Abstract:
Objective Hybrid sweetgum is an important timber and ornamental tree resources in China, but its genetic transformation system has not been established yet. Establishing genetic transformation system of hybrid sweetgum provides a useful approach for trait improvement and allows us to conduct a functional identification of gene in hybrid sweetgum.
Method Based on the efficient somatic embryogenesis of hybrid sweetgum, the embryogenic calluses were transformed by Agrobacterium tumefaciens-mediated genetic transformation. Factors influencing transformation were studied by orthogonal experiment, including hygromycin selective pressure, concentration of Agrobacterium, infection time, co-culture time and co-culture temperature.
Result Results showed that the minimum lethal concentration of hygromycin to embryogenic callus was 10 mg/L. The number of Gus positive spots was highest when bacterium solution (OD600) was 0.8, co-culture time was 3 d, infection time was 10 min, co-culture temperature was 25 ℃. The most transgenic positive resistant calluses were obtained when bacterium solution (OD600) was 0.2, co-culture time was 2 d, infection time was 10 min, co-culture temperature was 23 ℃. And the optimal treatment combination was 0.2 bacterium solution (OD600), 2 d co-culture time, 20 min infection time, 23 ℃ co-culture temperature.
Conclusion A total of 210 positive transgenic callus were obtained by molecular identification. The genetic transformation system was established, which provides more feasible foundation for the transformation of broadleaf tree callus.
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