Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 73(2): 111-119, 2020 Firenze University Press www.fupress.com/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-544 Citation: G. Kumar, S. Singh (2020) Induced cytomictic crosstalk behaviour among micro-meiocytes of Cyamopsis tetragonoloba (L.) Taub. (cluster bean): Reasons and repercussions. Caryolo- gia 73(2): 111-119. doi: 10.13128/cary- ologia-544 Received: July 14, 2019 Accepted: April 13, 2020 Published: July 31, 2020 Copyright: © 2020 G. Kumar, S. Singh. This is an open access, peer- reviewed article published by Firenze University Press (http://www.fupress. com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Induced cytomictic crosstalk behaviour among micro-meiocytes of Cyamopsis tetragonoloba (L.) Taub. (cluster bean): Reasons and repercussions Girjesh Kumar, Shefali Singh* Plant Genetics Laboratory, Department of Botany, University of Allahabad, India *Corresponding author. E-mail: shefalisingh.910@gmail.com Abstract. Cytomictic behaviour of chromosomes among pollen mother cells was observed in mutagenic studies in cluster bean (Cyamopsis tetragonoloba (L.) Taub.). The study of pollen mother cells (PMC) revealed various chromosomal aberrations among which cytomixis was notified due to its obtrusive peculiarity and is therefore given description in this article. Cytoplasmic and chromatin transmigration were dis- cernible among contiguous or slightly distant PMCs through recreation of passage via direct cell-to-cell fusion or channel formation. This cytomictic phenomenon was invariably more pronounced at meiosis I as compared to meiosis II. Plasmodesmatal connections play a paramount role in aiding this behaviour by establishing intercellu- lar crosstalks. The cellular intermingling resulted in syncyte cells which were identified due to their doubled size. Syncyte or unreduced PMC formation leading to unreduced fertile gametes is speculated to act as a possible way out for infraspecific polyploidi- zation of species. Pollen fertility was computed, alongwith this heterosized pollens of varying diameter were segregated. Large sized pollens were 2n pollens; where size dif- ference is a consequence of cytomixis. Cytoplasmic connections among pollens were also observed sporadically. It is opined that syncyte formation and 2n pollen produc- tion have evolutionary significance. Keywords: Cyamopsis tetragonoloba, cytomixis, heterosized pollens, infraspecific poly- poidy, PMCs, syncyte. INTRODUCTION Cytomixis is promiscuous intercellular interaction for exchange of nuclear material, dividing chromosomal bodies and other integral cytoplasmic orga- nelles. The credit for first description of this phenomenon was conferred to Arnoldy (1900) in gymnosperms. Later on, the behaviour was also explored in PMCs of Crocus sativus by Koernicke (1901); however, the term ‘cytomix- is’ was christened by Gates (1911) during his findings in Oenothera gigas and Oenothera biennis. Besides reproductive cells, cytomixis has also been witnessed in root meristematic cells (Jacob 1941), tapetal cells (Cooper 1952), shoot apex 112 Girjesh Kumar, Shefali Singh (Guzicka and Wozny 2004) and other diverse somatic cell systems. In angiosperms, it is invariably more frequent in family Poaceae (reported in 82 species) and Fabaceae (reported in 48 species) (Mursalimov et al. 2013a) Occurrence of cytomixis is speculated to be of patho- logical nature and is frequently documented in species with unbalanced genomes such as haploids, aneuploids, hybrids (de Nettancourt and Grant 1964), mutants (Gottschalk 1970), triploids (Salesses 1970). There are also few instances where cytomixis was more profound among polyploids than their diploid counterparts (Semyarkhina and Kuptsou 1974); where it is perceived to allow elimi- nation of extra DNA in order to stabilize the genome and produce balanced and/or reduced pollen grains (Zhou 2003). The phenomenon has also been documented