Effect Of Aspergillum Niger And P-coumaric Acid On Content Of Resveratol In Polygonum Cuspidatum Callus And Induction Of Hairy Roots

Polygonum Cuspidatum is a kind of herbage belongs to polygonaceae.It is a Chinese traditional medicine.The mostly activity component is Resveratrol in Polygonum Cuspidatum. Resveratrol and its derivatives are consider of a promising natural chemical anticarcinogen.This study dealt with the young stalks of perennial Polygonum Cuspidatum as explants and treating the callus induction as materials.By adding fungal elicitor derived from Aspergillum niger and precursor(p-coumaric acid),the experiment studied that the elicitor and the precursor affected the accumulation of resveratrol and Polydatin in callus.The studying results showed as follows:1.Effects of p-coumaric acid on content of resveratrol and polydatinThe precursor could raise the content of Res and PD in Polygonum Cuspidatum callus.The content of Res and PD were the highest with A3 treatment,the content of Res was 69.79μg·g^-1FW and the content of PD was 96.97μg·g^-1FW,respectively increased by 50.1%and 52.3%.In a growth period,the peak of Res content appeared on the 20th day.In a word,the best time to gain the callus was om the 20th day.2.Effects of fungal elicitor derived from Aspergillum niger on content of resveratrol and polydatinThe study screened the concentration and culture time of the elicitor derived from Aspergillum niger.The result showed that 100 mg·L^-1 of the elicitor was added into the medium on the fifteenth day and on the fifth day the amount of resveratrol and Polydatin in Polygonum cuspidatum callus.The content of Res and PD were 44.19μg·g^-1 FW and 75.41μg·g^-1 FW, respectively increased by 63.7%and 46.2%.3.Effects of fungal elicitor derived from Aspergillum niger and p-coumaric acid on content of resveratrol and PolydatinBy adding the different combinations with Aspergillum niger and p-coumaric acid,the result showed that they could promote the accumulation of resveratrol and Polydatin,the combination of elicitor derived from Aspergillum niger and p-coumaric acid(C2) had the highest inducing efficiency on the fifth day.The amount of resveratrol and Polydatin in Polygonum cuspidatum callus.The content of Res and PD were 171.04μg·g^-1 FW and 264.58μg·g^-1 FW,respectively increased by 77.50%and 55.30%.4.Effects of p-coumaric acid on POD,SOD and BGL activityBy the induction of p-coumaric acid and determining the POD,SOD and BGL activity in callus.The result showed that the p-coumaric acid had some effects on these three enzymes.By determining the POD activity,the result showed that the activity had different trend in the treatment and check,the treatment(CK,A1,A2) was rising-down trend and A3,A4 was down-rising- down trend.By determining the SOD activity,the result showed that the activity had different trend in the treatment and check,the treatment was rising-down-rising-down trend and the check was down-rising-down trend.By determining the BGL activity,the result showed that the activity had the same trend in the treatment and check,the trend was rising-down-rising-down; The content of H₂O₂ had the same trend in the treatment and check,the trend was rising-down-rising-down.5.Effects of fungal elicitor derived from Aspergillum niger on POD,SOD and BGL activityBy the induction of elicitor derived from Aspergillum niger and determining the POD activity, SOD activity and BGL activity in callus.The result showed that the elicitor derived from Aspergillum niger had some effects on these three enzymes.By determining the POD activity,the result showed that the activity had different trend in the treatment and check,the treatment was rising-down-rising-down trend,and the B4 was down-rising-down-rising trend.By determining the SOD activity,the result showed that the activity had the same treat,the treatment was down-rising-down trend.By determining the BGL activity,the result showed that the activity had different trend in the treatment and check,the treatment was down-rising-down-rising-down trend and the check was rising-down- rising trend;The content of H₂O₂ had the same trend in the treatment(C1,C2) and check,the trend was rising-down-rising-down.6.Effects of fungal elicitor derived from Aspergillum niger and the p-coumaric acid on POD,SOD and BGL activityBy the induction of elicitor derived from Aspergillum niger and determining the POD activity, SOD activity and BGL activity in callus.The result showed that the elicitor derived from Aspergillum niger had some effects on these three enzymes.By determining the POD activity,the result showed that the activity had the same trend in the treatment and check,the trend was rising-down.By determining the SOD activity,the result showed that the activity had the same trend in the treatment and check,the trend was down-rising-down.By determining the BGL activity,the result showed that the activity had the same trend in the treatment and check,the trend was down-rising-down. 7.Effects of inducible factor acetosyringone(AS) and the co-culture time on induced hairy roots of Polygonum CuspidatumIt was proved by PCR test that the genes rolB Ri plasmid of Agrobacterium rhizo gene were completely transformed into the hair-like roots.The result showed a hair roots induction rate as high as 40.0%and an induced-root density of 6.4;With the different transformation factors of concentration acetosyringone optimized,the induction rate of the hair roots of Reynoutria japonica could be raised to 67.5%and its hair-like root density could reach 13.7.By means of comprehensive comparison,the appropriate concentration of acetosyringone and the co-culture time were 50～100 mg·L^-1 and 2～3 d respectively.8.The content of Res and PD in fresh hairy roots of Polygonum CuspidatumThe content of Res and PD were determined by HPLC.The contents were 18.21μg·g^-1 and 215.33μg·g^-1 respectively in fresh hairy roots of Polygonum Cuspidatum.