Annua Hairy Root Growth And Artemisinin Biosynthesis Of Fungal Elicitors And Flowering And Artemisinin Biosynthesis Correlation Study

The dissertation is mainly composed of the following two parts:Part I. Effects of fungal elicitors on cell growth and artemisinin biosynthesis in hairy root cultures of Artemisia annua L.The artemisinin biosynthesis in the hairy root cultures of Artemisia annua L. was enhanced via a treatment of three fungal elicitors separately [Verticillium dahliae Kleb., Rhizopus stolonifer (Ehrenb. ex Fr.) Vuill and Colletotrichum dematium (Pers.) Grove]. Among these three elicitors, V. dahliae had the highest inducing efficiency, but neither of them manifests any noticeable effects on the cell growth of the hairy root cultures. The artemisinin content of the hairy root cultures treated with V. dehliae elicitor was 1.12 mg/g DW, which was 45% higher than the control (0.77 mg/g DW). The results showed that elicitation was dependent on the elicitor concentration, the incubation period and the physiological stage at which the hairy root cultures were treated. In addition, we found that for V. Dahliae, the optimum concentration was 0.4 mg carbohydrate per milliliter medium, the strongest response of A. annua hairy root cultures to the elicitation was at the late exponential growth stage, and the highest artemisinin content of the hairy root cultures was on the 4th day post treatment.Part II. Effects of flowering-promoting genes FPF1 -. CO on Artemisia annua flowering time and on the linkage between flowering and artemisinin biosynthesis.1. The Flowering Promoting Factorl (FPF1) gene from Arabidopsis thaliana was transferred into Artemisia annua L. via Agrobacterium tumefaciens. The FPF1 gene was placed in the binary vector pBI121 under the control of CaMV 35S promoter to construct the plant expression vector pBI121FPF7. Leaf explants of A. annua were infected with A. tumefaciens LBA4404 containing pBI121FPFl to induce the buds. Transgenic plants were obtained through the selection with kanamycin. PCR> PCR-Southern blot and Southern blot confirmed that the foreign FPF1 gene had been integrated into the A. annua genome. The results of RT-PCT and RT-PCR-Southern blot suggested that the foreign FPF1 gene had expressed at the transcriptional level. Under short-day conditions, the flowering time of FPF1 transgenic plant was about 20 days earlier man thenon-transgenic plant. But there were no significant differences in artemisinin content between the flowering transgenic plant and the non-flowering non-transgenic plant. The above results showed that early-flowering can not necessarily make increase the artemisinin content of the flowering plant, perhaps there is no direct linkage between flowering and artemisinin biosynthesis.2. The early flowering gene CONSTANS (CO) from Arabidopsis thaliana was transferred into Artemisia annua using the Agrobacterium tumefaciens-mediaied transformation system. The plant expression vector pBI121CO was constructed by inserted the CO gene into the binary vector pBI121 under the control of CaMV 35S promoter. Analysis of PCR> PCR-Southern blot and Southern blot revealed that the transgenic plants contained the foreign CO gene. The results of RT-PCT and RT-PCR-Southern blot suggested that the foreign CO gene had expressed at the transcriptional level. Under short-day conditions, the flowering time of CO transgenic plant was about 2 weeks earlier than the non-transgenic plant. But there were no significant differences in artemisinin content between the flowering transgenic plant and the non-flowering non-transgenic plant, that is the increase of artemisinin content before plant flowering is not the result of flowering itself, perhaps there is no direct linkage between flowering and artemisinin biosynthesis.