Ultrastructural Observation on the Intra-and Intercellular Microtrabecular Network of the Pollen Mother Cells in Onion ( Allium cepa )

Using DGD embedment_free electron microscopy, ultrastructural observation on the intra_ and intercellular microtrabecular network (MN) of the pollen mother cells (PMC) of the whole meiotic prophase Ⅰ in onion ( Allium cepa L.) was performed. Complex nuclear MN was observed in the nucleus of PMCs, spreading throughout the nuclear region. The nucleolus and chromosomes were connected with the MN filament network. The uniformity of nuclear MN changed with the development of the PMCs. A lamina_like structure surrounded the nucleus and joined the MN in nucleus and in cytoplasm, but it disassembled at the end of prophase Ⅰ. There was also a complex cytoplasmic MN in PMCs, without obvious variation during the prophase Ⅰ. Furthermore, MN in cytoplasmic connections (plasmodesmata and cytoplasmic channels) was noticed to link the frameworks in two neighboring PMCs into one entity. Cytomixis was observed at synizesis of prophase Ⅰ in this experiment, and MN in cytoplasm and in nucleus was noticed to distribute in these granules which migrated from one PMC into its neighboring cell. At this time the nucleus moved aside from center of the PMC, but the rest of the cell was still fulfilled with MN filaments. The relationships of nuclear MN with nucleolus and chromosomes, lamina with nucleus, as well as intra_ and intercellular MN with cytomixis are discussed in this paper.

Pollen Mother Cell Miosis and Male Gametophyte Development of Pumpkin

[Objective] Pollen mother cell miosis and male gametophyte development of pumpkin were observed in this study, to provide some cytological basis for pumpkin anther or microspore culture. [Method] Ehrlich＇s hematoxylin staining-methyl salicylate clearing technique was used for observation and research of the variation of cell structure and chromosomal behavior during pollen mother cell miosis and male gametophyte development of ‘Tianhong＇ pumpkin. [Result] The meiosis in pollen moth- er cells of pumpkin was simultaneous cytokinesis. In the process of nuclear division, nuclear membrane and nucleolus of pumpkin pollen mother cells gradually disappeared in the metaphase I and reappeared in telophase I , phragmoplast formed between the two generated crescent-shaped nuclei without cell wall, the phragmoplast gradually disappeared in the metaphase II and reappeared in telophase II. Phragmoplast spread outward from the center of spindle during the second division was connected with that formed on the central interface of two nuclei during the first division, cell wall of microspores generated from periphery to center. Most of the tetrads contained four sub-cells while a few contained extra small cells. During the period of uniuclete microspore at periphery, the single nucleolus split into 2-3 or more small nucleoli, mature pollen grain was two-celled. Mononucleate pollen cells were mostly appeared in the flower buds with length of 1.0-2.0 cm, which could be used as an important indicator to collect materials for anther or microspore culture. [Conclusion] This study laid the foundation for research of the cytogenetics of pumpkin.

Meiotic chromosome preparation techniques of pollen mother cells for laser micro-dissection in Populus spp.

In order to isolate meiotic chromosomes of Populus species meiotic chromosome preparation techniques of pollen mother cells for laser micro-dissection were studied. Pollen mother cells at diakinesis ofPopulus canadensis Moench were used as samples. Two methods were used to prepare meiotic chromosomes： in the first, cell suspensions were dropped on polyethylene-naphthalate or polyester membrane slides which had just been incubated at -20~C; in the second method, cell suspensions were also dropped on polyethylene-naphthalate or polyester membrane slides, but spread with the aid of high temperatures. The cells did not completely spread by the first method and chromosomes at diakinesis could not be individually distinguished. In contrast, well-spread diakinesis chromosomes were obtained by the second method, where chromosomes, connected with their nucleolus, were successfully isolated with the laser micro-dissection system. As well, we discuss the prospect of applications of laser micro-dissection in cytogeneties and molecular genetics in Populus species.

