Studies On Transforming GAI Gene Of Arabidopsis Thaliana Into Jatropha Curcas

Petioles and veins of barbadosnut were used as the explants to optimize its regeneration system. The results showed that:it was easy to induce callus and its optimal medium was MS+6-BA 0.05mg/L+NAA 0.05mg/L+sucrose 30g/L+agar 5.5g/L. The calli were cultured in bud differentiation medium MS+6-BA1.0mg/L+NAA0.05mg/L+sucrose 30g/L+agar 5.5g/L after one week. Then the 2 or 3 cm buds were cultured in rooting medium 1/2MS+IBA0.2mg /L+sucrose 20g/L+agar 5.5g/L. All that provide good basis for the barbadosnut transformation system.The Kan resistance was tested. The sensitive concentration of Kan was 200mg/L. The GAI gene of Arabidopsis thaliana which controls the plant stem elongation had been transferred into barbadosnut’s leaf callus by Particle Bombardment and Agrobacterium mediated method separately.Some factors which could impact the transgenic rate such as the acceptor materials, the co-culture time, co-culture temperature, the concentration of AS and the concentration of antibacterial antibiotics were studied in the Agrobacterium-mediated transformation method. The result showed that the calli could effectively resist the Agrobacterium contamination and they could continue to grow under the conditions that:leaf callus was cultured 5d, co-cultured at 20℃for 2d after infection, Cef concentration 200mg/L and AS 200μmol/L. The calli were cultured in the mediusms with selection pressure in them as the procedure described in the regeneration system two weeks later.In the Particle Bombardment method we found that:the pre-culture time 2d,650psi, shooting distance 6cm and 1 shoot for each sample was optimal for the transformation.Transformed seedlings were obtianed with the method of Kan selection. The PCR detection of leaves DNA from transgenic plants was positive. After being propagated several generations, the PCR testing of extracted leaves DNA was still positive.This result showed that the GAI gene had probably been integrated into the genome of barbadosnut and inherited stably. At last, genomic DNA of transformated seedlings was extracted for Southern Blot identification. The result showed that:there was no signal in the non-transformed plantlets’ genomic DNA and the transformed DNA dealt with XbaI enzyme had a clear signal in Southern blot hybridization. It showed that the GAI gene has been successfully integrated into the genome of the barbadosnut.