Induction of tetraploid ruzigrass (Brachiaria ruziziensis) plants by colchicine treatment of in vitro multiple‐shoot clumps and seedlings

Abstract Tetraploid plants of ruzigrass (Brachiaria ruziziensis) have been obtained by colchicine treatment of in vitro multiple‐shoot clumps and germinated seedlings. Multiple‐shoot clumps were induced and maintained on MS basal medium with 0.5 mg L−1 2,4‐dichlorophenoxyacetic acid (2,4‐D) and 2.0 mg L−1 6‐benzylaminopurine (BAP) according to a previous report. The colchicine treatment consisted of culturing multiple‐shoot clumps on MS basal medium containing 0.0125–0.1% colchicine for 12 and 48 h. Surviving multiple‐shoot clumps were regenerated on MS basal medium containing 2.0 mg L−1 BAP. The ploidy level of plants after colchicine treatment was determined by flow cytometry. Eight tetraploid plants were obtained from multiple‐shoot clumps treated with 0.1% colchicine for 12 h. This treatment was identified as the optimum treatment and resulted in the highest frequency (28.6%) of tetraploid plants among the treatments tested on multiple‐shoot clumps. Five tetraploid plants (31.3%) were obtained when seedlings were treated with 0.1% colchicine for 3 h. Although higher concentrations of colchicine and longer durations of colchicine treatment reduced the survival rate of the explants, such treatments increased the frequency of tetraploid plants. A total of 23 tetraploid plants were obtained from 900 different colchicine‐treated multiple‐shoot clumps, and a total of five tetraploid plants were obtained from 300 colchicine‐treated seedlings. Cytological analysis affirmed the results of flow cytometry. Significant differences in leaf blade stomata were observed between diploid and tetraploid ruzigrass.