Optimization Of The Hairy Root Inducing System Of Broccoli And Regulation Of Sulforaphanent Synthesis And Release By Signal Molecule

The sulforaphane in broccoli has anti-cancer efficacy,which can effectively reduce the occurrence of multiple cancers,such as liver cancer,stomach cancer,breast cancer,bladder cancer,lung cancer,etc.In recent years,researchers have carried out a large number of studies on the synthesis,metabolism,anticancer activity and antioxidation of sulforaphane in cruciferous vegetables,and obtained a series of research results.But at present,the extraction of sulforaphane mainly depends on the seeds of broccoli,while the seeds of broccoli are expensive and the yield is low,resulting in the high price of sulforaphane products.Organs in vitro hairy root scale production of plant secondary metabolites have been successful in some species,and at present,it is not reported sulforaphane synthesis mechanism and signal molecular regulatory mechanism in the hairy root of broccoli.Thus establishment of broccoli hairy root culture system and the hairy root proliferation system,adding elicitor to increase the sulforaphane release from the medium is necessary.It will lay the foundation for the industrial production of sulforaphane and for the production of sulforaphane drugs.In this paper,the inducing system of hairy root of broccoli was optimized,using three elicitors（SA,JA,Me JA）to regulate hairy root growth and sulforaphane synthesis and release in broccoli hairy root,the results as follow:1 Broccoli induction system op timization（1）Reasearch effect of subculture time on inducing rate of hairy roots,the inducing rate was 68.00%.The inducing rate was not significant when the subculture times were 4-11.（2）Through the experiment of effect of seedling age on inducing rate of hairy roots,the inducing rate was 73.68%when the seedling age was 20 d.the inducing rate were more than 68.00%when the seedling age were 18,19 and 21 d.（3）In the research of e ffect of size and placement way of leaves on inducing rate of hairy roots.The inducing rate was 66.70%,when the leaves size and placement way were1 cm² size and up-placing.（4）On the trial of effect of ATCC15834 concentration on inducing rate of hairy roots.The highest inducing rate was got and reached 69.23%,when the OD₆₀₀ value was 0.275.（5）In the study of effect of antibiotics on net growth of hairy roots.Adding 500 mg/L concentration Cef to the hairy roots of growth,Cef promoted the growth of hairy roots,and the net growth reached 2.31 cm.（6）In the research of effect of Cef concentration on net growth of hairy roots.Addition 300 mg/L concentration Cef to the hairy roots of growth,the hairy roots had the best growth,and the net growth was 3.32 cm.（7）The results showed that the T-DNA of the Agrobacterium Rhizogenes Ri plasmid containing rol B gene was integrated and expressed in the hairy root genome of broccoli.2 Regulation of Sulforaphane nt synthesis and release by three signal molecule（1）Effects of different concentrations of three signaling molecules on the proliferation of hairy roots in different growth periods:Adding 200μmol/L subsalicylic acid in the 9th days of culture system,proliferation multiple was 1.54.Adding 100μmol/L methyl jasmonate in the 0th days of culture system,proliferation multiple was1.39.Adding 150μmol/L jasmonic acid in the 3th days of culture system,proliferation multiple was 1.53.（2）Effects of different concentrations of three signaling molecules on the yield of sulforaphane in hairy roots and medium in different growth periods:Adding 200μmol/L subsalicylic acid in the 15th days of culture system,the release content of sulforaphane reached 47.91μg/flask,the synthesis content of sulforaphane reached 8680.07μg/flask,the yield of sulforaphane reached 8727.98μg/flask.Adding 100μmol/L methyl jasmonate in the 0th days of culture system,the release content of sulforaphane reached 3.4μg/flask,the synthesis content of sulforaphane reached 4455.27μg/flask,the yield of sulforaphane reached 4458.67μg/flask.Adding 150μmol/L jasmonic acid in the 12th days of culture system,the release content of sulforaphane reached 115.94μg/flask,the synthesis content of sulforaphane reached 1.12μg/flask,the yield of sulforaphane reached 117.06μg/flask.（3）In conclusion,the effects of different concentrations of three signaling molecules on the hairy root proliferation and sulforaphane synthesis and release in different growth periods:the synthesis effect of subsalicylic acid on sulforaphane in hairy root was the most obvious.Adding 200μmol/L subsalicylic acid in the 15th days of culture system,the hairy root proliferation was 1.01,the release content of sulforaphane reached 47.91μg/flask,the synthesis content of sulforaphane reached 8680.07μg/flask,the yield of sulforaphane reached 8727.98μg/flask.