Genotoxic Effects of Superconducting Static Magnetic Fields(SMFs)on Wheat(Triticum aestivum)Pollen Mother Cells(PMCs)

The effects of superconducting static magnetic fields （SMFs） on the pollen mother cells （PMCs） of wheat were investigated in order to evaluate the possible genotoxic effect of such non-ionizing radiation. The seeds of wheat were exposed to static magnetic fields with either different magnetic flux densities （0, 1, 3, 5 and 7 Tesla） for 5 h or different durations （1, 3 and 5 h） at a magnetic flux density of 7 Tesla. The seeds were germinated at 23℃ after exposure and the seedlings were transplanted into the field. The PMCs from young wheat ears were taken and slides were made following the conventional method. The genotoxic effect was evaluated in terms of micronucleus （MN）, chromosomal bridge, lagging chromosome and fragments in PMCs. Although the exposed groups of a low field intensity （below 5 Tesla） showed no statistically significant difference in the aberration frequency compared with the unexposed control groups and sham exposed groups, a significant increase in the chromosomal bridge, lagging chromosome, triple-polar segregation or micronucleus was observed at a field strength of 5 Tesla or 7 Tesla, respectively. The analysis of dose-effect relationships indicated that the increased frequency of meiotic abnormal cells correlated with the flux density of the magnetic field and duration, but no linear relationship was observed. Such statistically significant differences indicated a potential genotoxic effect of high static magnetic fields above 5 T.

High temperature treatment induced production of unreduced 2n pollen in Camellia oleifera

Unreduced gametes through chromosome doubling play a major role in the process of plant polyploidization.Our previous work confirmed that Camellia oleifera can produce natural 2n pollen,and it is possible to induce the 2n pollen formation by high temperature treatment.This study focused on the optimization of the 2n pollen induction technique and the mechanisms of high temperature-induced2n pollen formation in C.oleifera.We found that the optimal protocol for inducing 2n pollen via high temperature was to perform 45℃with4 h at the prophaseⅠstage of the pollen mother cells(PMCs).Meanwhile,high temperature significantly decreased the yield and fertility of2n pollen.Through the observation of meiosis,abnormal chromosome and cytological behaviour was discovered under high-temperature treatment,and we confirmed that the formation of 2n pollen is caused by abnormal cell plate.Based on weighted gene co-expression network analysis,fifteen hub genes related to cell cycle control were identified.After male flower buds were exposed to heat shock,polygalacturonase gene(CoPGX3)was significantly upregulated.We inferred that high temperature causes the CoPGX3 gene to be overexpressed and that CoPGX3 is redistributed into the cytosol where it degrades cytoplasmic pectin,which leads to an abnormal cell plate.Furthermore,abnormal cytokinesis resulted in the formation of dyads and triads,and PMCs divided to produce 2n pollen.Our findings provide new insights into the mechanism of 2n pollen induced by high temperature in a woody plant and lay a foundation for further ploidy breeding of C.oleifera.

芦笋超雄株花粉母细胞减数分裂观察

减数分裂是有性生殖的关键过程。为了明确芦笋超雄株花粉母细胞减数分裂的具体情况及异常行为对配子生长发育的影响,采用改良苯酚品红压片法对58株芦笋超雄株的986个花粉母细胞减数分裂过程进行了系统观察。结果表明,(1)超雄株花粉母细胞减数分裂为胞质分裂同时型,染色体构型为10个二价体,四分体为十字交叉型。(2)中期Ⅰ小型棒状二价体的出现比例为96.55%,四价体为8.62%,染色体粘连为22.41%。(3)后期Ⅰ落后染色体的出现比例为12.07%,染色体桥为13.79%;分裂期出现微核的比例为6.59%。(4)在中期Ⅱ~末期Ⅱ中分裂不同步的出现比例为34.45%。超雄株花粉母细胞在整个减数分裂进程中,前期Ⅰ所需时间最长。减数分裂配对成10个二价体,在倍性水平上属于二倍体。减数分裂异常行为主要出现在中期Ⅰ和后期Ⅰ。对超雄株花粉母细胞减数分裂过程的观察,不仅能够丰富芦笋的细胞遗传学内容,还可以为芦笋发育生物学、杂交育种、基因定位等方面提供必要的细胞遗传学信息。

