Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 77(3): 63-71, 2024 Firenze University Press https://riviste.fupress.net/index.php/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-2478 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Singh, S. & Kumar, G. (2024). Report of genomic doubling in Cya- mopsis tetragonoloba (L.) Taub. (Fabaceae): salient features and effects. Caryologia 77(3): 63-71. doi: 10.36253/caryologia-2478 Received: Mar 14, 2024 Accepted: Sep 28, 2024 Published: March 25, 2025 © 2024 Author(s). This is an open access, peer-reviewed article pub- lished by Firenze University Press (https://www.fupress.com) and distrib- uted, except where otherwise noted, under the terms of the CC BY 4.0 License for content and CC0 1.0 Uni- versal for metadata. 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. ORCID SS: 0000-0001-9670-7286 Report of genomic doubling in Cyamopsis tetragonoloba (L.) Taub. (Fabaceae): salient features and effects Shefali Singh1,*, Girjesh Kumar2 1 Department of Botany at K. N. Govt. P.G. College, Gyanpur, Bhadohi, India 2 Plant Genetics Laboratory, Department of Botany, University of Allahabad, India *Corresponding author. E-mail: shefalisingh.910@gmail.com Abstract. Leguminous plants have always been highly valued to meet the nutritional necessities of human. An attempt to for genome multiplication in a legume crop is present in this study. Induced polyploidy as a technique has opened immense future prospects for the agriculture world. In this regard, in-vivo autopolyploidization experi- ment was planned on Cluster bean (Cyamopsis tetragonoloba (L.) Taub.). Present study is the successful documentation of the artificial induction of genome doubling by the application of colchicine i.e.C1 and is the first report of its successful establish- ment in the next generation (C2). Colchicine treatment was given to the young seed- lings in two different concentrations of 0.2 and 0.4% for three different time durations. Results deciphered that chromosomal complement in case of diploid control plant is 2n=2x=14, whereas in the true polyploids it went on to be 2n=4x=28. Among the total of 148 seedlings that were treated with colchicine solution, 44 putative polyploid plants were monitored based on their distinctive morphological variations. Efficiency of 0.2% concentrations was found to be more than 0.4% concentrations. Palynological and anatomical evidences were also used for ascertaining polyploid organization to the putative plants with increment in size of stomata and pollen grains in the successful autotetraploids. Stomatal size is also an important determinant of ploidy where larger stomata with increase in chloroplast number was a typical feature among tetraploids. Cluster bean is a self-pollinated crop with narrow genetic base, but ploidy manipula- tion experiment might augment in broadening the genetic diversity. Procurement of large sized flower and seeds is a promising benchmark considering its improved aes- thetic value. Autopolyploidization via colchicine has bestowed with the splendiferous act that offers prudent significance for diverse fields. Keywords: Colchicine, chromosome, induced polyploidy, legume, meiosis. INTRODUCTION The seemingly proficient pace of morphological up-gradation required several editing at genetic level to augment a more robust genome constitu- tion. One such passage was attained via accomplishment of polyploidy has been suggested as a major driving force of plant evolution (Soltis and Soltis https://riviste.fupress.net/index.php/caryologia https://doi.org/10.36253/caryologia-2478 https://doi.org/10.36253/caryologia-2478 https://www.fupress.com https://creativecommons.org/licenses/by/4.0/legalcode https://creativecommons.org/publicdomain/zero/1.0/legalcode https://orcid.org/0000-0001-9670-7286 mailto:shefalisingh.910@gmail.com 64 Shefali Singh, Girjesh Kumar 2009). There is ample source of evidence in this relation since many sequenced genomes display the signature of polyploid ancestry (Comai 2005; Yu et al. 2005). Classic studies estimated that 30–50% of angiosperms are poly- ploids (Stebbins 1950). It is proposed to be the most pre- dominant mechanisms of sympatric speciation in plants (Sattler et al. 2016). The first