Preliminary Study On Callus Suspension Culture And Regulation Culture Technique For Synthesis And Accumulationin Of Two Kinds Of Coumarin In Fraxinus Rhynchophylla

The leaves from germinated dormant bud of Fraxinus rhynchophylla was as experimental material.The callus induction,subculture,establishment of suspension culture system were studied in this paper.In order to obtain more aesculin and aesculetin,series of experiments had been carried out to study their metabolism regulation techniques.After MeJA induction,relevant physiological indicators of suspension cultures were determined and contents of aesculin and aesculetin were examined.It have not been reported in the literatures that callus was inducted and plantlet was regenerated in vitro from leaf,and had no report about suspension culture of F.rhynchophylla also.So this research not only improved and perfected tissue culture technology of F.rhynchophylla,but also laid a foundation for the industrialized production of coumarin compounds by established bioreactors for suspension culture system.From the,study of explants disinfection method,we found that explant sterilization rate was high with the method of 75%alcohol disinfected 25-30 s and then 10%Ca(C10)2 disinfected 10 min.The browning degree of explants was low at the same time.On the medium of WPM+1.5 mg/L NAA+0.1 mg/L TDZ,the induction rate of callus could reach 60%.Callus grew vigorously,when its volume was larger and the color was green.Growth regulator combination of low concentration was easy to induce loose,yellowish callus.While induced by growth regulator combination with high concentration callus was dense and darkgreen,which often browninged highly.In callus subculture,growth regulator combinations of NAA and TDZ were used to test the callus proliferation multiples of F.rhynchophylla in different hormone concentration.The regression equation YNAA=-1.0762x2+4.0333x+1.6716(R2=0.9789)and yTDZ=-210.92x2+53.367x+ 2.0448(R2=0.9837)were established.Matching effect of two equations was good.Their first derivative extremum showed the optimum culture medium of WPM+1.87mg/L NAA+ 0.13mg/L TDZ.The callus of F.rhynchophylla was cultured in this medium for 30 d and its proliferation multiples was 5.45.There were many factors which had an influence on increment of callus in the suspension culture.We got the optimal suspension culture condition for callus of F.rhynchophylla in this study:the callus was cultured in the medium of 1/2 WPM+1.2 mg/L NAA+0.07 mg/L TDZ,with inoculation amount of 3 g/50 mL callus and the speed of shaking incubator of 100 rpm.We found that callus of F.rhynchophylla presented a sigmoid growth curve.1-3d was lag phase.4-7d was logarithmic growth phase.8-11d was stable phase.12d started to enter into the decline phase.Aesculin and aesculetin in suspension callus culture cycle of F.rhynchophylla had the same trend.Their content was low in delay phase and logarithmic phase.They increased after enter into stable phase.Throughout the culture cycle,the highest yield of aesculin and aesculetin was respectively 1.5 and 1.8 times of the lowest ones.The yield of aesculin reached 0.769 mg/g(dry weight)and yield of esculetin reached 0.0099 mg/g(dry weight)during each subculture.CAT and SOD were two kinds of defense enzymes in plant cells.Their enzyme activity was relatively lower in lag phase and logarithmic growth phase.Cell viability decreased after stable phase.Competition among cells became stronger.Two kinds of defense enzyme content began to increase.But it decreased along with a large number of cells died.PAL was the defense enzyme of cell stress resistance and the key enzyme of coumarin biosynthetic pathway.So the varying pattern was closed to the accumulation of aesculin and aesculetin.The varying trend of PAL in F.rhynchophylla suspension callus was reduce-increase-decrease.PAL activity was low in delay phase and logarithmic growth phase.But it increased in stable phase and decreased in decline phase.After adding different concentrations of MeJA(50?M,100?M and 200?M),fresh weight changes of F.rhynchophylla suspension callus were inhibited unobviously and only obviously at 48h.In general,cell activity under different concentrations MeJA had fluctuant decreased,which indicated that MeJA played a role of inhibitor on cell viability of F.rhynchophylla suspension callus has the effect of inhibition.We found that adding MeJA had obvious promotion to accumulation of aesculin and aesculetin in F.rhynchophylla.With the addition of 100?M MeJA,inducing effect of F.rhynchophylla suspension callus was the most significant.After 24h,aesculin accumulation amount increased 68.67%than the control and aesculetin accumulation amount increased by 36.36%.Three kinds of defense enzyme activity were increased after MeJA treatment,compared with control group.It showed that stress caused by MeJA stimulated F.rhynchophylla callus cells,the cells performed stress responsestress.The time of maximum activity of three defense enzymes arrived significantly ahead of CK after MeJA treatment,and the maximum of the enzyme activity appeared at 24h after MeJA treament in all concentrations.