in PMCs of transgenic tobacco plants (Sidorchuk et al. 2007). A unique pattern of B-chromosome pioneered cytomixis was observed in B-carrier plant poppy, where B-chro- mosome was the first entrants in the recipient cell and A-chromosomes followed them (Patra et al. 1988), for which it was argued that heterochroatin blocks of B-chro- mosomes played a facilitating role for cytomixis. Cautious contemplation has revealed that cytomixis is a sort of cell selection, which selects and preserves fit- ting variants but eliminates unbalanced and irreparable PMCs (Kravets 2013). There is a difference of opinion regarding its significance; however general consensus by authors configures an evolutionary trail (Boldrini et al. 2006; Li et al. 2009). According to Cheng et al. (1980), cytomixis acts as an additional facilitator in phylogenet- ic evolution of karyotypes by reducing or increasing the basic series. However Guan et al. (2012) opined contrary views by asserting its deleterious effects on fertility while Veilleux (1985) accredited cytomixis to be a potential means to conserve genetic heterozygosity of gametes. Plethora of study recruited on cytomictic behaviour suggests that the phenomenon is a resulting event regu- lated by genetic and environmental factors rather than being due to fortuitous causes such as artifact produced by fixation, mechanical injuries or pathological anoma- ly (Gottschalk 1970; Song and Li 2009). Factors such as partial or total inhibition of cytokinesis during micro- sporogenesis (Risuen˜o et al. 1969), effect of gamma radiation (Kumar and Yadav 2012; Dwivedi and Kumar 2018), action of chemical agents such as colchicine (Gau- tam and Kumar 2013), are reported to repercuss into cytomixis. Several environmental constraints such as thermal stress (Sidorchuk et al. 2016), cold harsh condi- tions also intrigue inter-meiocyte fusion and hence syn- cyte formation (Singhal et al. 2011). Depending upon the intensity and severity, cyto- mixis is categorized into three main types: weak (local), intensive, and destructive or pathological (Kravchen- ko 1977). The study is significant because cytomixis is linked to evolution since it may lead to change in ploidy as well as often leads to unreduced gamete. Further- more, the study is of great relevance in assessing rea- sons and process of its occurence, and the complex pro- cess of microsporgenesis which is substantially affected by cytomixis. Role of plasmodesmatal connections and callose insulation needs more detailed scrutiny. Ioniz- ing radiation i.e. gamma rays was used in the present study for exploiting its mutagenic role for improvement genetic characteristics of the plant system. Role of gam- ma rays has also been anticipated for its role in induc- ing polyploids and aneuploids via cytomixis in several reports. Gamma ray is ascribed to be most efficient fac- tor that results in imbalanced genetic system (Saras- wathy et al. 1990). The pla nt materia l cluster bea n [Cyamopsis tetragonoloba (L.) Taub.] is an important legumes, thriv- ing well in semi arid zones of Indian and Pakistan. The plant is highly valued for its guar gum that is extracted form the seed endosperm that add ons its economical value. Besides this, cluster bean occupies a decent posi- tion in traditional folklore medicines and is nutraceu- tically also very important. Cytomictic behaviour in Cyamopsis tetragonoloba has been previously described spontaneously (Sarbhoy 1980), but the present article is envisioned to reach new vistas by exploring multitude facets of gamma rays induced cytomixis. Salient features and repercussions entailed in relation to meiotic behav- iour and reproductive success will be ambit of this work. MATERIALS AND METHODS Plant material Seeds of Cluster bean [Cyamopsis tetragonoloba (L.) Taub.] were procured from Central Arid Zone Research Institute (CAZRI) Jodhpur, Rajasthan, India. After pre- liminary screening, accession number RGC-1038 was selected for cytogenetical work. Agroclimatic conditions of the experimental site Present study was conducted in an experimental cage in Roxburgh Botanical Garden, Department of Bot- any, University of Allahabad, Prayagraj, UP, India dur- ing kharif season in July to November. The geographical location is 25o27’43.01’’N, 81o51’10.42’’E. Prayagraj lies in sub-tropical climatic zone and receives an annual rain- fall of 958mm where relative humidity is 59%. 