Cytogenetic studies on microsporogenesis and male gametophyte development in autotriploid cucumber (Cucumis sativus L.): implication for fertility and production of trisomics

To understand the correlation between chromosomes behavior and fertility in autotriploid cucumber (Cucumis sativus L.), microsporogenesis in pollen mother cells (PMCs) and male gametophyte development were studied using improved staining and chromosome preparation techniques. Meanwhile, for more efficient selection of trisomics from the progeny of autotriploid-diploid crosses, fertilization rates of ovules from reciprocal crosses were counted to observe the transfer rate of gametes in the autotriploid cucumber. Variable chromosome configurations, e.g. multivalents, quadrivalents, trivalents, bivalents and univalents were observed in the most PMCs of the autotriploids at metaphase I. Chromosome lagging and bridges at anaphase in both meiotic divisions resulted from irregular chromosome separation and asynchronization was frequently observed as well, which led to formation of micronuclei and inviable gametes. The frequency of normal PMCs in autotriploids at the stage of tetrad was only 40.6%. Among those normal microspores, most of them (91.2%) could develop into normal gametophytes with 2 cells and 3 germ pores. Stainability and germination rate of pollen grains were only 18.8 and 13.5%, respectively. However, chromosomes separated to form gametes with 8 chromosomes at anaphase I, suggesting a possible method for the production of primary trisomics from the progeny of autotriploid-diploid crosses. Fruit set of 3n × 2n and 2n × 3n were 80 and 70%, respectively. It obtained an average of 6.2 plump seeds per fruit in 3n × 2n, while 4.9 in 2n × 3n crosses. Transfer rates of gametes through the gastrula or the pollen in autotriploids were 13.4 and 10.4%, respectively. Some aneuploid gametes (n + 1 = 8, n + 2 = 9) also have capability of setting seed and sexual reproduction besides normal gametes containing whole chromosome sets (n = 7, 2n = 14). Further, some primary trisomic plants were selected from the progeny of autotriploid-diploid crosses. Based on the results obtained we suggest that abnormal meiosis in PMCs was the cytogenetic reason for low fertility of autotriploid cucumber pollen. 3n × 2n cross was more efficient for selecting primary trisomic plants in cucumber.     

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。     

Meiotic restitution in wheat-barley hybrids

Meiotic restitution occurs in pollen mother cells (PMCs) of reciprocal F₁ hybrids between wheat and barley. In occasional PMCs, all or most of the 28 chromosomes assemble at the equatorial plate at metaphase I, but instead of undergoing anaphase I separation they reform into a mass of chromatin to form a restitution nucleus. Some of these restituted nuclei undergo a regular second division and dyads are produced among other non-restituted cells which have reached the tetrad stage of division. Other restituted nuclei fail to undergo a second division and then the PMCs appear as monads among neighbouring tetrads. Both the monads and dyads are expected to produce microsporocytes with the diploid complement of chromosomes. Chromosomes which fail to become included in the restituted nucleus form separate micronuclei and, depending on whether they undergo a regular second division or not, the PMCs containing them eventually appear as tetrads, triads or dyads. These partially restituted nuclei are expected to produce unreduced gametes, deficient for one or more chromosomes. It is postulated from these observations that restitution in wheatbarley F₁ hybrids depends on a high frequency of univalent accumulation at the equatorial plate at metaphase I and the subsequent failure of the chromosomes to undergo anaphase I separation.     

Cytogenetic studies on two F1 hybrids of autotetraploid rice varieties showing extremely high level of heterosis

Mechanisms of two F1 hybrids (D46A × DTP-4 and D46A × Dminghui63) of autotetraploid rice (2n = 4x = 48) showing extremely high pollen fertility 87.40% and 85.97%, respectively, seed set 82.00% and 79.00%, respectively and extremely high level of heterosis were analyzed cytologically. The chromosome pairing of D46A × DTP-4 and D46A × Dminghui63 was normal at metaphase I(MI), and had almost no I or III, with an average of 0.020I +14.36 II 6.44rod+7.91ring) +0.01III + 4.80 IV + 0.01VIII and 0.06 I + 17.67 II (11.01rod + 6.67ring)] + 0.06 III +3.10IV+0.01VI, respectively. The most frequent chromosome configurations were 10II+7IV and 12II+bIV. The bivalent frequency was less frequent in hybrids than that in restoring parents, and the same results were gained from univalents, trivalent and multivalents. However, the quadrivalent frequency was significantly higher in hybrids than that in restoring parents at MI. The other meiotic phases progressed normally, except for low percentages of PMCs with lagging chromosomes at AI and low percentages of PMCs with micronuclei at telophaseI (TI) and telophaseII (TII). PMCs with lagging chromosomes at AI and PMCs with micronuclei at TI and TII showed negative correlation between pollen fertility and seed set. Above 90% of the PMCs could form normal microspores, which resulted in the production of viable pollen grains, abnormal microspores were observed including penta-fission and hexa-fission. Based on these results we suggest that the two F1 hybrids had better behaviors of chromosome pairing and genetic stability than autotetraploid rice and other autotetraploid plants ever studied.     

