Large Pollen Grains Research Articles

Pollen dimorphism and androgenesis in Hordeum vulgare

Dimorphism of binucleate pollen grains of <em>Hordeum vulgare</em> has been confirmed. It is considered, however, in contrast to the accepted opinions, that some of the large pollen grains with dense cytoplasm lying close to the tapetum are the outset forms for embryoids, and not the small pollen grains with scarce cytoplasm lying in the pollen sac centre.

Morphological anomalies in pea (Pisum sativum L. cv. Hamil.) pollen grains under high doses of zinc

The morphology of pea (<i>Pisum sativum</i> L. cv. Hamil.) pollen grains was studied after adding zinc to the substrate in doses of. 0, 1, 500 and 1000 mg/pot. The observations were carried out using both light and scanning electron microscopy. The largest pollen grains were found at a dose of 500 mg/pot. Approximately 10% of the grains with such abnormalities in sporoderm structure as various types of swellings, mostly ring-shaped, were found under high concentrations of Zn (500 and 1000 mg/pot). The greatest number of deformed grains (over 40%), deeply plicated or strongly flattened, were observed at the dose of 1000 mg Zn. All of the described deformations related to excess Zn are documented photographically.

Styrian oil pumpkin pollen germinability at higher irradiation doses: optimization of the in vitro germination protocol and irradiation procedure

Protocols for haploid induction in cucurbits are based on pollination with irradiated pollen, but the induction frequency is low and the majority of obtained embryos are zygotic. The longevity of Cucurbita pepo L. pollen is short even under natural conditions; following irradiation, germinability is decreased even further. This study was initiated to develop an optimal in vitro germination medium for styrian oil pumpkin (Cucurbita pepo L. subsp. pepo var. styriaca Greb.) pollen, which would enable accurate germination testing. Different pH values and the addition of sucrose, mannitol, and polyethylene glycol to the Brewbaker and Kwack germination medium were tested. The optimum medium condition was pH 9 and 12.5% (w/v) sucrose, while other tested components were not efficient. Using the optimized medium, X-ray-irradiated (100-700 Gy) pollen germinability was assessed under 2 air humidity conditions. Germinability of pollen irradiated at room humidity (RH) was generally lower than that of pollen irradiated at high humidity (HH). A major variability in pollen size (diameters ranged from 79.2 to 196.5 µm) and 2 subgroups were found in the pollen population. Following irradiation, HH conditions allowed germination of larger pollen grains than those of the nonirradiated control and RH.

Morphological, cytological and metabolic consequences of autopolyploidization in Hylocereus (Cactaceae) species.

BackgroundGenome doubling may have multi-level effects on the morphology, viability and physiology of polyploids compared to diploids. We studied the changes associated with autopolyploidization in two systems of somatic newly induced polyploids, diploid-autotetraploid and triploid-autohexaploid, belonging to the genus Hylocereus (Cactaceae). Stomata, fruits, seeds, embryos, and pollen were studied. Fruit pulp and seeds were subjected to metabolite profiling using established gas chromatography-mass spectrometry (GC-MS) and ultra-performance liquid chromatography (UPLC) Q-TOF-MS/MS (time of flight)-protocols.ResultsAutopolyploid lines produced lower numbers of tetrads, larger pollen grains with lower viability, larger stomata with lower density, and smaller fruits with lower seed numbers and decreased seed viability. The abundance of sugars was lower in the fruits and seeds of the two duplicated lines than in their donor lines, accompanied by increased contents of amino acids, tricarboxylic acid (TCA) cycle intermediates, organic acids and flavonoids. Betacyanins, the major fruit pigments in diploid and triploid donors, decreased following genome doubling. Both autopolyploid Hylocereus lines thus exhibited unfavorable changes, with the outcome being more dramatic in the autohexaploid than in the autotetraploid line.ConclusionInduced autotetraploid and autohexaploid lines exhibited morphological and cytological characteristics that differed from those of their donor plants and that were accompanied by significant metabolic alterations. It is suggested that a developmental arrest occurs in the fruits of the autohexaploid line, since their pericarp shows a greater abundance of acids and of reduced sugars. We conclude that genome doubling does not necessarily confer a fitness advantage and that the extent of alterations induced by autopolyploidization depends on the genetic background of the donor genotype.