绣球小孢子母细胞减数分裂染色体行为及花粉特征

【目的】建立绣球花发育的形态学指标与减数分裂进程的对应关系,观测小孢子母细胞减数分裂过程中染色体行为及花粉特征,为绣球种质创新奠定工作基础。【方法】采集不同发育阶段的花序,卡宝品红压片后,观察可育花的花粉母细胞减数分裂情况,统计减数分裂异常现象出现的比例,利用液体培养基培养并测定花粉萌发力。【结果】1)绣球‘无尽夏’花序边缘的装饰花开放之前,可育花的花粉母细胞细胞质浓厚、核仁明显。当花序边缘的装饰花微微开放时,可育花的花蕾直径在3~3.5 mm,花药宽约0.5 mm,长约1 mm,此时花粉母细胞减数分裂进入粗线期。随着可育花进一步发育,可以观察到从终变期到花粉粒的各个时期。花序边缘的装饰花完全开放时,可育花的减数分裂已经结束,此时可育花的花蕾直径在4 mm左右,花药宽约1 mm,长约1.5 mm。2)绣球‘无尽夏’在减数分裂过程中染色体行为正常,2个杂种F1的小孢子母细胞减数分裂过程出现大量异常现象。Hydrangea macrophylla‘Magical Jade’×H.chinensis的杂种F1在小孢子母细胞减数分裂中期I有46.41%的细胞出现游离于赤道板之外的染色体,52.48%的细胞在后期I和末期I会出现落后染色体或形成染色体桥,35.11%的细胞在末期Ⅱ出现微核现象。H.macrophylla‘Taube’×H.macrophylla‘Veitchii’的杂种F1在小孢子母细胞减数分裂中期I出现未配对染色体的比率为15.31%,在后期I和末期I有17.30%的细胞出现落后染色体或形成染色体桥,末期Ⅱ未观察到微核现象。3)绣球‘无尽夏’花粉粒为球形,大小一致,直径为(16.72±0.75)μm,萌发率为(25.15±3.54)%。在2个杂种F1中观察到有球形和形态不规则的皱缩花粉,H.macrophylla‘Taube’×H.macrophylla‘Veitchii’杂种F1球形花粉粒的直径为(16.60±1.73)μm,萌发率为(16.26±1.72)%。H.macrophylla‘Magical Jade’×H.chinensis杂种F1的球形花粉粒可分为大小2类,小花粉粒的直径为(16.48±0.71)μm,大花粉粒的直径为(23.30±1.82)μm,是小花粉的1.41倍,推测为2n花粉,大花粉占花粉粒总量的(6.12±1.01)%。【结论】花序边缘的装饰花微微开放,可育花的花蕾直径3~4 mm,可以作为绣球离瓣组花粉母细胞减数分裂启动的标志。绣球‘无尽夏’及H.macrophylla‘Taube’×H.macrophylla‘Veitchii’的杂种F1花粉萌发率较高,在后期的育种工作中可以进一步用作杂交亲本。

银杏小孢子母细胞响应秋水仙碱低频多倍化的细胞学和转录组分析

银杏(Ginkgo biloba L.)三倍体具有很高的综合价值,但秋水仙碱诱导银杏有性多倍化仍较为困难,2n雄配子最高得率仅有7%。为初步探究秋水仙碱诱导银杏2n雄配子的形成机制,本研究使用0.6%的秋水仙碱对进入减数分裂粗线期的银杏小孢子母细胞进行避光瓶浸没处理,对不同处理时间下小孢子囊细胞中的微管骨架、生理生化变化以及转录本进行对比分析。结果显示,秋水仙碱处理的银杏小孢子母细胞游离脯氨酸、丙二醛含量及过氧化物酶活性在处理48 h时较高,可溶性蛋白含量和超氧化物酶活性低于对照组。与对照组相比,处理至24 h时,细胞微管蛋白开始减少,48 h呈弥散状,厚度变薄,荧光显微下几乎无微管蛋白显现。转录组分析共筛选到4690个差异表达基因,其中热激基因HSP11在48 h高差异表达。另外,在48 h鉴定到的41个差异转录因子中,有23个转录因子显著上调表达,其中最大的三个家族是AP2/ERF、MYB-related和NAC蛋白家族。上述结果表明,48 h是秋水仙碱诱导银杏小孢子母细胞加倍的关键时间点,HSP11基因、AP2/ERF、MYB-related和NAC转录因子家族成员在此过程中均起着关键的调控作用。本研究结果为探索与调控秋水仙碱诱导银杏2n雄配子形成有关的通路提供了参考依据,并为后续银杏有性多倍化分子机理研究奠定了理论基础。