113Induced cytomictic crosstalk behaviour among micro-meiocytes of cluster bean Treatment and Sowing Fresh seeds of Cyamopsis tetragonoloba were arranged into different packets that were irradiated with gamma rays at increasing dose (viz. 100 Gy, 200 Gy and 300 Gy) from a Co-62 source radioisotope inside gam- ma chamber at National Botanical Research Institute (NBRI), Lucknow, India at radiation speed of 2gy per second. These irradiated seeds were sown in respective pots in replicates in complete randomized block design (CRBD) to raise the generation alongwith a control set that was maintained as a standard. Bud Fixation Floral buds were fixed in carnoy’s fixative (solution constituting 3 parts of 90% alcohol: 1 part glacial acetic acid) for duration of 24 hours. Buds were preserved in 70% alcohol at 4°C in refrigerator for future use. Meiotic study Flower buds of appropriate size were teased in a drop of 70% alcohol, followed by staining and mount- ing in 2% acetocarmine. Squash of the bud was pre- pared using a tapper. After squash preparation, slides were observed under Olympus light microscope whereas important stages were captured using Nikon Phase Con- trast Research photomicroscope (Nikon Eclipse, E200, Japan) at 40X resolution. Pollen fertility was also com- puted on the basis of glycerine-acetocarmine stainability test using temporary mounts (Marks 1954). Adequately stained, globose, nucleated pollens were marked as fer- tile whereas sparsely stained, shrivelled and enucle- ated pollens were regarded as sterile. Variation in pollen diameter was recorded. Statistical calibration The data obtained were analysed using statisti- cal software SPSS 16 and means were compared using Duncan’s Multiple Range Test (DMRT) (P≤0.05). All the results were expressed in form of Mean ± Standard Error. RESULTS Cytogenetical screening of microsporogenic cells is a reliable test for in-depth view of in Cluster bean [Cya- mopsis tetragonoloba (L.) Taub.]. Cytogenetical studies revealed that chromosome complement set of the plant is n=7 (Fig. 2B showing metaphase I), confirming the somatic chromosomal configuration to be 2n=14. Meio- cytes, in control, were perfectly normal and bivalents morphology was canonical with no considerable indica- tion of aberrations; also there was no sign of cytomictic connections amongst PMCs. However, mutagenic treat- ment of gamma rays had impacted into a wide range of chromosomal anomalies alongwith cytomictic behaviour Fig. 1. Cytomixis via direct cell fusion (A-F) where A: Direct cell fusion at Diplotene; B: Cell fusion between Prophase 1 and meta- phase I; C: Chromatin transfer between two PMCs; D: Horizontal transfer of chromosomes where one PMC is chromatin deficient; E: A chromosomal fragment in the transition phase; F: Migrating chromatin pushed towards periphery as sticky chromatin band. Cytomictic transmigration via Channel formation (G–I) where G: Single channel bridging two meiocytes; H: Simultaneous transfer of chromatin from 1 PMC to 2 PMCs; Multiple channel formation. Group formation (J – L) where J and K: Transitory micronuclei pushed at ends of meiocytes; L: Association between cells at Ana- phase II stage. Scale bar: 10.45 µm. 114 Girjesh Kumar, Shefali Singh amidst dividing meiocytes. PMCs exhibiting Cytomictic events The frequency of cytomixis was computed to be 7.29±0.33 % at 100 Gy dose which increased from this value to 10.84±0.46% at 200 Gy and 14.67±0.60% at 300 Gy. It was witnessed to