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Evaluation of Colchicine Dosage Effects on Autopolyploid Induction in Diploid Medicago sativa ssp. Falcata

X
Xuefang Zhang1
R
Ruru Shi2
F
Fengling Shi1,*
1Key Laboratory of Grassland Resources, Ministry of Education, College of Grassland Science, Inner Mongolia Agricultural University, Hohhot 010018, China.
2Institute of Forest and Grassland Genetics and Breeding, Inner Mongolia Academy of Forestry Science, Hohhot 010018, China.

  • Submitted24-08-2025|

  • Accepted07-04-2026|

  • First Online 17-04-2026|

  • doi 10.18805/LRF-898

ABSTRACT

Background: Medicago sativa subsp.falcata (yellow-flowered alfalfa), a perennial leguminous forage crop, has inherent agronomic limitations, including slow growth, low biomass yield and difficulties in cultivation. The development of polyploid germplasm is a promising strategy for varietal improvement, requiring systematic optimization of colchicine induction protocols.

Methods: Using imbibed seeds of diploid Medicago sativa subsp. falcata (2 n=16), we performed colchicine soak treatments and systematically characterized mutagenized plants via stomatal morphology analysis and chromosome counting. Our quantitative assessment revealed concentration and exposure time-dependent effects on seed germination, seedling development and polyploidization efficiency.

Result: The results showed that increasing colchicine concentrations led to a progressive inhibition of seed germination and seedling growth. This was evidenced by a delay in germination initiation, a reduction in germination rates, an increase in hypocotyl diameter and a shortening of radicle length. After treatment with 0.15% colchicine for 6 hours, chromosome doubling was observed at a frequency of 10%, with a seedling survival rate of 39%.Simultaneously, the stomatal density of the treated polyploid plants reached the lowest. These results indicate that this method represents a feasible approach for polyploid breeding in this germplasm.

INTRODUCTION

Chemical mutagens are specialized compounds that alter DNA structure or function in organisms. These agents induce genetic mutations through diverse mechanisms, including chromosome doubling and the insertion or deletion of DNA segments (Ren, 2023). Among diverse chemical mutagens, colchicine exhibits a unique mode of action: it disrupts spindle formation during mitosis, thereby inducing chromosome doubling (Zhang et al., 2016). Owing to its low cost, high mutagenic efficiency and broad applicability across genetic materials, colchicine has become one of the most widely used chemical mutagens in practice. Following genome doubling, plants typically undergo a series of phenotypic and physiological modifications. These alterations include enhanced growth rates, improved genetic traits and increased metabolite accumulation (Li et al., 2024). Consequently, induced autotetraploids exhibit multidimensional differences compared to their diploid progenitors, spanning morphological, anatomical, physiological and agronomic characteristics. Polyploid plants exhibit characteristic phenotypic enhancements, including thicker, larger leaves with intensified pigmentation, elevated chlorophyll content, enlarged floral organs and fruits, thickened vascular bundles, as well as significantly improved stress tolerance and increased biomass and bioactive compound production (Lei et al., 2023; Veronesi et al., 1986; Zhang et al., 2024; Qiu et al., 2020).
       
Researchers have successfully induced polyploidy in various forage species-including Melilotoides ruthenica (Wang et al., 2013), Medicago sativa (Li et al., 2016) and Lolium perenne (Ferreres et al., 2019) -using colchicine and established corresponding mutant libraries (Darmadi et al., 2025). These mutagenized collections have not only provided valuable materials for functional genomic studies but also served as novel germplasm resources for cultivar development (Francis et al., 1989).
       
Yellow-flowered alfalfa (Medicago sativa subsp. falcata), a perennial leguminous forage crop (Wang et al., 2008), has been widely cultivated for its exceptional cold and drought tolerance. In China, extensive research efforts have focused on this species, including analyses of genetic diversity in germplasm resources (Li et al., 2022; Paun et al., 2024), improvement of local cultivars (Zhou et al., 2022; Li et al., 2025) and investigations into stress resistance mechanisms (Cui et al., 2019; Jia et al., 2025; Han et al., 2025). In this study, diploid Medicago sativa subsp. Falcata (2n = 16) germplasm from Xinjiang was used to investigate the effects of colchicine on polyploidization. Seeds were treated with various concentrations of colchicine for different durations to evaluate the following parameters: (i) seed germination rate, (ii) radicle growth dynamics, (iii) seedling survival rate, (iv) frequency of chromosome doubling and (v) stomatal morphological characteristics. This comprehensive profiling establishes an optimized colchicine induction protocol for efficient polyploid generation, providing a foundational mutagenized population for genetic improvement of this stress-tolerant forage crop.

MATERIALS AND METHODS

Plant material

The plant material used in this study consisted of wild Medicago sativa subsp.falcata. seeds (2n=16) collected from Xinjiang, China in 2021. Seeds were stored at 4°C with a thousand-seed weight of 0.9096 g. All experiments were conducted in 2024 at the Life Sciences Building ,Inner Mongolia Agricultural University (geographic coordinates: 40°83'N, 111°73'E).
 
Seed pre treatment
 
Plump, uniformly-sized diploid Medicago sativa subsp. falcata. seeds were selected and scarified with 30-grit sandpaper to disrupt the cuticular layer and remove surface contaminants. Seeds were subsequently surface-sterilized in 70% (v/v) ethanol for 30 s to mitigate fungal contamination during germination, followed by three thorough rinses with sterile distilled water and air-drying in sterile Petri dishes until no surface moisture remained. Pretreated seeds were transferred to sterile 90-mm dishes (100 seeds per dish; three biological replicates per treatment) and stratified at 4°C in a controlled climate chamber for 24 h to synchronize imbibition. Seeds exhibiting >90% volumetric expansion were identified as fully imbibed, blot-dried on Whatman ® No.1 sterile filter paper and immediately exposed to colchicine solutions to initiate polyploid mutagenesis.
 
Colchicine treatment
 
Imbibed Medicago falcata seeds were treated with colchicine solutions prepared in distilled water at concentrations of 0.05%, 0.1%, 0.15% and 0.2% (v/v), each supplemented with 1% DMSO as a penetration enhancer (Table 1 for experimental design). Seeds were immersed in sterile conical flasks containing 10 ml of the assigned colchicine solution per flask, with distilled water-treated samples serving as controls (CK). All flasks were sealed with Parafilm®, wrapped in aluminum foil for light exclusion and agitated at 100 rpm and 20°C for specified durations (3, 6, 9, 12, or 24 h). Post-treatment, seeds were rinsed under running water for 40 min followed by three sterile-water washes. Finally, seeds were plated on Petri dishes and incubated at 25°C under a 16-h light/8-h dark photoperiod.

Table 1: Colchicine treatment conditions.


 
Seed germination characteristics and radicle growth analysis
 
Germination was monitored daily over 7 days, with recordings of: (i) seed germination progress, (ii) cumulative number of germinated seeds and (iii) radicle development. Germination energy and seedling fresh weight were quantified on day 7. The experiment was terminated on day 15, at which point final germination percentages were calculated.
 






