Characteristics of the cytomixis in the pollen mother cells of transgenic tobacco plants (Nicotiana tabacum L.) with mutant phenotype

This paper represents the frequency characteristics and cytological analysis of cytomixis in the course of male meiosis in transgenic tobacco plants (Nicotiana tabacum L.) with altered flower morphology and male sterility. The influence of cytomixis on qualitative composition of the meiotic products is shoron (cytoplasts and polyads formation). It was established that doubling of chromosomes number increased cytomixis frequency in the investigated plants.     

Efficiency of the induction of cytomixis in the microsporogenesis of dicotyledonous (<Emphasis Type="Italic">N. tabacum</Emphasis> L.) and monocotyledonous (<Emphasis Type="Italic">H. distichum</Emphasis> L.) plants by thermal stress

The efficiencies of the induction of cytomixis in microsporogenesis by thermal stress are compared in tobacco (N. tabacum L.) and barley (H. distichum L.) It has been shown that different thermal treatment schedules (budding tobacco plants at 50°C and air-dried barley grains at 48°C) produce similar results in the species: the frequency of cytomixis increases, and its maximum shifts to later stages of meiosis. However, the species show differences in response. The cytomixis frequency increase in tobacco is more pronounced, and its maximum shifts from the zygotene–pachytene stages of meiotic prophase I to prometaphase–metaphase I. Later in the meiosis, aberrations in chromosome structure and meiotic apparatus formation typical of cytomixis are noted, as well as cytomixis activation in tapetum cells. Thermal stress disturbs the integration of callose-bearing vesicles into the callose wall. Cold treatment at 7°C does not affect cytomixis frequency in tobacco microsporogenesis. Incubation of barley seeds at 48°C activates cytomixis in comparison to the control, shifts its maximum from the premeiotic interphase to zygotene, and changes the habit of cytomictic interactions from pairwise contacts to the formation of multicellular clusters. Thermal treatment induces cytomictic interactions within the tapetum and between microsporocytes and the tapetum. However, later meiotic phases show no adverse consequences of active cytomixis in barley. It is conjectured that heat stress affects callose metabolism and integration into the forming callose wall, thereby causing incomplete closure of cytomictic channels and favoring intercellular chromosome migration at advanced meiotic stages.     

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.     

A rise of ploidy level influences the rate of cytomixis in tobacco male meiosis

The effect of plant ploidy level on the rate of cytomixis in microsporogenesis has been analyzed with the help of a unique model, the collection of tobacco plants of different ploidies (2n?=?2x?=?24, 4x?=?48, 6x?=?72, and 8x?=?96). As has been shown, the rate of cytomixis proportionally increases in 6x and 8x cytotypes, being rather similar in 2x and 4x plants. The rate of cytomixis is highly variable, differing even in the genetically identical plants grown under the same conditions. The cytological pattern of cytomixis in the microsporogenesis of control 4x plants has been compared with the corresponding patterns of 2x, 6x, and 8x plants. Involvement of cytomixis in production of unreduced gametes and stabilization of the newly formed hybrid and polyploidy genomes is discussed.     

Cytomixis and its role in the regulation of plant fertility

UV and gamma irradiation of barley seedlings induces an increase in the number of various pathologies in the male reproductive system of plants. The majority of cytological abnormalities are rather nonspecific. The main type of the observed pathologies of microsporogenesis is cytomixis, whose activation correlates with a callose hypersecretion in microsporocyte walls. A negative correlation between cytomixis and the sterility of microspores (in the case of gamma irradiation) or the sterility of mature pollen grains (in the case of UV-B irradiation) is revealed. It is supposed that cytomixis represents a kind of a premeiotic cell selection in plants characterized by an intraorganismic genetic heterogeneity (mosaics). The novelty of the idea is that the cytopathology that accompanies cytomixis is considered as a mechanism of the induced death of genetically imbalanced or nonrepairable cells, which is intended to keep the fertility of a male reproductive system. The activation of this mechanism has a threshold character.     

Induced cytomictic variations and syncyte formation during microsporogenesis in <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.