Mortality of pollen grains may result from errors of meiosis: study of pollen tetrads in Typha latifolia L

In the cattail Typha latifolia the four haploid products of meiosis remain attached and form the flat tetrad of pollen grains. Gametophytic lethals arisen de novo in diploid cells of sporophyte must manifest themselves as pollen tetrads with two dead grains. This could allow to estimate the rate of recessive lethals arresting pollen grain development. We studied pollen samples collected from 44 sprouts in two populations in the vicinity of Novosibirsk. The anomalous tetrads T1, T2, T3, and T4 carrying one, two, three, and four dead grains, respectively, were detected in each sampled individual. The mean frequency of all anomalous tetrads in the two populations was 3.4% and 8.7%. The frequencies of tetrad classes varied widely among the individuals with correlation coefficient up to 0.94, but their ratios remained nearly constant. The majority of anomalous tetrads were presented by T1 and T2 classes (their sum comprising 72.7 and 74.0% in two populations), T1 being a little more abundant. The observed pattern of frequencies of tetrads with dead grains can be explained by errors of male meiosis such as chromosome non-disjunction in both meiotic divisions. The tetrads with two dead pollen grains may result mostly from non-disjunction in anaphase I, and those with one pollen grain from non-disjunction in anaphase II, thus making tetrad analysis ineffective for estimating the rate of gametophytic lethals.     

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.     

鱼腥草花粉母细胞减数分裂特征及其育性研究

为深入了解鱼腥草花粉母细胞的减数分裂特征与花粉育性的关系,该研究采用卡宝品红染色法对2个鱼腥草居群花粉母细胞的减数分裂过程进行观察,并采用氯化三苯基四氮唑(TTC)染色法、I2-KI染色法、B-K培养基培养法及荧光显微镜观察法来检测鱼腥草花粉的活力及萌发率。结果发现:(1)鱼腥草减数分裂的进程与花序大小、花药颜色、花药长度均有密切的关系。(2)2个居群的鱼腥草中花粉母细胞减数分裂过程正常占88.2%,有11.8%的花粉母细胞减数分裂异常。(3)减数分裂异常表现在减数分裂过程中出现微核、落后染色体、染色体桥、不均等分离、多分体等现象,并发现在二分体阶段及单核花粉发育过程中存在细胞融合。(4)2个居群的鱼腥草花粉活力均不超过1.5%,花粉几乎不萌发。研究认为,鱼腥草花粉育性低的主要原因是单核花粉的发育过程异常,而非鱼腥草花粉母细胞减数分裂异常所致。     

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.     

Two novel meiotic restitution mechanisms in haploid maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

Two original mechanisms of nuclear restitution related to different processes of meiotic division of pollen mother cells (PMCs) have been found in male meiosis of the lines of maize haploids no. 2903 and no. 2904. The first mechanism, which is characteristic of haploid no. 2903, consists in spindle deformation (bend) in the conventional metaphase-anaphase I. This leads to asymmetric incomplete cytokinesis with daughter cell membranes in the form of incisions on the mother cell membrane. As a result, the chromosomes of the daughter nuclei are combined into a common spindle during the second meiotic division, and a dyad of haploid microspores is formed at the tetrad stage. The frequency of this abnormality is about 50%. The second restitution mechanism, which has been observed in PMCs of haploid no. 2904, results from disturbance of the fusion of membrane vesicles (plastosomes) at the moment of formation of daughter cell membranes and completion of cytokinesis in the first meiotic division. This type of cell division yields a binuclear monad. In the second meiotic division, the chromosomes of the daughter nuclei form a common spindle, and meiosis results in a dyad of haploid microspores. The frequency of this abnormality is as high as 15%. As a result, haploid lines no. 2903 and no. 2904 partly restore fertility.     