manifest either through direct cell to cell fusion or via cytoplasmic channels; where frequency of direct fusion was higher in comparison at all the three doses. Table 1 represents data on cytomic- tic frequency at various stages of meiosis. Traversing of cytoplasmic contents, chromatin material, cellular orga- nelles and other vital intrinsic trophic factors between proximate PMCs was witnessed. Onset of transitory events was witnessed by acquisition of cell polarization where nucleus was positioned towards the cell periph- ery i.e in between the communicating meiocytes unlike the non-cytomictic cells where nucleus was in the cen- tral space of PMC. Direct fusion was recorded at diverse stages of division with different degree of cytomictic intensities. For categorising the intensity, three levels of cytomixis were identified according to Kravchenko (1977). Cells at lower doses had loose wall connexion; these formed pairs and led to cause local cytomixis since no indication of chromatin transmigration was observed. Several PMCs deciphered rather intensive cytomictic phenomena where the migrating chromatin and micronuclei were encountered in between the asso- ciating PMCs or were pushed towards periphery of the parent PMC. Transferring content was seen to pass via cytoplasmic channels as sticky chromatin bands. Cyto- plasmic channels (CC) were also of distinct morphology. It was, either, in the form of single channels (Fig. 1G) or multiple bridging (Fig. 1I) architectures through which nuclear transaction occurred. Fig.1H shows simultane- ous transfer of chromatin from one PMC to two PMCs through channel formation. At some instances, cytomix- is occurred via group formation where multiple PMCs participated in the confluence (Fig. 1J to L). Distinguish- ing feature of such grouping was the attainment of chain transfer. Chain transfer was peculiar where one cell donates a nucleus to the recipient cell and this recipient, in turn, transacts its nucleus to the succeeding one and so on. Besides cytomixis, several other abnormalities were notifiable among which stickiness, univalents, disturbed polarity, unequal separation and laggards were more common alongwith less frequents anomalies such as bridges and micronuclei formation. An increasing trend for other chromosomal anomalies was recorded with respect to gamma irradiation i.e. from 9.80±0.29 at 100 Gy to 16.72±0.40 at 300 Gy gamma dose (Table 1). Syncyte manifestation A remunerative phenomenon of syncyte was wit- nessed at all the three doses of gamma irradiation viz. 100 Gy (0.25±0.25%), 200 Gy (0.55±0.28%) and 300 Gy (0.66 ±0.33%). Syncytes are recreated by complete con- fluence of two PMCs, where whole chromatin material is transferred to the recipient PMC. Therefore the recipient PMC is complemented with doubled chromatin comple- ment. Fig. 2C is a syncytic cell representing 14 bivalents in place of 7 bivalents. Conversely, hypoploid cells were also recorded with lesser number of bivalents (Fig. 2D is a hypoploid cell). Binucleate PMCs with supernumer- Fig. 2. Consequence of cytomixis on late meiotic phases and pol- len morphology. A: Supernumerary nucleoli; B: Normal PMC with seven bivalents; C: Syncyte with 14 bivalents; D: Hypoploid meio- cyte; E: Normal tetrad; F: Polyad; G: Normal fertile pollen; H: Two nucleated pollen; I: Two pollens fusing through wall dissolution; J: Heterosized pollens; K: Pollens with differential size and diameter; L: Fertile and sterile pollens. Scale bar: 10.45 µm. 115Induced cytomictic crosstalk behaviour among micro-meiocytes of cluster bean ary nucleolus were seen that might have been engineered due to nucleolar transfer via common meiocytic links. Abnormal sporads, pollen fertility and size variation Besides meiocytic fusion, tetrad and pollen grain fusion (Fig. 2I) were also recorded which is a rather uncommon and interesting phenomena to be reported in this study, since cytomictic behaviour is documented