 
Germination index (GI) = Σ (Gt /Dt )


 
 
(Gt = Number of seeds germinated on day t (dimensionless) Dt = Corresponding germination day (day)
 
Vigor index (%) = Germination index × Seedling fresh weight × 100%


Chromosomal analysis of root tip cells following colchicine treatment
 
(1) Root/shoot tip collection: Fresh root and shoot tips  (~25 mm in length) were excised at 09:00 local time and immediately transferred to ice-chilled centrifuge tubes filled with distilled water. Samples were held at 4°C for 24 h to synchronize cell cycles before fixation.
 
(2) Fixation: Samples were immersed in freshly prepared Carnoy’s fixative (95% ethanol: glacial acetic acid, 3:1 v/v) for 24 h at room temperature. After fixation, residual fixative was thoroughly removed by three 5-minute washes with distilled water under gentle agitation.
 
(3) Acid hydrolysis: Fixed root and shoot tips were transferred to 1.5-mL microcentrifuge tubes containing 1 M hydrochloric acid (HCl) and incubated at 60°C for 8 min in a precision water bath (Julabo SW23). Following hydrolysis, residual acid was removed by three 2-minute washes with distilled water under gentle agitation (100 rpm).
 
(4) Slide preparation: The excised root/shoot tips were placed on glass slides and treated with 45% acetic acid solution. Cover slips were then applied and the samples were gently tapped with forceps until the tips dispersed into a homogeneous cloud-like suspension.
 
(5) Cytogenetic analysis: Ten cells per root tip were analyzed systematically at 1000× magnification. Chromosome counts were recorded for each cell and the proportions of diploid (2 n), mixoploid and tetraploid (4 n) cells were calculated relative to the total cell population.
 
Treatment intensity (TI) = Exposure duration (h) × Colchicine concentration (%)
 
Seedling emergence rate
 
Germinated seeds were transplanted into 10-cm-deep nursery trays filled with growth substrate (fertilizer: vermiculite: volcanic rock, 1:1:1 w/v). The relative survival rate of seedlings was quantified 30 days after transplantation.



 
Stomatal density quantification
 
For stomatal observations, fully expanded leaves of comparable size were sampled simultaneously from identical nodal positions. The abaxial epidermis was prepared using transparent adhesive tape (Xu, 2020) and imaged under an Olympus BX53 microscope with a 40× objective. Wild-type diploid Medicago sativa subsp. falcata from Xinjiang was used as the control.
 
Statistical and computational analyses
 
Statistical analyses were performed using Microsoft Excel 2021 and SPSS 26.0, with data visualization conducted in Origin 2022. Chromosomal images of diploid Medicago sativa subsp.falcata were processed using Photoshop for enhanced clarity.

RESULTS AND DISCUSSION

Dose-and time-dependent effects of colchicine on germination in diploid M. sativa subsp.falcata
 
As shown in Fig 1, seeds treated with colchicine for 3 hours exhibited no significant difference in relative germination rate (RGR) compared to the untreated control group (P>0.05) at any of the tested concentrations, which may be attributed to the limited physiological impact of short-duration, low-concentration treatments (Xu, 2020). In contrast, extended exposure durations (6, 9, 12 and 24 hours) resulted in concentration-dependent decreases in RGR, with the most substantial inhibition observed at the highest concentration-time combination. The lowest RGR (46.40%) was recorded under the most severe treatment conditions (0.2% colchicine for 24 hours). Furthermore, within each concentration group, prolonged treatment also led to time-dependent reductions in germination performance. Similarly, relative germination energy (RGE) showed significant dose-and time-dependent declines (P<0.05), demonstrating a progressive decrease with increasing colchicine concentration and exposure duration. The inhibitory effects of prolonged high-concentration treatments on RGR, RGE and relative vigor index were consistent with previous findings in Medicago erecta (Wang, 2013), Medicago lupulina and Medicago sativa (Liu, 2014). The phytotoxicity of colchicine appears to be positively correlated with both concentration and treatment duration, wherein excessively high concentrations or overly extended exposure times cause substantial damage to seeds (Harbard, 2012).

Fig 1: Germination responses to colchicine concentration and duration.


 
Dose- and time-dependent effects of colchicine on seed vigor in diploid M.sativa subsp. falcata
 
Fig 2 demonstrates that the relative seed vigor index (RSVI) was significantly lower in all treatment groups compared to the control (P<0.05) The RSVI progressively decreased with increasing colchicine concentrations and prolonged exposure durations. The most severe suppression of RSVI (23.10%) was observed at the highest concentration and longest duration (0.2% for 24 h).

Fig 2: Seed vigor responses to colchicine gradients and exposure durations.


 
Dose- and time-dependent effects of colchicine on radicle growth in diploid M.sativa subsp.falcata
 
During polyploid induction in plants using the seed-soaking method, colchicine often causes significant toxic effects on the root system. In this study, treatment of Medicago falcata seeds with colchicine resulted in a significant reduction in the average radicle length-by more than 65% compared with the untreated control group (P<0.05; Fig 3). Concurrently, the hypocotyl diameter showed a gradual increase with higher colchicine concentrations and longer exposure durations. Under the treatment with 0.2% colchicine for 9 h, the average hypocotyl diameter reached a maximum value of 1.33 mm, representing an increase of 0.83 mm compared to the control group (Fig 4). These morphological responses are consistent with reports by (Zhang, 2006) in Psathyrostachys juncea, as well as in Agropyron cristatum (Qalby et al., 2020) and Medicago sativa subsp.falcata. (Liu, 2014), which described typical cytological abnormalities such as swollen coleoptiles and bulbous root tips. Further analysis indicated that higher concentrations and extended treatment durations exacerbated colchicine-induced damage to hypocotyl tissues. Treated radicles commonly exhibited yellowing and necrosis, loss of regenerative capacity and failure of hypocotyls to initiate new roots, ultimately leading to severely impeded root development and significantly reduced seedling survival. Similar phenomena have been reported in colchicine mutagenesis experiments in Taraxacum (Zhu, 2022). The underlying mechanism involves colchicine’s disruption of spindle formation, which inhibits normal cell division. Meanwhile, ongoing cellular metabolic activities promote abnormal cell expansion, resulting in structural thickening of the hypocotyl and radicle. Notably, the increased radicle diameter may serve as an indirect morphological indicator of chromosomal variation, providing preliminary phenotypic evidence for successful polyploid induction.

Fig 3: Radicle elongation under colchicine treatment.



Fig 4: Hypocotyl thickening rate under colchicine treatment.


 
Colchicine effects on chromosome numbers in diploid M.sativa subsp. falcata root tips
 
Polyploidy is inherently characterized by chromosome number doubling and chromosome microscopic observation and counting thus represent one of the most accurate identification methods (Li et al., 2016). The success of this technique hinges on the selection of an optimal fixation time to ensure that the observed materials are in an active stage of cell division. In this study, radicles were excised daily from 9:00 to 10:00, a time window corresponding to the peak of cell division during Medicago falcata seed germination (Li et al., 2015). By comparing the chromosome numbers in the root tips of M. falcata treated with colchicine at different concentrations and for various durations, the tetraploid induction rate was found to increase progressively with rising colchicine concentrations and extended treatment times. Specifically, the results (Table 2) showed that treatment with 0.15% colchicine for 6 h resulted in a tetraploid cell proportion of 10.00%, while the maximum tetraploid ratio (30.00%) was achieved at an elevated concentration of 0.2% with a prolonged treatment duration of 12 h. These findings are consistent with those reported in colchicine-induced polyploidy studies on Medicago sativa (Basu et al., 2018), Medicago sativa cv. Jinda (Ji et al., 2012) and Melilotoides ruthenica (Kong et al., 2010). Furthermore, mixoploids first appeared when the treatment intensity (concentration × time) reached or exceeded 0.0045, whereas tetraploids could be induced when the treatment intensity surpassed 0.009 (Fig 5).