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.     

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     

A case of early dissolution of the microsporocyte callose wall in male-sterile (CMS) sweet pepper (Capsicum annuum L.)

On squash preparations of anthers from pollen fertile and sterile plants of sweet pepper (Capsicum annuum L. cv. Severka) callose envelopes of microsporocytes, stained specifically with resorcin blue, were investigated microscopically. During normal course of microsporogenesis in fertile plants the envelopes remained intact up to the stage of microspore tetrads. Then callose begins to dissolve, and that from individual microspores towards the envelope periphery. In sterile analogues of the same cultivar the callose breakdown occurred precociously, usually in the course of the second, but sometimes as early as the first meiotic division of PMCs. Having completed meiosis sporadic microsporocytes formed microspore tetrads. Most PMCs contained an undivided four-nucleate protoplast rimmed with a narrow or wider unstained zone of dissolved callose. In certain cases more condensed callose septa pointing to the furrows on the surface of the PMC protoplast were well-observable in this lytic zone, as a residuum of normal mechanism of tetradogenesis.     

Two novel meiotic restitution mechanisms in haploid maize (Zea mays L.)

Two original mechanisms of nuclear restitution related to different processes of meiotic division of pollen mother cells (PMCs) have been found in male meiosis of the lines of maize haploids no. 2903 and no. 2904. The first mechanism, which is characteristic of haploid no. 2903, consists in spindle deformation (bend) in the conventional metaphase-anaphase I. This leads to asymmetric incomplete cytokinesis with daughter cell membranes in the form of incisions on the mother cell membrane. As a result, the chromosomes of the daughter nuclei are combined into a common spindle during the second meiotic division, and a dyad of haploid microspores is formed at the tetrad stage. The frequency of this abnormality is about 50%. The second restitution mechanism, which has been observed in PMCs of haploid no. 2904, results from disturbance of the fusion of membrane vesicles (plastosomes) at the moment of formation of daughter cell membranes and completion of cytokinesis in the first meiotic division. This type of cell division yields a binuclear monad. In the second meiotic division, the chromosomes of the daughter nuclei form a common spindle, and meiosis results in a dyad of haploid microspores. The frequency of this abnormality is as high as 15%. As a result, haploid lines no. 2903 and no. 2904 partly restore fertility.     

Inverted meiosis and its place in the evolution of sexual reproduction pathways

Inverted meiosis is observed in plants (Cyperaceae and Juncaceae) and insects (Coccoidea, Aphididae) with holocentric chromosomes, the centromeres of which occupy from 70 to 90% of the metaphase chromosome length. In the first meiotic division (meiosis I), chiasmata are formed and rodlike bivalents orient equationally, and in anaphase I, sister chromatids segregate to the poles; the diploid chromosome number is maintained. Non-sister chromatids of homologous chromosomes remain in contact during interkinesis and prophase II and segregate in anaphase II, forming haploid chromosome sets. The segregation of sister chromatids in meiosis I was demonstrated by example of three plant species that were heterozygous for chromosomal rearrangements. In these species, sister chromatids, marked with rearrangement, segregated in anaphase I. Using fluorescent antibodies, it was demonstrated that meiotic recombination enzymes Spo11 and Rad5l, typical of canonical meiosis, functioned at the meiotic prophase I of pollen mother cells of Luzula elegance and Rhynchospora pubera. Moreover, antibodies to synaptonemal complexes proteins ASY1 and ZYP1 were visualized as filamentous structures, pointing to probable formation of synaptonemal complexes. In L. elegance, chiasmata are formed by means of chromatin threads containing satellite DNA. According to the hypothesis of the author of this review, equational division of sister chromatids at meiosis I in the organisms with inverted meiosis can be explained by the absence of specific meiotic proteins (shugoshins). These proteins are able to protect cohesins of holocentric centromeres from hydrolysis by separases at meiosis I, as occurs in the organisms with monocentric chromosomes and canonical meiosis. The basic type of inverted meiosis was described in Coccoidea and Aphididae males. In their females, the variants of parthenogenesis were also observed. Until now, the methods of molecular cytogenetics were not applied for the analysis of inverted meiosis in Coccoidea and Aphididae. Evolutionary, inverted meiosis is thought to have appeared secondarily as an adaptation of the molecular mechanisms of canonical meiosis to chromosome holocentrism.     