to occur more frequently at meiosis I. Abnormal micro- spores including monads, dyad and polyads (Fig. 2F) were also encountered. Variation in pollen size (diam- eter) was also recorded which were accounted to be heterosized pollens. In case of control, pollen diameter was not variable but in treated sets, alongwith medium normal sized pollens, differential frequency of small and large sized ‘2n pollens’ were recorded. Fig. 2J and 2K are heterosized pollens. Smaller pollens were generally non-viable whereas larger ones had a differential degree of fertility. Table 2 is a representation of pollen fertility in control and treated sets; it also depicts mean diam- eter and relative frequency of large, medium and small sized pollen grains. Pollen fertility was also calculated and a sharp reduction was discernible from 97±0.57% at 100Gy to 74.33±0.88% at 300 Gy dose. DISCUSSION Meiosis is a beautifully manoeuvred cellular event that has been established to ensure efficient chromo- some partitioning, recombination and trait assort- ment. Furthermore, its proper disposition is essential in restoring gametic viability. Henceforth, it becomes seemingly important to follow and magnify knowledge on the meiotic cell cycle. A typical promiscuous cytom- ictic behaviour among PMCs of Cluster bean (Cyamop- sis tetragonoloba (L.) Taub.) was reported in response to mutagenic incidence of gamma rays. Significance for recruiting studies on this phenomenon is vested in the fact that cytomictic events provide a larger view onto the mechanism of intercellular activities and cellular con- veyance. Cytomixis was more profuse at meiosis I than at meiosis II and it appears to be active energy dependent process since KCN solution or decline in temperature checks it (Zhang et al. 1985). The perplexity surround- ing the mechanism through which chromatin transfer occurs was resolved after identification of the role of cytoplasmic connections (Gates 1911). These connec- tions form an important avenue for cytopasmic cross- talks among proximate PMCs. These are engineered Ta bl e 1. E ffe ct o f G am m a ir ra di at io n on th e in ci de nc e of C yt om ix is a nd S yn cy te fo rm at io n in C lu st er b ea n [C ya m op si s te tr ag on ol o (L .) Ta ub .]. Tr ea tm en t C yt om ix is Sy nc yt es (% ) Fr eq ue nc y of c el ls s ho w in g cy to m ix is a t v ar io us s ta ge s of m ei os is , % (M ea n ± SE ) O th er ab no rm al iti es (M ea n ± SE ) N N o. o f PM C s (( M ea n) Fr eq ue nc y of P M C s in vo lv ed in C yt om ix is Ty pe o f c yt om ix is M ei os is I M ei os is I I D F C C PI M I A I T I M II A II T II Pt D p D k C on tr ol 33 15 - - - - - - - - - - - - - - 10 0 G y 22 97 7. 29 ±0 .3 3 61 .6 6± 1. 66 38 .3 3± 1. 66 0. 25 ±0 .2 5 1. 91 ±0 .1 6 1. 70 ±0 .2 7 1. 00 ±0 .1 7 0. 77 ±0 .0 8 0. 67 ±0 .0 4 0. 99 ±0 .1 5 0. 45 ±0 .2 3 - - 9. 80 ±0 .2 9 20 0 G y 33 00 10 .8 4± 0. 46 71 .0 2± 2. 41 28 .9 6± 2. 41 0. 55 ±0 .2 8 2. 02 ±0 .1 7 1. 65 ±0 .0 6 1. 68 ±0 .1 4 1. 34 ±0 .1 1 1. 24 ±0 .2 0 0. 77 ±0 .0 7 0. 97 ±0 .1 1 0. 68 ±0 .2 0 0. 42 ±0 .2 5 12 .0 2± 0. 31 30 0 G y 22 93 14 .6 7± 0. 60 75 .1 1± 1. 78 26 .4 5± 1. 45 0. 66 ±0 .3 3 2. 61 ±0 .1 8 2. 15 ±0 .0 5 2. 04 ±0 .0 6 1. 59 ±0 .1 5 1. 47 ±0 .1 3 1. 59 ±0 .2 4 1. 46 ±0 .0 7 0. 89 ±0 .2 8 0. 79 ±0 .1 1 16 .7 2± 0. 40 A bb re vi at io ns : P M C s- Po lle n M ot he r C el ls , C C -C yt om ic tic c ha nn el , D F- D ir ec t f us io n, P I- Pr op ha se I , M I- M et ap ha se I , A I- A na ph as e I, T I- Te lo ph as e I, M II -M et ap ha se I I, A II -A na ph as e II , T II -T el op ha se I I, Pt -P ac hy te ne , D p- D ip lo te ne , D k- D ia ki ne si s. 116 Girjesh Kumar, Shefali Singh at prophase I of meiosis and are recognised as primary CC. Persisting plasmodesmata expands its extremities, it forms passage of large interconnecting cells, which is termed as cytoplasmic channels. Cell wall dissolution between the adjacent cells may also lead to cytoplasmic