Pollen morphology of some taxa of Vicia L. subgenus Vicia (Fabaceae) from Turkey

In this study, the pollen morphology of 11 taxa belonging to Atossa (Alef.) Asch. & Graebner, Hypechusa (Alef.) Asch. & Graebner, Peregrinae Kupicha, Wiggersia (Alef.) Maxted, Vicia L. and Narbonensis (Radzhi) Maxted sections of the genus Vicia L. subgenus Vicia (Fabeae, Fabaceae) naturally growing in Turkey has been studied using Light Microscopy (LM) and Scanning Electron Microscopy (SEM) to evaluate the taxonomic relevance of pollen characters. Twelve morphometric characters are analysed with one-way analysis of variance and Tukey’s honestly significant difference test for multiple comparisons. Of the taxa studied V. narbonensis var. narbonensis (sect. Narbonensis) has the largest pollen grains (P = 51.98 μm × E = 30.52 μm) and V. lathyroides (sect. Wiggersia) has the smallest pollen grains (P = 27.71 μm × E = 20.14 μm). The pollen grains are subprolate to perprolate (P/E = 1.16–2.11), but the prolate shape occurs in the majority of the taxa. The regular pollen grains of all taxa are trizonocolporate, isopolar, and released in monads. Ornamentation of the mesocolpium is psilate-perforate in V. lathyroides (sect. Wiggersia), reticulate-rugulate in V. narbonensis var. narbonensis (sect. Narbonensis), (micro)reticulate in V. sericocarpa var. sericocarpa (sect. Hypechusa), V. sativa subsp. sativa (sect. Vicia) and V. grandiflora var. grandiflora (sect. Vicia), and reticulate-perforate in the remaining taxa. The apocolpium and colpus area are psilate or perforate in all taxa except V. sericocarpa var. sericocarpa (sect. Hypechusa) and V. sativa subsp. sativa (sect. Vicia), which exhibit the obscurely reticulate-perforate pattern. Several palynological features have taxonomic importance in sectional level: polar axis, equatorial diameter, pollen shape (P/E ratio), colpus length, colpus width, size of pori, porus length/width ratio, lumina diameter, muri thickness and ornamentation. The results also indicate that pollen characters can be useful in distinguishing the examined taxa.

Pennsylvanian (mid-Bolsovian to Asturian) megaspores and large pollen of the Bristol Coalfield, UK

Abstract. A detailed account of assemblages of megaspores and large pollen grains from the mid-Bolsovian to late Asturian Warwickshire Group (Winterbourne, Pennant Sandstone and Grovesend formations) of the Bristol Coalfield is presented. The megaspore assemblages show certain similarities to those from the well-documented, and partly coeval, sequence in the nearby Forest of Dean Coalfield. However, the Bristol Coalfield megaspore assemblages are generally less diverse. We consider the palaeoecological implications of the megaspore assemblages and conclude that differing depositional settings had different vegetational successions (including differences in the composition and diversity of megaspore-producing plants). A consideration of megaspore biostratigraphy suggests that the uppermost Pennant Sandstone Formation may be of early Asturian age, suggesting that the postulated gap between the Pennant Sandstone Formation and overlying Grovesend Formation is smaller than previously suggested. We also report the first European occurrence of the large enigmatic pteridosperm pollen grain Parasporites maccabei Schopf, 1938.

Male fertility versus sterility, cytotype, and DNA quantitative variation in seed production in diploid and tetraploid sea lavenders (Limonium sp., Plumbaginaceae) reveal diversity in reproduction modes