Table 2: Chromosomal analysis of diploid M. falcata under colchicine treatment.



Fig 5: Ploidy identification by root tip squashing method.


 
Dose-and time-dependent effects of colchicine on relative seedling survival in diploid M.sativa subsp. falcata
 
However, seedling survival rate showed a negative correlation with tetraploid induction rate, that is, a higher tetraploid induction rate was associated with a lower survival rate (Wang et al., 2024). This finding is consistent with the results of Li for colchicine-treated common alfalfa (Medicago sativa), where a negative correlation between tetraploidy rate and seedling survival was likewise observed (Li et al., 2016). Therefore, when determining the optimal mutagenic conditions for chromosome doubling in yellow-flowered alfalfa (Medicago falcata), it is essential to not only consider the tetraploid induction rate, but also to comprehensively evaluate key indicators such as seed relative vigor index and seedling survival rate. A comprehensive analysis in this study indicated that treatment with 0.15% colchicine for 6 h effectively induced tetraploidy, while resulting in a higher seedling survival rate compared to other treatment combinations that also induced tetraploids. As shown in Fig 6, the relative survival rate of yellow-flowered alfalfa seedlings exhibited a declining trend with increasing colchicine concentration and extended treatment duration. When the concentration reached 0.2% with treatment times of 12 h or 24 h, nearly complete mortality of the seedlings was observed.

Fig 6: Seedling survival under colchicine treatment.


 
Dose- and time-dependent effects of colchicine on stomatal morphology in diploid M.sativa subsp.falcata
 
Polyploid plants commonly exhibit increased cell volume. Normal cell growth and function rely on a dynamic balance between the nucleus and cytoplasm (Pacey et al., 2020). From Fig 8.1 and 8.2, as plant ploidy increases, the nuclear volume expands accordingly. To maintain a stable nucleo-cytoplasmic ratio and cellular functions, the cytoplasmic volume must also increase, leading to an overall enlargement of the cell. Stomata are formed by a pair of guard cells and the increase in their size directly results in larger stomatal dimensions (Zhang et al., 2024). Previous studies have confirmed that stomatal size, guard cell volume and chloroplast number are significantly positively correlated with ploidy level, whereas stomatal density is negatively correlated with ploidy (Padoan et al., 2013). For instance, (Francis et al., 1974) demonstrated that stomatal traits can be used effectively to differentiate ploidy levels in Cucurbita species. In the present study, stomatal density in leaves of mutagen-treated seedlings showed a significant difference compared to the control group (P<0.05). As shown in Fig 7, the lowest stomatal density was observed after treatment with 0.1% colchicine for 9 h, suggesting a higher chromosome doubling efficiency under this treatment condition.

Fig 7: Stomatal density responses to colchicine treatment.



Fig 8.1: Stomatal morphology in untreated leaf epidermis of M. sativa subsp. falcata.



Fig 8.2: Stomatal morphology in colchicine-treated leaf epidermis of M. sativa subsp. falcata.

CONCLUSION

(1)   Colchicine treatment of imbibed diploid Medicago sativa subsp.falcata seeds (2 n=16) induced significant morphological alterations, including increased hypocotyl diameter (P<0.05) and reduced radicle length compared to untreated controls.
       
(2) Colchicine treatment induced chromosomal variations in the root tip cells of alfalfa, resulting in multiple cytotypes (2 n = 2x = 16 ± 3, 2x < T < 4x, 2 n = 4x = 32±3). Among the tested regimens, treatment with 0.15% colchicine for 6 h was identified as the optimal dosage for tetraploid induction.
       
Future research scope and directionsÿBased on the present findings, future research will focus on the following directions: First, the optimized treatment conditions will be adopted to induce polyploidy in diploid Medicago sativa subsp.falcata. seeds, with a systematic assessment of the intergenerational genetic stability of ploidy levels and key agronomic traits for screening stable, elite germplasm. Second, molecular markers coupled with whole-genome sequencing will be used to analyze genomic variation during colchicine-induced chromosome doubling and identify key genes associated with important agronomic traits.

ACKNOWLEDGEMENT

FL.S. designed the study; XF.Z. performed the experiments and wrote the manuscript. This work was financially supported by the Major Demonstration Project for Open Competition in Seed Industry Science and Technology Innovation of Inner Mongolia (Grant No. 2022JBGS0016). Additional support was provided by the Key Laboratory of Grassland Resources, Ministry of Education, China.
 
Disclaimers
 
The views and conclusions presented in this article are those of all authors and do not necessarily represent the official positions of their affiliated institutions.The authors are responsible for the accuracy and completeness of the information provided, but shall not be held legally liable for any direct or indirect losses arising from the use of this content.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

REFERENCES


  1. Basu, S. and Lodh, D. (2018). Artificial induction of polyploidy in Andrographis paniculata (Burm. F.) wall. ex nees. The Journal of Indian Botanical Society. 97(1 and 2): 21-28.

  2. Cui, G., Chai, H., Yin, H., Yang, M., Hu, G., Guo, M., Zhang, P. et al. (2019). Full-length transcriptome sequencing reveals the low-temperature-tolerance mechanism of Medicago falcata roots. BMC Plant Biology. 19(1): 575. 

  3. Darmadi, D. and Fathurrahman, F. (2025). Identification of Phenotypic Nature of Rainbow Corn (Zea mays L.) Due to Colchicine Treatment. In IOP Conference Series: Earth and Environmental Science. IOP Publishing. 1478(1): 012002.

  4. Ferreres, I., Ortega, M., López-Cristoffanini, C., Nogués, S. and Serrat, X. (2019). Colchicine and osmotic stress for improving anther culture efficiency on long grain temperate and tropical japonica rice genotypes. Plant Biotechnology. 36(4): 269-273. 

  5. Francis, A. and Jones, R.N. (1989). Heritable nature of colchicine induced variation in diploid Lolium perenne. Heredity. 62(3): 407-410. 

  6. Francis, R.R. and Bemis, W.P. (1974). Stomatal size in induced polyploids in cucurbita1. Hort Science. 9(2): 138-138.

  7. Han, B., Duan, X., Jiang, H., Zhao, Y.,  Wang, Y., Hu, T. and Yang, P. (2025). Molecular cloning and functional analysis of MsDUF from alfalfa (Medicago sativa L.). Legume Research. 48(2): 246-253. doi: 10.18805/LRF-821.

  8. Harbard, J.L., Griffin, A.R., Foster, S., Brooker, C., Kha, L.D. and Koutoulis, A. (2012). Production of colchicine-induced autotetraploids as a basis for sterility breeding in Acacia mangium Willd. Forestry: An International Journal of Forest Research. 85(3): 427-436.

