Study On Inducing Hairy Roots Of Pogostemon Cablin And Establishing The System Of Cell Suspension

Pogostemon cablin(Blanco) Benth is one of the Traditional Chinese Medicinal materials commonly used, also is the "top ten of Guangdong traditional Chinese medicinal", with properties of acridness and tepidity. Its aboveground part can be used as medicine. In this study, P. cablin leaves as explants induce aseptic seedlings and as experimental material. Study on Agrobacterium rhizogenes induced hairy roots of P. cablin and Cell suspension culture of P. cablin.The main results are as follows:(1) Inducing hairy roots of P. cablin and differentialIn this study, P. cablin aseptic seedling leaf explants infeeted by Agrobacterium rhizogenes ATCC15834 and C58C1. Study on the best infection time of Agrobacterium rhizogenes, acetosyringone concentration, pre culture time, infection time, co-culture time on hairy root induction rate of P. cablin and identify the induced hairy roots by PCR technology.Studies shows that Agrobacterium rhizogenes ATCC15834 at 140 r/min, 28℃, dark conditions oscillation cultured for 18 h; Agrobacterium rhizogenes C58C1 under the same conditions oscillation in cultured 19 h,OD600 value of 0.6, can be used to infect the explant. The optimum conditions of inducing hairy roots of P. cablin are 2 days-pre culture time, 15 mg/L acetosyringone, 25 min infection time and 2 days- co-culture time, the induction rate of Agrobacterium rhizogenes ATCC15834 and C58C1 infecting P. cablin explants were the highest, which reached to 83.3% and 80.5%. The PCR amplification results confirmed that the part of T-DNA from Ri plasmid of Agrobacterium rhizogenes was in P. cablin hairy root genome integration and expression, indicating that P. cablin hairy roots have been induced successfully.(2) Plant regeneration of P. cablin Hairy root and differentialThis study uses "two step method" and study on effects of callus induction of P. cablin hairy roots under different hormones and different culture conditions. Studies showed that the optimum conditions of induction of plant regeneration of P. cablin Hairy root are in the light of 14h/d, 25±2℃, MS+6-BA(2 mg/L) +NAA(0.1 mg/L) medium inducing callus, the induction rate was 100% after 30d; In the MS+6-BA(0.1 mg/L) +NAA(0.1 mg/L) medium induction differentiation of buds,the induction rate was 100% after 35d; rooting in 1/2MS media, rooting rate was 100% after 20 d. The PCR amplification results confirmed that the part of T-DNA from Ri plasmid of Agrobacterium rhizogenes was in plant regeneration of P. cablin Hairy root genome integration and expression.(3)P. cablin loose callus inductionThis study is based on MS medium, study on effects of induction of P. cablin loose callus under different hormones and different culture conditions. Studies showed that in the light of 14h/d, 25±2℃, MS + 2,4-D(0.05 mg/L) + KT(0.5 mg/L) medium, callus induction rate was the highest which is 87.5%. After several subcultures, can obtained loose callus in white or yellowish white.(4) Establish cell suspension culture system of P. cablin and assay of patchouli alcoholStudy on effects of growth in suspension cells of P. cablin under irradiation time, rotational speed, inoculation size, temperature, carbon sources and establish cell suspension culture system of P. cablin. Study on SA and MJ effects of growth in suspension cells cultures of P. cablin and content of patchouli alcohol.Studies shows that the growth curves of P. cablin cell suspension culture showed typical "S" curve, 9-15 d is the logarithmic phase, 24 d as growing cycle. The optimum conditions of cell suspension culture system of P. cablin are in the light of 24h/d, 135r/min rotational speed, 7g/100 ml inoculation size, 25±2℃, carbon sources is sucrose 30g/L. Oscillating culture for 18 d, the fresh weight of P. cablin suspension cells to 529.8795 g/L. The SA in the concentration of 1 mg/L, fresh weight was 546.1853 g/L and dry weight was 16.1237g/L with the promotion of the role of P. cablin suspension cells culture. Each concentration of MJ had inhibitory effect on the growth of P. cablin suspension cells culture. Each concentrations of SA and MJ can promote the Patchouli alcohol content of P. cablin suspension cells. At 20mg/L SA concentration, the Patchouli alcohol content of P. cablin suspension cells was the highest for 0.0976mg/g, which is 2.69 times that of the control. At 50mg/L MJ concentration, the Patchouli alcohol content of P. cablin suspension cells was the highest for 0.0638mg/g, which is 1.76 times that of the control.