The genus Limonium Miller, a complex taxonomic group, comprises annuals and perennials that can produce sexual and/or asexual seeds (apomixis). In this study, we used diverse cytogenetic and cytometric approaches to analyze male sporogenesis and gametogenesis for characterizing male reproductive output on seed production in Limonium ovalifolium and Limonium multiflorum. We showed here that the first species is mostly composed of diploid cytotypes with 2n = 16 chromosomes and the latter species by tetraploid cytotypes with 2n = 32, 34, 35, 36 chromosomes and had a genome roughly twice as big as the former one. In both species, euploid and aneuploid cytotypes with large metacentric chromosomes having decondensed interstitial sites were found within and among populations, possibly involved in chromosomal reconstructions. L. ovalifolium diploids showed regular meiosis resulting in normal tetrads, while diverse chromosome pairing and segregation irregularities leading to the formation of abnormal meiotic products are found in balanced and non-balanced L. multiflorum tetraploids. Before anther dehiscence, the characteristic unicellular, bicellular, or tricellular pollen grains showing the typical Limonium micro- or macro-reticulate exine ornamentation patterns were observed in L. ovalifolium using scanning electron microscopy. Most of these grains were viable and able to produce pollen tubes in vitro. In both balanced and unbalanced L. multiflorum tetraploids, microspores only developed until the “ring-vacuolate stage” with a collapsed morphology without the typical exine patterns, pointing to a sporophytic defect. These microspores were unviable and therefore never germinated in vitro. L. ovalifolium individuals presented larger pollen grains than those of L. multiflorum, indicating that pollen size and ploidy levels are not correlated in the Limonium system. Cytohistological studies in mature seeds from both species revealed that an embryo and a residual endosperm were present in each seed. Flow cytometric seed screens using such mature seeds showed quantitative variations in seeds ploidy level. It is concluded that male function seems to play an important role in the reproduction modes of Limonium diploids and tetraploids.Electronic supplementary materialThe online version of this article (doi:10.1007/s00497-012-0199-y) contains supplementary material, which is available to authorized users.

Viability, production and morphology of pollen grains for different species in the genus Manihot (Euphorbiaceae)

The objective of this work was to characterize the viability, production and morphology of pollen for different species in the genus Manihot. Floral buds from Manihot accessions were collected from two germplasm banks at Embrapa Cassava & Fruits. The viability of the pollen was assessed via colorimetric, in vitro and in vivo assays. The diameter of the pollen grains was determined by measuring the transversal length of the grain. The experimental design was entirely randomized. Studies on pollen ultrastructure were performed via scanning electron microscopy. Pollen viability was high in the colorimetric tests and intermediate in vivo tests; there was no germination in the in vitro tests. The average production for all accessions was 1,253 pollen grains per floral bud. The size of the pollen grains varied from 132 to 163 µm in the wild accessions, and 129 to 146 µm in the cultivated accessions. The pollen grains for all accessions were very large, apolar, spherical as well as inaperturate, with an exine ornamented with pila organized in a Croton pattern. The wild accessions, in general, produced more and larger pollen grains compared with the cultivated accessions.

High Pollen Sterility and 2<i>n</i> Pollen Grains in an Asynaptic 4<i>x</i> Cytotype (2<i>n</i>=48) of <i>Solanum nigrum</i> L.

Cytological investigations have been carried out on the 9 wild accessions of Solanum nigrum L., a medicinally important species having wide-range therapeutic properties. Male meiotic studies performed in 9 accessions revealed the presence of 3 intraspecific cytotypes, 2x (n=12), 4x (n=24) and 6x (n=36) in the species. The course of meiosis in 8 of the 9 accessions among the 2x, 4x, 6x cytotypes was noticed to be perfectly normal resulting in very high pollen fertility (95–100%). However, 1 accession of 4x cytotype scored from Palchan (Solang Valley, Kullu, Himachal Pradesh) depicted irregular chromosomal behaviour during meiosis due to the presence of all 48 chromosomes as univalents. The further course of meiosis in this individual was highly irregular with the presence of large number of lagging and unoriented chromosomes, eccentric positioned univalents in the PMCs and unequal distribution of chromosomes at anaphase-I. Microsporogenesis was also abnormal which is characterized by the presence of irregular sporads, such as dyads, triads and polyads. Lagging chromosomes constituted micronuclei at sporad stages. Consequent to all these meiotic irregularities, 95% of the pollen grains were observed to be sterile. Pollen grains were also recorded to be of different sizes. In meiotically abnormal 4x accession, pollen grains were of 2 different types compared to normal 4x plants showing perfectly normal meiosis. The large pollen grains, which were measured to be 1.25–1.5 times bigger than normal sized pollen grains, were considered as unreduced or 2n pollen grains. The role of such 2n (unreduced) pollen grains in producing polyploid genotypes through sexual polyploidization in a chromosomally variable S. nigrum (2x, 3x, 4x, 5x, 6x, 8x, 9x, 12x) has also been discussed.

