In vitro-induced tetraploidy in sugarcane (Saccharum officinarum L. cv. CP57) using colchicine: protocol optimization and cytomorphological characterization

In vitro-induced tetraploidy in sugarcane (Saccharum officinarum L. cv. CP57) using colchicine: protocol optimization and cytomorphological characterization | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article In vitro-induced tetraploidy in sugarcane (Saccharum officinarum L. cv. CP57) using colchicine: protocol optimization and cytomorphological characterization Mahsa Fayazi Zade, Payam Pour Mohammadi, Karim Sorkheh Tamimi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7660881/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract An efficient in vitro protocol was developed for inducing stable tetraploidy in sugarcane ( Saccharum officinarum L. cv. CP57), a cultivar with restricted flowering that hinders conventional breeding. In vitro -grown plantlets were treated with colchicine (0, 50, 100, and 200 mg L⁻¹) for 24, 48, or 72 h. The 72 h treatments resulted in complete mortality. The optimal treatment for polyploidy induction was 50 mg L⁻¹ colchicine for 24 h, which yielded the highest survival rate (83.3 ± 4.4%) and successfully produced tetraploids. Morpho-physiological analysis 60 days post-acclimatization revealed that this treatment significantly reduced tiller number (by 58%) and plant height (by 18%) while increasing chlorophyll content (by 18%) compared to diploid controls. Stomatal density decreased by 76%, while stomatal length and width increased by 150% and 120%, respectively, serving as reliable indicators of polyploidy. Cytological analysis confirmed tetraploidy (2n = ~230 chromosomes) in 68% of cells from plantlets treated with 50 mg L⁻¹ colchicine for 24 h. Karyotype analysis showed a 58% increase in total chromosome length and enhanced karyotype symmetry in tetraploids. The established protocol effectively induces tetraploidy in sugarcane cv. CP57 and provides a valuable biotechnological tool for breeding programs aimed at enhancing genetic diversity and improving agronomic traits. polyploidy induction in vitro culture colchicine stomatal traits karyotype analysis sugarcane Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Introduction Saccharum officinarum L., commonly known as sugarcane, is a perennial C₄ grass within the Poaceae family (tribe Andropogoneae) and ranks among the world’s ten most strategically important crops. It is widely cultivated across tropical and subtropical regions (Verma et al. 2024 ). Sugarcane contributes approximately 70% of global sugar production (FAO 2023 ) and yields a range of value-added co-products, including bioethanol, pulp and paper, animal feed, and various chemical derivatives, highlighting its critical role in both food and energy security (Cardona et al. 2010 ; Dias et al. 2012). Consequently, sugarcane breeding programs focus on developing high-yielding, genetically diverse cultivars to meet escalating global demand. In numerous sugarcane-growing regions, including Iran, conventional breeding is hindered by the species’ limited flowering and seed production under local environmental conditions (Piperidis and D’Hont 2020 ). This necessitates the exploration of biotechnological approaches, such as in vitro culture and mutagenesis, to create novel genetic variation and overcome the narrow genetic base among commercial cultivars (Dhooghe et al. 2011 ). Among these strategies, artificial chromosome doubling via induced polyploidy is a powerful technique to enhance genetic diversity and agronomic performance (Sattler et al. 2016 ). Artificial polyploidy modifies genome size, influencing morphological, physiological, and cytological characteristics, thereby facilitating both evolutionary research and crop improvement (Van de Peer et al. 2017 ). Induced polyploids often exhibit superior attributes compared to diploids, such as increased biomass, higher levels of metabolites, and greater resilience to biotic and abiotic stresses, although these outcomes vary by genotype and species (Salma et al. 2017 ). In vitro polyploidy induction is particularly advantageous as it allows for precise control of antimitotic agent concentration and exposure time on a large number of uniform explants under aseptic conditions. This is typically achieved using agents such as colchicine, trifluralin, or oryzalin, with colchicine being the most commonly used due to its effectiveness in inhibiting spindle formation during mitosis (Blakeslee and Avery 1937 ). Various explant types including apical meristems, microshoots, and callus cultures have been successfully treated with colchicine to produce polyploid plants (Nhut et al. 2022 ). However, optimal colchicine concentrations and exposure durations differ widely depending on species, cultivar, and explant type, typically ranging from 0.006% to 0.3% (w/v) (Dhooghe et al. 2011 ). To date, no research has specifically investigated the Iranian sugarcane cultivar CP57 ( S. officinarum L. cv. CP57) under local conditions. Like many cultivated Saccharum lines in Iran, CP57 exhibits poor flowering, restricting traditional breeding efforts. This study aimed to establish an optimized in vitro protocol for inducing polyploidy in CP57 and to assess its effects on morphological, physiological, and cytological properties. We hypothesized that colchicine treatment would reliably induce tetraploid individuals, identifiable through changes in chromosome number, stomatal traits, plant morphology, and chlorophyll content. Our specific objectives were to: Establish and optimize an efficient in vitro protocol for inducing stable tetraploid plants of sugarcane cv. CP57. Characterize the morphological effects of chromosome doubling (tiller number, plant height). Evaluate stomatal density and dimensions as indicators of ploidy level. Measure changes in chlorophyll content following colchicine treatment. Conduct detailed karyotype analyses to verify ploidy status and assess genomic changes. By establishing this optimized protocol, this research seeks to provide a reliable biotechnological tool to broaden the genetic foundation of sugarcane, enhancing key agronomic traits and stress tolerance. Materials and Methods Plant material and in vitro culture establishment Shoot tips excised from lateral buds of sugarcane ( Saccharum officinarum L. cv. CP57) were surface-sterilized and cultured on Murashige and Skoog (MS) basal medium (Murashige and Skoog 1962 ) supplemented with 3% (w/v) sucrose, 2 mg L⁻¹ 6-benzylaminopurine (BAP), and 0.5 mg L⁻¹ kinetin. The pH was adjusted to 5.8 prior to adding 0.8% (w/v) agar and autoclaving at 121°C for 20 min. Cultures were maintained in a growth chamber at 25 ± 1°C under a 16-h photoperiod with a light intensity of 45–75 µmol m⁻² s⁻¹ provided by cool white fluorescent lamps. After four weeks, uniform plantlets approximately 4–5 cm in height were selected for colchicine treatment. Experimental design and colchicine treatment The experiment was arranged in a completely randomized design (CRD) with a 4 × 3 factorial arrangement. The factors were colchicine concentration (0, 50, 100, and 200 mg L⁻¹) and exposure duration (24, 48, and 72 h). Each treatment consisted of six replicates, with five plantlets per replicate. For treatment, uniform plantlets were placed in 250 mL culture vessels containing 100 mL of liquid MS medium with the corresponding colchicine concentration. The vessels were placed on a rotary shaker at 30 rpm and maintained in complete darkness at 25°C for the designated durations. Following exposure, plantlets were rinsed three times with sterile distilled water and transferred to solid MS rooting medium containing 3 mg L⁻¹ naphthaleneacetic acid (NAA). Acclimatization of plantlets After two weeks on rooting medium, plantlets with well-developed roots were transplanted into 10-cm pots filled with a sterilized 1:1 (v/v) mixture of cocopeat and perlite. The plantlets were acclimatized in a greenhouse at 25 ± 2°C and 60–70% relative humidity. They were irrigated as needed and fertilized weekly with half-strength Hoagland’s solution. Morphological and stomatal analysis Sixty days post-acclimatization, five plants per replicate (n = 30 per treatment) were evaluated for the following traits: Tiller number: The total number of new tillers per plant was counted. Plant height: Measured from the substrate surface to the tip of the youngest fully expanded leaf (cm). Stem diameter: Measured 5 cm above the substrate surface using a digital caliper (mm). For stomatal analysis, impressions were taken from the abaxial surface of the third fully expanded leaf between 09:00 and 11:00 h using clear nail varnish. The dried impressions were mounted on glass slides and observed under a light microscope (Olympus CX43). Stomatal density was determined by counting the number of stomata per mm² in three randomly selected fields of view at 400x magnification. Stomatal length and width were measured for ten stomata per field across three fields per plant