Induction Of Hairy Root By Agrobacterium Rhizogenes And Establishment Of Culture System Of Broccoli

Broccoli is rich in nutrients including protein, lipid, vitamin C and carotene, the content of minerals, such as calcium, phosphorus, iron, potassium, zinc, manganese are higher than that of brassica oleracea. And broccoli is rich in sulforaphane, which is a kind of anti-cancer substances and has high cancer prevention and anti-cancer effects, in addition, broccoli also can enhance immunity, promote growth, improve blood vessel toughness and other effects. Because of extensive pharmacologic action in broccoli, the demand of it is growing on the market. However, due to the complexity of breeding and long cycle, and limited by the natural growing conditions, the traditional cultivation technique has a significant impact on yield and quality of broccoli. In the field of medicinal plants, in order to cultivate new varieties of plants and extract the active ingredients, the way of that agrobacterium rhizogenes disseminated explants to induce and culture hairy root has become a hot research and hairy root is equal or even higher rich in secondary metabolites compared with original plants. This paper established a system of broccoli hairy root induction by optimizing the conditions of agrobacterium rhizogenes strain, explant types, pre-incubation time, bacterial concentration, immersion time, co-culture temperature and time, sterilization methods and so on, while the hairy root growth conditions, such as media types, medium carbon, medium PH and exogenous hormones were optimized, which laid the foundation of the study of broccoli hairy root. In addition, the anti-cancer substance sulforaphane in broccoli hairy root has a crucial role in the study of future research includin medicine and other fields. The following major conclusions were got in this study: 1. Establishment of broccoli hairy root culture system(1) Broccoli was infested with agrobacterium rhizogenes strain ATCC15834 and ATCC11325, according to the induction rate of hairy root, found that the suitable strain inducing hairy root was ATCC15834, and the induction rate was 64.82%.(2) When leaves and stem segment explants of broccoli were infested with ATCC15834 strain, a higher induction rate was got in the more mature but not aging leaves, which reached 64.03%.(3) The leaves of broccoli seedlings of 15 days were cut into 0.5 cm2 and inoculated in MS medium for pre-incubation of 0,1,2,3,4,5,6 d, and leaves had the highest hairy root induction rate, 69.12%, in the 4th day.(4) After pre-incubation, the leaves were disseminated by agrobacterium ATCC15834 whose OD600 value were 0.1, 0.3, 0.5, 0.7, 0.9, and the highest induction rate was got and reached 64.18% when the OD600 value was 0.5(5) Through the experiments of immersion time and co-culture time, a high induction rate was obtained when immersion time was 5min, and co-culture time was 4 d, and the inducation rate were 71.82% and 65.45% respectively.(6) When the disseminating leaves were placed in MS medium adding acetyl acetosyringone(AS) and co-cultured, the appropriate concentration of AS could promote transformation of plant explants. When the adding concentration of AS were 0, 50, 100, 150, 200, 250 μmol/L, the result indicated that the highest induction rate reached 66.59% when the AS concentration was 100 μmol/L.(7) After the dissemination and co-culture, leaves with agrobacterium should be cultured in MS+250mg/L carbenicillin disodium(cab) sterilization medium, and subculture was every 5 days once, the fully sterilized hairy roots were got after 2-3 times subculture, and used as material cultured in liquid medium. 2. Broccoli hairy root culture in vitro(1) The hairy roots with 2-3cm were transplanted to different liquid media, and found that broccoli hairy roots in different media types had quite different growth conditions, and the MS medium were conducive to growth and maximum proliferation times reached 9.46.(2) By adding sucrose and glucose into MS medium found that hairy roots had the highest induction times, which was 9.43, when the sucrose dosage was 30 g/L.(3) By adding different exogenous hormones into medium found that 0.5 mg/L of IBA was benefit to hairy roots growth, the biggest proliferation times reached 10.82. 3. Determination of Sulforaphane content in explants of broccoliSulforaphane content in leaves, ordinary roots and hairy roots were determined by HPLC method, the result found that sulforaphane content in hairy roots reached 2939.159 μg/g DW and significantly higher than that of leaves and ordinary roots.