Inhibition Of ACC Synthase Gene Expression In Fustoma Grandiflorum ’Double Mariachi Pink’using RNA Interference Technology

Eustoma grandiflorum is Gentianceae biennial herbs, which was one of the popular cut flowers in the world, and has high economic, viewing and admiring value. Flower’s viewing life, especially the vase life of cut flower, is an extremely important quality, which directly affect the commodity value of the flowers. How to extend the viewing life of flowers has been the key issue concerned the experts at home and abroad.In the research, a lisianthus (Eustoma grandiflorum) cultivar ’Double Mariachi Pink’ is used. An efficient and stable Agrobacterium-mediated genetic transformation system of Eustoma grandiflorum has been established. The interference vector pCAMBIA1301-gfp-(GUS+ACS) i-NPTII is transformed into lisianthus via Agrobacterium tumefaciens. Through the ACS interference vector transformation to the plants, we expect to cultivate a new variety of Eustoma grandiflorum which has longer vase life. The infection conditions as follows:Resuspend the Eustoma grandiflorum seedlings’leaves which have been cultured for three days with the concentration of OD600≈0.5of Agrobacterium for10min, then culture five days in the dark under the conditions of24℃, and then transferred to selection medium for selected training.First, use65resistant seedlings for root testing.46transformed seedlings root out of65resistant seedlings. The rooting rate is70.6%. Secondly, design a pair of primers based on the GFP reporter gene sequence for the PCR analysis.46lines detected by PCR,11lines could be amplified with the differential band. And, RT-PCR analysis demonstrates that3transformed plants are positive.3lines are detected by PCR GFP activation,2lines could be detected with GFP activation.Extracted protein from3positive transformed plants, using the SDS-PAGE method to detect whether gfp gene expressed in the Eustoma grandiflorum.In addition, we do some existing studies about the relevant factors which affecting the transplantation survival rate and flowering of Eustoma grandiflorum, such as the effective number of leaves impacting on the survival rate. The results show that the survival rate of the plants more than6roots is greater than the roots less than6. Compared with control Eustoma grandiflorum flowering10d, the transformation of the anti-ACS plants are flowering24d, and the transformation of the ACSi interference plants are flowering27d. The transformation of the ACSi interference plants extend ornamental cycle.