| Neem tree (Azadirachta indica A.Juss) is one of the promising resources for botanical insecticides. Azadirachtin is mainly active component, being the outstanding characteristics of both a broad-spectrum bioactivity and peculiar mode of action, eco-friendly and non-toxic effects towards mostly beneficial organisms. Currently, the azadirachtin used in the market is extracted from the neem seeds, by which it is difficult to meet the needs of the growing market. Using the cell engineering technique, we try to explore a new way to produce the pesticide. On the cell culture of medicinal plants, there are many advantages, such as cells growing fast, controllable production conditions, and accumulation of target components through biological transformation.In this study, young leaves, stem segments and seeds from neem were used as explants, respectively, to be induced and subcultured into callus for establishing neem cell suspension culture system. With cell biomass and active ingredient content as the main indexes, cultural conditions were optimized by single factor analysis during cell suspension in shake flask, All results are indicated as follows. By the comparsion among three levels (nothing but the types of medium, two kinds), Azadirachta indica callus induction medium is MS (Murashige and Skoog)+2,4-D (2,4-Dichlorophenyloxy) 1 mg/L+6-BA (6-Benzylaminopurine) 0.5 mg/L. The dynamic changes of pH, conductivity, carbon source, nitrogen source and phosphorus source of the medium were preliminarily analyzed during the cultivation of cell suspension, respectively.The process conditions of cell suspension culture of Azadirachta indica were optimized by single factor analysis as follows. Using MS medium, inoculum size is 30 g/L, initial pH of 5.8, photoperiod light/dark to 16/8 h,3% sucrose as carbon source, the ratio of NH4+ /NO3- 12:48 and phosphate concentration 0.625 mmol/L of phosphate. After 20 days of culture, the cell dry weight increment ratio reached 3.79 ± 0.19, and the content of the limonoids reached 3.62 ±0.14 mg/g.By one step, organic solvent was used to immerse callus, suspension culture and suspention culture cells to obtain their extracts, respectively. In accordance with the standard containing 0.003% azadirachtin, the extracts were diluted to pest control regents, being applied to treat Tenebrio molitor larvaes. The results showed, given that the lethal rates of all three kinds of extracts are higher than that of negative control, which the maximum lethal rate of the extract reachs over 20% when culture for 9 days, while they are lower than that of 0.003%Azadirachtin EC. The performance of three kinds of extracts against the larvaes are superior to the negative control on body weight, antifeedant and repellent rates, reaching or exceeding the pesticide pest control level of Azadirachtin Emulsion.In conclusion, the cell culture of neem can not only meet the industrialized condition for large-scale production, but also harvest a large number of cell cultures. Moreover, the extracting method of neem cell cultures by one step, can be directly used for pest control agent, reducing the production costs of Azadirachtin separation and purification. Thus, this study is a useful trial for industrialized production of azadirachtin pesticide abtained from cell suspention culture. |
PDF Full Text Request