Agrobacterium-mediated Transformation Of Chinese Jujube

Chinese jujube (Ziziphus jujuba Mill.) is an important native fruit tree of China. Conventional croos is very difficult in Chinese jujube for reasons of very low fruit-setting, very high embryo abortion as well as very small flower. With the development of biotechnology, genetic engineering has become an effective means for modern plant breeding improvement.This study is aimed at to establishing an Agrobacterium-mediated genetic transformation system and laying a foundation for genetic engineering in Chinese jujube. The main results of the study are as follows:1. An optimized regeneration system for anther wall of Z. Jujuba Mill.'Dongzao'was established. The optimal medium for inducing callus from anther is 1/2MS+1.0mg/L 2,4-D+20g/L maltose+5.5g/L agar. The optimal medium of callus proliferation is MS+0.4mg/L TDZ+0.2mg/L NAA+20g/L maltose+5.5g/L agar. The combination of 1.0 mg/L TDZ and 0.4 mg/L NAA is the best one to shoots induction from callus with the highest shoot induction rate (100%) and a high number of shoots per callus (3.9). The combination of 0.2 mg/L BA, 0.1 mg/L IBA and 1.5 mg/L PVA promoted the highest rate of hyperhydric shoots restoration (48.9%).The highest rooting (43.3%), and highest root length (4.2 cm) was recorded at 1.5 mg /l IAA.2. A high efficient regeneration system using anther wall of Z. Jujuba Mill.'Yueguang'was firstly established. The optimal medium for inducing callus for anther wall was 1/2MS+1.0mg/L 2,4-D+20g/L maltose+5.5g/L agar. The optimal medium of callus proliferation is MS+0.6mg/L TDZ+0.2mg/L NAA+20g/L maltose+5.5g/L agar. The optimal differentiation medium of callus was MS+5.5g/L agar+20g/L maltose+0.8mg/L TDZ+4.0mg/L AgNO3with the highest shoot induction response (100%) and highest number of shoots per callus (10.4). The combination of 0.2 mg/L BA, 0.1 mg/L IBA and 1.5 mg/L PVA promoted the highest rate of hyperhydric shoots restoration. The highest rooting (43.3%), and highest root length (4.2 cm) was recorded at 2.0 mg /L IBA. The addition of GA3 inhabited the growth of callus and hyperhydric shoots restoration.3.The genetic transgenic system of'Dongzao'leaf mediated by Agrobacterium tumefacienswas investigated and the optimal conditions were determined as follows: bacterial concentration OD600 0.8, infection time 20 min, co-culture 3 days and the co-culture medium MS+20g/L maltose+5.5g/L agar+1.0mg/L TDZ+0.1mg/L IBA+200μΜAS.Two insect-resistant genes (Bt gene and protease inhibitor gene, API) were firstly into'Dongzao'and . 8 transgenic lines have been obtained.4. The genetic transgenic system of Z. Jujuba Mill.'Dongzao'and'Dongzao'with witches'broom disease (JWB) mediated by Agrobacterium rhizogenes were firstly established. For'Dongzao'leaf, the optimal conditions were determined as follows: bacterial concentration OD600 0.8, infection time 20 min, co-culture 3-5 days, the co-culture medium was 1/2MS+30g/L sugar+5.5g/L agar+4.0mg/L IBA and the medium of inducing hairy root was 1/2MS+30g/L sugar+5.5g/L agar+100mg/L CRO under a 14-h light and 10-h dark cycle. For stems of diseased'Dongzao', the optimal conditions were determined as follows: bacterial concentration OD600 0.8 with activation of 50μΜAS for 1-2h before infection, infection time 5-10 min, co-culture 5 days, the co-culture medium was MS+30g/L sugar+5.5g/L agar and the medium of inducing hairy root was MS+30g/L sugar+5.5g/L agar+100mg/L CRO under under a 14-h light and 10-h dark cycle. This is the first time to thansform Chinese jujube with JWB into healthy one via Agrobacterium rhizogenesinfection. 30 recovering plantlet lines have been obtained.