Syncytes during male meiosis resulting in 2n pollen grain formation in Lindelofia longiflora var. falconeri

Abstract Lindelofia longiflora (Royle ex Benth.) Baill. var. falconeri (Cl.) Brand (Family: Boraginaceae) is investigated cytologically (n= 12) for the first time from the cold deserts of Pangi Valley, Chamba District (Himachal Pradesh) in India. We report the formation of syncytes and 2n pollen grains in the species. During meiosis, the majority of the pollen mother cells (PMCs) exhibited 12 bivalents, equal segregation of chromosomes during anaphases, regular tetrads, and normal‐sized pollen grain formation. Occasionally, two proximate PMCs fused during the early stages of prophase‐I and resulted in the formation of syncytes. The frequency of syncytes in the accession is rather low, at 25 out of 1866 (1.33%). Such syncyte PMCs are detectable during meiosis due to their larger size compared to typical PMCs. The syncytes or polyploid cells showed normal 24 bivalents and depicted perfectly regular meiotic course. But the products of such PMCs yield 2n or larger sized pollen grains that are almost double the size of typical normal or n pollen grains. The origin of syncytes as a consequence of the fusion of meiocytes during the early stages of meiosis‐I could be attributed to low temperature stress conditions prevailing in the Pangi Valley, where temperature during May and June dip to below freezing, the time the plants enters the reproductive/flowering bud stage. It is possible that such apparently fertile 2n pollen grains originating from syncytes might play a role in the origin of intraspecific polyploids in the species.     

Cytomixis and associated meiotic abnormalities affecting pollen fertility in Clematis orientalis

Present cytological investigations from the cold desert regions of Lahaul-Spiti and Kinnaur (India) record the first ever tetraploid (2n=32) chromosome count and cytomixis in Clematis orientalis L. var. acutifolia Hook. f. et Thoms. The phenomenon of cytomixis (9.33–29.80 %) involving chromatin transfer among 2–3 proximate pollen mother cells (PMCs) during male meiosis occurs through narrow and broad cytoplasmic channels from early prophase to tetrad stage. However, frequency of its occurrence during the later meiotic stages is rather low. Chromatin transfer results into the formation of hypo-, hyperploid and enucleated PMCs. Various meiotic abnormalities associated with cytomixis such as chromatin stickiness, pycnotic chromatin, interbivalent connections, out of plate bivalents, late disjunction of bivalents, and laggards and bridges resulted into some pollen sterility (16.33–49.30 %) and heterogeneous pollen grains size.     

Methomyl,imbraclaobrid and clethodim induced cytomixis and syncytes behaviors in PMCs of Pisum sativum L: Causes and outcomes

Cytomixis is a common phenomenon observed in meiotic cells such as anther which is influenced by various factors. Use of pesticides is a common practice in agriculture. However, it is not known whether pesticides can induce cytomixis in plant cells and induce genetic variation. To understand this, the present study was planned to assess the cytomixis and syncytes behaviors in PMCs of Pisum sativum L. Seeds of P. sativum (Family: Fabaceae) were treated with different concentrations of commonly used pesticides methomyl (ME), imbraclaobrid (IM) and clethodim (CL). Seeds were treated with various concentrations (0.1, 0.2, 0.3, 0.4 and 0.5% of ME, IM and CL prepared in water) for 1 and 3 h. Effect of pesticides on pollen fertility, frequency of cytomixis, and kind of cytomixis cells was assessed. In the cytomixis cells, the cytomictic channel (CC) and direct fusion (DF), and various stages of meiosis (PI, MI, AI and TI) with cytomixis cells were observed. In addition, frequency of syncytes cell and their various stages of meiosis I (PI, MI, AI and TI) in pollen mother cells (PMCs) was assessed. During the microsporogenesis in P. sativum, the occurrence of cytomixis and syncytes at various stages of meiosis I were seen. The formation of cytoplasmic channels and direct fusing of pollen mother cells (PMCs) were both seen to cause cytomixis, with the former being more common than the latter. The percentage of PMCs with cytomixis and syncytes cells increased with increase in the concentration of pesticides. The result of the present investigation indicates that commonly used pesticides ME, IM, and CL have a significant effect on pollen fertility, frequency of cytomixis, and kind of cytomixis cells, the cytomictic channel (CC) and direct fusion (DF), in addition, frequency of syncytes cell and their various stages of meiosis I (PI, MI, AI and TI) in pollen mother cells (PMCs) on P. sativum.     

