Effect Of Soybean Idioplasmic Material And Culture Conditions On Accumulation Of Isoflavone In Glycine Max(L.) Merr. Callus

Soybean isoflavones are a large class of secondary metabolites found mostly in legumes. Recently, the isoflavones which have been associated with several health benefits, have been the one of the hottest topics in secondary metabolites. Isoflavones are usually extracted from soybean seeds and the waste water of the processing, so isoflavones resource is relatively limited. The use of plant cell cultures for large-scale production of active compounds is of interest from scientific and economical points of view. Many secondary metabolites have been reported to be produced by plant cell cultures. So soybean cell cultures can be used to provide high concentrations of bioactive isoflavones.Compared with HPLC, HPCE was improved to detect the contents of isoflavones with the method of internal standard analyze. The effect of soybean varieties and the explants on the growth of callus and accumulation of isoflavnones has been investigated. Meantime, dynamic changes of the accumulation of isoflavnones and the activity of PAL (EC 4.3.1.5) have been studied. Then the culture conditions which influence the synthesis of isoflavones in soyben callus has been explored. The main results were as follows:1. High performance capillary electrophoresis was improved to quickly analyze contents of isoflavones in soybean seed and callus. The biochanin was used as an internal standard. Electrophoresis conditions were as follows: a capillary tube with 40mmol/L sodium borate containing 5% methanol as the running buffer(pH10.0) and an applied voltage of 16.5kV, at 25℃, detected at a wavelength of 254 nm. Under the optimum conditions, 6 kinds of isoflavones were separated successfully within 21 minutes. The corrected peak areas of isoflavones increased linearly with components of isoflavones, and the recoveries were ranged from 97.2%～103.3%. The analytical results demonstrate the method is simple, quick and well reproducible, and can be used for the determination of isoflavones.2. There were significant differences of the biomass, content and yield of isoflavones among soybean callus from difference varieties and the interplay between variety and soybean callus. There were significant differences of the content and yield of isoflavones, but no significant difference of the biomass among the calluses from different explants. It was found that there was very significant positive correlation between the content of isoflavones of different varieties and the content and the yield of isoflavones in soybean callus. In a word, the content of isoflavones in difference varieties has significant effect on accumulation of isoflavones in soybean callus. By the experiments in this paper, there always has high contents and yield of isoflanoves in soybean callus from Jianyangqiudadou hypocotyls. So it can be used as a potential material for producing isoflavones in the future.3. The growth of soybean callus was approximately consistent with the accumulation of isoflavones by studying the dynamic changes of the accumulation of isoflavnones and the activity of PAL (EC 4.3.1.5). The shape of dynamic trends like a "S" curve, but there was little difference among the different explants. So there was close connection between accumulation of isoflavones and primary metabolism. During the entire course of culture of callus, the activity of PAL changed in a double-wave. The activity of PAL was activated before the accumulation of isoflavones. There was a short lag phase between activation of PAL and synthesis of isoflavones. Taken together, the activity of PAL has significant affect on the synthesis of isoflavones in soybean callus, and the PAL is a key enzyme for it.4. The effect of culture conditions on growth of callus and accumulation of isoflavones:(1) The illumination has great influence on the growth of callus and the accumulation of isoflavones. Comparede to illumination (24h) and darkness (24h), the contents and yield of isoflavones with the combination of illumination and darkness (16/8h) increased up to 11.09 (mg/g) and 12.04 (mg/Flask).(2) It was showed that improving sucrose concentration enhanced the growth of callus and stimulated accumulation of isoflavones. In comparison with 2% sucrose, the contents and yield of isoflavones with 4% sucrose in the medium increased by 30 % and 61%.(3) There was concentration effect on the growth of callus and the accumulation of isoflavones for 2,4-D,NAA and 6-BA. In comparison with control, 1.5mg/L 2,4-D was the best concentration, it had made the biomass of callus and the yield of isoflavones improve synchronistically. Among the 6-BA concentrations tested, 0.4 mg/L 6-BA enhanced the growth of callus and stimulated accumulation of isoflavones markedly.(4) The phenylalanine stimulated the accumulation of isoflavones markedly; however, it restrained the growth of callus remarkably. The results showed that among the phenylalanine concentrations tested, 0.120 mmol/L phenylalanine improved the accumulation of isoflavones, and the yield of isoflavones reached 12.36mg/Flask, which has increased by 7 % compared to the control.(5) By the experiments in this paper, SA which was chosen as an effective elicitor for isoflavones synthesis enhanced the growth of callus and stimulated accumulation of isoflavones remarkably. In comparison with control, the contents and yield of isoflavones with 120μmol/L SA in the medium increased by 35 % and 58 %.