connections (Falistocco et al. 1995). Hydrolytic enzymes released by endoplasmic reticulum and golgi bodies are involved in CC formation (Yu et al. 2004). These pri- mary CC may form via fusion of several plasmodesma- ta or through enlargement of single plasmodesmata or de novo in the region where no plasmodesmata occurs (Wang et al. 1998; Mursalimov et al. 2013a). However the cellulose-pectin wall is gradually replaced by cal- lose layer at subsequent stages, as explained by Kravets (2013). The callose deposition insulates the cellular crosstalks and ceases the primary CC. It is for this rea- son that cytomixis is more profusely recorded at mieois I rather than meiosis II. However cytomictic behaviour may still persits by the genesis of secondary CC which is formed by action of enzymes callase that acts on callose wall. Specific organelles-spherosome like vesicles secre- ate callase and points at which callose catalyzes destruc- tion of callose, secondary CC originates (Mursalimov et al. 2013b). These secondary CC remain available for cytomixis at the later stages of meiosis. Local cytomixis represents association of meiocytes into groups via cytomictic channels in the early pro- phase of meiosis without any participation of migrating chromatin. Severe cytomictic channels such as Fig. 1H was also seen where cytoplasmic content of one PMC emanates in two PMCs. This has also been reported in Vicia faba (Bhat et al. 2017). Cytopathological symptoms are evident in intensive cytomixis, where transaction of chromatin; migration of the cytoplasmic content, nuclei etc are witnessed whereas destructive cytomixis involves complete destruction of the donor cells and severe path- ological signs the filling of the anther cavity with agglu- tinated chromatin, and the impairment of remaining microsporocytes during meiosis. Actually, destructive cytomixis represents rather the way of the MSC autoly- sis than the way of communication between microsporo- genic cells (Kravets 2013). Actin filaments play a key role in cytomixis since migration of cell contents through cytomictic channels is stopped due to cytochalasin B, a chemical that prevents the growth of actin filaments (Zhang et al. 1985). Several pertinent questions regarding function- al state of the transferring chromatin were answered by conducive histone modification experiments using immunostaining technique Mursalimov et al. (2015). Migrating chromatin had no signs of selective hetero- chromatinization and was decrypted to be in transcrip- tionally active state. Ultrastructural studies indicate that neither nucleus nor chromatin is damaged while traversing through cytomictic channel (Mursalimov and Deineko 2011). These arguments implicits ample evidence that cytomixis is a genetically controlled enig- matic phenomenon occurring due to environmental or physiological factors (Bellucci et al. 2003); which has been installed in cells to facilitate inter-cellular trans- migration of vital cellular components. Several reports elucidates that cytomictic behaviour is linked to meiotic segregation and aberrant gene functioning at preceding meiotic or mitotic stages subverts to both chromosomal aberration as well as cytomixis. Thus, cytomixis regula- tion may be controlled by genes responsible for the chro- mosome segregation such as the DIF1 gene in Arabidop- sis thaliana (Bhatt et al. 1999). An intriguing aspect revealed was presence of more than one nucleus in PMCs which displayed coenocytic behaviour. This behaviour is persistently encountered in intergeneric hybrids for example in Meconopsis aculeate (Singhal and Kumar 2008). Consequently, one cell gets an extra nucleus, leaving behind the other nucleus defi- cient cell. Such coenocytes lead in formation of abnor- mal-sized pollen grains as suggested earlier by Mendes- Bonato et al. (2001). Furthermore, fusion of two PMCs led to syncyte formation, also documented in Chrysan- themum (Kim et al. 2009), Mertensia echioides (Malik et al. 2014). Frequency of syncytes is quite low but it is easily detectible due to its invariably larger size com- pared single meiocyte. The product of such meiocytes resulted into the formation of ‘2n’ or large-sized pollen grains. Jones and Reed (2007) approved that presence of Table 2. Impact of Gamma rays on Pollen fertility and Relative pollen size frequency in Cluster bean [Cyamopsis tetragonoloba (L.) Taub.]