Proliferation Of Broccoli Cell In Vitro And Metabolism Regulation Of Sulforaphane

The use of plant cell culture technology for the production of plant secondary metabolites has broad application prospects because of its unique advantages and rapid development. The production of secondary metabolites increases when it is regulated by elicitors, which is an effective way to produce secondary metabolites in factory. Broccoli is rich in anti-cancer substances, sulforaphane, and has been more and more attention. This article used broccoli callus as experimental material and made use of plant tissue culture techniques to inspect effects of different nutrients and concentration ratios of NH4+ / NO3-, and different concentrations of Ca2 +, Mg2+ on cell growth and physiological and biochemical indices. And the effects of different elicitors, SA, SNP, La3+ and Ce4+, on cell growth and synthesis of sulforaphane. In addition, the exogenous SA mediated by NO signaling pathway to promote the synthesis of sulforaphane was studied. The results were as follows:1. Effects of different nutrients on cell growth and physiological and biochemical indices(1) Callus could be induced by explants of root, stem and leaf of broccoli, callus induced by root grow best, while callus induced by stem grow worst.(2) When the ratio of NO3-/NH4+ was 40/20(mmol/L), callus cells induced from root and leaf had the maximum biomass and was significantly higher than that of other concentrations; when the ratio was 45/15(mmol/L), callus cells induced from stem had the maximum biomass; the callus cells induced from root, stem and leaf had maximum biomass when Ca2+ concentrations were 1.5, 1.5 and 0.5mmol/L, respectively, and the biomass were 17.6 g/flask, 5.5 g/flask and 10.8 g/flask, and respectively increased by 10.7%, 67% and 28.6% compared with CK; when Mg2+ concentrations were 1.5, 1.0 and 1.0mmol/L, the callus biomass induced from root, stem and leaf increased by 21.1%, 54.8%, 13.8% when compared with CK.(3) NO3-/NH4+ proportion, Ca2+ and Mg2+ could cause cell changes of PAL activity in broccoli in vitro cells, the increase of NH4+ concentration had significant effects on PAL activity, in which the biggest changes of callus were induced from stem, while the smallest changes of callus were induced from root.(4) NO3-/NH4+ proportion, Ca2+ and Mg2+ acting on broccoli in vitro cell could cause the changes of SOD, POD and CAT activities, and changes of enzyme activity are often correlated with cell biomass and cell viability. When NO3- and NH4+ were added to the cells in Specific concentration proportions, SOD, POD and CAT activities decreased, while those activities would increase at any other proportions. When cells were treated by low or high concentrations of Ca2+ and Mg2+ would lead to the increase of the three protective enzyme activities.2. The effects of exogenous SNP, SA and La, Ce4+ on sulforaphane synthesis of broccoli in vitro cells(1) Exogenous SA and SNP inhibited cell growth, but could promote the synthesis of sulforaphane. when SA and SNP concentrations were 10mg/L and 0.1mmol/L, respectively, the sulforaphane content increased by 59% and 43.4% compared with CK. When the concentration of La(NO3)3 and Ce(NH4)2(NO3)6 were 40mg/L and 5mg/L respectively, the cell biomass increased most significantly and increased by 39.5% and 22.5% compared with CK; La(NO3)3 could inhibit sulforaphane synthesis in cells, causing significant reduction of sulforaphane level; when Ce(NH4)2(NO3)6 concentration was 20mg/L, the sulforaphane content increased most significantly and increased by 51.6% compared with CK.(2) Cells treated by exogenous SA, SNP and La(NO3)3 would decrease the yield of sulforaphane, and the difference was significant. While cells treated with exogenous Ce(NH4)2(NO3)6 would increase the yield of sulforaphane, the sulforaphane yield increased by 63.7% compared with CK when adding 5mg/L Ce(NH4)2(NO3)6.3. NO signaling pathways mediated by exogenous SA promoted biosynthesis of sulforaphane in broccoli in vitro cells(1) The impact of exogenous SA on the accumulation of NO and the SF synthesis in cells. When SA concentration was 10mg/L, the increase amplitude of NO content was the biggest, which was 3.34 times of CK, and also had the highest content of sulforaphane, which was 1.69 times of CK.(2) The NO, SF synthetic dynamic in cells treated by exogenous SA showed that NO content after adding SA for 12 h, and was 5.0 times of CK; SF content reached peak value after adding SA for 30 h, and was 1.7 times of CK.(3) When adding specific inhibitor of NOS and NR at the same time, NO content in the cells decreased by 89.1% compared with CK; adding specific NOS inhibitor L-NAME(5mmol/L), NO decreased by 47.1% compared with CK; while adding NR specific inhibitor Tungstate(5mmo/L), NO decreased by 74.4% compared with CK.(4) Adding NO scavenger or inhibitor before SA treatment would also reduce the content of NO in the cell, but the decline amplitude was smaller than that of single addition of NO inhibitors or scavengers, while the content of SF still increased in a certain extent, but the increase amplitude was smaller than that of single addition of SA. But after adding the NO donor SNP, the content of NO and SF would increase in a large extent, but the content was still lower than that of the SA treatment separately. When adding NO donor SNP alone, NO level in cell significantly increased, SF content rose to 82.9% of SA-treated group. When adding SNP, L-NAME and Tungstate, NO content was significantly higher than that in CK; while adding SNP and c-PTIO, the content of NO and SF decreased, which indicated that NO was cleared by c-PTIO, leading to the synthesis of SF was partially inhibited.