The intercellular translocation of chromatin material along with other cytoplasmic contents among the proximate meiocytes lying in close contact with each other commonly referred as cytomixis was reported during microsporogenesis in Phaseolus vulgaris L., a member of the family Fabaceae. The phenomenon of cytomixis was observed at three administered doses of gamma rays viz. 100, 200, and 300 Gy respectively in the diploid plants of Phaseolus vulgaris L. The gamma rays irradiated plants showed the characteristic feature of inter-meiocyte chromatin/chromosomes transmigration through various means such as channel formation, beak formation or by direct adhesion between the PMC’s (Pollen mother cells). The present study also reports the first instance of syncyte formation induced via cytomictic transmigration in Phaseolus vulgaris L. Though the frequency of syncyte formation was rather low yet these could play a significant role in plant evolution. It is speculated that syncyte enhances the ploidy level of plants by forming 2n gametes and may lead to the production of polyploid plants. The phenomenon of cytomixis shows a gradual inclination along with the increasing treatment doses of gamma rays. The preponderance of cytomixis was more frequent during meiosis I as compared to meiosis II. An interesting feature noticed during the present study was the channel formation among the microspores and fusion among the tetrads due to cell wall dissolution. The impact of this phenomenon is also visible on the development of post-meiotic products. The formation of heterosized pollen grains; a deviation from the normal pollen grains has also been reported. The production of gametes with unbalanced chromosomes is of utmost importance and should be given more attention in future studies as they possess the capability of inducing variations at the genomic level and can be further utilized in the improvement of germplasm.     

First detection of cytomixis and its consequences in <Emphasis Type="Italic">Thalictrum cultratum</Emphasis> Wall. (Ranunculaceae)

In current paper, we studied the meiotic chromosome number and details of cytomixis recorded for the first time in Thalictrum cultratum Wall. from the high altitude areas of northwest Himalaya (India). Cytomixis, the phenomenon of inter PMC (pollen mother cell) migration of chromatin material has been reported in many angiosperms and other groups of plants, but there is no published report in Thalictrum cultratum. All the presently studied accessions of the species existed at diploid level (x = 7). The present chromosome count of n = 14 has been reported for the first time from the study area. The cytological stability of any plant is an important consideration in view of its existence in nature. Meiotic disturbances can impose threat to the reproductive potential of plant. Current study reveals the occurrence of inter PMC migration of chromatin material. In present investigation, the phenomenon of cytomixis can be observed between 2 to 9 PMCs. Chromatin migration occurs through narrow and broad cytoplasmic channels or occasionally fused together during metaphase-I and anaphase-I. Cytomixis resulted in the formation of hypo- (n = 2, 2 + 1 small fragment, n = 3 + 3 small fragments, 4, 5 + extra mass of chromatin, 13) and hyperploid (n = 7 + 2 small fragments, 8 + 2 small fragments, 14, 14 + 2 small fragments, 15) PMCs, variable sized pollen grains and pollen sterility (20%). The current study reveals that cytomixis is responsible for formation of PMCs with variable chromosomes (hypoand hyperploid) and pollen grains of heterogeneous sizes and pollen sterility in T. cultratum. This is the first report of cytomixis in the species.     

Male meiosis and behaviour of sex chromosomes in different populations of Rumex acetosa L. from the Western Himalayas, India

Detailed meiotic analysis in 28 North West Himalayan populations of dioecious plant Rumex acetosa L. was carried out. The species is generally discussed as an important plant having sex chromosomes. Male meiosis in all the studied populations clearly showed the formation of six bivalents and one trivalent during diakinesis and metaphase-I. The sex chromosomes in male plants exhibit a chain of trivalent (Y₁–X–Y₂). In addition, among the presently investigated populations ring-shaped trivalents were also observed for the first time in the species. Varied frequency of abnormal segregation of sex trivalent was also observed leading to XY:Y segregation instead of normal X:Y₁Y₂ segregation. A majority of the populations exhibit normal meiosis. Plants of six populations show meiotic abnormalities like cytomixis, laggards, bridges, chromatin stickiness, etc., leading to reduced pollen fertility. Translocation between an autosome and sex chromosomes was also observed in some of the populations. 0–1B chromosomes were noticed in one population. This is the first ever meiotic analysis of the species from India.     