using image analysis software (ImageJ, NIH, USA). Cytological examination and karyotype analysis Root tips (~ 1 cm) were collected from control and treated plants, pre-treated with 0.002 M 8-hydroxyquinoline for 4 h at 4°C, and fixed in Carnoy’s solution (3:1 ethanol:glacial acetic acid) for 24 h. The root tips were hydrolyzed in 1 N HCl at 60°C for 10 min, stained with Schiff’s reagent for 1 h, and squashed in a drop of 45% acetic acid. Metaphase spreads were examined under a light microscope (Nikon Eclipse E200). Chromosome counts were performed on fifty well-spread metaphase cells per treatment. Karyotype analysis was conducted on five complete metaphase plates per ploidy level using KaryoType software (v2.0). The following parameters were determined: long arm (L) and short arm (S) lengths, total chromosome length (TCL), arm ratio (AR), centromeric index (CI), intrachromosomal asymmetry index (A₁), interchromosomal asymmetry index (A₂), and total form percentage (TF%). Chromosomes were classified based on arm ratio, and karyotype symmetry was assessed according to Stebbins (1971). Statistical analysis Data were subjected to two-way analysis of variance (ANOVA) using R software (v4.3.1), with colchicine concentration and exposure duration as fixed factors. The assumptions of homogeneity of variances were verified using Levene's test. Mean comparisons were performed using Tukey’s Honestly Significant Difference (HSD) test at a 5% probability level (p ≤ 0.05). Data are presented as mean ± standard error (SE). Results Survival and growth post-treatment Exposure to colchicine for 72 h at all concentrations (50, 100, and 200 mg L⁻¹) resulted in 100% mortality of Saccharum officinarum cv. CP57 plantlets, and these treatments were excluded from further analysis. Survival rates for 24 h and 48 h treatments are presented in Table 1 . After 24 h, plantlets treated with 50 mg L⁻¹ colchicine exhibited the highest survival rate (83.3 ± 4.4%), decreasing to 50.0 ± 6.5% at 100 mg L⁻¹ and 16.7 ± 3.2% at 200 mg L⁻¹ (p ≤ 0.05). Extending exposure to 48 h further reduced survival: 66.7 ± 5.3% at 50 mg L⁻¹, 33.3 ± 4.4% at 100 mg L⁻¹, and 0% at 200 mg L⁻¹. Control plantlets (0 mg L⁻¹) maintained 100% survival across both durations. Morphological Changes A two-way ANOVA revealed highly significant main effects of colchicine concentration and exposure duration (p < 0.001) on tiller number and plant height, with significant interaction effects (p < 0.01) for both traits (Table 1 ). Control plantlets yielded a mean tiller count of 12.0 ± 1.0 per plant. Following 24 hours of colchicine treatment, tiller numbers exhibited a concentration-dependent reduction: 5.0 ± 0.8 (–58%) at 50 mg L⁻¹, 3.5 ± 0.6 (–71%) at 100 mg L⁻¹, and 1.8 ± 0.5 (–85%) at 200 mg L⁻¹. Prolonging the exposure to 48 hours resulted in a further decrease in tiller counts (Fig. 1 ). The mean height of control plants was 30.5 ± 1.2 cm. Following 24 hours of colchicine treatment, plant heights decreased in a concentration-dependent manner: 25.0 ± 1.0 cm (–18%) at 50 mg L⁻¹, 20.0 ± 1.0 cm (–34%) at 100 mg L⁻¹, and 15.2 ± 0.8 cm (–50%) at 200 mg L⁻¹. A 48-hour exposure further reduced plant height (Fig. 2 ). Table 1 Two-way ANOVA summary (MS values) for morphological, stomatal, and physiological traits. Source of Variation df Tiller Number Plant Height (cm) Chlorophyll Content (SPAD) Stomatal Density Stomatal Width* Stomatal Length* Colchicine (C) 3 232.38** 441.08** 294.76** 180.18** 1496.36** 1186.26** Duration (D) 1 0.74 (ns) 108.79** 70.67* 134.32** 401.57** 339.38** C × D 3 10.44** 28.96** 35.11** 85.38** 224.59** 360.60** Error 35 0.42 3.10 1.69 1.57 11.87 15.45 CV (%) 13.68 8.05 7.31 9.43 7.79 10.72 Chlorophyll Content The highest chlorophyll content (SPAD value) was recorded in plants treated with 50 mg L⁻¹ colchicine for 24 hours (45.8 ± 1.3), representing an 18% increase compared to the 24-hour untreated control group (38.9 ± 1.1). Conversely, the lowest chlorophyll content was observed in plants treated with 200 mg L⁻¹ colchicine for 48 hours (8.7 ± 0.5). All treatments differed significantly from each other and the controls (Fig. 3 ). Stomatal Traits ANOVA indicated significant effects of colchicine concentration and exposure duration (p < 0.001) on all stomatal traits, with significant interaction effects (p < 0.05). In control leaves, stomatal density averaged 22.5 ± 1.2 stomata mm⁻². Following 24 h of colchicine treatment, density dropped to 5.4 ± 0.8 stomata mm⁻² (–76%) at 50 mg L⁻¹, 4.1 ± 0.7 mm⁻² (–82%) at 100 mg L⁻¹, and 2.0 ± 0.5 mm⁻² (–91%) at 200 mg L⁻¹. Forty-eight-hour treatments further reduced density (Fig. 4 ). Control stomata measured 19.8 ± 0.6 µm (length) and 24.2 ± 0.7 µm (width). The 50 mg L⁻¹/24 h treatment enlarged stomata to 49.5 ± 1.0 µm (+ 150%) in length and 53.2 ± 1.2 µm (+ 120%) in width. The 200 mg L⁻¹/24 h treatment produced the largest stomata (60.2 ± 1.5 µm length, + 204%; 65.3 ± 1.5 µm width, + 170%) (Fig. 5 , 6 , 7 ). Chromosome Number and Karyotype Analysis All control metaphase spreads consistently displayed a diploid chromosome count of 2n = 114 (Fig. 8 ). In the 50 mg L⁻¹/24 h treatment, 68% of metaphase cells (34/50) exhibited approximately 2n ≈ 230 chromosomes; the remainder retained the diploid number or showed minor aneuploidy. At 100 mg L⁻¹/24 h, only 42% of cells (21/50) reached the tetraploid count (Fig. 9 ). Karyotype parameters for confirmed tetraploid plants (50 mg L⁻¹/24 h) versus diploid controls are summarized in Table 2 . The detailed measurements for all individual chromosomes of the tetraploid are provided in Supplementary Tables. Total chromosome length (TCL) averaged 207.0 ± 1.8 µm in diploids and 328.2 ± 2.1 µm in tetraploids (+ 58%). The intrachromosomal asymmetry index (A₁) decreased significantly from 0.52 ± 0.01 to 0.48 ± 0.01 (p < 0.05), indicating a more symmetric karyotype. Stebbins’ symmetry class shifted from B2 (diploid) to B1 (tetraploid). Cytological analysis confirmed the ploidy level of both control and treated plants. Metaphase spreads from control plantlets consistently revealed a diploid chromosome count of 2n = 114 (Fig. 10 ). In contrast, root tip cells from plantlets treated with 50 mg L⁻¹ colchicine for 24 h exhibited a tetraploid count of approximately 2n ≈ 230 chromosomes (Fig. 11 ). The karyogram of the control plant (Fig. 12 ) further illustrates the organized diploid set, while the karyogram of the treated plant (Fig. 13 ) visually demonstrates the successful chromosome doubling and the increased karyotype symmetry characteristic of the induced tetraploids. Table 2 Karyotype parameters comparing diploid (2n = 114) and tetraploid (2n ≈ 230) CP57 sugarcane. Row Index Control Treated 1 Number of Chromosomes (2n) 114 230 2 Total Genome Length (TL) 207.01 327.16 3 Coefficient of Variation (CV) 22.42 19.70 4 Asymmetry Index (AI) 5.65 7.68 5 Stebbins Symmetry Class (SC) B2 B1 6 Relative Chromosome Length (RL) 1.74 0.87 7 Centromeric Index (CI%) 31.70 33.01 8 Total Form of Karyotype (TF%) 33.99 36.30 9 Chromosome Form Percentage (F%) 0.59 0.31 10 Relative Chromatin Content (VRS) 3.63 2.84 11 Average Chromosome Length (CL) 3.63 3.84 12 Relative Length of Shortest Chromosome (S%) 1.19 0.96 13 Range of Relative Chromosome Length (DRL) 2.37 1.88 14 Interchromosomal Asymmetry Index (A2) 0.16 0.21 15 Average Length of Long Chromosome Arms (L) 2.39 1.81 16 Average Length of Short Chromosome Arms (S) 1.23 1.03 17 Intrachromosomal Asymmetry Index (A1) 0.52 0.48 18 Karyotype Asymmetry Index (ASK) 66.02 63.88 19 Difference Index of Two Chromosome Arms (d-Value) 1.46 1.52 20 Ratio of Long to Short Chromosome Arms (AR) 1.94 1.76 21 Value of Short to Long Chromosome Arms (r-Value) 0.51 0.56 Cellular Abnormalities Some colchicine-treated samples exhibited interphase nuclear abnormalities, including multinucleate cells (up to six nuclei per cell of varying sizes) and disrupted chromosome migration, reflecting colchicine-induced spindle inhibition (Fig. 14 ). Discussion This study successfully established an efficient in vitro protocol for tetraploid induction in sugarcane cv. CP57, with treatment using 50 mg L⁻¹ colchicine for 24 h proving highly effective in inducing stable tetraploidy. The 100% mortality observed at 72 h exposures corroborates earlier findings that prolonged colchicine exposure severely compromises cell viability (Touchell et al. 2020 ). Our results agree with previous studies in sugarcane hybrids, where moderate concentrations and shorter durations balanced survival and induction efficiency (Khan et al. 2008; D Hont, 2005). The morphological alterations—reduced tillering and plant height—are characteristic of artificial polyploids, often resulting from mitotic disruption and altered phytohormone transport (Chen et al. 2007). The dramatic changes in stomatal traits (increased size, decreased density) serve as reliable phenotypic markers for polyploidy in sugarcane, consistent with findings in other specie (Zhang et al. 2024 ). Wang et al ( 2021 ) Reports that the chlorophyll