  9. Ji, R.H. (2012). Study on Chromosome Doubling Technique of M. sativa subsp. Caerulea (Less. Ex Ledeb.) Schmalh cv. Jindera. [Inner Mongolia Agricultural University Master’s Thesis]. China National Knowledge Infrastructure.

  10. Jia, M., Mu, Y., Yang, Z., Shao, J., Shi, F. and Yun, L. (2025). Cold resistance evaluation of alfalfa during the germination and seedling stages and analysis of its relationship with fall dormancy. Legume Research: An International Journal. 48(10): 1656-1662. doi: 10.18805/LRF-875.

  11. Kong, S.Y. (2010). Studies on Polyploid Induction of Melilotoides Ruthenica Strains. [Inner Mongolia Agricultural University Master’s Thesis]. China National Knowledge Infrastructure.

  12. Lei, L., Liu, G., Yan, D., Zhang, M., Cui, Q., Zhao, Q., Xu, G. et al. (2023). Manipulation of ploidy for blueberry breeding: in vitro chromosome doubling of diploid vaccinium duclouxii (Lévl.) Hand.-Mazz by trifluralin. Scientia Horticulturae. 317: 112056. 

  13. Li, C.J., Sun, Y.L., Wang, Y.X., Cheng, A.P. and Zhang, B. (2022). Genetic diversity of agronomic traits of 50 Medicago falcata L. germplasm. Journal of Xinjiang Agricultural University. 45(3): 173-181.

  14. Li, H., Wang, L., Li, J., Sun, X., Lin, D. and Wang, Z. (2025). Alfalfa autotoxicity: Role of cultivar renovation in mitigating germination and seedling growth under continuous cropping systems. Legume Research: An International Journal. 48(5): 828-834. doi: 10.18805/LRF-837.

  15. Li, Y. and Shi, S.L. (2016). The mutagenic effects of colchicine treatment on chromosome ploidy and the leaf ep-idermal cells of Medicag sativa cv qingshui. Acta Prataculturae Sinica. 25(2): 141-149.

  16. Li, Y., Yin, L., He, X., Hu, C., Wu, R., Long, Q., Yuan, D. et al. (2024). Ploidy and fruit trait variation in oil-tea Camellia: Implications for ploidy breeding. Journal of Integrative Agriculture. 23(8): 2662-2673. 

  17. Liu, Q. (2014). 6 Polyploidy Induction and Ploidy Identification of 6 medicago SPP. Germplasms. [Inner Mongolia Agricultural University Master’s Thesis]. China National Knowledge Infrastructure.

  18. Liu, S.R., Lin, Y. and Luo, J.Z. (2015). Effect of different pretreatment methods of corymbia citriodora chromosome technology. Eucalypt Science and Technology. 32(4): 27-31.

  19. Pacey, E.K., Maherali, H. and Husband, B.C. (2020). The influence of experimentally induced polyploidy on the relationships between endopolyploidy and plant function in Arabidopsis thaliana. Ecology and Evolution. 10(1): 198-216. 

  20. Padoan, D., Mossad, A., Chiancone, B., Germana, M.A. and Khan, P.S.S.V. (2013). Ploidy levels in Citrus clementine affects leaf morphology, stomatal density and water content. Theoretical and Experimental Plant Physiology. 25: 283- 290. 

  21. Paun, G., Neagu, E., Alecu, A., Albu, C., Seciu-Grama, A.M. and Radu, G.L. (2024). Evaluating the antioxidant and antidiabetic properties of Medicago sativa and Solidago virgaurea polyphenolic-rich extracts. Molecules. 29(2): 326.

  22. Qalby, N., Sjahril, R., Dachlan, A. and Sakae, A. (2020). Colchicine Induced Polyploidy in Common Ice Plant Mesembryanthemum crystallinum L. In: IOP Conference Series: Earth and Environmental Science. IOP Publishing. 486(1): 012097.

  23. Qiu, Y., Hamernick, S., Ortiz, J.B. and Watkins, E. (2020). DNA content and ploidy estimation of Festuca ovina accessions by flow cytometry. Crop Science. 60(5): 2757-2767.

  24. Ren, W.B., Fu, C.X., Lin, H., Guo, H.Q., Yuan, F., Liu, X.Q. and Liu, Y.L. (2023). Technological Innovation and Application of Alfalfa Mutagenesis Breeding Technology.

  25. Veronesi, F., Mariani, A. and Bingham, E.T. (1986). Unreduced gametes in diploid Medicago and their importance in alfalfa breeding. Theoretical and Applied Genetics. 72(1): 37-41. 

  26. Wang, J.J., Yun, J.F. and Lv, S.J. (2008). Preliminary study on physiological responses of salt tolerance in Sickle Alfalfa. Journal of Arid Land Resources and Environment. 22(12): 158-163. 

  27. Wang, L., Shi, F.L., Bian, X.Y., Yi, F.X. and Gao, X. (2013). Analyzing the Mutative Effects of Medicago ruthenica (L.) sojak. cv. zhilixing treated by colchicine. Seed. 32(10): 50-53.

  28. Wang, L., Zheng, P., Ge, H., Zhao, X., Kou, Y., Yang, S., Jia, R. et al. (2024). Colchicine-induced tetraploidy in protocorms of Aerides rosea Lodd. ex Lindl. and Paxton. and its identification. Plants. 13(24): 3535. 

  29. Xu, B. (2020). Cultivation of Alfalfa Haploid and Analysis of the Hybrid Fertility and Heterosis of Main Characters. [Doctoral  Dissertation, Inner Mongolia Agricultural University]. China National Knowledge Infrastructure.

  30. Zhang, J., Cheng, C., Xiao, F., Zhang, X., Zhang, C., Zhao, Y., Wang, X. et al. (2024). Effects of ploidy level on leaf morphology, stomata and anatomical structure of Hibiscus syriacus L. BMC Plant Biology. 24(1): 1133. 

  31. Zhang, Q., Zhang, F., Li, B., Zhang, L. and Shi, H. (2016). Production of tetraploid plants of Trollius chinensis bunge induced by colchicine. Czech Journal of Genetics and Plant Breeding. 52(1): 34-38. 

  32. Zhang, X.L. (2006). Studies on The Polyploid Induction of Psathyrostachys perennis Keng. cv. Shandan. [Inner Mongolia Agricultural University Master’s Thesis]. China National Knowledge Infrastructure.

  33. Zhang, Y., Du, A., Tong, L., Yan, G., Lu, L., Yin, Y., Tu, S. et al. (2024). Genome resequencing for autotetraploid rice and its closest relatives reveals abundant variation and high potential in rice breeding. International Journal of Molecular Sciences. 25(16): 9012. 

  34. Zhou, X.Y., Liu, Y.F., Qiu, R., Han, H.J., Liu, Y.L. and Zhao, Y. (2022). Cloning and expression analysis of MfMYB30 gene from Medicago falcata L. cv. hulunbuir. Acta Bot. Boreal. Occident. Sin. 42(9): 1495-1503.