Persistent occurrence of meiotic abnormalities in a new hexaploid cytotype of <Emphasis Type="Italic">Thalictrum foetidum</Emphasis> from Indian cold deserts

Thalictrum foetidum L. (Ranunculaceae), a morphologically variable and widely distributed species of temperate and alpine Himalayas is worked out cytologically for the first time from India. Earlier studies from outside India were restricted to chromosome counts and karyotypic analysis. We studied the male meiosis, microsporogenesis and pollen viability in the wild accessions from the cold deserts of Lahaul-Spiti, Kinnaur and Pangi Valley of Himachal Pradesh. Present cytomorphological surveys in the species record the existence of two distinct morphotypes involving plant size; colour and size of leaf/leaflet; dentation of leaflet lobes; and degree of leaf pubescence. All the accessions in the two morphovariants share the same meiotic chromosome number (n = 21) and adds a new intraspecific hexaploid cytotype. The accessions show the phenomenon of cytomixis involving transfer of chromatin material among proximate pollen mother cells (PMCs) and associated meiotic abnormalities like, out of plate bivalents, interchromosomal connections, and laggards, bridges and micronuclei at anaphases/telophases. Microsporogenesis results into abnormal sporads (tetrads with micronuclei, dyads, triads and polyads). The products of such sporads resulted into some pollen sterility and pollen grains of heterogeneous sizes. The persistent occurrence of phenomenon of cytomixis and associated meiotic abnormalities and consequently pollen sterility and pollen grains of heterogeneous sizes in the hexaploid cytotype of T. foetidum seems to be under some genetic factors associated with the genome.     

Cytological studies on meiosis and male gametophyte development in autotetraploid cucumber

With improved staining and chromosome preparation techniques, meiosis of pollen mother cells (PMCs) and male gametophyte development in autotetraploid cucumber (Cucumis sativus L.) was studied to understand the correlation between chromosomes behaviour and fertility. Various chromosome configurations, e.g. multivalent, quadrivalents, trivalents, bivalents and univalents were observed in most PMCs at metaphase I. Lagging chromosomes were frequently observed at anaphase in both meiotic divisions. In addition, chromosomes segregations were not synchronous and equal in some PMCs during anaphase II and telophase II. Dyads, triads, tetrads with micronuclei and polyads were observed at tetrad stage, and the frequencies of normal tetrad with four microcytes were only 55.4 %. The frequency of abnormal behaviour in each stage of meiosis was counted, and the average value was 37.2 %. The normal meiotic process could be accomplished to form the microspore tetrads via simultaneous cytokinesis. Most microspores could develop into fertile gametophytes with 2 cells and 3 germ pores through the following stages: single-nucleus early stage, single-nucleus late stage and 2-celled stage. The frequency of abnormalities was low during the process of male gametophyte development. The germination rate of pollen grains was 46.9 %. These results suggested that abnormal meiosis in PMCs was the reason for low pollen fertility in the autotetraploid cucumber.     

麦蓝菜的花粉母细胞减数分裂及核型分析

采用压片法,对麦蓝菜的花粉母细胞减数分裂及体细胞核型进行了研究。结果表明,麦蓝菜的绝大多数花粉母细胞减数分裂中染色体的行为正常,在终变期同源染色体配对后可形成15个二价体;在少数花粉母细胞减数分裂中观察到落后染色体、染色体桥等异常行为;其减数分裂为同时型,其花粉粒育性为88.04%。麦蓝菜的染色体数目为2n=2X=30,核型公式为K(2n)=2X=30=22m(2SAT)+8 sm,染色体相对长度组成为2n=16M2+14M1,其核型为"1A"型。     

Cytology of higher polysomics in Coix gigantea (Poaceae): Pentasomy and hexasomy

Among a natural population of Coix gigantea a pentasomic (2n+3; 2n=23) and a hexasomic (2n+4; 2n=24) were isolated and studied cytologically for the first time. The extra homologous chromosomes in the pentasomic plant did not disturb the behaviour of the rest of the bivalents through meiosis except for the rare occurrence of a few univalents. The majority of PMCs at diakinesis showed nine bivalents and a pentavalent. The various anaphase segregations led to the formation of sub- and super-haploid male gametes with from n–1 (n=9) to n+4 (n=14) chromosomal constitutions. The four extra homologous chromosomes in the hexasomic plant, however, disturbed meiosis leading to sticky chromosomal configurations and sterility. At diakinesis, the majority of the PMCs showed nine bivalents and a hexavalent.The origin of these higher polysomics is considered to be through fusion of male and female super-haploid gametes produced by the trisomics that occurred in large numbers in the population. The occurrence and dominance of nullisomics together with other aneuploids in the diploid population of C. gigantea make it quite certain that the 2n=20 form of the species is at a tetraploid level.     

