Proliferation Of Broccoli Hairy Roots And Regulation Of Sulforaphane Accumulation By Fungal Elicitor

Sulforaphane is an isothiocyanate ester compound obtained by myrosinase enzyme, with a strong anti-cancer activity. Although broccoli seeds are rich in sulforaphane, still can not meet the heavy demand of market as a result of traditional breeding methods were complicated, long period and expensive seeds. And synthesis of sulforaphane is difficult to achieve due to the complex process of industrial production. Hairy roots grow rapidly, can synthesize a large number of secondary metabolites, so optimizing broccoli hairy root culture system and regulating the metabolic of sulforaphane to increase sulforaphane content in hairy root are more and more concerned. The extraction of sulforaphane from broccoli is complicated, if sulforaphane can release into the medium, the extraction from the medium will be more convenient, which can promote mass production of sulforaphane. Through the optimization of broccoli hairy root culture conditions?inoculum size, dissolved oxygen, p H, rotation speed, temperature, IBA concentrations?, this article established dynamics of hairy root growth and sulforaphane synthesis, based on the dynamics and using of fungal elicitors?species effect, concentration effect, time effect, synergistic effect?,we regulate sulforaphane accumulation in broccoli hairy root, the results as follow:1. The establishment of broccoli hairy root proliferation system?1? The inoculum size of 2 mg·m L^-1 FW was most beneficial for biomass accumulation of broccoli hairy root, the proliferation multiples was up to 10.45.?2? The proliferation multiples was 24.14 under the condition of 0.2 mg·L^-1 of IBA.?3? The biomass of broccoli hairy root had larger accumulation with p H was between 5.2 and 6.4 in liquid medium, and the proliferation rate was between 23.95²4.14.?4? When the volume ratio of liquid medium and culture bottle was 1:2, the amount of dissolved oxygen was the most suitable for hairy root biomass accumulation, and the proliferation multiple was up to 26.24.?5? Different rotate speeds had large influence on hairy root proliferation multiple, the lower and higher speeds were not suitable for growth of broccoli hairy root, when the speed was 110 r·min^-1, broccoli hairy root had the biggest proliferation multiple which reached to 26.25.?6? Low and high temperatures were not suitable for the growth of broccoli hairy root, when the culture temperature was 25 ? in temperature control shaker, the proliferation multiple was biggest and up to 26.25. In large-scale culture, the culture temperature could be controlled between 25²8 ?, proliferation multiple of hairy root was between 25.87²6.25.2. The study of broccoli hairy root growth and sulforaphane synthesis?1?The growth of broccoli hairy roots was divided into four periods, which were delay phase?0⁹ d?, fast growth phase?9¹8 d?, stable phase?18²1 d? and decline phase?21 d after?.?2? The metabolism of sulforaphane in broccoli hairy root began 12 th d?fast growth phase? after culturing; the rapid accumulation was at 18²1th d?stable phase?; then sulforaphane content began to decrease and a small amount of sulforaphane were released.3. Effects of fungal elicitors on hairy root growth and sulforaphane accumulation?1? Aniger elicitor of 5 ?g·m L^-1 promoted hairy roots accumulation, proliferation multiple was 37.81, Aspergillus oryzae elicitor and Aspergillus fumigatus elicitor could inhibit the growth of broccoli hairy root, Penicillium elicitor had no significant effect on the proliferation rate of hairy roots.?2? Aniger elicitor of 10 ?g·m L^-1 could increase the content of sulforaphane of broccoli hairy roots, and reached 1297.47 ?g·flask^-1. Aspergillus fumigatus elicitor promoted the release of sulforaphane from roots into the media.?3? Synergy effects of fungal elicitors: the synergy of different concentrations Aspergillus niger and Aspergillus fumigatus promoted the biomass accumulation of broccoli hairy root and sulforaphane synthetic, adding 5 ?g·m L^-1 Aspergillus niger elicitor in the 9th days of culture system and induced for 144 h, the proliferation multiple of hairy root was up to 39.18, then transferred to MS liquid medium containing 10 ?g·m L^-1 Aspergillus elicitor and 30 ?g·m L^-1 Aniger elicitor, and cultured to 21 d, the sulforaphane content was up to 2517.15 ?g·flask^-1.