  35. Zhu, M.X., Liu, M., Zhang, J., Wu, Y.F. and Huo, X.Z. (2022). Effects of colchicine on seed germination and growth characteristics of Taraxacum officinalis. Journal of Yibin University. 22(6): 106-109. 
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    Full Research Article

    Evaluation of Colchicine Dosage Effects on Autopolyploid Induction in Diploid Medicago sativa ssp. Falcata

    X
    Xuefang Zhang1
    R
    Ruru Shi2
    F
    Fengling Shi1,*
    1Key Laboratory of Grassland Resources, Ministry of Education, College of Grassland Science, Inner Mongolia Agricultural University, Hohhot 010018, China.
    2Institute of Forest and Grassland Genetics and Breeding, Inner Mongolia Academy of Forestry Science, Hohhot 010018, China.

    • Submitted24-08-2025|

    • Accepted07-04-2026|

    • First Online 17-04-2026|

    • doi 10.18805/LRF-898

    ABSTRACT

    Background: Medicago sativa subsp.falcata (yellow-flowered alfalfa), a perennial leguminous forage crop, has inherent agronomic limitations, including slow growth, low biomass yield and difficulties in cultivation. The development of polyploid germplasm is a promising strategy for varietal improvement, requiring systematic optimization of colchicine induction protocols.

    Methods: Using imbibed seeds of diploid Medicago sativa subsp. falcata (2 n=16), we performed colchicine soak treatments and systematically characterized mutagenized plants via stomatal morphology analysis and chromosome counting. Our quantitative assessment revealed concentration and exposure time-dependent effects on seed germination, seedling development and polyploidization efficiency.

    Result: The results showed that increasing colchicine concentrations led to a progressive inhibition of seed germination and seedling growth. This was evidenced by a delay in germination initiation, a reduction in germination rates, an increase in hypocotyl diameter and a shortening of radicle length. After treatment with 0.15% colchicine for 6 hours, chromosome doubling was observed at a frequency of 10%, with a seedling survival rate of 39%.Simultaneously, the stomatal density of the treated polyploid plants reached the lowest. These results indicate that this method represents a feasible approach for polyploid breeding in this germplasm.

    INTRODUCTION

    Chemical mutagens are specialized compounds that alter DNA structure or function in organisms. These agents induce genetic mutations through diverse mechanisms, including chromosome doubling and the insertion or deletion of DNA segments (Ren, 2023). Among diverse chemical mutagens, colchicine exhibits a unique mode of action: it disrupts spindle formation during mitosis, thereby inducing chromosome doubling (Zhang et al., 2016). Owing to its low cost, high mutagenic efficiency and broad applicability across genetic materials, colchicine has become one of the most widely used chemical mutagens in practice. Following genome doubling, plants typically undergo a series of phenotypic and physiological modifications. These alterations include enhanced growth rates, improved genetic traits and increased metabolite accumulation (Li et al., 2024). Consequently, induced autotetraploids exhibit multidimensional differences compared to their diploid progenitors, spanning morphological, anatomical, physiological and agronomic characteristics. Polyploid plants exhibit characteristic phenotypic enhancements, including thicker, larger leaves with intensified pigmentation, elevated chlorophyll content, enlarged floral organs and fruits, thickened vascular bundles, as well as significantly improved stress tolerance and increased biomass and bioactive compound production (Lei et al., 2023; Veronesi et al., 1986; Zhang et al., 2024; Qiu et al., 2020).
           
    Researchers have successfully induced polyploidy in various forage species-including Melilotoides ruthenica (Wang et al., 2013), Medicago sativa (Li et al., 2016) and Lolium perenne (Ferreres et al., 2019) -using colchicine and established corresponding mutant libraries (Darmadi et al., 2025). These mutagenized collections have not only provided valuable materials for functional genomic studies but also served as novel germplasm resources for cultivar development (Francis et al., 1989).
           
    Yellow-flowered alfalfa (Medicago sativa subsp. falcata), a perennial leguminous forage crop (Wang et al., 2008), has been widely cultivated for its exceptional cold and drought tolerance. In China, extensive research efforts have focused on this species, including analyses of genetic diversity in germplasm resources (Li et al., 2022; Paun et al., 2024), improvement of local cultivars (Zhou et al., 2022; Li et al., 2025) and investigations into stress resistance mechanisms (Cui et al., 2019; Jia et al., 2025; Han et al., 2025). In this study, diploid Medicago sativa subsp. Falcata (2n = 16) germplasm from Xinjiang was used to investigate the effects of colchicine on polyploidization. Seeds were treated with various concentrations of colchicine for different durations to evaluate the following parameters: (i) seed germination rate, (ii) radicle growth dynamics, (iii) seedling survival rate, (iv) frequency of chromosome doubling and (v) stomatal morphological characteristics. This comprehensive profiling establishes an optimized colchicine induction protocol for efficient polyploid generation, providing a foundational mutagenized population for genetic improvement of this stress-tolerant forage crop.

    MATERIALS AND METHODS

    Plant material

    The plant material used in this study consisted of wild Medicago sativa subsp.falcata. seeds (2n=16) collected from Xinjiang, China in 2021. Seeds were stored at 4°C with a thousand-seed weight of 0.9096 g. All experiments were conducted in 2024 at the Life Sciences Building ,Inner Mongolia Agricultural University (geographic coordinates: 40°83'N, 111°73'E).
     
    Seed pre treatment
     
    Plump, uniformly-sized diploid Medicago sativa subsp. falcata. seeds were selected and scarified with 30-grit sandpaper to disrupt the cuticular layer and remove surface contaminants. Seeds were subsequently surface-sterilized in 70% (v/v) ethanol for 30 s to mitigate fungal contamination during germination, followed by three thorough rinses with sterile distilled water and air-drying in sterile Petri dishes until no surface moisture remained. Pretreated seeds were transferred to sterile 90-mm dishes (100 seeds per dish; three biological replicates per treatment) and stratified at 4°C in a controlled climate chamber for 24 h to synchronize imbibition. Seeds exhibiting >90% volumetric expansion were identified as fully imbibed, blot-dried on Whatman ® No.1 sterile filter paper and immediately exposed to colchicine solutions to initiate polyploid mutagenesis.
     
    Colchicine treatment
     
    Imbibed Medicago falcata seeds were treated with colchicine solutions prepared in distilled water at concentrations of 0.05%, 0.1%, 0.15% and 0.2% (v/v), each supplemented with 1% DMSO as a penetration enhancer (Table 1 for experimental design). Seeds were immersed in sterile conical flasks containing 10 ml of the assigned colchicine solution per flask, with distilled water-treated samples serving as controls (CK). All flasks were sealed with Parafilm®, wrapped in aluminum foil for light exclusion and agitated at 100 rpm and 20°C for specified durations (3, 6, 9, 12, or 24 h). Post-treatment, seeds were rinsed under running water for 40 min followed by three sterile-water washes. Finally, seeds were plated on Petri dishes and incubated at 25°C under a 16-h light/8-h dark photoperiod.

    Table 1: Colchicine treatment conditions.


     
    Seed germination characteristics and radicle growth analysis
     
    Germination was monitored daily over 7 days, with recordings of: (i) seed germination progress, (ii) cumulative number of germinated seeds and (iii) radicle development. Germination energy and seedling fresh weight were quantified on day 7. The experiment was terminated on day 15, at which point final germination percentages were calculated.
     