The role of microtubular cytoskeleton and callose walls in the cytomixis process in tobacco (Nicotiana tabacum L.) pollen mother cells

We have studied the microtubule cytoskeleton structure and callose walls deposition in the course of meiosis at the cytomictic and normal tobacco (N. tabacum L.) PMCs. It was ascertained that microtubule cytoskeleton did not play an evident part in the process of cytomixis. Increased cytomixis frequency probably is determined by irregular callose walls deposition. The possible reasons of nuclear material passage between tobacco PMCs at the cellular level are discussed.     

Cytomixis in pollen mother cells of Medicago sativa L

Cytomixis (i.e., chromatin migration between meiocytes) has been detected in many plant species, but not in Medicago sativa spp. In the present study we report the identification of a few cytomictic alfalfa plants. Those plants, the "mother plants," were selfed and crossed with a normal control plant. Microsporogenesis analysis was performed on the mother plants, on the S(1) and F(1) plants, and on controls. The S(1) and F(1) plants, like the mother plants, were found to be cytomictic. Single or multiple chromatin bridges between two or more meiocytes were observed almost exclusively in prophase I. Some completely empty meiocytes were also observed. In addition to cytomixis, other meiotic abnormalities were found. Control plants showed an almost regular meiosis. The highest values of cytomixis were observed in the mother plants, and the lowest in their F(1) progenies. Variability of cytomixis in the F(1) plants is probably due to a heterozygotic condition of the parents for this trait. No significant correlation was found between cytomixis and pollen viability, even if the cytomictic plants showed low values of pollen viability.     

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.     

Cytomixis in plants: facts and doubts

The migration of nuclei between plant cells (cytomixis) is a mysterious cellular phenomenon frequently observable in the male meiosis of higher plants. Cytomixis attracts attention because of unknown cellular mechanisms underlying migration of nuclei and its potential evolutionary significance, since the genetic material is transferred between the cells that form pollen. Although cytomixis was discovered over a century ago, the advance in our understanding of this process has been rather insignificant because of methodological difficulties. The data that allowed for a new insight into this phenomenon were obtained by examining the migrating nuclei with electron and confocal laser microscopy, immunostaining, and fluorescence in situ hybridization. As has been shown, the chromatin migrating between cells is surrounded by an undamaged nuclear membrane. Such chromatin does not undergo heterochromatization and contains normal euchromatin markers. The condensation degree of the migrating chromatin corresponds to the current meiotic stage, and normal structures of synaptonemal complex are present in the migrating part of the nucleus. The cells involved in cytomixis lack any detectable morphological and molecular markers of programmed cell death. It has been shown that individual chromosomes and genomes (in the case of allopolyploids) have no predisposition to the migration between cells, i.e., parts of the nucleus are involved in cytomixis in a random manner. However, the fate of migrating chromatin after it has entered the recipient cell is still vague. A huge amount of indirect data suggests that migrating chromatin is incorporated into the nucleus of the recipient cell; nonetheless, the corresponding direct evidences are still absent. No specific markers of cytomictic chromatin have been yet discovered. Thus, the causes and consequences of cytomixis are still disputable. This review briefs the recent data on the relevant issues, describes the classical and modern methodological approaches to analysis of the intercellular migration of nuclei, and discusses the problems in cytomixis research and its prospects.     