example of a natural plant polyploid was the gigas mutant of Oenothera lamarkiana catalogued by De Vries (Lutz 1907). Lateron, Winkler (1916) recorded first artificial polyploid in Solanum via callus regeneration from the surfaces of stem explants and coined the term ‘polyploidy’ for these types of plant. Several anti-mitotic agents are known to engineer polyploids artificially such as oryzalin, trifluralin and colchicine. However, in the present study colchicine has been selected. Colchicine is an alkaloid extracted from meadow saffron (Colchicum autumnale L.) which binds tubulin dimers in vitro and results in the formation of a tubulin–colchicine complex acting primarily to prevent microtubule (MT) assembly (Panda et al. 1995). It has been successfully used to modify the chromosome num- bers in diverse plants species including ornamentals, medicinal and cereals. Cluster bean or Guar (Cyamopsis tetragonoloba is a chief leguminous vegetable crop of semi-arid regions of the Indian sub-continent. It is an economically impor- tant gum yielding plant and a wonderful green manure crop with magnificient soil replenishing properties. The sterols of guar seeds include campesterol, avenasterol, stigmasterol, sitosterol and traces of Delta-7-avenasterol, stigmast-7-enol, brassicasterol and cholesterol (Mukhtar et al. 2006). The plant contains many important nutri- ents and phytochemicals such as saponin and flavonoids and is well-known traditional plant used in folklore medicine (Mukhtar et al. 2006; Wang and Morris 2007). Considerable levels of saponin help in normalizing cholestrol levels in body. Gum yielded from seeds is natural polysaccha- ride galactomannan rich constituents found effective in osteoarthritis, transdermal drug delivery systems (Mur- thy et al. 2004). These pharmacological properties and immense economic importance are the compelling forc- es to enhance the productivity of clusterbean. Attempts to elevate endogenous phytochemical constituents as well as protein content of this excellent nutraceutical plant shall have excellent contribution. This has been the foundation for conceptualising autopolyploidization experiment on cluster bean. Three crucial parameters including cytogenetical, morphological and anatomi- cal were explored for granting confirmatory elucidation regarding successful polyploid induction. MATERIAL AND METHODS Plant material Accessions of seeds of Cyamopsis tetragonoloba were procured from regional station of National Bureau of Plant Genetics Resources i.e. Central Arid Zone Research Institute, Jodhpur Rajasthan and among these RGC- 1038 was selected for experimental work. Agroclimatic conditions of the experimental site This study was conducted in an experimental cage in Roxburgh Botanical Garden, Department of Botany, University of Allahabad, Prayagraj, UP, India during kharif season (July to November). The geographical loca- tion is 25o27’43.01’’N, 81o51’10.42’’E. Prayagraj lies in sub-tropical climatic zone and receives an annual rain- fall of 1027mm where relative humidity is 59%. Colchicine treatment Previous literature mentioning standardised proto- cols for colchicine treatment were referred. Fresh seeds of cluster bean were sown in triplicates in pots. After the emergence of two-cotyledonary stage of seedlings, colchicine treatment (C22H25NO6) was applied on their apical meristem. For this, good quality of sterilized absorbent cotton was utilised for making small spherical balls. Cotton balls dipped in colchicine solution at two different concentrations viz. 0.2% and 0.4%, were placed in between cotyledons carefully for three different time duration of 12 hours (1 day), 24 hours (2 consecutive days) and 36 hours (3 consecutive days), respectively. The plants were covered with earthen pots to prevent evaporation of colchicine solution from the cotton balls. For 24 hours and 36 hours, the treatment was repeated for second and third day also. After completion of each treatment, cotton balls were removed and the growing apical tips were washed thoroughly with distilled water. Plants were carefully monitored in their developing stage in normal field condition. Morphological studies and anatomical studies Recording of the various morphological characteris- tics in the