Studies On Culture And Regulation Of Nicotine Biosynthesis Of Hairy Root Transformed By Ri Plasmid Of Tobacco

Factors affecting the transformation of tobacco by Ri plasmid of Agrobacterium rhizogene were studied and the optimum transformation condition was defined. The structure of the hairy root tip and the dynamic characteristics of growth of a selected hairy root line were studied. The transformation of hairy root was verified by PCR test and GUS histochemistry test. The physical and chemical factors that affect the growth and nicotine biosynthesis of hairy root were investigated. The main results were as follows:1. Hairy roots were induced from tobacco leaves with Agrobacterium rhizogenes LBA1334 harboring agropin type PRil855 and binary vector PIG121HM carrying a kanamycin resistant gene nptll and GUS gene and the good hairy root clones were selected. The root with many root hairs and branches grew vigorously toward all the direction on MS medium without plant hormone and on kanamyain containing medium (km:30mg/L). The structure of the hairy root tip is different from that of ordinary root. Witch was not typical root cap maybe the main cause of losing the geotropism of hairy root. Plantlet regenerated from hairy root when cultured on Ms medium without plant hormone. The nicotine content of the hairy root was little higher than that in the natural roots. The culture system of hairy root could release nicotine into the medium and the amount of nicotine was 81.1% of the total content of nicotine produced by the culture system.2. A 560 bp fragment was amplified from the genomic DNA of hairy root by PCR using primes of rolC which was one of the four genes related to hairy root induction located on TL-DNA of Ri plasmid. Blue dots were discovered on the hairy root and the leaves of planted regenerated from hairy root after dipped into X-gluc solution at 37℃ for one night. These results proved tobacco hairy root could be an efficient culture system to accumulate valuable substance and an ideal pathway for plant breeding through exogenous genes expressing.3. Factors affecting the transformation of tobacco by Ri plasmid of Agrobacterium rhizogene were studied. Tobacco varieties. Agrobacterium rhizogene strains and basal medium influenced the efficiency of hairy root formation strikingly. While the PH of medium and acetosyringone had little effects. MS medium and strain LBA1334 give the best result on tobacco transformation. Sha Mu Xun was more sensitive to Agrobacterium rhizogene than K326 and NC89. Leaf discs dipped into bacterium solution diluted 5 times at exponential phase for 5 minutes, and pre-cultured on MS medium for two days was very useful preparation for transformation.484. The growth of the hairy root and nicotine biosynthesis were influenced by various physical and chemical factors. It was found that the optimum pH value of medium was 6.0. Fast growth of the hairy root and maximal production of nicotine was observed in l/2medium and in the presence of 2-3% sucrose. The best volume of liquid medium for the growth and nicotine synthesis of the hairy root was 2/5 of the volume of container at 110rpm. IAA(lmg/L)and LH(2g/L)also enhanced the weight and nicotine content of the hairy root.5. Phytohormones affected obviously the growth and nicotine content of the hairy root. GA3 improved remarkably the growth speed of the hairy root but the nicotine synthesis was inhibited. IAA could raise the weight and the nicotine content of the hairy root when the concentration of IAA was 1mg/L. The growth and nicotine production in hairy root cultures weren't greatly promoted by addition of NAA.6. The medium pH value was studied during the culture process. The results show that the medium pH value was 5.0 at the beginning of cultivation, and the pH could be kept till the stationary stage. The medium pH increased linearly relative to the biomass accumulation of the hairy root.