content in tetraploid is significantly increased compared with the diploid variety, contributing to higher photosynthetic capacity. This enhancement suggests a potential for improved photosynthetic efficiency and biomass yield, though field validation is necessary. Cytological analysis confirmed the protocol's efficacy, with a high rate (68%) of tetraploid cells achieved at the optimal treatment. The observed karyotype changes, including increased total chromosome length and enhanced symmetry, are consistent with genomic restructuring following polyploidization (Sattler et al. 2016 ). The presence of aneuploid cells and chimeras is a typical challenge in colchicine-induced polyploidy (Dhooghe et al. 2011 ), underscoring the need for careful selection or additional mitotic cycles to achieve stable, homogeneous tetraploid lines. Conclusion The optimized in vitro protocol employing 50 mg L⁻¹ colchicine for 24 h effectively induces tetraploidy in sugarcane cv. CP57 with high survival and induction rates. The tetraploid lines were unequivocally characterized through a combination of stomatal, morphological, physiological, and detailed cytological analyses. This protocol provides a valuable biotechnological tool for sugarcane breeding programs, enabling the creation of novel genetic diversity for the development of cultivars with enhanced yield potential and stress tolerance. Future work will focus on the agronomic evaluation of these tetraploid lines under field conditions and the use of flow cytometry for high-throughput ploidy screening. Declarations Funding: This work was supported by the Agricultural Sciences and Natural Resources University of Khuzestan. Conflict of interest: The authors declare they have no financial or non-financial competing interests. Ethics approval: Not applicable. Consent to participate: Not applicable. Consent for publication: Not applicable. Availability of data and materials: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Code availability: Not applicable. Authors' contributions: MFZ: Investigation, Methodology, Formal analysis. PPM: Conceptualization, Supervision, Writing – original draft, Writing – review & editing. KST: Methodology, Investigation (Cytogenetic analysis). Acknowledgements: The authors would like to thank the Agricultural Sciences and Natural Resources University of Khuzestan for providing the laboratory facilities and technical support to conduct this research. We are also grateful to the Karun Agro Industry Incorporation for generously supplying the plant materials. References Blakeslee AF, Avery AG (1937) Methods of inducing doubling of chromosomes in plants. J Hered 28:393–411. https://doi.org/10.1093/oxfordjournals.jhered.a104294. Cardona CA, Quintero JA, Paz IC (2010) Production of bioethanol from sugarcane bagasse: status and perspectives. Bioresour Technol 101:4754–4766. https://doi.org/10.1016/j.biortech.2009.10.097. Wang Y, Jia B, Ren H, Feng Z (2021) Ploidy level enhances the photosynthetic capacity of a tetraploid variety of Acer buergerianum Miq. PeerJ 11:e12620. doi: 10.7717/peerj.12620 Chen ZJ (2007) Genetic and epigenetic mechanisms for gene expression and phenotypic variation in plant polyploids. Annu Rev Plant Biol 58:377–406. doi: 10.1146/annurev.arplant.58.032806.103835. Dhooghe E, Van Laere K, Eeckhaut T, Leus L, Van Huylenbroeck J (2011) Mitotic chromosome doubling of plant tissues in vitro . Plant Cell Tissue Organ Cult 104:359–373. https://doi.org/10.1007/s11240-010-9786-5. FAO (2023) FAOSTAT statistical database. Food and Agriculture Organization, Rome. Nhut, D.T., Tam, D.T.T., Luan, V.Q., Hien, N.T.T., Tung, H.T. (2022). A Protocol of Shoot Regeneration and Polyploid Plantlet Production in Paphiopedilum villosum . In: Nhut, D.T., Tung, H.T., YEUNG, E.CT. (eds) Plant Tissue Culture: New Techniques and Application in Horticultural Species of Tropical Region. Springer, Singapore. https://doi.org/10.1007/978-981-16-6498-4_16 D'Hont A. Unraveling the genome structure of polyploids using FISH and GISH; examples of sugarcane and banana. Cytogenet Genome Res. 2005;109(1-3):27-33. doi: 10.1159/000082378. PMID: 15753555. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497. Zhang J, Cheng C, Xiao F, Zhang X, Zhang C, Zhao Y, Xu J, Zhang S, Wang X (2024) Effects of ploidy level on leaf morphology, stomata, and anatomical structure of Hibiscus syriacus L. BMC Plant Biol 24, 1133. https://doi.org/10.1186/s12870-024-05778-y Piperidis G, D’Hont A (2020) Sugarcane genome architecture decrypted with chromosome-specific oligo probes. Plant J 103:2139–2151. doi: 10.1111/tpj.14881. Salma U, Kundu S, Mandal N (2017) Artificial polyploidy in medicinal plants: advancement in the last two decades and impending prospects. J Crop Sci Biotechnol 20:9–19. https://doi.org/10.1007/s12892-016-0080-1 Sattler MC, Carvalho CR, Clarindo WR (2016) The polyploidy and its key role in plant breeding. Planta 243:281–296. https://doi.org/10.1007/s00425-015-2450-x. Verma KK, Song X-P, Singh M, RDM P, Wu J-M, Li Y-R (eds) (2024) Sugarcane cultivationand management: challenges and opportunities, 1st edn. Apple Academic Press. https://doi.org/10.1201/9781003504122 Touchell, D. H., Palmer, I. E., Ranney, T. G. (2020). In vitro ploidy manipulation for crop improvement. Frontiers in Plant Science, 11, 722. https://doi.org/10.3389/fpls.2020.00722 Van de Peer Y, Mizrachi E, Marchal K (2017) The evolutionary significance of polyploidy. Nat Rev Genet 18:411–424. https://doi.org/10.1038/nrg.2017.26 Supplementary Files SupplementaryTable.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7660881","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":521077475,"identity":"f1998eb9-6c05-40a7-9910-c49ba0b226e9","order_by":0,"name":"Mahsa Fayazi Zade","email":"","orcid":"","institution":"Agricultural Sciences and Natural Resources University of Khuzestan","correspondingAuthor":false,"prefix":"","firstName":"Mahsa","middleName":"Fayazi","lastName":"Zade","suffix":""},{"id":521077476,"identity":"8e1585ae-9ebf-46d4-ae5a-0e3c602fe002","order_by":1,"name":"Payam Pour Mohammadi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA70lEQVRIiWNgGAWjYBACgwM8YJqxjb0BImAAoXBrsYRr4TlApBZ7mJYGiQRkLXiA2fHeg59u5tjJ9kk+fvy5cM9hY3MG5ocfGAru4dZy5lyydO62ZOM26TQz6RnPDptZNrAZSzAYFOPWciPHAKiFObFNOsGMmefAYRuDAwxmQPcl4NRicCPH+HfutvrENsnjnz9DtLB/I6TFDGjL4cQ2CR4DaaAWM2CwE7DlzBkz69xtx43beHLKgFrSjQ0O8xRLJODTcrzH+HbutmrZ+e3HNwMdZm244Xj7xg8f/uDWggUwAzFJGkbBKBgFo2AUYAAAwwBVFXz0024AAAAASUVORK5CYII=","orcid":"","institution":"Agricultural Sciences and Natural Resources University of Khuzestan","correspondingAuthor":true,"prefix":"","firstName":"Payam","middleName":"Pour","lastName":"Mohammadi","suffix":""},{"id":521077477,"identity":"886097d3-fcd0-4337-bd4f-c253835553e3","order_by":2,"name":"Karim Sorkheh Tamimi","email":"","orcid":"","institution":"Shahid Chamran University of Ahvaz","correspondingAuthor":false,"prefix":"","firstName":"Karim","middleName":"Sorkheh","lastName":"Tamimi","suffix":""}],"badges":[],"createdAt":"2025-09-19 19:00:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7660881/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7660881/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":93071290,"identity":"6cc6de4b-9a91-402a-95a8-ce01b6a7dff2","added_by":"auto","created_at":"2025-10-08 17:55:51","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1248793,"visible":true,"origin":"","legend":"","description":"","filename":"VISUALDIAGRAM.png","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/a5cf0ea6b1adc9911395ccbb.png"},{"id":93071188,"identity":"b9ee151a-c511-4869-ba02-9cb0ff6b6df8","added_by":"auto","created_at":"2025-10-08 17:55:45","extension":"xml","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":9171,"visible":true,"origin":"","legend":"","description":"","filename":"pctoPCTOD2500686.xml","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/e91396cf9d17943842c314af.xml"},{"id":93071212,"identity":"75912167-0ddb-4925-af9d-9ca153784364","added_by":"auto","created_at":"2025-10-08 17:55:46","extension":"xml","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1076,"visible":true,"origin":"","legend":"","description":"","filename":"PCTOD250068619471.go.xml","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/79ee7a15e88c0d5eb9778d79.xml"},{"id":93071234,"identity":"2a67b0b5-739f-441f-a9d1-93d87082f560","added_by":"auto","created_at":"2025-10-08 17:55:48","extension":"xml","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":947,"visible":true,"origin":"","legend":"","description":"","filename":"PCTOD2500686Import.xml","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/facaf3cc194ea42679877544.xml"},{"id":93071246,"identity":"fad66b11-bfbe-4671-b97e-e6b84c43ff94","added_by":"auto","created_at":"2025-10-08 17:55:49","extension":"xml","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":74111,"visible":true,"origin":"","legend":"","description":"","filename":"PCTOD25006860enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/66b01c192a70e9def28f8ca5.xml"},{"id":93072233,"identity":"7f0ea61f-37fc-4a93-b0ed-950ec18d6fae","added_by":"auto","created_at":"2025-10-08 