A Comparative Study on Colchicine Application Methods in Obtaining Doubled Haploids of Tobacco (Nicotiana tabacum L.)

In order to obtain androgenic doubled haploids of tobacco (Nicotiana tabacum cv. Karabalar 6265) colchicine was applied at 3 different stages of anther culture. Before culture, anthers were treated with 0.4% aqueous solution of colchicine for 0, 2, 4, 6, 8, 10, and 12 h. Culture response of anthers decreased as the treatment duration increased (except 12 h) and the highest diploidization of 29.7% was obtained with 6 h. During culture, when macroscopic embryoids appeared in dehisced anthers, they were wrapt up in sterile cotton saturated with 0.2% colchicine for 3 days and transferred to fresh medium. This application resulted in 60% diploidization, but plant recovery from treated embryoids was low and only 5 plants could survive. When plantlets with 4 to 8 leaves immersed in 0.2% colchicine for 0, 7, 24 and 48 h on a shaker, besides 4.3%, 42.3%, 37.8% and 33.3% doubled haploids, respectively, haploids, tetraploids, aneuploids, and mixedploids were also found among the treated plants. The main advantage of this method is that treated plantlets can be transplanted directly into pots in order to grow androgenic plants. When chromosome doubling rate and viability are taken into consideration, among the 3 methods tested, plantlet treatment with 0.2% colchicine for 7 h appeared to be more efficient with 42.3% dihaploids. Durations shorter than 7 h must be tested in order to optimize the application.