东乡野生稻与栽培稻正反交种间杂种F1的雄配子发生与发育

该文以东乡野生稻与栽培稻(美国光壳稻P1003)的正反交种间杂种F₁(正交为光壳稻P1003×东乡野生稻; 反交为东乡野生稻×光壳稻P1003)为试材, 研究其各个减数分裂时期的染色体行为特征、染色体交换重组及雄配子发育特点。结果表明: 正反交杂种F₁花粉母细胞细胞核减数分裂的同步性较高, 细胞质为连续型胞质分裂类型。在细胞核分裂的过程中, 核仁在前期I到中期I逐渐消失。染色体在前期I到中期I逐渐收缩, 变得更加清晰可见。在终变期和中期I, 90.54%以上的花粉母细胞能形成12个二价体(含少数棒状二价体和8字型二价体), 部分花粉母细胞(正交9.46%, 反交7.65%)出现少量的单价体、三价体和四价体。后期I观察到1.27%–1.35%的花粉母细胞含有1至数条落后染色体。最终有92.6%–94.8%的小孢子能发育成形态正常、染色能力强的成熟花粉粒。另外, 在正交杂种F₁的粗线期中存在2个核仁, 而反交杂种F₁及其双亲的粗线期只有1个核仁。这些研究结果可为作物品种改良、种质资源创新以及种间亲缘关系研究提供细胞学证据。     

Investigation on the Ontogeny of Pollen in Pinus thunbergii Parl

The microsporogenesis in Pinus thunbergii occurs in early to middle March, and the development of pollen in late March to early April. A mature pollen grain consists of four cells. The starch grains aggregate in the equators during meiosis Ⅰ to Ⅱ. The callose materials appear first in inner side of the wall of meiocyte and then in the equator. Sometimes the 3-or 4-sac- cate pollen grains have been found. In the ontogeny of pollen in Pinus thunbergii following events are noteworthy. (1) During meiosis the equatorial aggregation of starch grains in me iocyte becomes conspicuous. (2) During formation of tetrad the callose wall appears in regular activity. It is noteworthy that there is itself feature for the accumulation of callose in Pinus thunbergii. After formation of prothallial cells the callose appears mainly in the area between body and saccus. So the prominent symmetry of distribution of callose occurs in the pollen grains. But almost no callose accumulation takes place i,n tenuity in distal face of pollen grain. On the contrary, there are certain callose accumulation around the prothallial cells in the proximal face, forming the polar distribution of callose in pollen grains.     

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。     

Cytogenetic Studies on Five Intermediate Forms of Progeny of Triticum aestivum Agropyron glaucum

A stuty has been made of the chromosome number of root-tip cells and the chro- mosome configuration of PMCs in five intermediate forms of progeny of Triticum aestivum × Agropyron glaucum. The somatic cells in each of these intermediate forms have 28 pairs of chromosomes (2n=56) and the PMCs at diakinesis or metaphase Ⅰ of meiosis have 28 bivalents. The process of meiosis in most PMCs is regular. Lagging chromosomes and fragments at anaphase Ⅰ or telophase Ⅰ were observed only in few PWCs. According to our results, all the five intermediate forms of progeny of T. aestivum × Ag. glaucum are allooctoploids. The application of these allooctoploids is also briefly discussed.     

Meiosis in a triploid hybrid of <Emphasis Type="Italic">Gossypium</Emphasis>: high frequency of secondary bipolar spindles at metaphase II

Studies on meiosis in pollen mother cells (PMCs) of a triploid interspecific hybrid (3x = 39 chromosomes, AAD) between tetraploid Gossypium hirsutum (4n = 2x = 52,AADD) and diploid G. arboreum (2n = 2x = 26,AA) are reported. During meiotic metaphase I, 13 AA bivalents and 13 D univalents are expected in the hybrid. However, only 28% of the PMCs had this expected configuration. The rest of the PMCs had between 8 and 12 bivalents and between 12 and 17 univalents. Univalents lagged at anaphase I, and at metaphase II one or a group of univalents remained scattered in the cytoplasm and failed to assemble at a single metaphase plate. Primary bipolar spindles organized around the bivalents and multivalents. In addition to the primary spindle, several secondary and smaller bipolar spindles organized themselves around individual univalents and groups of univalents. Almost all (97%) of the PMCs showed secondary spindles. Each spindle functioned independently and despite their multiple numbers in a cell, meiosis I proceeded normally, with polyad formation. These observations strongly support the view that in plant meiocytes bilateral kinetochore symmetry is not required for establishing a bipolar spindle and that single unpaired chromosomes can initiate and stabilize the formation of a functional bipolar spindle.     