18:03:48","extension":"jpeg","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":473052,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/e7cc4cb8e1ff640374b1946b.jpeg"},{"id":93071287,"identity":"87c4fd78-21f1-475d-a307-365e2398efca","added_by":"auto","created_at":"2025-10-08 17:55:51","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":457087,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/eb88c24e37f5146eb6b39db4.png"},{"id":93071275,"identity":"f89b7490-3d1c-4dec-9766-29b91053d7a5","added_by":"auto","created_at":"2025-10-08 17:55:50","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":503494,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/397bef90cf6bb63b2917e533.png"},{"id":93071286,"identity":"2a9e14e1-2f5a-4804-be2f-5096013b10a4","added_by":"auto","created_at":"2025-10-08 17:55:50","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":214943,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/df95c50afa9cb84985157726.png"},{"id":93072235,"identity":"c07dccc4-0095-4b94-8f3b-3ab6c2831ad0","added_by":"auto","created_at":"2025-10-08 18:03:50","extension":"jpeg","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":199124,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/db3f683147c14d8cbf4ed11a.jpeg"},{"id":93071292,"identity":"d7175c94-f423-49a6-971a-040516a426c8","added_by":"auto","created_at":"2025-10-08 17:55:52","extension":"jpeg","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":39630,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/6a1ab6f4fc89dee07f7a51a0.jpeg"},{"id":93071213,"identity":"5a7f843e-689f-4a78-a8c6-636ed20c18eb","added_by":"auto","created_at":"2025-10-08 17:55:46","extension":"jpeg","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":58692,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/55d7f088d9a16661d889046c.jpeg"},{"id":93072237,"identity":"a33e0073-2eb2-4e19-9990-ea73e27ba93b","added_by":"auto","created_at":"2025-10-08 18:03:50","extension":"jpeg","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":357307,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/259682d0dcc88c377d7ae484.jpeg"},{"id":93071183,"identity":"933a9b50-1fd3-4a9b-8a07-a0ad753e2b1f","added_by":"auto","created_at":"2025-10-08 17:55:45","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":165937,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/b00e5177f474e315208387c4.png"},{"id":93071272,"identity":"d4ab478d-b9f7-4b0c-8d6e-533ebe5a2b4c","added_by":"auto","created_at":"2025-10-08 17:55:50","extension":"png","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":70077,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/c818376373ee209fae5ac4c2.png"},{"id":93071244,"identity":"9f91b511-4400-47db-adb5-f89520332cd5","added_by":"auto","created_at":"2025-10-08 17:55:49","extension":"png","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":65830,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/1de81647c8d4ffd1ec56ff59.png"},{"id":93071279,"identity":"87f87d84-4e1a-43fb-a294-c43f2324fd4d","added_by":"auto","created_at":"2025-10-08 17:55:50","extension":"png","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":31208,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/6d58818bc2fb3cad8d20658e.png"},{"id":93071235,"identity":"fe1ce676-a742-41f0-b963-8ce54eb90930","added_by":"auto","created_at":"2025-10-08 17:55:48","extension":"png","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":66642,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/1d3425307d13cacdadc2d965.png"},{"id":93071208,"identity":"2ba096da-18b0-4d3f-a1bb-b32387a79277","added_by":"auto","created_at":"2025-10-08 17:55:46","extension":"png","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":13771,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/5cf465b230b7346f77f5297a.png"},{"id":93071211,"identity":"1e0899f1-74bd-4f00-a4f1-d9355e3e6775","added_by":"auto","created_at":"2025-10-08 17:55:46","extension":"png","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":25788,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/507827be0519b7dcdda3d169.png"},{"id":93071216,"identity":"9f0e1a12-a6a0-4b96-8442-f2e9fcfbbed0","added_by":"auto","created_at":"2025-10-08 17:55:46","extension":"png","order_by":29,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":107141,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/20d2f015f9e879babf0e9117.png"},{"id":93071207,"identity":"623b8c4b-6950-4297-a116-109968dce4ab","added_by":"auto","created_at":"2025-10-08 17:55:46","extension":"xml","order_by":30,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":72690,"visible":true,"origin":"","legend":"","description":"","filename":"PCTOD25006860structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/8dfe8931813180e2eff28033.xml"},{"id":93072230,"identity":"5b958c36-371d-4f8f-9afe-c5d1f29de1be","added_by":"auto","created_at":"2025-10-08 18:03:46","extension":"html","order_by":31,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":81263,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/fe75a7384f8cde1bc8410de5.html"},{"id":93071209,"identity":"ae429c20-8397-4e26-a489-715ed72c89c4","added_by":"auto","created_at":"2025-10-08 17:55:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":33788,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Different Concentrations and Application Durations of Colchicine Treatment on tiller number.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/bcc5d4d232cec708b07ece01.png"},{"id":93071288,"identity":"3f141acf-53e4-49a6-bfc0-37bb78e73f30","added_by":"auto","created_at":"2025-10-08 17:55:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":40356,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Different Concentrations and Application Durations of Colchicine Treatment on plant height\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/ee4f0c2a8fac66bc7b5ee898.png"},{"id":93071259,"identity":"133125c9-97cc-47e6-b033-ca7e182f13cd","added_by":"auto","created_at":"2025-10-08 17:55:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":35190,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Different Concentrations and Application Durations of Colchicine Treatment on Chlorophyll Content\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/9418f6eaef7908281cbfe2a8.png"},{"id":93071214,"identity":"c7561fa7-15e1-4b81-8b58-cc4995371417","added_by":"auto","created_at":"2025-10-08 17:55:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":37065,"visible":true,"origin":"","legend":"\u003cp\u003eStomatal density variations under different colchicine concentrations and durations.\u003c/p\u003e\n\u003cp\u003eChromosome Number and Karyotype Analysis\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/dfc8e3fba0b034dab205532e.png"},{"id":93071239,"identity":"b740a699-ce20-4c42-a32c-6b4552030340","added_by":"auto","created_at":"2025-10-08 17:55:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":35122,"visible":true,"origin":"","legend":"\u003cp\u003eStomatal width variations under different colchicine concentrations and durations.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/a919992c6d5d85f9addbc053.png"},{"id":93071229,"identity":"00d6fac5-9a61-47f8-826c-c61b811432be","added_by":"auto","created_at":"2025-10-08 17:55:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":36617,"visible":true,"origin":"","legend":"\u003cp\u003eStomatal length variations under different colchicine concentrations and durations.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/bb4d36c1a9074daa42958d45.png"},{"id":93071186,"identity":"053f6fc2-cb89-4d66-b9a8-aae12b1f3f11","added_by":"auto","created_at":"2025-10-08 17:55:45","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":527153,"visible":true,"origin":"","legend":"\u003cp\u003eStomatal morphology at ×100 magnification. (a) Stomatal structure in control (untreated) plant. (b) Stomatal structure in colchicine-treated plant (50 mg L⁻¹ colchicine for 24 h).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/feab63964cfa405741a48e4b.png"},{"id":93071278,"identity":"e9823af8-f8e3-4930-bc9f-a50e670ba8fc","added_by":"auto","created_at":"2025-10-08 17:55:50","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":433253,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative diploid (2n = 114) metaphase spread (Control).\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/9d8072d7da2296d5322f943a.png"},{"id":93071220,"identity":"14d56344-ddf1-4c7b-9126-d64e97f09a6d","added_by":"auto","created_at":"2025-10-08 17:55:47","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":451892,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative tetraploid (2n ≈ 230) metaphase spread (50 mg L⁻¹ colchicine for 24 h).\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/04c5d53a1f7984054fa3fd5a.png"},{"id":93072236,"identity":"98bc4dd1-a7e6-4e50-b419-42296c577637","added_by":"auto","created_at":"2025-10-08 18:03:50","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":188933,"visible":true,"origin":"","legend":"\u003cp\u003eKaryotype of sugarcane cultivar CP-57 (control sample), 2n = 114.