苦荞二倍体及其同源四倍体减数分裂过程的稳定性与花粉育性

【目的】探明苦荞二倍体及其同源四倍体苦荞花粉母细胞减数分裂过程的稳定性与花粉育性,为苦荞多倍体育种提供参考。【方法】观察和统计八宿苦荞、WN047、WN090、武岗苦荞、WN065、川荞1号、二季早、黔威1号、晋苦2号、六荞1号、KP9920和黔威2号12个苦荞二倍体和苦荞四倍体花粉母细胞减数分裂过程中期I(MI时期)染色体配对构型、末期Ⅱ(TⅡ时期)的细胞异常程度及花粉粒败育性指标,分析比较二倍体和苦荞四倍体差异性。【结果】12个苦荞二倍体染色体配对构型主要为2n=2x=8II,占比95.42%,无双核仁;同源四倍体染色体构型多数为2n=4x=8IV,占比79.05%,有双核仁(占比18.6%)。12个苦荞二倍体在TⅡ期异常细胞频率平均为4.34%,细胞平均微核数为0.0434个,不同材料间差异不显著;同源四倍体异常细胞频率平均为24.09%,细胞平均微核数平均0.4949,不同材料间存在显著(P<0.05)或极显著(P<0.01)差异。12个苦荞二倍体花粉败育率平均9.54%,不同材料间存在显著(P<0.05)或极显著(P<0.01)差异;同源四倍体花粉败育率平均18.70%,不同材料间存在显著(P<0.05)或极显著(P<0.01)差异。苦荞四倍体细胞平均微核数及花粉粒败育率与苦荞二倍体均有极显著差异(P<0.01)。【结论】苦荞同源四倍体比二倍体具更复杂的染色体配对构型,苦荞四倍体在TⅡ时期的细胞异常性及花粉败育率均极显著高于二倍体,是导致苦荞四倍体比二倍体遗传稳定性差的主因。

通辽杨花粉母细胞减数分裂及其染色体行为研究

在温室水培条件下对通辽杨（Populus simonii Carr．×P．nigra L,‘Tongliao’）花粉母细胞减数分裂进程及其染色体行为进行了研究。结果表明：（1）通辽杨花粉母细胞减数分裂与其雄花芽／花序外部特征和花药颜色有着密切关系，前期Ⅰ作为减数分裂过程中最关键而复杂的一个阶段，大约占整个减数分裂进程90％的时间；中期Ⅰ存在单价体，后期Ⅰ有落后染色体出现，表现出较强的遗传杂合性，而且中期Ⅱ平行纺锤体的出现与天然花粉中大花粉的存在之间可能有着一定的联系；（2）在通辽杨减数分裂过程中核仁数目存在1～8个的动态变化，这种现象可能与杨属植物古多倍性起源有关，并推测通辽杨染色体组内至少存在8对带有次缢痕的染色体；（3）在同一个花芽，同一个小花，乃至同一个花药中，往往能同时观察到5～9个不同的分裂相，这种减数分裂不同步性是通辽杨适应环境的一种进化表现，对其种群繁殖具有重要意义。

