Genetic transformation of protoplasts from Peucedanum terebinthaceum and regeneration of herbicide-resistant plantlets

Calli produced from the segments of young petioles of Peucedanum terebinthaceum Fisch. ex Turcz. were transfered into liquid medium for suspension culture. The protoplasts were isolated from cells of clusters by enzymes digestion. The mutant als(acetolactate synthase) gene of Arabidopsis thaliana (L.) Heynh. was transferred into protoplasts of P. terebinthaceum with PEG method. A transformation medium containing 40 similar to 50 mu g/mL DNA and 10% PEG and an incubation condition of 26 degrees C for 25 minutes in dark revealed the best result. Supplementation with 0 similar to 60 mu g/mL ctDNA into the medium gave no remarkable effect on transformation frequency. After cell clumps formed from the treated protoplasts in liquid medium, they were transferred onto proliferation medium containing 0.01 mg/L chlorsulfuron. The resistant clones grew 20 days later, and were transferred onto differentiation medium containing 0.03 mg/L chlorsulfuron. Plantlets regenerated via embryoid pathway. Southern blot hybridization with the P-32 labeled 2.5 kb fragments of p35s-als demonstrated that the mutant als gene had inserted into P. terebinthaceum genome. Chlorsulfuron-resistant calli which grew normally were produced when segments of transgenic plantlets were cultured on the proliferation medium containing 0.01 mg/L chlorsulfuon, whereas the growth of untransgenic explants were inhibited. Meanwhile, the transgenic plants could stand high density of chlorsulfuron up to 0.06 mg/L, but not the wild plants. These results indicated that the exogenous als gene had integrated into P, terebinthaceum genome and could transcript and translate stably.