Impaired Male Meiosis due to Irregular Synapsis Coupled with Cytomixis in a New Diploid Cytotype of Dianthus angulatus (Caryophyllaceae) from Indian Cold Deserts

Dianthus angulatus (Caryophyllaceae) is cytologically examined here for the first time for the area of India. The diploid chromosome count of 2n?=?30, ascertained here, represents a new cytotype, supplementing the earlier report of a hexaploid cytotype with 2n?=?90 from outside of India. We report here the occurrence of two plants showing impaired meiosis due to irregular synapsis and cytomixis collected from Kinnaur district, Himachal Pradesh (India). The other plants of this species collected in Lahaul-Spiti region showed normal male meiosis (n?=?15) with a high (95%?100%) pollen fertility and normal seed set, and they reproduce sexually. Irregular synapsis in two plants from the Kinnaur region is characterized by the complete absence of chromosome pairing and the presence of 30 univalents at diakinesis and meta-anaphase. In addition, other meiotic irregularities were found, such as unoriented chromosomes, laggards, precocious movements of univalents at anaphase-I and micronuclei at telophase. Microsporogenesis was also abnormal, resulting in the formation of monads, dyads, triads, polyads, and tetrads with micronuclei. The occurrence of intra- and intermicrosporal chromatin material transfer during microsporogenesis was observed, which is a rather rarely observed phenomenon. The synaptic irregularities coupled with chromatin transfer in these plants seem to be responsible for the high pollen sterility (38%?42%) and heterogeneously sized pollen grains. In these plants no seeds were set, and plants reproduced vegetatively through root suckers.     

Assessment of mutagenicity induced by MMS and DES in Capsicum annuum L.

Seeds of Capsicum annuum L. var. G4 were subjected to different concentrations of methyl methane sulphonate (MMS) and diethyl sulphate (DES). The effects of different mutagenic treatments on meiosis, chiasma frequency, and pollen fertility have been studied in M₁ generation. Various types of meiotic aberrations such as univalent, multivalent, stickiness, bridge, laggards, cytomixis etc. were observed in all the treatments. However, the MMS treatments proved to be more effective in inducing meiotic aberrations as compared to DES. Moreover, the frequency of meiotic aberrations was at its maximum at metaphase followed by anaphase and telophase stages. As the concentrations increase, reduction in chiasma frequency and pollen fertility was observed in all the treatments and, MMS again was found to be more effective than DES treatments.     

Cytomixis, its nature, significance and the cytological consequences

Cytomixis is the widespread natural process of intercellular interaction which is characteristic for vegetative and generative tissues in both normal and pathological conditions. The origin significance and genetic control cytomixis still remain not completely clear. The popularity of view of the pathological nature of cytomixis based on its peculiar plants with genetic instability and impaired homeostasis. In the genetic control of cytomixis seem to be involved meiotic genes which are responsible for segregation and organization of chromosomes. Their activity is modified by environmental factors through signal transduction. It is assumed via cytomixis, from one side, the informational contact can be reached and meiosis and gametogenesis are synchronized, with another, increase of the genetic variety and level of the heterozygosis of microsporocytes. The activity of cytomixis varies over wide limits. The greatest influence on its activity have mutagenesis hybridization and polyploidy. In this context cytomixis can fulfill the function of cell selection which is activated by exceeding of the threshold level of the microsporocyte damages (or genetical disbalance).     

Cytogeography and phenomenon of cytomixis in Silene vulgaris from cold regions of Northwest Himalayas (India)

Silene vulgaris is an important ethnobotanical species, but so far no thorough meiotic analysis has been performed, despite its wide occurrence in temperate and alpine Himalayas, this prompted us to undertake present study. We investigated original meiotic chromosome number and effect of cytomixis on meiotic course, microsporogenesis and pollen grain size and fertility. The paper also summarizes the previously published chromosome counts in the species and cytogeography of 2x and 4x cytotypes in the Indian Himalayas and elsewhere. Meiocytes preparations were made through standard squashed technique in 1 % acetocarmine. Two ploidy levels have been determined during the cytomorphological surveys, diploid (2n = 2x = 24) and tetraploid (2n = 4x = 48). Most of the presently scored populations were found to be diploid (2n = 2x = 24). Morphologically 2x and 4x individuals were indistinguishable from each other. Meiosis was perfectly normal in 4x cytotype. However, some of the 2x plants showed the phenomenon of cytomixis and associated meiotic abnormalities. Consequent to cytomixis, hypo-, hyperploid and enucleated pollen mother cells (PMCs) were resulted. Such PMCs combined with other meiotic abnormalities yielded sterile and variable sized pollen grains. On the basis of overall information, it is clear that 2x cytotype is the most widely distributed cytotype in the Indian Himalayas as well as elsewhere. On the other hand, the 4x cytotype seems to be less frequently present in the Indian Himalayas and outside of India.     