diploid and tetraploids was an important pur- view of this work. Henceforth, parameters such as plant height, Days to 50% flowering, Days to 50% maturity, leaf length, leaf breadth, cluster per plant, pods per clus- ter and seed weight were calculated for C1 and C2 gen- 65Report of genomic doubling in Cyamopsis tetragonoloba (L.) Taub. (Fabaceae): salient features and effects eration. With respect to the control (diploid), morpho- logically distinct features such as leathery texture and excessive hairy outgrowth were marked, based on this several plants were suspected to be of polyploid organi- zation. The epidermal layer from the abaxial surface of fully expanded leaves was stripped with a razor blade for stomatal study. Stomata of these leaves were observed under microscope by preparing temporary glycerine mounts. Differences in the size of suspected polyploid and diploid plants were taken into account by measur- ing three parameters i.e. stomatal index, stomatal length and stomatal breadth on micrometer scale in Dewinter Bio-wizard software at 40X resolution. For length and breadth measurement, data from 20 microscopic views were recorded from each slide for diploid and polyploid leaf samples for C1 and C2 generation. Meiotic study With the arrival of blooming season, young floral buds were fixed in Carnoy’s fixative (Glacial Acetic Acid and Absolute alcohol in proportion of 1:3, v/v) which were transferred to pure alcohol next day for preserva- tion at 4oC. These buds were utilised for performing microsporogenic studies where anthers were teased in 2% acetocarmine stain with traces of Iron acetate. Slides were observed under Olympus CH20i at 40X resolu- tion and photography was done using Pinnacle soft- ware under Nikon phase contrast microscope. Chromo- some counting, as prescribed to be the usual method for ploidy determination (Maluszynska 2003), was per- formed in the meiocytes of suspected polyploids and diploids was done. Pollen fertility was also calculated as assessment of viability is imperative in relation to the reproductive success of plants. Adequately stained glo- bose pollen grains were marked as fertile against those pollens which appear to be pale yellow with shrunken cytoplasm. Statistical analysis Statistical calibration was done using SPSS 16.0 version of software. The means were compared at p≤0.5 applying Post hoc and Duncan Multiple Range Test (DMRT). RESULTS With the help of detailed study, six successful poly- ploids were isolated on the basis of morphological, cytogenetical and anatomical studies. Morphological observations After treatment, an instant retardation in growth was discernible in the seedlings unlike that of the of control set. Emergence of the third leaf was delayed by a week in the treated sets, while in case of control it appeared normally within a span of three to four days after seedling emergence. Survivability was also affected, especially at 0.4% concentration, as some of the seed- lings collapsed plausibly because apical meristem was damaged due to cellular necrosis. After initial hindranc- es, these plants got acclimatized and normalized towards growth and development. However, their rate of growth was still slower than that of the diploid ones which had attained normal plant height and were profusely devel- oped. An array of variations were visible in leaves and this is demonstrated in Figure 1. Leaves were highly deformed and appeared to be invariably concentrated in a whorl at the first node. Leaves were peculiarly leathery, thickened and of fairly large sized as compare to the dip- loid ones. Large number of trichomes were present on the leaf surface which imparted glabrous texture to the stem and leaf surface, as shown in Figure 1. There were certain plants which initially displayed morphological variations and were prudently examined, however they reverted later on. Table 1. Colchicine treatment on the apical meristems of seedlings of Cyamopsis tetragonoloba. Concentration Durations of treatment (hrs) Number of seedlings Plants survived Expected polyploids Reverted polyploids Confirmed Number % 0.2% 12 24 23 - - - 0 24 24 21 8 6 2 8.33% 36 24 18 15 12 3 12.50 0.4% 12 24 17 9 9 - - 24 24 15 10 9 1 4.16 36 24 12 - - - - 66 Shefali Singh, Girjesh