An original meiotic mutation in Paspalum regnellii

 Cytogenetic studies carried out over a period of 2 consecutive years on a native Brazilian accession of Paspalum regnellii (2n=40) revealed a meiotic mutation that has not been previously reported for any other species. Among 13 inflorescences investigated during the first collection year, three presented anomalous meiotic behavior starting from metaphase I. At the beginning of this phase, the chromosomes occupied the entire equatorial plate in a membrane-to-membrane arrangement, and the spindle fibers, which were clearly visible, did not converge towards the poles. Degeneration of spindle fibers occurred at the end of metaphase I. Chromosome segregation did not occur and the bivalents were left scattered at random in the cytoplasm. Remnants of chromosome fibers could be seen close to the centromere during this stage. The bivalents gave origin to micronuclei in telophase I, with extremely wide variations in number and size among cells. With the absence of spindle formation during meiosis II, metaphase and anaphase II were not observed. Second cytokinesis occurred in prophase II cells after the occurrence of first cytokinesis. The final product of meiosis was completely abnormal, with a predominance of polyads with microspores of different sizes that resulted in abortive pollen grains. In the affected inflorescences, all microsporocytes presented this anomaly, which caused total sterility. Received: 27 March 1997 / Revision accepted: 7 July 1997     

Studies on Chromosome Behaviour of F1 and Fertility Restoration in Hybrid of Gossypium hirtutum ×G. bickii

By means of dripping GAs(50 ppm) and NAA(40 ppm) on the hybrid boll-embryo culture in vitro, one F1 plant of G. hirsuturn × G. bickii was obtained in 1982, the Fx plant flowered profusely but failed to produce any seeds when selfed or back crossed. In meiosis of the pollen mother cell (PMC), mean chromosome coniugation was 33.24 Ⅰ +2.67 Ⅱ +0.095 Ⅲ + 0.048 Ⅳ with high frequence (77.6%) of one chiasma attenuated bivalents. Mean number of chiasmta per bivalents was 1.23. The univalents were scattered over the achromatic figure for most PMCs. Although a few of the bivalents were located in' the equatorial region, but they did not form a definite plate. At the second anaphase the distribution of chromosomes was very irregular. In the majority of cases, multipolar distripution of chromosomes was observed. At the completion of meiosis highly abnormal sporads occurred, which contained from 2 ro 13 spores of various sizes. Thus, all of the pollen grains produced were sterile. When the F1 branches were grafted onto the upland cotton and thence they were backcrossed under short day (12 hours) and cold night(15–18℃) exposure, BC1 seeds could be harvested. BC1 and BC2 plants could grow up later. In the BC, generation, the fertility of the hybrid was restored. By 1988, ten pure lines of hybrid with the characers of both male parent (viz.red petal with purple spot and strong fibre) and female parent (white fibre, high yield, earliness ect.) were selected for the first time.     

黄芩的花粉母细胞减数分裂及核型分析

采用压片法,对黄芩花粉母细胞减数分裂及核型进行了研究。结果表明:黄芩的大多数花粉母细胞减数分裂中染色体的行为正常,在终变期同源染色体配对后可形成9个二价体,后期Ⅰ染色体以9∶9的方式向细胞两极分离,其减数分裂为同时型;在少数花粉母细胞减数分裂中观察到落后染色体、染色体桥等异常行为;其花粉粒育性为76.49%。黄芩的染色体数目为2n=2X=18,核型公式为K(2n)=2X=18=16m+2 sm,染色体相对长度组成为2n=1 s+4M1+3M2+1L,其核型为"1A"型。     