Polymorphism in pollen mother cells of Valeriana wallichii DC. (Valerianaceae)

While studying meiosis in the male tract of Valeriana wallichii DC (Valerianaceae). polymorphism in size as well as chromosome number was observed in its pollen mother cells. The present report is based on study undertaken in ten populations of the species; 8 from J&K (India) and 2 from Uttrakhand (India). In all the anthers studied from plants of these populations, pollen mother cells could be differentiated as small (20–30 μm), medium (30–50 μm) and large sized (50–65 μm). In two populations (Noorichamb, J&K and Chakrata, UK), besides size, PMCs with different ploidy level were also observed. While the small and medium PMCs had normal chromosome number with 16 bivalents, some of the large PMCs were tetraploid having 64 chromosomes. Rarely, some pmcs at early prophase- I were also seen in a seemingly fusing state in some of the anthers. Size difference in PMCs also got reflected in the size of pollen grains and these could be classified as small (<30 μm), medium (30–50 μm) and large (50–60 μ) in diameter. Pollen of different sizes differed for their stainability and viability. While stainability of small pollen was 74%, it was 95.5% and 100% in medium and large sized pollen grains respectively. Pollen viability (in FDA + 50% sucrose) was 55.8%, 84% and 100% for small, medium and large pollen grains respectively.

Morphological changes and self-incompatibility breakdown associated with autopolyploidization in Hylocereus species (Cactaceae)

Chromosome duplication—autopolyploidization—may affect plant morphology and breeding systems, ultimately enabling the production of improved genotypes. In this study, the autotetraploid lines obtained from the self-incompatible diploid Hylocereus monacanthus and the autooctapolyploid lines obtained from the self-compatible tetraploid H. megalanthus were studied and compared with the donor accessions. The resulting H. monacanthus autotetraploids exhibit lower fruit weight, seed number, and pollen viability than the donor plant, but it has larger pollen grains. Although the resulting H. megalanthus autooctaploids had larger pollen grains and lower pollen viability compared with the donor plant, only aborted fruits were obtained from these lines. The most valuable change observed was the breakdown of the self-incompatibility system in the H. monacanthus autotetraploid lines. This research provides important information on the horticultural value of vine cacti autopolyploid lines.

Pollen grain size variation in Caryophylloideae: a mixed strategy for pollen deposition along styles with long stigmatic areas?

The literature supports a positive correlation between pollen size and style length and that pollen size may be affected by a trade-off with pollen number. A correlation between pollen size and pollinator type has been hypothesized by some authors but not confirmed. To assess these associations, pollen grain number, pollen grain diameter and style length were measured in 53 species of Caryophylloideae that had been classified beforehand as being diurnal, nocturnal or self-pollinated. Surprisingly, many species showed two, and a few even three, distinct pollen grain size classes per individual anther. Therefore, further analyses were based on the different pollen size classes and respective pollen grain numbers. Firstly, we found no indication of a trade-off between pollen size and number. Secondly, a positive linear correlation between style length and pollen grain diameter was found using conventional analysis methods and phylogenetically independent contrasts. Thirdly, when using conventional analysis methods a strong relationship between style length and pollination mode was found, with nocturnal species having significantly longer styles and larger pollen grains than diurnal and selfing species. These results were not supported, however, when calculating comparisons of phylogenetically independent pairs. We discuss our findings in relation to intra-specific pollen polymorphism and pollen degeneration, both of which have been associated with different pollen grain sizes in Caryophylloideae. Furthermore, we discuss the possibility that pollen polymorphism might be a mixed strategy to optimise the success of pollen grains deposited at different pollen–ovule distances on long styles.

Gametes with somatic chromosome number and their significance in interspecific hybridization in Fuchsia

Sexual polyploidization has both a theoretical as well as an applied significance. Morphological screening for large pollen grains and shape of pollen produced by the individual, cytological investigation of hybrid progeny, and unbalanced separation of chromosomes at anaphase I in pollen mother cells were used to detect the gametes with somatic chromosome number in Fuchsia. The interspecific hybrids of F. fulgens (sect. Ellobium) × F. magellanica (sect. Quelusia), F. fulgens (sect. Ellobium) × F. splendens (sect. Ellobium), and F. triphylla (sect. Fuchsia) × F. splendens (sect. Ellobium) produced at the University of Auckland, New Zealand, showed both large and normal pollen grains in the same anther indicating the presence of unreduced gametes. Cytological investigation carried out on the hybrid progeny of F. fulgens (diploid, 2n=22, sect. Ellobium) × F. magellanica (tetraploid, 2n=44, sect. Quelusia) and F. triphylla (diploid, sect. Fuchsia) × F. arborescens (diploid, sect. Schufia) revealed unexpected chromosome numbers of 2n=44 and 2n=33, respectively. In general, the hybrids showed low fertility caused by genetically unbalanced gametes resulted from random disjunction of chromosomes at anaphase I. Studies on meiosis together with the presence of different shapes and sizes of pollen grains in Fuchsia proved indirectly that unreduced gametes are the products of first division meiotic nuclear restitution. These unreduced gametes were viable irrespective of pollen shape, their predominance in the hybrids, nuclear DNA amount and species phylogenetic position.