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.     

“Cuckoo” Aegilops addition chromosome in wheat ensures its transmission by causing chromosome breaks in meiospores lacking it

In monosomic additions of Aegilops sharonensis to Chinese Spring wheat (2n=42 wheat chromosomes + 1 homoeologous group 4 Aegilops chromosome known as 4S¹), probably all functional gametes carried one Aegilops chromosome. Such preferential transmission is unusual in monosomic alien additions. Male and female meiosis seemed usually normal, but about 75% of embryo sacs and 28% of pollen grains were visibly abnormal near anthesis. Before the first gametophyte mitosis, up to about 13% of megaspores and pollen grains showed abnormalities usual in wheat aneuploids. However, in first mitosis, 50% of megaspores at metaphase and anaphase and 41% of pollen grains at anaphase and telophase contained acentric chromosome (or chromatid) segments of various sizes, up to about 32 in a cell, which were separated from the rest of the chromosome (or chromatid) by a gap or a thin Feulgen-positive thread. Such separated segments (SSs) are not normally seen in wheat and its aneuploids. The data indicat that alien and wheat chromosomes interact in meiocytes so that meiospores with the alien chromosome develop into normal gametophytes, but meiospores lacking the alien chromosome have SSs at first mitosis by whose loss or unequal distribution between daughter nuclei sterilizing deficiencies arise. Thus only gametophytes with the alien chromosome are competent.     

Meiosis and pollen mitosis in diploid and triploid Colocasia antiquorum Schott.

The relationship between diploid and triploid forms of Colocasia antiquorum Schott. was assessed through comparative meiotic and pollen mitotic studies. Owing to poor spreading of the chromosomes of both materials, karyological observations on pachytene nuclei were limited to a few chromosomes. Among the two nucleolar chromosomes and a metacentric, telochromomere-bearing chromosome of the diploid, the latter and one of the nucleolar chromosomes characterized by a heteropycnotic short arm were identified in both bivalent and trivalent associations in the triploid. The homologues in these cases were homomorphic and intimately paired. Two types of heteromorphic bivalents exhibiting partial pairing of homomorphic segments were also recorded in the triploid. Among the 14 bivalents of the diploid at diakinesis, two were nucleolus-associated. In the triploid, chromosomal associations at diakinesis included trivalents (2 to 9), bivalents and univalents, and the chiasma frequency per paired chromosome was lower than in the diploids. In 21.6 percent of the PMCs at this stage intragenomic pairing of one or two chromosomes was observed. Post-diakinesis stages in the diploid were regular while in the triploid they were marked by various irregularities in a majority of the cells. However, fertility (stainability), size and divisional frequency of pollen in both materials were remarkably similar. Chromosome numbers in pollen nuclei in the triploid ranged from 8 to 25. Based on these data an autopolyploid origin for the triploid Colocasia and a lower base number than the gametic chromosome number for this genus are advanced.     

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

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 ofprophase-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.     

Cellular organisation in meiotic and early post-meiotic rice anthers

We have used fluorescent, confocal laser and transmission electron microscopy (TEM) to examine cellular organisations, including callose (1,3-beta-glucan) behaviour, in meiotic and early post-meiotic rice anthers. These features are critical for pollen formation and provide information to better understand pollen sterility caused by abiotic stress in rice and other monocotyledonous species. Among organelles during meiosis, abundant plastids, mitochondria and nuclei of the anther cells show distinctive features. Chloroplasts in the endothecium store starch and indicate a potential for photosynthetic activity. During meiosis, the middle layer cells are markedly compressed and at the tetrad stage are either vacuolated or filled with degenerating electron-opaque organelles. Viable mitochondria, stained with Rhodamine 123, are seen in the endothecium and tapetum, but the mitochondria in the middle layer are not stained during meiosis. The radial walls of the tapetum are disorganised and degenerating, indicating the formation of a syncytium; pro-orbicules are located at the locular walls at the tetrad stage. Immunohistochemical studies show that the sporogenous cells are entirely enveloped by a thick callosic layer at early meiosis. Cell plate callose was assembled in a plane between the dyad cells. In the tetrads, however, callose formed only at the centre, showing that the tetrad microspores are not enveloped but separated by callose walls. Thick, undulating electron-opaque walls around the tetrads indicate the beginning of exinous microspore wall differentiation.