The Establishment Of Cajanus Cajan Hairy Root Culture System And Correlation Physiology And Biochemistry Study

Hairy root infected by agrobacterium had the adwantage of rapid growth, much branching, ageotropism and hereditary relatively stable. It had a very active metabolism capacity, and even the similar secondary metabolic way compared with original plants. Thus, hairy root could synthesize the compounds the same as original plants, and also may accumulate the intermediates and derivatives of the compounds, had the equivalent or higher content than original ones, and even a new compound.In the study about medicinal plants, it has become a hot, which by means of induction and culture of hairy roots for extracting the active ingredients of medicinal herbs. In my study, the Agrobacterium Rhizogenes LBA9402and leaves of Cajanus cajan were used as materials. The leaves of Cajanus cajan were infected by the agrobacterium, then the hairy root was produced. After co-culture, sterilization and expand training, and finally set up the Cajanus cajan hairy root induction and culture system. Then, the growth percentage of hairy roots and apigenin and genistein contents, as well as the influence of protective enzyme (CAT, POD, SOD) activity and soluble protein content were detected in suspension culture. After that, three elicitors, including UV irradiation, Ca2+and IBA, were used to influence the physiological and biochemical indexes. And this provides the research basic to the hairy roots. The main results are shown as following:1. The system of hairy roots from Cajanus cajan Hairy roots of Cajanus cajan was induced by Agrobacterium rhizogenes LBA9402with direct inoculation. The best condition of induction of hairy roots by this test was, pre-culture for2days, the concentration of bacterium was OD=0.6-0.8, disseminated for20min, co-culture for3days, the rate of the hairy roots was60%. AS could promote the transformation of Agrobacterium rhizogenes, improve the induction frequency of pigeonpea hairy roots. PCR analysis at molecular level obtained expected specific band proved that T-DNA of Ri plasmid of Agrobacterium rhizogenes LBA9402has successfully transferred into the genome of hairy roots of Cajanus cajan. Experiment also proved that, N6liquid culture medium,30g/L sucrose and exogenous hormone IBA all could improve the growth of pigeonpea hairy roots.2. Studies on changes of physiology and biochemistry indexes of hairy root of Cajanus cajan during growth cycle The growth percentage of pigeonpea hairy roots and the content of the sucrose in culture media have inverse relationship; the growth of hairy root experienced delayed periods, rapid growth periods and moderate growth periods,; the three protective enzyme activity was increased before and then decreased, overall; soluble protein content had a trendy of always increasing; the content of apigenin and genistein in hairy roots both increased with the time passed by, until the growth cycle and then became flat, metabolic cycle of the hairy root of Cajanus cajan was20days.3. Influence of elicitors to the hairy roots physiological and biochemical indexes UV irradiation20min,1mmol/L Ca2+,0.5mg/L IBA could make the hairy roots growth best. When the concentration of IBA was0.5mg/L, the enzyme activity reached maximum, and maybe most beneficial to accumulate apigenin and genistein. UV irradiation could inhibit the activity of POD and SOD, under the treatrment of Ca2+, the changing trend of POD and SOD acitivity was the same as growth rate of the hairy root, but the changing of CAT was lack of regularity. The change of soluble protein content was not significant.