The use of cyanobacteria as filler in nitrocellulose capillaries improves ultrastructural preservation of immature barley pollen upon high pressure freezing

The high pressure freezing (HPF) followed by freeze substitution technique has advantages over chemical fixation in the context of preserving sample ultrastructure. However, when HPF is applied to cultured pollen grains, the large intercellular spaces present lead to a poor level of ultrastructure preservation. We report here that the mixing of cyanobacteria with immature barley pollen grains succeeded in greatly reducing the volume of liquid present between the large pollen grains, and so improved the loading of the sample into a nitrocellulose capillary. The use of yeast or cyanobacteria paste to surround the filled capillaries was beneficial in speeding the transfer of heat during the freezing process. This modification of the HPF method resulted in a greatly improved level of ultrastructure preservation.

Cytoplasmic connection of sperm cells to the pollen vegetative cell nucleus: potential roles of the male germ unit revisited

The male germ cells of angiosperm plants are neither free-living nor flagellated and therefore are dependent on the unique structure of the pollen grain for fertilization. During angiosperm male gametogenesis, an asymmetric mitotic division produces the generative cell, which is completely enclosed within the cytoplasm of the larger pollen grain vegetative cell. Mitotic division of the generative cell generates two sperm cells that remain connected by a common extracellular matrix with potential intercellular connections. In addition, one sperm cell has a cytoplasmic projection in contact with the vegetative cell nucleus. The shared extracellular matrix of the two sperm cells and the physical association of one sperm cell to the vegetative cell nucleus forms a linkage of all the genetic material in the pollen grain, termed the male germ unit. Found in species representing both the monocot and eudicot lineages, the cytoplasmic projection is formed by vesicle formation and microtubule elongation shortly after the formation of the generative cell and tethers the male germ unit until just prior to fertilization. The cytoplasmic projection plays a structural role in linking the male germ unit, but potentially plays other important roles. Recently, it has been speculated that the cytoplasmic projection and the male germ unit may facilitate communication between the somatic vegetative cell nucleus and the germinal sperm cells, via RNA and/or protein transport. This review focuses on the nature of the sperm cell cytoplasmic projection and the potential communicative function of the male germ unit.

Palynology of the Genus Stachytarpheta Vahl. (Verbenaceae)

The exine morphology of pollen grains of Stachytarpheta indica (Linn.) Vahl, Stachytarpheta cayennensis (Rich.) Vahl and Stachytarpheta angustifolia (Mill.) Vahl is reported. This study was carried out with a light microscope. Pollen grains from fresh anthers were collected and aceolysed. Statistical analysis used to analyse the data collected include cluster analysis, correlation analysis, similarity and distance indices. The pollen grains are spheroidal to oblate to sub-oblate in shape. They are aperturate, both colpate and porate. Tricolpate types occur most frequently, acolpate, monocolpate, bicolpate and tetracolpate types less frequently. The multicolpate and multiporate attributes in all the species indicate that the genus is not primitive in evolutionary history and this species probably, evolved around in the same time. According to the size, the pollen grains of the genus falls into groups permagna (pollen diameter 100-200 μm) and giganta (pollen diameter greater than 200 μm). S. cayennensis and S. anguistifolia belong to group permagna and S. indica only in the group giganta. This separates S. indica from the other two species. The large pollen grain size in the genus clearly supports the fact that the flowers in the genus are more insect-and-bird pollinated than wind pollinated. The similarity and distance indices of the species showed that S. cayennensis and S. angustifolia are the closest. S. indica is closer to S. angustifolia but farther from S. cayennensis.