     
    Germination index (GI) = Σ (Gt /Dt )


     
     
    (Gt = Number of seeds germinated on day t (dimensionless) Dt = Corresponding germination day (day)
     
    Vigor index (%) = Germination index × Seedling fresh weight × 100%


    Chromosomal analysis of root tip cells following colchicine treatment
     
    (1) Root/shoot tip collection: Fresh root and shoot tips  (~25 mm in length) were excised at 09:00 local time and immediately transferred to ice-chilled centrifuge tubes filled with distilled water. Samples were held at 4°C for 24 h to synchronize cell cycles before fixation.
     
    (2) Fixation: Samples were immersed in freshly prepared Carnoy’s fixative (95% ethanol: glacial acetic acid, 3:1 v/v) for 24 h at room temperature. After fixation, residual fixative was thoroughly removed by three 5-minute washes with distilled water under gentle agitation.
     
    (3) Acid hydrolysis: Fixed root and shoot tips were transferred to 1.5-mL microcentrifuge tubes containing 1 M hydrochloric acid (HCl) and incubated at 60°C for 8 min in a precision water bath (Julabo SW23). Following hydrolysis, residual acid was removed by three 2-minute washes with distilled water under gentle agitation (100 rpm).
     
    (4) Slide preparation: The excised root/shoot tips were placed on glass slides and treated with 45% acetic acid solution. Cover slips were then applied and the samples were gently tapped with forceps until the tips dispersed into a homogeneous cloud-like suspension.
     
    (5) Cytogenetic analysis: Ten cells per root tip were analyzed systematically at 1000× magnification. Chromosome counts were recorded for each cell and the proportions of diploid (2 n), mixoploid and tetraploid (4 n) cells were calculated relative to the total cell population.
     
    Treatment intensity (TI) = Exposure duration (h) × Colchicine concentration (%)
     
    Seedling emergence rate
     
    Germinated seeds were transplanted into 10-cm-deep nursery trays filled with growth substrate (fertilizer: vermiculite: volcanic rock, 1:1:1 w/v). The relative survival rate of seedlings was quantified 30 days after transplantation.



     
    Stomatal density quantification
     
    For stomatal observations, fully expanded leaves of comparable size were sampled simultaneously from identical nodal positions. The abaxial epidermis was prepared using transparent adhesive tape (Xu, 2020) and imaged under an Olympus BX53 microscope with a 40× objective. Wild-type diploid Medicago sativa subsp. falcata from Xinjiang was used as the control.
     
    Statistical and computational analyses
     
    Statistical analyses were performed using Microsoft Excel 2021 and SPSS 26.0, with data visualization conducted in Origin 2022. Chromosomal images of diploid Medicago sativa subsp.falcata were processed using Photoshop for enhanced clarity.

    RESULTS AND DISCUSSION

    Dose-and time-dependent effects of colchicine on germination in diploid M. sativa subsp.falcata
     
    As shown in Fig 1, seeds treated with colchicine for 3 hours exhibited no significant difference in relative germination rate (RGR) compared to the untreated control group (P>0.05) at any of the tested concentrations, which may be attributed to the limited physiological impact of short-duration, low-concentration treatments (Xu, 2020). In contrast, extended exposure durations (6, 9, 12 and 24 hours) resulted in concentration-dependent decreases in RGR, with the most substantial inhibition observed at the highest concentration-time combination. The lowest RGR (46.40%) was recorded under the most severe treatment conditions (0.2% colchicine for 24 hours). Furthermore, within each concentration group, prolonged treatment also led to time-dependent reductions in germination performance. Similarly, relative germination energy (RGE) showed significant dose-and time-dependent declines (P<0.05), demonstrating a progressive decrease with increasing colchicine concentration and exposure duration. The inhibitory effects of prolonged high-concentration treatments on RGR, RGE and relative vigor index were consistent with previous findings in Medicago erecta (Wang, 2013), Medicago lupulina and Medicago sativa (Liu, 2014). The phytotoxicity of colchicine appears to be positively correlated with both concentration and treatment duration, wherein excessively high concentrations or overly extended exposure times cause substantial damage to seeds (Harbard, 2012).

    Fig 1: Germination responses to colchicine concentration and duration.


     
    Dose- and time-dependent effects of colchicine on seed vigor in diploid M.sativa subsp. falcata
     
    Fig 2 demonstrates that the relative seed vigor index (RSVI) was significantly lower in all treatment groups compared to the control (P<0.05) The RSVI progressively decreased with increasing colchicine concentrations and prolonged exposure durations. The most severe suppression of RSVI (23.10%) was observed at the highest concentration and longest duration (0.2% for 24 h).

    Fig 2: Seed vigor responses to colchicine gradients and exposure durations.


     
    Dose- and time-dependent effects of colchicine on radicle growth in diploid M.sativa subsp.falcata
     
    During polyploid induction in plants using the seed-soaking method, colchicine often causes significant toxic effects on the root system. In this study, treatment of Medicago falcata seeds with colchicine resulted in a significant reduction in the average radicle length-by more than 65% compared with the untreated control group (P<0.05; Fig 3). Concurrently, the hypocotyl diameter showed a gradual increase with higher colchicine concentrations and longer exposure durations. Under the treatment with 0.2% colchicine for 9 h, the average hypocotyl diameter reached a maximum value of 1.33 mm, representing an increase of 0.83 mm compared to the control group (Fig 4). These morphological responses are consistent with reports by (Zhang, 2006) in Psathyrostachys juncea, as well as in Agropyron cristatum (Qalby et al., 2020) and Medicago sativa subsp.falcata. (Liu, 2014), which described typical cytological abnormalities such as swollen coleoptiles and bulbous root tips. Further analysis indicated that higher concentrations and extended treatment durations exacerbated colchicine-induced damage to hypocotyl tissues. Treated radicles commonly exhibited yellowing and necrosis, loss of regenerative capacity and failure of hypocotyls to initiate new roots, ultimately leading to severely impeded root development and significantly reduced seedling survival. Similar phenomena have been reported in colchicine mutagenesis experiments in Taraxacum (Zhu, 2022). The underlying mechanism involves colchicine’s disruption of spindle formation, which inhibits normal cell division. Meanwhile, ongoing cellular metabolic activities promote abnormal cell expansion, resulting in structural thickening of the hypocotyl and radicle. Notably, the increased radicle diameter may serve as an indirect morphological indicator of chromosomal variation, providing preliminary phenotypic evidence for successful polyploid induction.

    Fig 3: Radicle elongation under colchicine treatment.



    Fig 4: Hypocotyl thickening rate under colchicine treatment.


     
    Colchicine effects on chromosome numbers in diploid M.sativa subsp. falcata root tips
     
    Polyploidy is inherently characterized by chromosome number doubling and chromosome microscopic observation and counting thus represent one of the most accurate identification methods (Li et al., 2016). The success of this technique hinges on the selection of an optimal fixation time to ensure that the observed materials are in an active stage of cell division. In this study, radicles were excised daily from 9:00 to 10:00, a time window corresponding to the peak of cell division during Medicago falcata seed germination (Li et al., 2015). By comparing the chromosome numbers in the root tips of M. falcata treated with colchicine at different concentrations and for various durations, the tetraploid induction rate was found to increase progressively with rising colchicine concentrations and extended treatment times. Specifically, the results (Table 2) showed that treatment with 0.15% colchicine for 6 h resulted in a tetraploid cell proportion of 10.00%, while the maximum tetraploid ratio (30.00%) was achieved at an elevated concentration of 0.2% with a prolonged treatment duration of 12 h. These findings are consistent with those reported in colchicine-induced polyploidy studies on Medicago sativa (Basu et al., 2018), Medicago sativa cv. Jinda (Ji et al., 2012) and Melilotoides ruthenica (Kong et al., 2010). Furthermore, mixoploids first appeared when the treatment intensity (concentration × time) reached or exceeded 0.0045, whereas tetraploids could be induced when the treatment intensity surpassed 0.009 (Fig 5).