Microsporogenesis and pollen formation in Zingiber officinale Roscoe

Zingiber officinale Roscoe, the common ginger, is an invaluable horticultural crop cultivated majorly in China, India, Brazil, Jamaica and Nigeria. Its pungent aromatic rhizome is used all over the world as a spice, culinary herb, condiment, home remedy and medicinal agent. This species does not produce seeds and so is difficult to breed its new genotypes through sexual hybridization. Despite the economic and genetic value, ginger has not been subjected to detailed cytogenetic research, which could lead to a better understanding of its reproduction for future genetic improvement. Therefore, the present work is undertaken for the first time to study microsporogenesis and pollen formation in Z. officinale. Pre-meiotic stages were observed under light microscope using anther squashes and staining by either acetocarmine or carbol fuchsin, whereas, the post meiotic stages were visualized under fluorescence microscope using either isolated microspores or pollen grains stained by 4′, 6′-diamidino-2-phenylindole. The combination of these two different cytological techniques allowed the study of the complete microsporogenesis and pollen formation processes step by step starting from pollen mother cells (PMCs) stage to mature pollen grain. Assessment of meiotic behavior was also performed by evaluating all possible PMCs on each slide and at all stages of meiosis. Cytological analysis revealed that only anthers from flowers of small spikes presented appropriate PMCs to analyze meiotic irregularities. The present paper enriches the database of cytology and pollen viability estimations for supporting sexual hybridization. The present study demonstrated a good relationship between the development stage of microspore and the size of the spike. This basic information will hopefully allow the development of protocol for production of androgenic haploids to accelerate breeding and genetic improvement of ginger. Further, research is under progress for evaluation of several media and other cultural factors to achieve microspore embryogenesis and plant regeneration in this species.     

Meiotic behavior of pollen mother cells in relation to ploidy level of somatic hybrids between Solanum tuberosum and S. chacoense

Potato somatic hybrids obtained by protoplast fusion between Solanum tuberosum (4x) and Solanum chacoense (2x) were investigated for genome stability and meiotic behavior associated with the pollen viability in order to elucidate the mechanism influencing the fertility of the somatic hybrids. The ploidy level detections conducted in 2004 and 2007 demonstrated that 68 out of 108 somatic hybrids had their ploidy level changed to be uniform and euploidy after successive in vitro subcultures, which mainly occurred in octaploids, aneuploids, and mixoploids, while 74% hexaploids were still stable in their genome dosage in 2007. Different types of abnormal meiotic behavior were observed during the development of pollen mother cells (PMCs) including the formation of univalents, multivalents, laggard chromosomes, and chromosomal bridges, as well as triads and polyads. A higher proportion of abnormal meiosis seemed to be accompanied with a genome dosage higher than the hexaploids expected in this study. A significant positive correlation between defective PMCs and the number of small pollen grains and negative correlation between number of small pollen grains and pollen viability strongly suggested that abnormal meiosis could be a causal factor influencing the fertility of the somatic hybrids. The hexaploids with stable genome dosage and a certain level of fertility will have great potential in a potato breeding program.     

二倍体和四倍体栽培苦荞的细胞学比较研究

观察和统计了二倍体和四倍体苦荞 (Fagopyrum tataricum)花粉母细胞减数分裂过程 TI、T 时期的异常程度及花粉粒败育性等多个指标 ,并比较分析了二倍体和四倍体苦荞在这些指标上的差异显著性。结果显示 :四倍体苦荞的异常 TI细胞频率、异常 T 细胞频率、TI细胞平均微核数、T 细胞平均微核数及花粉败育率均显著高于二倍体苦荞 ,这表明四倍体苦荞的遗传稳定性和花粉可育性都比二倍体苦荞差。     

萝卜与甘蓝属间杂种基因组原位杂交分析

用基因组原位杂交方法（Genomic in situ hybridization, 简称GISH）研究了萝卜( Raphanus sativus,2n=18,RR)和甘蓝（Brassica oleracea , 2n=18, CC）属间杂种F1减数分裂过程。结果表明杂种体细胞染色体组成为RC,2n=18,但花粉母细胞有三种不同类型：1. RC,2n=18, 终变期染色体平均配对构型为14.87Ⅰ+1.20Ⅱ+0.04Ⅲ+0.06Ⅳ, 染色体配对主要发生在萝卜和甘蓝染色体之间, 后期Ⅰ9条萝卜染色体主要以5/4和6/3的分离比移向两极, 所形成配子的染色体数目和组成均不平衡,配子败育; 2. RRCC,4n=36, 终变期染色体形成18个二价体,后期Ⅰ染色体均衡分离,形成RC不减数配子;3. RRCC缺体,4n=30－34, 少数萝卜染色体丢失,形成的配子具有全套的甘蓝染色体和部分萝卜染色体。     

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