A new cytotype of Jacobaea vulgaris (Asteraceae): frequency, morphology and origin

Jacobaea vulgaris subsp. vulgaris constitutes an intricate polyploid complex distributed in Europe. Four chromosome numbers have been reported in this species: 2n=20, 40, 80 and 32. Chromosome number 2n=20 reported from Bulgaria is due to misidentification with J. aquatica. We discovered new hexaploid (2n=6x=60) cytotype occuring within four sympatric populations of otherwise tetraploid and octoploid plants in the Czech Republic, Slovakia and Ukraine. The frequency of hexaploid individuals within 76 studied populations is very low (only 10 of 693 analysed plants), and hexaploids probably represent hybrids between tetraploid and octoploid plants. Multivariate morphometric analysis reveals partial separation of tetraploid and octoploid plants, whereas hexaploid individuals are similar to octoploids. Octoploids and hexaploids exhibit slightly longer ray florets, involucral bracts and tubular florets and more hairy outer achenes. Hexaploid plants display larger pollen grains and lower pollen fertility.

Contribution to the cytotaxonomy of Oryzopsis (Poaceae)

Meiotic studies were performed in twelve populations of four Oryzopsis species (O. pubiflora, O. lateralis, O. holciformis var. longiglomis and O. barbellata) to obtain data on the ploidy level and cytological evolution of the genus. The chromosome number 2n=2x=24 was revealed in all the species and populations studied. The present and other studies show the occurrence of two basic chromosome numbers in the genus, i.e. x=11 and x=12. Although Oryzopsis species and populations studied are diploid and are expected to form only bivalents in metaphase of meiosis-I, quadrivalents were observed in O. pubiflora and O. lateralis, possibly due to the occurrence of heterozygote translocations. B-chromosomes of 0–2 were observed in all species and populations studied. This is the first report of the occurrence of B-chromosomes in the genus Oryzopsis. Several meiocytes showed the presence of double chromosome number in O. lateralis, and multipolar cells were observed in populations of O. barbellata, O. lateralis and O. holciformis var. longiglomis. The occurrence of large pollen grains (possibly unreduced) was observed along with smaller (normal) pollen grains in these species. Significant differences observed in chiasma frequency and distribution among studied species may be of use in species delimitation. The Kakan population differed significantly from the other populations of O. lateralis in meiotic characteristics. If such cytological differences are accompanied by morphological variation (under investigation), we may consider this population as a new variety or subspecies.

Palynological insights of the eastern Asian and eastern North American disjunct genus Symplocarpus (Araceae)

Symplocarpus L. (Araceae) is a disjunct genus including S. foetidus (L.) Nutt. var. latissimus (Makino) S. Hara, S. nipponicus Makino, and S. nabekuraensis Otsuka & K. Inoue in temperate eastern Asia; S. egorovii N. S. Pavlova & V. A. Nechaev in the Russian far east; and S. foetidus (L.) Nutt. in temperate northeastern North America. For the first time, pollen morphology of Symplocarpus was investigated by light and scanning electron microscopes. Symplocarpus foetidus var. latissimus and S. foetidus had reticulate surfaces and rounded (obtuse), long, equatorial axis tips, whereas S. nipponicus had microreticulate surfaces and acute tips. Symplocarpus foetidus in North America had larger pollen grains than S. foetidus var. latissimus in eastern Asia. Thus, based on pollen characteristics, S. foetidus var. latissimus is more closely related to S. foetidus than to S. nipponicus. Other lines of evidence, such as molecular phylogenetic studies and phenology, support the phylogenetic relationships among these species proposed in this study. The slightly modified acetolysis procedure conducted in this paper is a promising application to study weak exine or exineless pollen grains.

Meiotic Chromosome Numbers in 9 Iranian Species of Oryzopsis (Poaceae)

Meiotic studies were performed in 9 Oryzopsis species of O. pubiflora, O. lateralis, O. holciformis, O. barbellata, O. munroi, O. microcarpa, O. sphacelata, O. virescense and O. molinioides showing the presence of 2n=2x=24 in all nine species studied. The species studied were diploid and had 12 chromosome number, and in O. microcarpa, O. sphacelata, O. molinioides and O. virescense chromosomes are new count. B-chromosomes 0–2 were observed in all species studied, which were smaller than A-chromosomes and did not pair with them. T-test analysis between the cells with B-chromosomes and those devoid of Bs showed significant difference in chiasma frequency and distribution as well as in chromosomes pairing only in O. munroi, with significant reduction in meiotic values in the presence of B-chromosomes. The occurrence of large pollen grains (possibly unreduced pollen grains) was observed along with small (normal) pollen grains in these species. Significant difference observed in chiasma frequency and distribution among species studied may be of use in species delimitation. Moreover species relationships obtained based on cytological data partly supports taxonomic treatment of the genus.