Colchicine-induced tetraploid plant in Carissa carandas L.: insights from cytological, morphological, biochemical, and molecular marker analyses

Karonda ( Carissa carandas L.) is an underutilized nutraceutical fruit crop valued for its high vitamin, mineral, and antioxidant contents as well as its adaptability to marginal environments. However, genetic improvement in this species has been limited due to its narrow genetic base and the scarcity of improved cultivars. Artificial polyploidization offers an effective approach to generate novel genetic variation and enhance desirable traits in perennial fruit crops. The present study aimed to induce tetraploidy in karonda using colchicine and to characterize the resulting plants through cytological, morphological, biochemical, and molecular analyses. Colchicine treatment significantly reduced seed germination and seedling survival in a concentration-dependent manner, indicating the cytotoxic effects of the antimitotic agent during early development. Flow cytometric analysis confirmed the successful induction of tetraploidy in a single seedling obtained from the 0.3% colchicine treatment, showing a two-fold increase in nuclear DNA content relative to the diploid control. The induced tetraploid plant exhibited distinct morphological alterations including increased plant height, stem diameter, internodal length, leaf width, leaf area, and leaf thickness. Biochemical analyses revealed higher total chlorophyll content, phenolic compounds, proline accumulation, and catalase activity in the tetraploid plant compared with diploid plants, whereas flavonoid content and activities of peroxidase and superoxide dismutase showed slight decreases. Stomatal analysis indicated larger stomata and reduced stomatal density, consistent with typical polyploid characteristics. Molecular marker analysis using ISSR and SRAP primers revealed distinct polymorphic banding patterns between diploid and tetraploid plants, suggesting genomic alterations associated with chromosome doubling. The present study provides an initial report of colchicine-induced tetraploidy in Carissa carandas and describes morphological, physiological, stomatal, and molecular characteristics observed in the recovered tetraploid plant. However, because only one confirmed tetraploid individual was obtained and the material originated from seed-derived plants, the observed responses should be interpreted cautiously as preliminary and should not be generalized to the species as a whole without validation using additional independently derived tetraploid lines. These findings highlight the potential of induced polyploidy as a promising strategy for genetic improvement of underutilized fruit crops with limited natural variability. Because only one confirmed tetraploid plant was recovered, the observed responses should be considered preliminary and require validation using additional independently derived polyploid lines.