    Table 2: Chromosomal analysis of diploid M. falcata under colchicine treatment.



    Fig 5: Ploidy identification by root tip squashing method.


     
    Dose-and time-dependent effects of colchicine on relative seedling survival in diploid M.sativa subsp. falcata
     
    However, seedling survival rate showed a negative correlation with tetraploid induction rate, that is, a higher tetraploid induction rate was associated with a lower survival rate (Wang et al., 2024). This finding is consistent with the results of Li for colchicine-treated common alfalfa (Medicago sativa), where a negative correlation between tetraploidy rate and seedling survival was likewise observed (Li et al., 2016). Therefore, when determining the optimal mutagenic conditions for chromosome doubling in yellow-flowered alfalfa (Medicago falcata), it is essential to not only consider the tetraploid induction rate, but also to comprehensively evaluate key indicators such as seed relative vigor index and seedling survival rate. A comprehensive analysis in this study indicated that treatment with 0.15% colchicine for 6 h effectively induced tetraploidy, while resulting in a higher seedling survival rate compared to other treatment combinations that also induced tetraploids. As shown in Fig 6, the relative survival rate of yellow-flowered alfalfa seedlings exhibited a declining trend with increasing colchicine concentration and extended treatment duration. When the concentration reached 0.2% with treatment times of 12 h or 24 h, nearly complete mortality of the seedlings was observed.

    Fig 6: Seedling survival under colchicine treatment.


     
    Dose- and time-dependent effects of colchicine on stomatal morphology in diploid M.sativa subsp.falcata
     
    Polyploid plants commonly exhibit increased cell volume. Normal cell growth and function rely on a dynamic balance between the nucleus and cytoplasm (Pacey et al., 2020). From Fig 8.1 and 8.2, as plant ploidy increases, the nuclear volume expands accordingly. To maintain a stable nucleo-cytoplasmic ratio and cellular functions, the cytoplasmic volume must also increase, leading to an overall enlargement of the cell. Stomata are formed by a pair of guard cells and the increase in their size directly results in larger stomatal dimensions (Zhang et al., 2024). Previous studies have confirmed that stomatal size, guard cell volume and chloroplast number are significantly positively correlated with ploidy level, whereas stomatal density is negatively correlated with ploidy (Padoan et al., 2013). For instance, (Francis et al., 1974) demonstrated that stomatal traits can be used effectively to differentiate ploidy levels in Cucurbita species. In the present study, stomatal density in leaves of mutagen-treated seedlings showed a significant difference compared to the control group (P<0.05). As shown in Fig 7, the lowest stomatal density was observed after treatment with 0.1% colchicine for 9 h, suggesting a higher chromosome doubling efficiency under this treatment condition.

    Fig 7: Stomatal density responses to colchicine treatment.



    Fig 8.1: Stomatal morphology in untreated leaf epidermis of M. sativa subsp. falcata.



    Fig 8.2: Stomatal morphology in colchicine-treated leaf epidermis of M. sativa subsp. falcata.

    CONCLUSION

    (1)   Colchicine treatment of imbibed diploid Medicago sativa subsp.falcata seeds (2 n=16) induced significant morphological alterations, including increased hypocotyl diameter (P<0.05) and reduced radicle length compared to untreated controls.
           
    (2) Colchicine treatment induced chromosomal variations in the root tip cells of alfalfa, resulting in multiple cytotypes (2 n = 2x = 16 ± 3, 2x < T < 4x, 2 n = 4x = 32±3). Among the tested regimens, treatment with 0.15% colchicine for 6 h was identified as the optimal dosage for tetraploid induction.
           
    Future research scope and directionsÿBased on the present findings, future research will focus on the following directions: First, the optimized treatment conditions will be adopted to induce polyploidy in diploid Medicago sativa subsp.falcata. seeds, with a systematic assessment of the intergenerational genetic stability of ploidy levels and key agronomic traits for screening stable, elite germplasm. Second, molecular markers coupled with whole-genome sequencing will be used to analyze genomic variation during colchicine-induced chromosome doubling and identify key genes associated with important agronomic traits.

    ACKNOWLEDGEMENT

    FL.S. designed the study; XF.Z. performed the experiments and wrote the manuscript. This work was financially supported by the Major Demonstration Project for Open Competition in Seed Industry Science and Technology Innovation of Inner Mongolia (Grant No. 2022JBGS0016). Additional support was provided by the Key Laboratory of Grassland Resources, Ministry of Education, China.
     
    Disclaimers
     
    The views and conclusions presented in this article are those of all authors and do not necessarily represent the official positions of their affiliated institutions.The authors are responsible for the accuracy and completeness of the information provided, but shall not be held legally liable for any direct or indirect losses arising from the use of this content.

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

    REFERENCES


    1. Basu, S. and Lodh, D. (2018). Artificial induction of polyploidy in Andrographis paniculata (Burm. F.) wall. ex nees. The Journal of Indian Botanical Society. 97(1 and 2): 21-28.

    2. Cui, G., Chai, H., Yin, H., Yang, M., Hu, G., Guo, M., Zhang, P. et al. (2019). Full-length transcriptome sequencing reveals the low-temperature-tolerance mechanism of Medicago falcata roots. BMC Plant Biology. 19(1): 575. 

    3. Darmadi, D. and Fathurrahman, F. (2025). Identification of Phenotypic Nature of Rainbow Corn (Zea mays L.) Due to Colchicine Treatment. In IOP Conference Series: Earth and Environmental Science. IOP Publishing. 1478(1): 012002.

    4. Ferreres, I., Ortega, M., López-Cristoffanini, C., Nogués, S. and Serrat, X. (2019). Colchicine and osmotic stress for improving anther culture efficiency on long grain temperate and tropical japonica rice genotypes. Plant Biotechnology. 36(4): 269-273. 

    5. Francis, A. and Jones, R.N. (1989). Heritable nature of colchicine induced variation in diploid Lolium perenne. Heredity. 62(3): 407-410. 

    6. Francis, R.R. and Bemis, W.P. (1974). Stomatal size in induced polyploids in cucurbita1. Hort Science. 9(2): 138-138.

    7. Han, B., Duan, X., Jiang, H., Zhao, Y.,  Wang, Y., Hu, T. and Yang, P. (2025). Molecular cloning and functional analysis of MsDUF from alfalfa (Medicago sativa L.). Legume Research. 48(2): 246-253. doi: 10.18805/LRF-821.

    8. Harbard, J.L., Griffin, A.R., Foster, S., Brooker, C., Kha, L.D. and Koutoulis, A. (2012). Production of colchicine-induced autotetraploids as a basis for sterility breeding in Acacia mangium Willd. Forestry: An International Journal of Forest Research. 85(3): 427-436.

