In vitro tetraploid plants regeneration from leaf explants of multiple genotypes in Populus

The present study develops a protocol for tetraploids of multiple genotypes induced from in vitro leaf explants of diploid full-sib progeny [(P. pseudo-simonii × P. nigra ‘Zheyin3#’) × (P. × beijingensis)] after a colchicine treatment. Leaf explants from a ten genotypes full-sib progeny were cultured in MS basal medium containing with 1.78 μM BA and 0.27 μM NAA for 4, 5, and 6 days and transferred to the same liquid MS medium containing different concentrations of colchicine (50, 75, or 100 μM) for 2, 3, and 4 days, respectively. The results indicate that preculture duration, colchicine concentration, and exposure time had significative impacts in tetraploid induction rate but no significant correlation with the genotype. The feasible protocol involves treating a leaf explants pre-culture for 5 days followed by their transfer to liquid MS with 75 μM colchicine for 3 days, for most genotypes of full-sib progeny which could regenerate from leaf explants in the shoot regeneration medium. Tetraploid plantlets are identified using flow cytometric analysis and further confirmed after chromosome counts. Size and frequency of leaf stomata was significantly distinguish the tetraploid from the diploid plants.