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/5655b885c0dd2828f51ef98f.png"},{"id":93071247,"identity":"4c9694f4-1284-4d82-a167-29c3314b997c","added_by":"auto","created_at":"2025-10-08 17:55:49","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":214637,"visible":true,"origin":"","legend":"\u003cp\u003eKaryotype of sugarcane cultivar CP-57 treated sample (50 mg L⁻¹ colchicine for 24 h), 2n = 230.\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/d60a137fed83ea9371f513ec.png"},{"id":93071240,"identity":"76e2aa4c-cbab-4cd1-bc4d-aded9f72cb97","added_by":"auto","created_at":"2025-10-08 17:55:49","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":40429,"visible":true,"origin":"","legend":"\u003cp\u003eKaryogram of sugarcane cultivar CP-57 (control sample), 2n = 114.\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/dfefa90ddef85804024d88fc.png"},{"id":93072231,"identity":"f06a29e8-31ce-44bc-bb98-81abf9965ef1","added_by":"auto","created_at":"2025-10-08 18:03:46","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":42892,"visible":true,"origin":"","legend":"\u003cp\u003eKaryogram of sugarcane cultivar CP-57 treated sample (50 mg L⁻¹ colchicine for 24 h), 2n = 230.\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/5168db364275c1640d8816a8.png"},{"id":93071227,"identity":"058204ff-1584-4007-ad23-8b4e351eed54","added_by":"auto","created_at":"2025-10-08 17:55:48","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":245707,"visible":true,"origin":"","legend":"\u003cp\u003eInterphase nuclear abnormalities observed in colchicine-treated CP57 cells.\u003c/p\u003e","description":"","filename":"14.png","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/f94d7026b52695cb17093a0a.png"},{"id":94470453,"identity":"b175c74c-e36e-4759-8541-40af291ec74e","added_by":"auto","created_at":"2025-10-27 15:32:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3375953,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/83f062d3-0bc8-4737-aa65-7bb5a2c5d197.pdf"},{"id":93071284,"identity":"65b1584a-a3b4-493d-a1fa-2c4e8f6da2b7","added_by":"auto","created_at":"2025-10-08 17:55:50","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":31734,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable.docx","url":"https://assets-eu.researchsquare.com/files/rs-7660881/v1/442123688971dcd1af0db563.docx"}],"financialInterests":"","formattedTitle":"In vitro-induced tetraploidy in sugarcane (Saccharum officinarum L. cv. CP57) using colchicine: protocol optimization and cytomorphological characterization","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eSaccharum officinarum\u003c/em\u003e L., commonly known as sugarcane, is a perennial C₄ grass within the Poaceae family (tribe Andropogoneae) and ranks among the world\u0026rsquo;s ten most strategically important crops. It is widely cultivated across tropical and subtropical regions (Verma et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Sugarcane contributes approximately 70% of global sugar production (FAO \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and yields a range of value-added co-products, including bioethanol, pulp and paper, animal feed, and various chemical derivatives, highlighting its critical role in both food and energy security (Cardona et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Dias et al. 2012). Consequently, sugarcane breeding programs focus on developing high-yielding, genetically diverse cultivars to meet escalating global demand.\u003c/p\u003e\u003cp\u003eIn numerous sugarcane-growing regions, including Iran, conventional breeding is hindered by the species\u0026rsquo; limited flowering and seed production under local environmental conditions (Piperidis and D\u0026rsquo;Hont \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This necessitates the exploration of biotechnological approaches, such as \u003cem\u003ein vitro\u003c/em\u003e culture and mutagenesis, to create novel genetic variation and overcome the narrow genetic base among commercial cultivars (Dhooghe et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Among these strategies, artificial chromosome doubling via induced polyploidy is a powerful technique to enhance genetic diversity and agronomic performance (Sattler et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Artificial polyploidy modifies genome size, influencing morphological, physiological, and cytological characteristics, thereby facilitating both evolutionary research and crop improvement (Van de Peer et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Induced polyploids often exhibit superior attributes compared to diploids, such as increased biomass, higher levels of metabolites, and greater resilience to biotic and abiotic stresses, although these outcomes vary by genotype and species (Salma et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e polyploidy induction is particularly advantageous as it allows for precise control of antimitotic agent concentration and exposure time on a large number of uniform explants under aseptic conditions. This is typically achieved using agents such as colchicine, trifluralin, or oryzalin, with colchicine being the most commonly used due to its effectiveness in inhibiting spindle formation during mitosis (Blakeslee and Avery \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1937\u003c/span\u003e). Various explant types including apical meristems, microshoots, and callus cultures have been successfully treated with colchicine to produce polyploid plants (Nhut et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, optimal colchicine concentrations and exposure durations differ widely depending on species, cultivar, and explant type, typically ranging from 0.006% to 0.3% (w/v) (Dhooghe et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTo date, no research has specifically investigated the Iranian sugarcane cultivar CP57 (\u003cem\u003eS. officinarum\u003c/em\u003e L. cv. CP57) under local conditions. Like many cultivated Saccharum lines in Iran, CP57 exhibits poor flowering, restricting traditional breeding efforts. This study aimed to establish an optimized \u003cem\u003ein vitro\u003c/em\u003e protocol for inducing polyploidy in CP57 and to assess its effects on morphological, physiological, and cytological properties. We hypothesized that colchicine treatment would reliably induce tetraploid individuals, identifiable through changes in chromosome number, stomatal traits, plant morphology, and chlorophyll content. Our specific objectives were to:\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eEstablish and optimize an efficient \u003cem\u003ein vitro\u003c/em\u003e protocol for inducing stable tetraploid plants of sugarcane cv. CP57.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eCharacterize the morphological effects of chromosome doubling (tiller number, plant height).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eEvaluate stomatal density and dimensions as indicators of ploidy level.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eMeasure changes in chlorophyll content following colchicine treatment.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eConduct detailed karyotype analyses to verify ploidy status and assess genomic changes.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003eBy establishing this optimized protocol, this research seeks to provide a reliable biotechnological tool to broaden the genetic foundation of sugarcane, enhancing key agronomic traits and stress tolerance.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003ePlant material and \u003cem\u003ein vitro\u003c/em\u003e culture establishment\u003c/p\u003e\u003cp\u003eShoot tips excised from lateral buds of sugarcane (\u003cem\u003eSaccharum officinarum\u003c/em\u003e L. cv. CP57) were surface-sterilized and cultured on Murashige and Skoog (MS) basal medium (Murashige and Skoog \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1962\u003c/span\u003e) supplemented with 3% (w/v) sucrose, 2 mg L⁻\u0026sup1; 6-benzylaminopurine (BAP), and 0.5 mg L⁻\u0026sup1; kinetin. The pH was adjusted to 5.8 prior to adding 0.8% (w/v) agar and autoclaving at 121\u0026deg;C for 20 min. Cultures were maintained in a growth chamber at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C under a 16-h photoperiod with a light intensity of 45\u0026ndash;75 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1; provided by cool white fluorescent lamps. After four weeks, uniform plantlets approximately 4\u0026ndash;5 cm in height were selected for colchicine treatment.\u003c/p\u003e\u003cp\u003eExperimental design and colchicine treatment\u003c/p\u003e\u003cp\u003eThe experiment was arranged in a completely randomized design (CRD) with a 4 \u0026times; 3 factorial arrangement. The factors were colchicine concentration (0, 50, 100, and 200 mg L⁻\u0026sup1;) and exposure duration (24, 48, and 72 h). Each treatment consisted of six replicates, with five plantlets per replicate.