Kumar With the onset of flowering, several notifiable dif- ferences in the reproductive stages were also observed and monitored. Flowers are usually present in clusters but the number of flowers in the cluster was quite less in these putative polyploids; however the size of the flowers (Figure 2E) alongwith the reproductive organs (Figure 2G) was distinctively and conspicuously enlarged with respect to the diploid ones. This behaviour is perceived in relation to the ‘gigas’ effects of polyploidy. A unique flower with splitting of the two fused petals was spotted (Figure 2F). Days to 50% flowering was recorded, data of which implicits delay in flowering in case of tetraploid plants as mentioned in Table 2. Days to 50% maturity was also delayed by significant margin in tetraploids as compared to the diploids. Seed setting was affected in the polyploids; however the size of seed was enlarged as shown in Figure 2H. Anatomical observations Compared to stomata of control plants, stomatal appa- ratus of several suspected polyploid plants were encoun- tered to be larger, that were surrounded by jumbo sized accessory cells (Figure 3). Number of chloroplast per sto- mata was also increased. Both stomatal length (22.18±0.38 micrometer) and stomatal breadth (13.45±0.65 microm- eter) were remarkably increased in polyploid plants as compare to diploids where calculated stomatal length was 15.93±0.28 micrometer and stomatal breadth was 10.54±0.15 micrometer (Figure 3A and 3B). Data of these parameters is mentioned in Table 2. Cytogenetical and palynological observations Previous cytogenetical illustration at mitotic stages has proved that chromosomal complement in Cyamop- Figure 1. Mutants/variants in leaf shape A: Control; B: Waxy thick- ened seedling; C: Stunted growth at seedling stage; D: Leathery sinuous leaf; E: Surface extension from one side of leaf; F: Sinulate leaf; G: Leaf fusion among two leaflets of trifoliar leaf; H: Leathery coated leaf; I:Triapiculated leaf; J: Elliptical leaf protrusion from leaf midrib; K: Glabrous assymetrical leaf; L: Tomentosa leaf. Figure 2. Morphological traits in diploid and autotetraploid plants of Cluster bean [Cyamopsis tetragonoloba (L.) Taub.]. A: Seedling of diploid (2n=14); B: Dark green thick texture of putative polyploid seedling; C: Comparative trend of diploid and polyploid plants; D: Leaves of diploid and tetraploid plants; E: Flowers from diploid and tetraploid plants; F: Flower with splitting of petals; G: Reproductive unit of diploid and polyploids; H: Diploid and tetraploid seeds. 67Report of genomic doubling in Cyamopsis tetragonoloba (L.) Taub. (Fabaceae): salient features and effects sis tetragonoloba is 2n=14. Haploid or the base chro- mosome number of this plant is n=x=7. Figure 4 is the cytological plate which denotes various stages of meio- sis in Pollen mother cells of diploid as well as in poly- ploid cells. Size of PMC was enlarged in case of poly- ploid cells as it was measured to be 27.97±1.15 microm- eter against the diploid cells 16.18±0.74 micrometer. Figure 4A is metaphase I and Figure 4B is anaphase I in case of control. Microsporogenic studies in the sus- pected polyploids were performed, where diakinesis and metaphase I (Figure 4C) stages revealed that num- ber of chromosomes bivalents was 14. Chromosomal counting was also done at anaphase I where 14:14 pole- ward separation was recorded (as mentioned in Fig- ure 4D) against the diploid chromosome segregation of 7:7. This provides affirmation to the chromosomal doubling. Several prominent multivalents configura- tions were recorded, data of which is documented in Table 3. Figure 4E to 4J is the illustrations of multiva- lent configurations. Laggards (Figure 4N) at anaphase I and unequal separation at anaphase were also recorded. Figure 4M shows 12:16 unequal separation of chromo- somes at anaphase I towards opposite poles. Palynological study was executed to assess viability of pollens in the diploids and polyploids. A noteworthy increment in pollen size was registered in the pollen grains of polyploids. Pollen size of diploid pollen grains was 18.93±0.57 micrometer whereas in polyploids, it was measured to be 33.01±0.83 micrometer. Figure 3C and 3D is diploid and polyploid pollens. Pollen