Colchicine-Induced Tetraploidy in Protocorms of Lodd. ex Lindl. and Paxton. and Its Identification.

(Orchidaceae) boasts high ornamental value due to its pleasant aroma, foxtail spike, and elegant floral morphology. Inducing to become tetraploid enhances horticultural traits and facilitates fertile intergeneric hybrids through crosses with other market-available tetraploid species. The experimental design involved the application of colchicine at varying concentrations-0.05%, 0.1%, and 0.2%-to a solid medium. Exposure durations were 5, 10, and 15 days, with treatments conducted under sterile conditions on 6-week-old protocorms post-germination. Results indicated that the protocorms were sensitive to colchicine concentrations exceeding 0.05%, with high concentrations leading to a mortality rate exceeding 50%. Flow cytometry (FCM) with 4',6-diamidino-2-phenylindole (DAPI) staining confirmed a doubling of chromosome numbers in tetraploid plants (2n = 4x = 76) compared to diploid controls (2n = 2x = 38). Induction efficiency was significantly influenced by colchicine concentration and treatment duration. A 10-day treatment with 0.2% colchicine yielded a 70.00% tetraploid induction rate; however, considering protocorm survival, a 5-day treatment with 0.05% colchicine was preferable, achieving a 63.55% survival rate and a 56.67% tetraploid induction rate. Tetraploid plants exhibited distinct morphological traits, such as a more compact growth habit, thicker leaves, and increased stem and root thickness. Leaf morphology changes included larger stomata with reduced density, denser spongy mesophyll, and more pronounced venation. Tetraploids also demonstrated a 1.94-fold increase in genome size compared to diploids. The tetraploid genotypes developed in this study hold significant potential for future breeding programs.