Studies On The Culture Of Pueraria Phaseoloides Hairy Roots And Its Isoflavones Production

Genetic transformation of Pueraria phaseolioides with Agrobacterium rhizogenes strain ATCC15834 and effects of sucrose concentrations, calcium and light on the growth and the production of isoflavones in hairy roots were studied. The main results were as follows:Hairy roots of P. phaseoloides could be obtained from the leaf explants 20 days after inoculation with Agrobacterium rhizogenes ATCC 15834 and could have a rapid growth on solid or liquid growth regulator-free MS medium. The transformation of hairy roots was confirmed by PCR amplification of rolC genes of Ri plasmid from A .rhizogenes. 30 days after cultivation, contents of isoflavones and puerarin in hairy root cultures were 1.6 times, 5.1 times, respectively, as much as that in control roots .High concentrations of sucrose in the medium stimulate the growth of hairy roots. The proliferation times of dry weight of hairy roots cultured for 16 days in the MS medium with 5%, 4%, 3% and 2% sucrose were 11.7,11.9, 10.1 and 5.9, respectively. Similar to the growth of hairy roots, the content of isoflavones in hairy roots was increased with the time during culturing.3% sucrose in the medium was most effective for accumulation of isoflavones and puerarin in the hairy roots. In this case, the highest content of puerarin, 5.15mg/gDW was obtained 12 days after cultivation, and the highest content of isoflavones, 27.76mg/gDW after cultivation for 16 days. Sucrose in the medium was utilized slowly at the still stage, but consumed very rapidly at fast growing stage and during this stage, the content of soluble sugar in hairy roots was increased with time and the highest sulable content was also obtained 12 days after cultivation. Sucrose in the medium was almost used up 16 days after cultivation.P. phaseolioides hairy roots couldn't grow in the calcium-free MS liquid medium containing EGTA. Ca2+ at the concentration of 5.986 mmol/L and 8.979mmol/L dramatically inhibited the growth and puerarin accumulation of hairy root cultures, but was suitable for accumulation of isoflavones in hairy root cultures.In comparison with dark culture, white light and blue light significantly inhibited the growth of hairy root cultures, their dry weight was decreased by 18.87% and 27.04%, respectively, but could enhence the content of isoflavones in hairy roots, especially culturing in white light. The content of isoflavones in puerariaphaseolioides hairy roots cultured in white light was 1.17 and 1.23 times, respectively, as much as that in hairy roots cultured in the dark and in blue light. When cultured in the white light and blue light, the hairy roots of P.phaseoloides looked light green, but white or light red-brown in the dark.