fertility was considerably reduced in polyploids as it declined to a very low percentage of 61.66±0.88 compared to 97.66 ± 0.88% of control. C2 generation observations The plants of C2 generation were comparatively stronger, resistant, healthier and larger than those of C1 generation. The morphological traits such as plant Table 2. A comparative analysis of various morphological parameters in diploid and autotetraploid plants of Cyamopsis tetragonoloba (L.) Taub. in C1 and C2 generation. Characteristics Diploid plants (2n=14) (Mean ± SE) Autotetraploid plants C1 generation (2n=4x=28) (Mean ± SE) Autotetraploid plants C2 generation (2n=4x=28) (Mean ± SE) Plant height (cm) 86.33±1.91 45.66±1.56 51.70±2.07 Days to 50% flowering 48.66±0.88 65.66±1.45 57.33±1.76 Days to 50% maturity 87.66±1.20 117.33±1.40 113.33±3.84 Leaf length (cm) 7.37±0.52 9.77±0.78 9.60±0.85 Leaf breadth (cm) 4.43±0.26 5.50±0.28 5.87±0.64 Length of Stomatal guard cells (micrometer) 15.93±0.28 22.18±0.38 20.71±0.72 Breadth of Stomatal guard cells (micrometer) 10.54±0.15 13.45±0.65 12.89±0.56 Size of Pollen Mother Cells (micrometer) 16.18±0.74 27.97±1.15 28.08±1.18 Size of Pollen grains (micrometer) 18.93±0.57 33.01±0.83 33.68±0.87 Pollen fertility 97.66±0.88 61.66±0.88 64.33±2.40 Cluster per plant 12.33±1.20 6.33±0.66 6.66±0.33 Pods per cluster 10.00±0.57 4.33 ± 0.88 5.67±0.66 Seed weight (gm) 1.57±0.11 2.37±0.15 2.18±0.14 S.E. = Standard Error. Table 3. Metaphase I configuration of all induced autotetraploids in Cyamopsis tetragonoloba (L.) Taub. Chromosomal Associations Percent frequency (Mean ± SE) 8II+2IV+1III+1I 2.97±0.21 7II +3IV+ 2I 2.19±0.1 7II+1IV+1VIII+2I 1.55±0.09 5II+3IV+1VI 0.92±0.24 4II+2IV+1III+1VIII+1I 1.55±0.09 4II+2IV+3III+31 1.25±0.16 3II+2IV+2III +1VII+1I 1.08±0.11 3II+1VIII+1VI+1V+1III 0.77±0.16 3II+1X+1V+1IV+1III 0.95±0.29 3II+2IV+1III+2V+1I 1.25±0.16 2II+2VI+1V+1IV+1III 0.93±0.04 1II+1VI+3IV+1VIII 0.96±0.31 1II+1X+2IV+1V+1III 0.63±0.18 1II+2VI+2V+1IV 0.46 ± 0.02 68 Shefali Singh, Girjesh Kumar height, length displayed a slight increment while leaf breadth, stomatal guard cell length and breadth dis- played a slight decrement in C2 generation as compared to C1 generation (Table 2). The morphological param- eters such as days to 50% flowering and days to maturity of C2 generation of colchicine induced autotetraploids were registered as 57.33 and 113.33 days which was con- siderably earlier than C1 generation. DISCUSSION Polyploidization, in a simple sense, is the heritable condition of possessing more than two complete sets of chromosomes (Comai 2005) which is regarded as an important speciation mechanism for all eukaryotes and has a profound impact on biodiversity dynamics and ecosystem functioning (Ainouche and Jenczewski 2010). Dewey (1980) highlights that each crop species responds differently to polyploidization, depending on their origi- nal ploidy level, genome structure, reproduction mode, perenniality and the plant organ for which the crop is cultivated. Induced tetraploids were conspicuously identifi- able among the other diploid population owing to their distinctive morphological features such as vigorousity Figure 3. Stomatal and pollen morphology in diploids and auto- tetraploids of Clusterbean. A: Stomata (diploid) at 10X; B: Stomata (autotetraploid); C: Stomata (diploid) at 40X; D: Stomata (autotetra- ploid at 40X; E: Diploid pollen; F: Tetraploid pollen (40X). Figure 4. Chromosomal complement in diploids and polyploidy plants. a and b are PMCs of diploid plants- A: Normal meta- phase I (seven bivalents); B: Normal anaphase I (7:7 separations), C-P are stages of autotetraploid PMCs- C: Metaphase I with 14 bivalents; D: Anaphase I with 14:14 separations; E-J are multi- valents- E: 2II+6I+3III+1IV+1V; F: 4II+2IV+1III+1VIII+1I; G: 1II+1VI+3IV+1VIII; H: 3II+ 2IV+2III+1VII+1I; I: 1X+ 2III+3IV; J: 3II+2IV +1III+ 2V +1I; K: Precocious movement with sticki- ness at one side at Metaphase II; L: Two precocious at Metaphase II; M: Unequal separation at Anaphase I where 12:16 chromosomes at opposite poles; N: 4 Laggards at Anaphase I; O: Asynchronous division; P: unoriented Anaphase II. Scale bar – diploid cell (16.18 micrometer), polyploid (27.15 micrometer). 