\u003c/p\u003e\u003cp\u003eFor treatment, uniform plantlets were placed in 250 mL culture vessels containing 100 mL of liquid MS medium with the corresponding colchicine concentration. The vessels were placed on a rotary shaker at 30 rpm and maintained in complete darkness at 25\u0026deg;C for the designated durations. Following exposure, plantlets were rinsed three times with sterile distilled water and transferred to solid MS rooting medium containing 3 mg L⁻\u0026sup1; naphthaleneacetic acid (NAA).\u003c/p\u003e\u003cp\u003eAcclimatization of plantlets\u003c/p\u003e\u003cp\u003eAfter two weeks on rooting medium, plantlets with well-developed roots were transplanted into 10-cm pots filled with a sterilized 1:1 (v/v) mixture of cocopeat and perlite. The plantlets were acclimatized in a greenhouse at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and 60\u0026ndash;70% relative humidity. They were irrigated as needed and fertilized weekly with half-strength Hoagland\u0026rsquo;s solution.\u003c/p\u003e\u003cp\u003eMorphological and stomatal analysis\u003c/p\u003e\u003cp\u003eSixty days post-acclimatization, five plants per replicate (n\u0026thinsp;=\u0026thinsp;30 per treatment) were evaluated for the following traits:\u003c/p\u003e\u003cp\u003eTiller number: The total number of new tillers per plant was counted.\u003c/p\u003e\u003cp\u003ePlant height: Measured from the substrate surface to the tip of the youngest fully expanded leaf (cm).\u003c/p\u003e\u003cp\u003eStem diameter: Measured 5 cm above the substrate surface using a digital caliper (mm).\u003c/p\u003e\u003cp\u003eFor stomatal analysis, impressions were taken from the abaxial surface of the third fully expanded leaf between 09:00 and 11:00 h using clear nail varnish. The dried impressions were mounted on glass slides and observed under a light microscope (Olympus CX43).\u003c/p\u003e\u003cp\u003eStomatal density was determined by counting the number of stomata per mm\u0026sup2; in three randomly selected fields of view at 400x magnification.\u003c/p\u003e\u003cp\u003eStomatal length and width were measured for ten stomata per field across three fields per plant using image analysis software (ImageJ, NIH, USA).\u003c/p\u003e\u003cp\u003eCytological examination and karyotype analysis\u003c/p\u003e\u003cp\u003eRoot tips (~\u0026thinsp;1 cm) were collected from control and treated plants, pre-treated with 0.002 M 8-hydroxyquinoline for 4 h at 4\u0026deg;C, and fixed in Carnoy\u0026rsquo;s solution (3:1 ethanol:glacial acetic acid) for 24 h. The root tips were hydrolyzed in 1 N HCl at 60\u0026deg;C for 10 min, stained with Schiff\u0026rsquo;s reagent for 1 h, and squashed in a drop of 45% acetic acid. Metaphase spreads were examined under a light microscope (Nikon Eclipse E200).\u003c/p\u003e\u003cp\u003eChromosome counts were performed on fifty well-spread metaphase cells per treatment. Karyotype analysis was conducted on five complete metaphase plates per ploidy level using KaryoType software (v2.0). The following parameters were determined: long arm (L) and short arm (S) lengths, total chromosome length (TCL), arm ratio (AR), centromeric index (CI), intrachromosomal asymmetry index (A₁), interchromosomal asymmetry index (A₂), and total form percentage (TF%). Chromosomes were classified based on arm ratio, and karyotype symmetry was assessed according to Stebbins (1971).\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eData were subjected to two-way analysis of variance (ANOVA) using R software (v4.3.1), with colchicine concentration and exposure duration as fixed factors. The assumptions of homogeneity of variances were verified using Levene's test. Mean comparisons were performed using Tukey\u0026rsquo;s Honestly Significant Difference (HSD) test at a 5% probability level (p\u0026thinsp;\u0026le;\u0026thinsp;0.05). Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SE).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eSurvival and growth post-treatment\u003c/p\u003e\n\u003cp\u003eExposure to colchicine for 72 h at all concentrations (50, 100, and 200 mg L⁻\u0026sup1;) resulted in 100% mortality of \u003cem\u003eSaccharum officinarum\u003c/em\u003e cv. CP57 plantlets, and these treatments were excluded from further analysis. Survival rates for 24 h and 48 h treatments are presented in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. After 24 h, plantlets treated with 50 mg L⁻\u0026sup1; colchicine exhibited the highest survival rate (83.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.4%), decreasing to 50.0\u0026thinsp;\u0026plusmn;\u0026thinsp;6.5% at 100 mg L⁻\u0026sup1; and 16.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2% at 200 mg L⁻\u0026sup1; (p\u0026thinsp;\u0026le;\u0026thinsp;0.05). Extending exposure to 48 h further reduced survival: 66.7\u0026thinsp;\u0026plusmn;\u0026thinsp;5.3% at 50 mg L⁻\u0026sup1;, 33.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.4% at 100 mg L⁻\u0026sup1;, and 0% at 200 mg L⁻\u0026sup1;. Control plantlets (0 mg L⁻\u0026sup1;) maintained 100% survival across both durations.\u003c/p\u003e\n\u003cp\u003eMorphological Changes\u003c/p\u003e\n\u003cp\u003eA two-way ANOVA revealed highly significant main effects of colchicine concentration and exposure duration (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) on tiller number and plant height, with significant interaction effects (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) for both traits (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eControl plantlets yielded a mean tiller count of 12.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 per plant. Following 24 hours of colchicine treatment, tiller numbers exhibited a concentration-dependent reduction: 5.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 (\u0026ndash;58%) at 50 mg L⁻\u0026sup1;, 3.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 (\u0026ndash;71%) at 100 mg L⁻\u0026sup1;, and 1.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 (\u0026ndash;85%) at 200 mg L⁻\u0026sup1;. Prolonging the exposure to 48 hours resulted in a further decrease in tiller counts (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe mean height of control plants was 30.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 cm. Following 24 hours of colchicine treatment, plant heights decreased in a concentration-dependent manner: 25.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 cm (\u0026ndash;18%) at 50 mg L⁻\u0026sup1;, 20.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 cm (\u0026ndash;34%) at 100 mg L⁻\u0026sup1;, and 15.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 cm (\u0026ndash;50%) at 200 mg L⁻\u0026sup1;. A 48-hour exposure further reduced plant height (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eTwo-way ANOVA summary (MS values) for morphological, stomatal, and physiological traits.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSource of Variation\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003edf\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTiller Number\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePlant Height (cm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eChlorophyll Content (SPAD)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStomatal Density\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStomatal Width*\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStomatal Length*\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eColchicine (C)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e232.38**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e441.08**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e294.76**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e180.18**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1496.36**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1186.26**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDuration (D)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.74 (ns)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e108.79**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e70.67*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e134.32**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e401.57**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e339.38**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC \u0026times; D\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.44**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e28.96**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e35.11**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e85.38**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e224.59**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e360.60**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eError\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e15.45\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCV (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10.72\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eChlorophyll Content\u003c/p\u003e\n\u003cp\u003eThe highest chlorophyll content (SPAD value) was recorded in plants treated with 50 mg L⁻\u0026sup1; colchicine for 24 hours (45.