空间条件对几种粮食作物的同工酶和细胞学特性的影响

搭载１９８７年发射的两颗返地卫星（８月５—１０日和９月９—１７日）的几种小麦、大麦种子，经回收后初步研究表明：（１）对发芽率的影响：空间处理的种子，其发芽率与对照无差别；（２）对茎及生育期的影响：空间处理的种子长成的幼苗比对照强壮，植株高度有些品种明显的矮于对照。生育期有不同程度延长；（３）对染色体结构的影响：花粉母细胞减数分裂观察表明搭载卫星的种子当代可诱导出比地面更多的染色体桥、落后染色体及染色体数目异于正常体细胞数目的现象。小麦品种Ｃｏｃｏｒｉｔ—７１经空间处理后花粉母细胞的染色体数目异常可高达２０．５１％，而地面对照组其花粉母细胞基本正常（２ｎ＝２８）；（４）对同工酶的影响：经空间处理的小麦种子酯酶、过氧化物酶的同工酶谱带空间处理的比地面对照减少。由此可见，空间处理可诱导纯合植物种子发生变异，为农作物育种创造一个新途径。

‘凤丹白’牡丹核型分析与减数分裂的细胞遗传学观察

对江南牡丹品种‘凤丹白’的核型和花粉母细胞减数分裂行为进行研究。结果表明:‘凤丹白’为二倍体,染色体数2n=10,核型公式:2n=2x=10=6m+2sm+2st,属于2A型,没有观察到随体。推测‘凤丹白’和杨山牡丹不是同物异名,主要表现在随体的有无。与杨山牡丹的核型进行比较发现:从杨山牡丹演化到‘凤丹’系列是由纯合到不同等级的杂合。‘凤丹白’减数分裂过程中具有不同步性,在后期Ⅰ、末期Ⅰ观察到染色体桥,后期Ⅱ、末期Ⅱ分别观察到“对角线”桥、不均等分裂、双桥等现象。上海地区观察‘凤丹白’花粉母细胞减数分裂的最佳取材时间为2月底～3月初。

杜仲大小孢子母细胞减数分裂进程对应关系的研究

以杜仲雌雄花芽为材料,通过对杜仲大、小孢子母细胞减数分裂时期的观察对比发现:①杜仲小孢子母细胞减数分裂一般在3月下旬到4月上旬之间进行,花药从团聚开始向四周伸展时,小孢子母细胞开始进入减数分裂,前期持续时间较长,中期Ⅰ到末期Ⅱ分裂进行较快.杜仲花粉为2细胞型,发育过程经历小孢子单核期(早期、中期、晚期)、二核期(前期后期)、成熟花粉期,自减数分裂结束后至散粉历时15d左右.②杜仲大、小孢子母细胞减数分裂时间相差很长,大孢子母细胞减数分裂开始的时间相对较晚、延续的时间较长.大约在小孢子母细胞减数分裂结束后10d左右,大孢子母细胞才刚刚开始进入减数分裂,此时花粉发育至二核期;恰好当花粉成熟散粉时,大孢子母细胞发育到减数分裂的细线末期到粗线期.根据雌雄配子发生、发育的时序性对应关系,通过相同培养条件下小孢子发育进程的观察,可以对杜仲大孢子母细胞减数分裂进程进行即时判别.

木立芦荟小孢子母细胞减数分裂与花粉育性关系的初步研究

文章从减数分裂过程、小孢子发育两方面,探讨了木立芦荟(AloearboresensMill.)花粉败育的原因。木立芦荟花粉母细胞染色体数目为2n=14,由4对长染色体和3对短染色体组成,属二型性核型。其减数分裂异常,发现存在单价体和多价体、染色体桥、落后染色体、不均等分离、微核等。同时观察到木立芦荟染色体具有极度的黏质性,使减数分裂各阶段的染色体不易散开。统计各种异常现象出现的频率并分析了这些异常现象形成的可能机制及对正常小孢子形成的影响,推测染色体间的丝状粘连可能是木立芦荟小孢子母细胞减数分裂异常并导致败育花粉产生的主要因素。成熟花粉粒中90%以上为败育花粉,属碘败型。