69Report of genomic doubling in Cyamopsis tetragonoloba (L.) Taub. (Fabaceae): salient features and effects in growth, robust nature with thick waxy coated large sized dark green leaves and flowers, excessive trichomes and hairs on stem and leaf surface. This robust nature is explained by the larger number of gene copies which compound into ‘gigas’ effect to the polyploid (Sattler et al. 2016) which also complements with a higher toler- ance to environmental stress (Kermani et al. 2003; Tossi et al. 2022). It appears that there are environmental driv- ers for coordinated variation in cell size and that chang- es in genome size can facilitate generic changes in cell size (Jordan et al. 2015). However, gigas effect is not a universal feature of all autopolyploids. Plants survivability was highly affected due to col- chicine and it consistently decreased with the increasing colchicine concentration. Colchicine brings mitotic arrest (Bakar-ates et al. 2018) by inhibiting mitosis by prevent- ing the polymerization of tubulin. This results in a failure of spindle formation, thus, preventing normal chromo- somal movement and replication (Kamath et al. 2008). The slower growth rate could be attributed to the lower rate of metabolic activities in the colchitetraploids (Joshi and Verma 2004). Also, rate of cell division was reduced since larger genomes require longer time in cell division, particularly in the S phase (Doyle and Coate 2019). Stomatal length assessment is regarded to be a reli- able and convenient method comparing diploids and polyploids (Jeloudar et al. 2019). Stoma size is associ- ated with CO2 gain and water discharge in plant pho- tosynthetic and transpiration processes and can be an indicator of ploidy levels (Moghbel et al. 2015). Com- pare to control, polyploids had enlarged stomatal aper- tures than those of diploid plants. However there was a reduction in the stomatal density in the tetraploids. Anatomical studies imply that reduction in stoma is a measure to check transpiration discharge. The tetraploid plants had significantly larger stomata and higher chlo- rophyll content indices, suggesting that the tetraploid plants may have higher photosynthetic and transpiration capacities (Zhang et al. 2018). These adaptive features of a polyploid genome explain their natural invasive- ness to extreme environmental conditions and the rea- son for their greater stabilisation over diploid genome. Number of chloroplast in the stomatal guard cells was also increased in the tetraploids. Polyploid leaves are of dark green texture which might be due to the increase in chloroplast number with quantitative increase in DNA content in tetraploids (Butterfass 1983). Significance of genomic doubling can be easily real- ized since several ancient polyploidization events existed at the base of evolution and several of these events gave rise to species-rich groups (Otto 2007). There are several salient features of a genome multiplication which estab- lished superlinear advancement of polyploids over their diploid counterparts. For instance, gene redundancy is a very peculiar characteristic of a polyploid cell where redundant copies of the genes have a possible chance for functional diversification (Comai 2005). These redun- dant set of genes posses the ability of shielding the poly- ploids from deleterious effects of recessive mutations (Joshi and Verma 2004; te Beest et al. 2012) by providing “buffering actions” in which extra copies of wild-type alleles masks the expression of harmful recessive coun- terparts. Polysomic inheritance confers higher level of heterozygosity to the polyploid individuals as compare to the diploids (Osborn et al. 2003). Heterozygosity is intimately associated to enhanced vigour. Transition of vegetative stage into reproductive is associated with activation of floral meristem identity genes. Delay in flowering in polyploids might be relat- ed to late triggering of floral responsive genes. Slower growth rates at initial stages results in delay in flower- ing. Apparently, this delay may also cause reproductive isolation of the neopolyploid. Polyploids are recognised with longer petals, deeper corolla tubes, fewer flowers per inflorescence as found in present work; therefore, attract different assemblages of pollinators compared