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3), representing an 18% increase compared to the 24-hour untreated control group (38.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1). Conversely, the lowest chlorophyll content was observed in plants treated with 200 mg L⁻\u0026sup1; colchicine for 48 hours (8.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5). All treatments differed significantly from each other and the controls (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eStomatal Traits\u003c/p\u003e\n\u003cp\u003eANOVA indicated significant effects of colchicine concentration and exposure duration (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) on all stomatal traits, with significant interaction effects (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003cp\u003eIn control leaves, stomatal density averaged 22.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 stomata mm⁻\u0026sup2;. Following 24 h of colchicine treatment, density dropped to 5.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 stomata mm⁻\u0026sup2; (\u0026ndash;76%) at 50 mg L⁻\u0026sup1;, 4.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 mm⁻\u0026sup2; (\u0026ndash;82%) at 100 mg L⁻\u0026sup1;, and 2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 mm⁻\u0026sup2; (\u0026ndash;91%) at 200 mg L⁻\u0026sup1;. Forty-eight-hour treatments further reduced density (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eControl stomata measured 19.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 \u0026micro;m (length) and 24.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 \u0026micro;m (width). The 50 mg L⁻\u0026sup1;/24 h treatment enlarged stomata to 49.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 \u0026micro;m (+\u0026thinsp;150%) in length and 53.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 \u0026micro;m (+\u0026thinsp;120%) in width. The 200 mg L⁻\u0026sup1;/24 h treatment produced the largest stomata (60.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 \u0026micro;m length, +\u0026thinsp;204%; 65.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 \u0026micro;m width, +\u0026thinsp;170%) (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eChromosome Number and Karyotype Analysis\u003c/p\u003e\n\u003cp\u003eAll control metaphase spreads consistently displayed a diploid chromosome count of 2n\u0026thinsp;=\u0026thinsp;114 (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e). In the 50 mg L⁻\u0026sup1;/24 h treatment, 68% of metaphase cells (34/50) exhibited approximately 2n\u0026thinsp;\u0026asymp;\u0026thinsp;230 chromosomes; the remainder retained the diploid number or showed minor aneuploidy. At 100 mg L⁻\u0026sup1;/24 h, only 42% of cells (21/50) reached the tetraploid count (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eKaryotype parameters for confirmed tetraploid plants (50 mg L⁻\u0026sup1;/24 h) versus diploid controls are summarized in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. The detailed measurements for all individual chromosomes of the tetraploid are provided in Supplementary Tables. Total chromosome length (TCL) averaged 207.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8 \u0026micro;m in diploids and 328.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1 \u0026micro;m in tetraploids (+\u0026thinsp;58%). The intrachromosomal asymmetry index (A₁) decreased significantly from 0.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 to 0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating a more symmetric karyotype. Stebbins\u0026rsquo; symmetry class shifted from B2 (diploid) to B1 (tetraploid).\u003c/p\u003e\n\u003cp\u003eCytological analysis confirmed the ploidy level of both control and treated plants. Metaphase spreads from control plantlets consistently revealed a diploid chromosome count of 2n\u0026thinsp;=\u0026thinsp;114 (Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e). In contrast, root tip cells from plantlets treated with 50 mg L⁻\u0026sup1; colchicine for 24 h exhibited a tetraploid count of approximately 2n\u0026thinsp;\u0026asymp;\u0026thinsp;230 chromosomes (Fig. \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e). The karyogram of the control plant (Fig. \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003e) further illustrates the organized diploid set, while the karyogram of the treated plant (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e13\u003c/span\u003e) visually demonstrates the successful chromosome doubling and the increased karyotype symmetry characteristic of the induced tetraploids.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab2\" border=\"1\" class=\"fr-table-selection-hover\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eKaryotype parameters comparing diploid (2n\u0026thinsp;=\u0026thinsp;114) and tetraploid (2n\u0026thinsp;\u0026asymp;\u0026thinsp;230) CP57 sugarcane.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRow\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIndex\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTreated\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNumber of Chromosomes (2n)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e114\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e230\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTotal Genome Length (TL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e207.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e327.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCoefficient of Variation (CV)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAsymmetry Index (AI)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStebbins Symmetry Class (SC)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eB2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eB1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRelative Chromosome Length (RL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCentromeric Index (CI%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTotal Form of Karyotype (TF%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eChromosome Form Percentage (F%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRelative Chromatin Content (VRS)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAverage Chromosome Length (CL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRelative Length of Shortest Chromosome (S%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRange of Relative Chromosome Length (DRL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eInterchromosomal Asymmetry Index (A2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAverage Length of Long Chromosome Arms (L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAverage Length of Short Chromosome Arms (S)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIntrachromosomal Asymmetry Index (A1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKaryotype Asymmetry Index (ASK)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e66.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e63.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDifference Index of Two Chromosome Arms (d-Value)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRatio of Long to Short Chromosome Arms (AR)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eValue of Short to Long Chromosome Arms (r-Value)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eCellular Abnormalities\u003c/p\u003e\n\u003cp\u003eSome colchicine-treated samples exhibited interphase nuclear abnormalities, including multinucleate cells (up to six nuclei per cell of varying sizes) and disrupted chromosome migration, reflecting colchicine-induced spindle inhibition (Fig. \u003cspan class=\"InternalRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study successfully established an efficient \u003cem\u003ein vitro\u003c/em\u003e protocol for tetraploid induction in sugarcane cv. CP57, with treatment using 50 mg L⁻\u0026sup1; colchicine for 24 h proving highly effective in inducing stable tetraploidy. The 100% mortality observed at 72 h exposures corroborates earlier findings that prolonged colchicine exposure severely compromises cell viability (Touchell et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Our results agree with previous studies in sugarcane hybrids, where moderate concentrations and shorter durations balanced survival and induction efficiency (Khan et al. 2008; D Hont, 2005).