<p>Artificial polyploidy is a powerful tool for overcoming genetic bottlenecks in crops. However, the immediate effects of genome doubling on the structure and function of already complex polyploid genomes, such as sugarcane, remain poorly understood. Chromosome doubling (tetraploidy) was induced in sugarcane (<i>Saccharum officinarum</i> L. cv. CP57) via an optimized in vitro colchicine treatment (50 mg L⁻ ¹ for 24 h). Survival of treated explants was 83.3%, from which confirmed chromosome-doubled (tetraploid) plants were obtained with a 3.6% induction efficiency (percentage of treated explants yielding tetraploids) as confirmed by flow cytometry. Strikingly, detailed cytogenetic analysis of the synthetic tetraploids (2n ≈ 230) revealed not only genome doubling but also a profound 58% increase in total chromosome length and a shift towards enhanced karyotype symmetry. This genomic restructuring was accompanied by systemic phenotypic reprogramming, including a 76% reduction in stomatal density, a 150% increase in stomatal length, a 58% reduction in tillering, and an 18% increase in chlorophyll content. This work successfully establishes the first confirmed tetraploid sugarcane line and demonstrates that chromosome doubling induces immediate and dramatic karyotype reorganization alongside developmental reprogramming. Our study provides both a foundational resource for sugarcane breeding and novel insights into the rapid genomic consequences of polyploidization in a complex crop genome.</p>