. Treatment Pollen fertility (%) Diameter(mm) Relative frequency of different Pollen size (%) Small Medium Large Small Medium Large Control 97.00±0.57 - 19.17±0.61 - - 100 - 100 Gy 91.33±1.20 15.24±0.36 19.48±0.36 31.72±0.64 7.33±0.33 83.00±1.15 9.66±0.88 200 Gy 82.66±0.66 15.94±0.50 19.98±0.40 32.83±0.57 11.66±0.33 71.00±0.57 17.33±0.88 300 Gy 74.33±0.88 15.48±0.57 19.66±0.70 33.02±0.29 12.66±0.88 64.66±0.66 22.66±0.33 117Induced cytomictic crosstalk behaviour among micro-meiocytes of cluster bean ‘giant’ pollen to be associated with 2n status. Unreduced (diploid) gametes such as 2n pollen are good source for inducing polyploids (Ghaffari 2006; Latoo et al. 2006). Syncytes are concluded to oblige with imperative signifi- cance since it results into aneuploids which are assets for cytogeneticists. It may have serve as an exemplary model for intergeneric polyploids production. It is witnessed that this additional supernumerary chromatin mass do not pair with main chromatin material of the recipient cell, instead it remains as a separate identity, which may later on from micronuclei or micropollen (Bhat et al. 2006). However, its synthesis is of great future prospects since there induction is remunerative of infraspecifc polyploidization. It that may serve novel in the field of genetic variation and crop improvement. Hypoploid cells are also quite prodigious tool from one viewpoint since they might become deficient in cer- tain intrinsic genetic factors and their scrutiny is thus imperative. Cytoplasmic connection among pollens, although a rare phenomenon, was also witnessed here. Such connections among pollen grains had already been noticed in the intergeneric hybrids of Roegneria tsukush- iensis x Psathyrostachys huashanica (Sun et al. 1994) and in Meconopsis aculeata (Singhal and Kumar 2008). Het- erosized pollens of varying diameter were also recorded. Genesis of heterosized pollens stems from the aneuploid PMCs post cytomixis. Pollen fertility was documented to decline with increasing gamma rays. The descending fer- tility is apparently an outcome of all the cumulative fac- tors that led to cytogenetical aberrations which eventual- ly affected the reproductive success of microsporogenesis. This study is succesfull documentation on gamma rays induced cytomixis in Cyamopsis tetragonoloba (L.) Taub. It also validates the efficacy of the ionizing radia- tion for inducing useful cytological variants such as ane- uploids and infraspecific polyploids. Gamma rays, plau- sibly has a substantial role in maintaining genetic het- erogeneity (Kravets 2013) or restoring and balancing the unbalanced genomes within the developing male game- tophyte, as highlighted by (Falistocco et al. 1995; Ghaf- fari 2006; Song and Li 2009). If cytomixis is a means for synthesising infraspecific polyploids, it is also char- acterized as genome stabilizing, cell sorting checkpoint. For clearing all the mysteries and to expand our level of knowledge, we hope arrival of more concrete techniques, which might help in furthering our vision. ACKNOWLEDGEMENTS Authors express gratitude to CAZRI, India for pro- viding seeds of Clusterbean. Authors extend sincere thanks to NBRI, India for providing facilities for gamma irradiation on seeds. Thanks to members of Plant Genet- ics Laboratory for their suggestions and reviews. FUNDING Corresponding author was financially supported by University Grant Commission. REFERENCES Arnoldy W. 1900. 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