Cytomorphological investigations in Oxyria digyna Hill. from the Kashmir Himalaya,India

In the present paper, detailed cytomorphological investigations in Oxyria digyna Hill. from Kashmir Himalaya—India have been reported for the first time. All the 14 investigated populations of O. digyna are diploid based on x = 7. Out of these in two populations 0–2B chromosomes have been recorded for the first time while 6 populations differed significantly in their meiotic characteristics. Meiotic abnormalities during male meiosis observed include inter PMC chromatin transfer (cytomixis). Non-synchronous disjunction of some bivalents, laggards and bridges at anaphases and telophases. Consequent to these meiotic anomalies, microsporogenesis in meiocytes is abnormal resulting in to dyads, triads and polyads with or without micronuclei. The overall effect is seen in reduced pollen fertility. Unreduced pollen grains were observed in some populations, which differed significantly in their size compared to the normal (reduced) pollen grains. It is observed that a smaller frequency of pollen grains differed morphologically in Aharbal and Yosmarg populations. The remaining eight populations showed regular meiotic course, normal microsporogenesis and high percentage of pollen fertility (95.09–99.09 %).     

Role of microtubular cytoskeleton and callose walls in the manifestation of cytomixis in pollen mother cells of tobacco Nicotiana tabacum L.

The structure and dynamics of microtubular cytoskeleton and of callose walls in normal pollen mother cells (PMC) of tobacco N. tabacum L. and in cells with intercellular translocation of nuclear material (cytomictic) was studied in the course of the cell cycle. The microtubular cytoskeleton was established as playing no obvious role in the process of cytomixis. The elevated level of cytomictic seems to be due to disturbances of synthesis of callose walls as a result of their attenuation and perforation. Possible causes of cytomictic in tobacco PMC at the cellular level are discussed.     

Induction of cytomixis affects microsporogenesis in Sesamum indicum L. (Pedaliaceae)

Cytomixis was recorded during microsporogenesis in sesame (Sesamum indicum L.), a member of the family Pedaliaceae. The phenomenon of cytomixis was observed at various stages of meiosis in 0.5% sodium azide (SA) treated populations of Sesamum indicum L. Cytomixis was observed to occur through various methods, i.e. by forming cytoplasmic channels and direct fusion of pollen mother cells (PMCs), the former was more frequent than the latter. The migration of nuclear content involved all the chromatin/chromosomes or part of it from donor to recipient cell/cells. Some completely empty meiocytes were also observed. Stickiness, precocious movement, laggards, unorientation and micronuclei were observed in almost all the sets treated with various doses of SA. Increase in the doses of SA had a positive effect on the percentage of PMCs showing cytomixis and chromosomal abnormalities. The impact of cytomixis on meiotic behaviour, reduced pollen viability and heterogeneous sized pollen grains were observed.     

Nature, significance, and cytological consequences of cytomixis

Cytomixis is a widespread natural process of intercellular interaction, which is common for vegetative and generative tissues in both normal and pathological conditions. The origin, significance, and genetic control of cytomixis still remain partially unclear. The widespread point of view regarding the pathological nature of cytomixis is based on its singularity for plants with genetic instability and impaired homeostasis. Meiotic genes responsible for the segregation and organization of chromosomes, the activity of which is modified by environmental factors through the signal transduction system, seem to be involved in the genetic control of cytomixis. It is assumed that, via cytomoxis, on the one hand, an information contact can be reached as well as meiosis and gametogenesis are synchronized and, on the other hand, the genetic variety and heterozygosis of microsporocytes increase. The activity of cytomoxis varies within a wide interval. Mutagenesis, hybridization, induction, and polyploidy have the greatest influence on this activity. In this context, cytomixis may have a function of cell selection, which is activated by exceeding the threshold level of microsporocytes damage (or genetic disbalance).