Abstract: In vitro induction of tetraploid in apple and the technique of chimera separation were studied by treating openpollinated mature seeds of Golden Delicious with colchicine. The most efficient condition for inducing tetraploids seemed to be treating embryos with the solution including of 0.5% colchicine and 1% dimethyl sulfone in darkness for 48 hours, and then inoculation on MS medium supplemented with TDZ 1.0 mg·L^-1, IBA 0.5 mg·L^-1, sucrose 30 g·L^-1 and agar 6 g·L^-1, with 10.6% mutation rate. Compared with the diploid, the tetraploid plants grew slowly, with shorter internode, larger leaves with larger guard cell and lower density of stomata, and smaller leaf shape index. Verification of chromosome number in shoot tip cells by pressing cover slip method and detection of relative nuclear DNA content by flow cytometry proved that most mutated plantlets are homogeneous tetraploid. It was an effective method of separating chimeras into their homogeneous ploidy by in vitro regeneration plantlets from leaf pieces.

Key words: Apple, Colchicine, In vitro, Polyploid, Tetraploid, Separation of chimera, Multiploid breeding