Study On Mechanism Of The Co-culture Of Dioscorea Zingiberensis Cell And Endophytic Fungi

Dioscorea zingiberensis C.H.Wright of dioscoreaceae is an important medicinal plant growing only in China by reason that diosgenin in its rhizome is a widely used starting material for hormone drugs. Recently, it is find that endophytic fungus isolated from host plant can produce the same physiological active substances as products of the hosts. In our laboratory, co-culture system for Dioscorea zingiberensis C.H.Wright cell and its endophytic fungi was established to produce diosgenin in order to increase the synthesis ability of diosgenin and to solve the shortage of diosgenin resources. In this paper, taking Peroxidase(POD), Polyphenol oxidase(PPO), Phenylalnine ammonia–lyse (PAL) and Easterase (EST) as indices, the physiological and biochemical mechanisms of the co-culture of Dioscorea zingiberensis C.H.Wright cell and its endophytic fungi were studied. The main results were as follows:1. MS supplemented with NAA 1.6mg/L, BA 2.0mg/L, 30g/L sucrose, 7g/L agar, pH 5.7 was the optimum medium for Dioscorea zingiberensis C.H cell culture. The optimum inoculum size was 0.8g FW/flask. The growth curves were all "S" shape. The growth cycle of Dioscorea was divided into three phases: lag phase, exponential phase and decelerating phase.2. Three co-culture systems were set for Dioscorea zingiberensis C.H.Wright cells and endophytic fungi RE0105, E0326 and RE1127 separately. When RE0105, E0326 and RE1127 were inoculated in each system, the activities of POD, PPO and PAL of co-culture were all increased and they were positive correlative with the biomass of cells. In the co-culture system of cells and RE0105, the biomass of cells and fungi were both higher than that of cells and fungi separately cultured. The cells were yellowy, round shape and loose. The activities of POD, PPO and PAL of co-culture were highest in the three systems. New isoenzyme bands appeared in co-culture without losing intrinsic isoenzyme bands of cells and fungi separately cultured. In the co-culture system of cells and E0326, the biomass of cells and fungi were both lower than that of cells and fungi separately cultured. The cells were brown and long shape. The activities of POD, PPO and PAL of co-culture were lowest in the three systems. Some isoenzyme bands consisted in cells and fungi separately cultured lost in co-culture. In the co-culture system of cells and RE1127, the growth of RE1127 was in the ascendant, and some isoenzyme bands consisted in cells and fungi separately cultured lost in co-culture. Therefore, the co-culture system of cells and RE0105 was adopted. It was optimum that Dioscorea zingiberensis C.H.Wright cell and endophytic fungi RE0105 were inoculated at the same time for cell growth. The second generation callus of co-culture of cell and RE0105 was stable in culture character, POD, PPO and PAL activities and isoenzyme bands of POD, PPO, EST.3. In the different culture phases of same medium and the different medium of same culture phases, the activities of POD and PPO were positive correlative with the growth rate of cells. PPO activities rose in the cultural period. 4. Isoenzyme bands of POD, PPO, EST of cells grown in different mediums were changed. In mediums fit for cell growth, or in exponential growth phase isoenzyme bands of POD, PPO and EST of cells were more and the shade of color of isoenzyme bands was darker. 5. In co-culture course with host cells, new isoenzyme band of POD that was the same as host cells appeared in endophytic fungi RE0105.