Colchicine in vitro tetraploid induction of Populus hopeiensis from leaf blades

Abstract

A protocol for tetraploid in vitro induction of diploid Populus hopeiensis leaf blades by colchicine was established. Leaf blades were pre-cultured in MS basal medium containing 1.78 μM 6-BA, 0.07 μM TDZ, and 0.53 μM IAA for 5, 6, and 7 days, and then transferred to a liquid MS medium supplemented with colchicine at concentrations of 50, 75, and 100 μM for 48, 72, and 96 h. We found that pre-culture duration and exposure time had moderate effects on the tetraploid induction rate, and colchicine concentration had highly significant effects on tetraploid induction rate. The highest tetraploid induction rate (21.2%) was observed when leaf blades were pre-cultured for 7 days and cultured for 96 h in liquid MS with 100 μM colchicine. Flow cytometry analysis was used to detect putative tetraploids from the regenerated plants, and chromosome number counting was employed to confirm the polyploidy level. A total of 54 tetraploids were obtained. Significant differences in stomatal length, stomatal width, and density of leaf stomata between diploid and tetraploid plants were observed, suggesting that tetraploid plants present modified morphological characteristics. Optimization of shoot regeneration system from Populus hopeiensis leaf blades and tetraploids induction by colchicine treatment.