs。     

欧洲黑杨花粉母细胞减数分裂观察及其不同步性分析

以欧洲黑杨的雄花枝为材料,采用温室切枝水培和醋酸洋红染色制片法,观察花粉母细胞减数分裂进程及其与花芽发育外部形态的关系。结果表明:(1)欧洲黑杨花粉母细胞的减数分裂进程快慢与温度高低关系密切;(2)减数分裂发生时其花芽的外部形态发育没有明显变化;(3)花粉母细胞减数分裂不同步性突出,同一花药内的花粉母细胞处于不同的减数分裂时期,且同一花芽尖端发育明显滞后于中部和基部;(4)减数分裂过程中有少量的核仁异常现象,虽观察到较多平行纺锤体现象,但未发现天然2n花粉。该研究结果揭示了欧洲黑杨减数分裂的遗传变异规律,为诱变育种、倍性育种以及分子水平的进一步研究奠定了细胞学基础。     

高效烟草遗传转化体系的建立及甜蛋白基因的导入

以烟草无菌茁叶片为外植体,通过根癌农杆菌LBA4404介导法,将Thamnatin基因导人烟草中,经梯度卡那霉素(Kana-mycin,Km)筛选,获得可在含75mg／L、100mg／L Km选择生根培养基上再生的抗性植株,其中部分Km抗性植株经PCR检测为阳性,转化率为31．3％,初步鉴定已成功地建立了烟草遗传转化系统,为进一步探讨甜蛋白在植物中的转化和表达情况奠定基础。     

外源钙调素和钙调素拮抗剂对烟草离体花粉管生长和生殖核分裂的调节

外源花椰菜钙调素（CaM）、牛脑CaMagarose以及CaM拮抗剂TFP对花粉管生长与生殖核分裂的作用均具有浓度和时间效应,CaM在花粉管生长早期,较低浓度能促进花粉管生长和生殖核分裂;但后期,较高浓度则有抑制作用。ITP在花粉管生长早0000000000000期,较高浓度抑制花粉管生长和生殖核分裂。     

An in vitro molecular study of the Nicotiana tabacum L. genome in the presence or absence of the herbicide atrazine

  Nicotiana tabacum L. somaclones both selected and not selected for tolerance to the triazine herbicide atrazine were used to compare tissue culture-induced variability in the presence or absence of stress. Two types of repeated sequences (rDNA and a randomly cloned, anonymous sequence) were analysed both qualitatively and quantitatively, and overall genome variation was assessed by RAPDs. Multiplicity differences were found for the two sequences both between the tolerant and susceptible group and within each group with respect to leaf DNA, but no qualitative differences were detected with either RFLPs or RAPDs. Moreover, we investigated whether stressinduced variation in the atrazine target gene, the chloroplast psbA gene, was responsible for herbicide tolerance by analysing two possible resistance mechanisms: the presence of a specific point mutation in the gene and its amplification and/or increased expression. Some somaclones were shown to be a mosaic for psbA gene mutation, but the number of cells or plastid genomes involved seemed too low to account for tolerance in the whole tissue. Atrazine tolerance could then be due to an increase in the number of plastids/plastid genomes or/and to a permanent response to respiration inhibition whose basis is, up to now, unknown. Received: 18 July 1997/Accepted: 22 August 1997     

Isolation of Pollen Protoplasts in Nicotiana tabacum

A new method for isolation of quantities of mature pollen protoplasts in Nicotiana tabacum has been established. The first step was to germinate mature pollen in Brewbaker and Kwack medium containing 20% sucrose. When most of the pollen grains had just germinated short pollen tubes, they were transferred to an enzymatic solution for the second step. The enzymatic solution contained 1% pectinase, 1% cellulase, 0.5% potassium dextran sulfate, 1 mol/L mannitol, 0.4 mol/L sorbitol in Dx medium with or without 15% Ficoll. The enzymes firstly degraded the pollen tube wall and then the intine. As a result, intact pollen protoplasts were released with the isolation rate up to 50%-70%. Factors affecting pollen protoplast isolation during the germination and maceration of pollen grains were studied. The suceees depended on two key points:pollen germination duration and osmotieum concentration. The optimal germination duration was 30 rain at 30℃. When it was too long, long pollen tubes formed and subsequently, large number of subprotoplasts instead of whole protoplasts were yielded, as the case reported by previous investigators. The optimal concentration of mannitol and sorbitol in enzyme solution was as high as 1.4 mol/L in total. Lowering of the osmoticum concentration resulted in decrease of percentage of pollen protoplasts.     

Intergeneric Somatic Hybrid Plants of Nicotiana tabacum L. and Lycium barbarum L. by Protoplast Electrofusion

Mesophyll protoplasts of Nicotiana tabacum L. and protoplasts from cell suspension of Lycium barbarum L. were heterofused by electrofusion with a frequency of ca. 4%--5%. One hundred cell lines were selected at random identified by isozyme analysis with peroxidase and superoxide dismatase, and the differences from their parent were found. Results indicated that 9 cell lines expressed enzymatic bands characteristic of both parents. Five of the 9 cell lines were highly morphogenic and regenerated numerous young shoots that manifested morphological traits specific to both parents. However, these shoots never grew up or regenerate roots. Esterase analysis of leaf material from the regenerants of 5 morphogenic hybrid cell lines demonstrated that two of them (NL4 and NL8) expressed an unique hybrid band which were not shown in either parents. Cytological observation on parental and NL4 hybrid cell lines revealed that the somatic chromosome number of NL4 varied from 58 to 80, significantly higher than that of either parents. Ribosomal DNA analysis of NL4 and NL8 showed that NL8 covered all fragments of both parents: NL4 did not have the fragments characteristic of Lyciurn barbarurn L. Both hybrids had new fragments, suggestive of intermolecular recombination of rDNAs of the parents. Four normal plants morphologically similar to tabacco parent were obtained from hybrid cell hne NL4, which survived after being transferred to soil. Cytological analysis of root-tips from one of the plants indicated that it has ca. 58 chromosomes. This paper also discussed the problems on the production frequency and incompatibility of somatic cell hybrid.