Abstract:Liquidambar is an important forestry species used to generate many commercial wood products, such as plywood. Inducing artificial polyploidy is an effective method to encourage genetic enhancements in forestry breeding. This report presents the first in vitro protocol for the induction of genus Liquidambar tetraploids based on the established in vitro regeneration system of hybrid sweetgum (Liquidambar styraciflua × Liquidambar formosana). The leaves and petioles from three genotypes were pre-cultured in woody plant medium (WPM) supplemented with 0.1 mg/L thidiazuron (TDZ), 0.8 mg/L benzyladenine (BA), and 0.1 mg/L α-naphthalene acetic acid (NAA) for a variable number of days (4, 6 or 8 days), and exposed to varying concentrations of colchicine (120, 160, 200 mg/L) for 3, 4 or 5 days; the four factors were investigated using an orthogonal experimental design. Adventitious shoots were rooted in 1/2 WPM medium supplemented with 2.0 mg/L indole butyric acid (IBA) and 0.1 mg/L NAA. The ploidy level was assessed using flow cytometry and chromosome counting. Four tetraploids and nine mixoploids were obtained from the leaves. Pre-treatment of the leaves for 8 days and exposure to 200 mg/L colchicine for 3 days led to the most efficient tetraploid induction. Producing 11 tetraploids and five mixoploids from petioles, the best tetraploid induction treatment for petioles was almost the same as that with the leaves, except that pre-culturing was required for only 6 days. In total, 15 tetraploids were obtained with these treatments. This study described a technique for the induction of tetraploid sweetgum from the leaves or petioles of parental material. Based on the success of polyploid breeding in other tree species, the production of hybrid sweetgum allotetraploids constitutes a promising strategy for the promotion of future forestry breeding.