Production Of Curculigoside By Suspension Culture Of Curculigo Orchioides

Curculigo orchioides Gaertn is a perennial herb of Amaryllidaceae family,also known as the brown,monosal,and citronella.Its roots are used as medicine for the treatment of kidney disease,pain in the lumbar spine,frequent urination,arthralgia and myalgia.With the deep understanding of the medicinal value of C.orchioides,the demand for C.orchioides is gradually increasing,and the wild resources of C.orchioides are destroyed and greatly reduced.The artificial cultivation of C.orchioides is also caused by land resources,pesticide residues,seasonal effects,and differences in active ingredients.The influence of factors is limited.Therefore,this project decided to use plant cell engineering technology to produce curculigoside,which can shorten the utilization cycle of C.orchioides resources,and more effectively protect the germplasm resources of C.orchioides from damage,which is conducive to the sustainable development and industrial upgrading of C.orchioides industry.In this paper,the C.orchioides was used as the material to induce callus formation using young leaves as explants.After repeated subculture,MS+2.0 mg/L 6-BA +0.5 mg/L NAA was used as the medium at 25 ?.The suspension cell was successfully established under the condition of 120 r/min rotation,12 h/d illumination and light intensity of 1 200 lx for 21 days.The biomass,pH,PAL activity and metabolites of the suspension cell line during the culture process were studied.The results showed that the suspension cell could produce metabolites such as curculigoside in a certain culture period,but the content was low.And it is related to cell biomass,PAL enzyme activity,and pH change.Bsaed on the effects of inoculation density,oscillation frequency,carbon source concentration,and growth regulator 2,4-D on cell proliferation were studied by response surface test design.The results showed that the optimal response level of each factor to promote cell proliferation was 73.94 g/L,2,4-D0.59 mg/L,shaking speed 103.75 r/min,carbon source concentration 40.28 g/L.Under these conditions,the obtained suspension cells grew well and the maximum biomass was 15.26.±0.3 X 10~5 cells/mL.Base on the effects of different light,temperature and pH conditions on the growth of C.chinensis cells and the synthesis of C.chinensis,the results showed that the light conditions were 12h/d,1200x,and the initial pH was 5.8-6.2 when the temperature was 26?.Conducive to the growth of curculigo cells and the synthesis of curculin.Base on the effects of different precursors and elicitor conditions on the growth of Curculigo latifolia cells and the synthesis of curculin showed that the addition of L-phenylalanine,methyl jasmonate and salicylic acid could promote the biosynthesis of curculin.When L-phenylalanine was 100 mg/L,the content of curculin was 3.32±0.18 mg/g dry weight,which was 2.48 times that of the control group.When methyl jasmonate was 100 ?mol/L,the highest content of curculin was 2.54±0.24 g/L dry weight,which was 1.85 times of the control group.Salicylic acid was 50?mol/L,and the highest content of curculin was 3.088 mg/g dry weight,which was 2.36 times that of the control group.Base on the effects of precursor and elicitor treatment on PAL activity,total sugar consumption and pH change of suspension cell culture medium,the results showed that the precursor L-phenylalanine,the elicitor methyl jasmonate and salicylic acid were both.It can increase the sugar consumption of cells,and the inducer methyl jasmonate and salicylic acid can increase the PAL activity of suspension cells and the pH of suspension culture.