with diploids. Large sized flowers enhance the aesthetic properties and it may increase pollinator visitation fre- quencies (Kennedy et al. 2006). This may in turn pro- vide opportunities for diversification in both plant and insect taxa (Nuismer and Thompson 2001). Polyploids also escape pathogenic attack compared to the diploids. Changes in disease resistance genes between polyploids and diploids also point towards altered pathogen resist- ance (Innes et al. 2008). The plausible causes of the chromosomal abnormali- ties encountered in polyploids complement is perhaps the struggle among the doubled chromosome number in the neo-polyploid. Diploid PMCs oblige seven bivalents of the cell perfectly but the tetraploid PMCs have to accom- modate more bivalents. This chromosomal doubling sets- up an inter-repulsive hindrance among the chromosomes which reciprocates into aberrant meiosis. Occurrence of multivalents particularly quadrivalents was conspicuous in varying frequencies in the present tetraploids, since the latter are derived from a single genome resulting in four homologous sets of chromosomes in tetraploids (Stace 1980). These multivalents create abnormal patterns during anaphase such as ‘3:1’ or ‘2:1 plus one laggard (Comai 2005). Such type of improper segregation results in formation of abnormal gametes with unbalanced ploidy that lead to aneuploids and sterility. A noteworthy feature recorded during palynological assessment was the increment in pollen grain diameter 70 Shefali Singh, Girjesh Kumar among the polyploids. However, there was a substan- tial deceleration in pollen viability in the tetraploids, as also reported in Pinellia ternata (Thunb.) (Liu et al. 2012). Parthasarathy and Rajan (1953) have advocated that chromosome doubling in the tetraploid may upset the balance of polygenes or modifying genes which may probably control the sterility. As a consequence of chro- mosomal aberrations, some sort of sterility takes place resulting to lower seed yield which is a barrier for the inheritance of polyploid (Kumar and Dwivedi 2017). Reduction in seed yield is also an after-effect of the pol- len sterility; since reproductive success largely relies on the viability of gametes. However, the seed size and weight were increased. Change in ploidy may also have substantial effect in altering quantity and quality of secondary metabolites. Polyploidization can be artificially induced to increase the production and/or improve the quality of impor- tant medicinal compounds, such as pharmaceuticals and aroma chemicals (Dhawan and Lavania 1996). Medicinal aromatic polyploids viz. Carum carvi L. (Dijkstra and Speckman 1980), Ocimum kilimandscharicum Gürke (Bose and Choundhury 1962) had increased terpene lev- els and elevated essential oil concentration. These chang- es in phytochemical composition might be associated with difference in triggering of metabolic cascade in the polyploid cell. Science of polyploidization has been adopted by researchers and plant breeders for attaining superior genotypes. Natural autopolyploidization is well proven to be proximal drivers in specie diversification and dif- ferentiation whereas inception of in vivo polyploidiza- tion bears the potential to revolutionize the face of agri- cultural and pharmaceutical areas. Leguminous crops such as cluster bean has narrow genetic base, but ploidy manipulation experiment might augment in broaden- ing the genetic diversity. Genomic doubling opens a new passage for neo-polyploids to evolve and diver- sify, but there is a need to further the level of knowl- edge on the mechanism behind this as it is still not very clearly understood. Gigas effect and robust nature of established polyploids are important characteristics that might confer environmental tolerance to the neo- polyploid. Shorter plant height, as observed in the pre- sent documentation, can act as a boon for plants with lodging issues. Increased flower size is also significant attribute that elevates the aesthetic properties which is of profound interest in floriculture. These traits had played pivotal role in past and may also have crucial influence in future on plant diversification and invasion to hostile realms. 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