Establishment Of Regeneration System Of Broccoli And Preliminary Exploration Of Genetic Transformation System

Glucosinolates are a class of plant secondary metabolites containing nitrogen, sulfur, distributed mainly incruciferae. Glucosinolates and its metabolites have extensive biological effects. In recent years, glucosinolates in plantdefense responses and anticancer etc. caused wide attention. When glucosinolates are catalysed by myrosinase, theycan hydrolysis to generates a series of active substances, which play a role in the invasive process in plant defenseagainst insect, herbivores animal and pathogenic bacteria; some aliphatic glucosinolate hydrolysis products showedstrong antitumor activity, in which sulforaphane-the hydrolysis product of4-methylsulfinylalkyl glucosinolates is thechemical composition that have the strongest anticancer activity in plants so far. It is a question worthy of further studyto increase the content of sulforaphane and cultivate vegetables with anticancer activity functions.In the past ten years,a large number of domestic and foreign research confirmed that often eating cruciferousvegetables, such as broccoli（Brassica oleracea var. italica）, cauliflower, radish, cabbage and can reduce the peoplesuffering from various different tumor disease risk, the anticancer active substances present in these plants issulforaphane and the precursor of sulforaphane is catalyzed by the enzyme expressed by FMO_GS-OX1gene.Incruciferous vegetables, the anticancer activity of sulforaphane in broccoli in far more than other vegetables, andyoung tissues is the highest,especially.In this study, use broccoli hypocotyl as explants, discussed the factors influencing regeneration system of broccoliand established regeneration system of broccoli, results showed that different genotype of explants have greatinfluence in bud differentiation rate. In this experiment the hypocotyls differentiation rate of Zhu Lv and Qing Xiu is92.5%and91.1%, which are higher than three other varieties; the differentiation rate of adventitious bud also affectedby seeding ages, we compared the adventitious bud differentiation rate of Zhu Lv and Qing Xiu in in different seedingages, and we obtained the highest differentiation rate of Zhu Lv and Qing Xiu with seeding ages of six days, which are94.1%and92.1%; The hormone was an important factor on the regeneration of adventitious buds, differentiation rateof adventitious buds of Zhu Lv and Qing Xiu was the highest when we added3mg/L6-BA and0.2mg/LIAA to MSmedium, which are95.2%and94.6%; Adding AgNO₃to MS medium can significantly improve the adventitious buddifferentiation rate. Differentiation rate of adventitious buds of Zhu Lv and Qing Xiu was the highest when we added4mg/LAgNO₃,, which are97.6%and95.2%; Adding0.2mg/L IBA to MS medium had good effect on rooting period,the differentiation of adventitious root of Zhu Lv and Qing Xiu are87.5%and82.6%. Construct the plant expressionvector35S：FMO_GS-OX1. We also optimized genetic transformation system of broccoli, the most suitable selectionpressure of kanamycin for Zhu Lv is7mg/L, and the smost suitable election pressure of kanamycin for Qing Xiu is 8mg/L; we optimized the infection time, the results show that the most suitable infection time for explant is3-5min;200mg/L carbenicillin can completely inhibiting the growth of Agrobacterium, the differentiation rate of adventitiousbud was the highest.