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。

华北落叶松小孢子母细胞(PMC)减数分裂和花粉的发育

采用染色体压片法系统地观察了华北落叶松Larix principis-rupprechtii小孢子母细胞(PMC)减数分裂的细胞学特征和花粉发育过程。结果表明:华北落叶松PMC减数分裂始于当年秋季,以弥散双线期的特殊状态休眠越冬,次年春天又重新恢复其后续的减数分裂过程。主要特点是在同一花药中,减数分裂不同步,且各阶段分裂速度较快,3天内全部停留在四分体阶段;二价体构型主要为两臂共发生过两次以上交换的环形二价体,少数细胞中偶见一臂发生交换的棒状二价体;其平均构型为10.62II+1.38II;中期II核分裂相既有平行式样,又有互为垂直式样,形成左右对称型和四面体型四分体,兼具单、双子叶植物四分体主要类型特点。四分体持续一周后,细胞壁开始溶解,4个小孢子分离并游离在药室中,成为独立的单核花粉。其后细胞核开始有丝分裂,最后形成由2个原叶细胞、1个管细胞、1个柄细胞和1个体细胞共5个细胞组成的成熟花粉。此外,还观察到成熟花粉当中有13.6%是由4个细胞组成的。

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 Liliumdavidiivar.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.

萝卜花粉母细胞减数分裂及其雄配子体发育

采用染色体压片法和爱氏苏木精染色一冬青油透明法研究了萝卜花粉母细胞减数分裂及其雄配子体发育过程。结果表明：萝卜花粉母细胞减数分裂的胞质分裂方式为同时型，终变期二价体多为棒状和环状，有3％的异型二价体，中期Ⅰ出现少量染色体落后现象，中期Ⅱ纺锤体的排列方式有垂直型、平行型和八字型，四分体排列按比例依次为十字交叉型、四面体型和左右对称型。成熟花粉为3核型，包括1个营养核及2个精核，具3个萌发孔。2．0-4．5mm的花蕾处于单核期，可作为花药培养或小孢子培养的选材依据。花粉可染率为87．9％，败育多发生在单核期。

青花菜花粉母细胞减数分裂及雄配子体发育

采用染色体制片及爱氏苏木精染色-冬青油透明技术对青花菜花粉母细胞减数分裂及雄配子体发育过程进行了细胞学研究.结果表明:花粉母细胞减数分裂的细胞质分裂为同时型,四分体为正四面体型或十字交叉型;中期Ⅰ和中期Ⅱ,少数细胞可见赤道板外染色体;后期Ⅰ和后期Ⅱ部分细胞出现染色体桥及落后染色体;四分体时期可观察到二分体、三分体及含微核的异常四分体.雄配子体发育过程包括2次有丝分裂,成熟花粉为3细胞型,具3个萌发孔.减数分裂过程中染色体行为异常的花粉母细胞约占10.28%;雄配子体发育过程中异常频率约为3.2%,败育主要发生在单核期.

黑芥与花椰菜体细胞杂种花粉母细胞的减数分裂不同步及染色体行为异常

本研究对黑芥与花椰菜体细胞杂种及其双亲的花粉母细胞减数分裂行为进行了观察和比较,结果表明黑芥与花椰菜的花粉母细胞减数分裂同步性很高,而其体细胞杂种表现出明显的减数分裂不同步,各杂种不同株系间减数分裂不同步的程度与跨度各异。减数分裂不同步发生是由于为部分花粉母细胞在减数分裂某一时期发生停滞或推迟,这一时期(或第一个停滞时期)主要发生在第一次减数分裂的前期。花粉母细胞发育虽不同步,但最后均完成减数分裂形成花粉粒,所形成的花粉粒的活性相差甚远。此外,研究结果还显示,体细胞杂种有着较高的减数分裂染色体行为异常频率,尤其是在中期Ⅰ后。主要表现为减数分裂中期Ⅰ的染色体的早迁、后期Ⅰ和后期Ⅱ的染色体滞后及染色体不均等分离、四分体时期的异常等现象。花粉粒的活性与减数分裂时期染色体行为异常程度有关,染色体行为异常及微核现象出现的频率越高,相对的花粉粒活力也越低。