    9. Ji, R.H. (2012). Study on Chromosome Doubling Technique of M. sativa subsp. Caerulea (Less. Ex Ledeb.) Schmalh cv. Jindera. [Inner Mongolia Agricultural University Master’s Thesis]. China National Knowledge Infrastructure.

    10. Jia, M., Mu, Y., Yang, Z., Shao, J., Shi, F. and Yun, L. (2025). Cold resistance evaluation of alfalfa during the germination and seedling stages and analysis of its relationship with fall dormancy. Legume Research: An International Journal. 48(10): 1656-1662. doi: 10.18805/LRF-875.

    11. Kong, S.Y. (2010). Studies on Polyploid Induction of Melilotoides Ruthenica Strains. [Inner Mongolia Agricultural University Master’s Thesis]. China National Knowledge Infrastructure.

    12. Lei, L., Liu, G., Yan, D., Zhang, M., Cui, Q., Zhao, Q., Xu, G. et al. (2023). Manipulation of ploidy for blueberry breeding: in vitro chromosome doubling of diploid vaccinium duclouxii (Lévl.) Hand.-Mazz by trifluralin. Scientia Horticulturae. 317: 112056. 

    13. Li, C.J., Sun, Y.L., Wang, Y.X., Cheng, A.P. and Zhang, B. (2022). Genetic diversity of agronomic traits of 50 Medicago falcata L. germplasm. Journal of Xinjiang Agricultural University. 45(3): 173-181.

    14. Li, H., Wang, L., Li, J., Sun, X., Lin, D. and Wang, Z. (2025). Alfalfa autotoxicity: Role of cultivar renovation in mitigating germination and seedling growth under continuous cropping systems. Legume Research: An International Journal. 48(5): 828-834. doi: 10.18805/LRF-837.

    15. Li, Y. and Shi, S.L. (2016). The mutagenic effects of colchicine treatment on chromosome ploidy and the leaf ep-idermal cells of Medicag sativa cv qingshui. Acta Prataculturae Sinica. 25(2): 141-149.

    16. Li, Y., Yin, L., He, X., Hu, C., Wu, R., Long, Q., Yuan, D. et al. (2024). Ploidy and fruit trait variation in oil-tea Camellia: Implications for ploidy breeding. Journal of Integrative Agriculture. 23(8): 2662-2673. 

    17. Liu, Q. (2014). 6 Polyploidy Induction and Ploidy Identification of 6 medicago SPP. Germplasms. [Inner Mongolia Agricultural University Master’s Thesis]. China National Knowledge Infrastructure.

    18. Liu, S.R., Lin, Y. and Luo, J.Z. (2015). Effect of different pretreatment methods of corymbia citriodora chromosome technology. Eucalypt Science and Technology. 32(4): 27-31.

    19. Pacey, E.K., Maherali, H. and Husband, B.C. (2020). The influence of experimentally induced polyploidy on the relationships between endopolyploidy and plant function in Arabidopsis thaliana. Ecology and Evolution. 10(1): 198-216. 

    20. Padoan, D., Mossad, A., Chiancone, B., Germana, M.A. and Khan, P.S.S.V. (2013). Ploidy levels in Citrus clementine affects leaf morphology, stomatal density and water content. Theoretical and Experimental Plant Physiology. 25: 283- 290. 

    21. Paun, G., Neagu, E., Alecu, A., Albu, C., Seciu-Grama, A.M. and Radu, G.L. (2024). Evaluating the antioxidant and antidiabetic properties of Medicago sativa and Solidago virgaurea polyphenolic-rich extracts. Molecules. 29(2): 326.

    22. Qalby, N., Sjahril, R., Dachlan, A. and Sakae, A. (2020). Colchicine Induced Polyploidy in Common Ice Plant Mesembryanthemum crystallinum L. In: IOP Conference Series: Earth and Environmental Science. IOP Publishing. 486(1): 012097.

    23. Qiu, Y., Hamernick, S., Ortiz, J.B. and Watkins, E. (2020). DNA content and ploidy estimation of Festuca ovina accessions by flow cytometry. Crop Science. 60(5): 2757-2767.

    24. Ren, W.B., Fu, C.X., Lin, H., Guo, H.Q., Yuan, F., Liu, X.Q. and Liu, Y.L. (2023). Technological Innovation and Application of Alfalfa Mutagenesis Breeding Technology.

    25. Veronesi, F., Mariani, A. and Bingham, E.T. (1986). Unreduced gametes in diploid Medicago and their importance in alfalfa breeding. Theoretical and Applied Genetics. 72(1): 37-41. 

    26. Wang, J.J., Yun, J.F. and Lv, S.J. (2008). Preliminary study on physiological responses of salt tolerance in Sickle Alfalfa. Journal of Arid Land Resources and Environment. 22(12): 158-163. 

    27. Wang, L., Shi, F.L., Bian, X.Y., Yi, F.X. and Gao, X. (2013). Analyzing the Mutative Effects of Medicago ruthenica (L.) sojak. cv. zhilixing treated by colchicine. Seed. 32(10): 50-53.

    28. Wang, L., Zheng, P., Ge, H., Zhao, X., Kou, Y., Yang, S., Jia, R. et al. (2024). Colchicine-induced tetraploidy in protocorms of Aerides rosea Lodd. ex Lindl. and Paxton. and its identification. Plants. 13(24): 3535. 

    29. Xu, B. (2020). Cultivation of Alfalfa Haploid and Analysis of the Hybrid Fertility and Heterosis of Main Characters. [Doctoral  Dissertation, Inner Mongolia Agricultural University]. China National Knowledge Infrastructure.

    30. Zhang, J., Cheng, C., Xiao, F., Zhang, X., Zhang, C., Zhao, Y., Wang, X. et al. (2024). Effects of ploidy level on leaf morphology, stomata and anatomical structure of Hibiscus syriacus L. BMC Plant Biology. 24(1): 1133. 

    31. Zhang, Q., Zhang, F., Li, B., Zhang, L. and Shi, H. (2016). Production of tetraploid plants of Trollius chinensis bunge induced by colchicine. Czech Journal of Genetics and Plant Breeding. 52(1): 34-38. 

    32. Zhang, X.L. (2006). Studies on The Polyploid Induction of Psathyrostachys perennis Keng. cv. Shandan. [Inner Mongolia Agricultural University Master’s Thesis]. China National Knowledge Infrastructure.

    33. Zhang, Y., Du, A., Tong, L., Yan, G., Lu, L., Yin, Y., Tu, S. et al. (2024). Genome resequencing for autotetraploid rice and its closest relatives reveals abundant variation and high potential in rice breeding. International Journal of Molecular Sciences. 25(16): 9012. 

    34. Zhou, X.Y., Liu, Y.F., Qiu, R., Han, H.J., Liu, Y.L. and Zhao, Y. (2022). Cloning and expression analysis of MfMYB30 gene from Medicago falcata L. cv. hulunbuir. Acta Bot. Boreal. Occident. Sin. 42(9): 1495-1503.

    35. Zhu, M.X., Liu, M., Zhang, J., Wu, Y.F. and Huo, X.Z. (2022). Effects of colchicine on seed germination and growth characteristics of Taraxacum officinalis. Journal of Yibin University. 22(6): 106-109. 
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