\u003c/p\u003e\u003cp\u003eThe morphological alterations\u0026mdash;reduced tillering and plant height\u0026mdash;are characteristic of artificial polyploids, often resulting from mitotic disruption and altered phytohormone transport (Chen et al. 2007). The dramatic changes in stomatal traits (increased size, decreased density) serve as reliable phenotypic markers for polyploidy in sugarcane, consistent with findings in other specie (Zhang et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWang et al (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) Reports that the chlorophyll content in tetraploid is significantly increased compared with the diploid variety, contributing to higher photosynthetic capacity. This enhancement suggests a potential for improved photosynthetic efficiency and biomass yield, though field validation is necessary.\u003c/p\u003e\u003cp\u003eCytological analysis confirmed the protocol's efficacy, with a high rate (68%) of tetraploid cells achieved at the optimal treatment. The observed karyotype changes, including increased total chromosome length and enhanced symmetry, are consistent with genomic restructuring following polyploidization (Sattler et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The presence of aneuploid cells and chimeras is a typical challenge in colchicine-induced polyploidy (Dhooghe et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), underscoring the need for careful selection or additional mitotic cycles to achieve stable, homogeneous tetraploid lines.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe optimized \u003cem\u003ein vitro\u003c/em\u003e protocol employing 50 mg L⁻\u0026sup1; colchicine for 24 h effectively induces tetraploidy in sugarcane cv. CP57 with high survival and induction rates. The tetraploid lines were unequivocally characterized through a combination of stomatal, morphological, physiological, and detailed cytological analyses. This protocol provides a valuable biotechnological tool for sugarcane breeding programs, enabling the creation of novel genetic diversity for the development of cultivars with enhanced yield potential and stress tolerance. Future work will focus on the agronomic evaluation of these tetraploid lines under field conditions and the use of flow cytometry for high-throughput ploidy screening.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Agricultural Sciences and Natural Resources University of Khuzestan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare they have no financial or non-financial competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMFZ: Investigation, Methodology, Formal analysis. PPM: Conceptualization, Supervision, Writing – original draft, Writing – review \u0026amp; editing. KST: Methodology, Investigation (Cytogenetic analysis).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the Agricultural Sciences and Natural Resources University of Khuzestan for providing the laboratory facilities and technical support to conduct this research. We are also grateful to the Karun Agro Industry Incorporation for generously supplying the plant materials.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBlakeslee AF, Avery AG (1937) Methods of inducing doubling of chromosomes in plants. J Hered 28:393\u0026ndash;411. https://doi.org/10.1093/oxfordjournals.jhered.a104294.\u003c/li\u003e\n\u003cli\u003eCardona CA, Quintero JA, Paz IC (2010) Production of bioethanol from sugarcane bagasse: status and perspectives. Bioresour Technol 101:4754\u0026ndash;4766. https://doi.org/10.1016/j.biortech.2009.10.097.\u003c/li\u003e\n\u003cli\u003eWang Y, Jia B, Ren H, Feng Z (2021) Ploidy level enhances the photosynthetic capacity of a tetraploid variety of \u003cem\u003eAcer buergerianum\u003c/em\u003e Miq. PeerJ 11:e12620. doi: 10.7717/peerj.12620\u003c/li\u003e\n\u003cli\u003eChen ZJ (2007) Genetic and epigenetic mechanisms for gene expression and phenotypic variation in plant polyploids. Annu Rev Plant Biol 58:377\u0026ndash;406. doi: 10.1146/annurev.arplant.58.032806.103835.\u003c/li\u003e\n\u003cli\u003eDhooghe E, Van Laere K, Eeckhaut T, Leus L, Van Huylenbroeck J (2011) Mitotic chromosome doubling of plant tissues \u003cem\u003ein vitro\u003c/em\u003e. Plant Cell Tissue Organ Cult 104:359\u0026ndash;373. https://doi.org/10.1007/s11240-010-9786-5.\u003c/li\u003e\n\u003cli\u003eFAO (2023) FAOSTAT statistical database. Food and Agriculture Organization, Rome.\u003c/li\u003e\n\u003cli\u003eNhut, D.T., Tam, D.T.T., Luan, V.Q., Hien, N.T.T., Tung, H.T. (2022). A Protocol of Shoot Regeneration and Polyploid Plantlet Production in \u003cem\u003ePaphiopedilum villosum\u003c/em\u003e. In: Nhut, D.T., Tung, H.T., YEUNG, E.CT. (eds) Plant Tissue Culture: New Techniques and Application in Horticultural Species of Tropical Region. Springer, Singapore. https://doi.org/10.1007/978-981-16-6498-4_16 \u003c/li\u003e\n\u003cli\u003eD\u0026apos;Hont A. Unraveling the genome structure of polyploids using FISH and GISH; examples of sugarcane and banana. Cytogenet Genome Res. 2005;109(1-3):27-33. doi: 10.1159/000082378. PMID: 15753555.\u003c/li\u003e\n\u003cli\u003eMurashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473\u0026ndash;497.\u003c/li\u003e\n\u003cli\u003eZhang J, Cheng C, Xiao F, Zhang X, Zhang C, Zhao Y, Xu J, Zhang S, Wang X (2024) Effects of ploidy level on leaf morphology, stomata, and anatomical structure of \u003cem\u003eHibiscus syriacus\u003c/em\u003e L. BMC Plant Biol 24, 1133. https://doi.org/10.1186/s12870-024-05778-y\u003c/li\u003e\n\u003cli\u003ePiperidis G, D\u0026rsquo;Hont A (2020) Sugarcane genome architecture decrypted with chromosome-specific oligo probes. Plant J 103:2139\u0026ndash;2151. doi: 10.1111/tpj.14881.\u003c/li\u003e\n\u003cli\u003eSalma U, Kundu S, Mandal N (2017) Artificial polyploidy in medicinal plants: advancement in the last two decades and impending prospects. J Crop Sci Biotechnol 20:9\u0026ndash;19. https://doi.org/10.1007/s12892-016-0080-1\u003c/li\u003e\n\u003cli\u003eSattler MC, Carvalho CR, Clarindo WR (2016) The polyploidy and its key role in plant breeding. Planta 243:281\u0026ndash;296. https://doi.org/10.1007/s00425-015-2450-x.\u003c/li\u003e\n\u003cli\u003eVerma KK, Song X-P, Singh M, RDM P, Wu J-M, Li Y-R (eds) (2024) Sugarcane cultivationand management: challenges and opportunities, 1st edn. Apple Academic Press. https://doi.org/10.1201/9781003504122\u003c/li\u003e\n\u003cli\u003eTouchell, D. H., Palmer, I. E., Ranney, T. G. (2020). \u003cem\u003eIn vitro\u003c/em\u003e ploidy manipulation for crop improvement. Frontiers in Plant Science, 11, 722. https://doi.org/10.3389/fpls.2020.00722\u003c/li\u003e\n\u003cli\u003eVan de Peer Y, Mizrachi E, Marchal K (2017) The evolutionary significance of polyploidy. Nat Rev Genet 18:411\u0026ndash;424. https://doi.org/10.1038/nrg.2017.26\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"polyploidy induction, in vitro culture, colchicine, stomatal traits, karyotype analysis, sugarcane","lastPublishedDoi":"10.21203/rs.3.rs-7660881/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7660881/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAn efficient \u003cem\u003ein vitro\u003c/em\u003e protocol was developed for inducing stable tetraploidy in sugarcane (\u003cem\u003eSaccharum officinarum\u003c/em\u003e L. cv. CP57), a cultivar with restricted flowering that hinders conventional breeding. \u003cem\u003eIn vitro\u003c/em\u003e-grown plantlets were treated with colchicine (0, 50, 100, and 200 mg L⁻¹) for 24, 48, or 72 h. The 72 h treatments resulted in complete mortality. The optimal treatment for polyploidy induction was 50 mg L⁻¹ colchicine for 24 h, which yielded the highest survival rate (83.3 ± 4.4%) and successfully produced tetraploids. Morpho-physiological analysis 60 days post-acclimatization revealed that this treatment significantly reduced tiller number (by 58%) and plant height (by 18%) while increasing chlorophyll content (by 18%) compared to diploid controls. Stomatal density decreased by 76%, while stomatal length and width increased by 150% and 120%, respectively, serving as reliable indicators of polyploidy. Cytological analysis confirmed tetraploidy (2n = ~230 chromosomes) in 68% of cells from plantlets treated with 50 mg L⁻¹ colchicine for 24 h. Karyotype analysis showed a 58% increase in total chromosome length and enhanced karyotype symmetry in tetraploids. The established protocol effectively induces tetraploidy in sugarcane cv. CP57 and provides a valuable biotechnological tool for breeding programs aimed at enhancing genetic diversity and improving agronomic traits.\u003c/p\u003e","manuscriptTitle":"In vitro-induced tetraploidy in sugarcane (Saccharum officinarum L. cv. CP57) using colchicine: protocol optimization and cytomorphological characterization","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-08 17:54:43","doi":"10.21203/rs.3.rs-7660881/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"25140b82-9b4e-411c-aed8-dc2155521982","owner":[],"postedDate":"October 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-27T13:58:05+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-08 17:54:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7660881","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7660881","identity":"rs-7660881","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}