| Magnolia officinalis is the valuable tree in China. It’s resources are in high utilization but in natural are scarce. With blind felling by human beings,the resources of M.officinalis are sharply declining and it has been included in the national level 3 of rarely endangered plants. Plant cell culture is regarded as a promising way to provide an attractive alternative source for limited natural resources. It’s characters are that under the condition of full manual control, the influence from the region, season, soil and harmful organisms is not work. Besides, it has short production cycle. So we can get metabolites which can surpass the traditional plants cultivation by choosing good training system and improving cultivation conditions. The experiment through the optimization of tissue culture condition of M. officinalis and established the cell cultures of M. officinalisand mainly focused on elicitor-stimulated biosynthesis of its secondary metabolites. These studies would offer the expeirmental basic for the culture and production about medicine ingredients of M. officinalis. The main results were shown as follows:1. The most suitable condition for callus induction of M. officinalis was found through selection of basic mediums, explants and composition of the hormone. The callus induction was significantly impacted by all of three factors. B5 medium was suitable for callus induction of M. officinalis in B5ã€MSã€1/2MS 3 kinds of basic medium.Three kinds of explants of M. officinalis, which can be used to induct callus, are stems, phyllophores and leaves. The callus induction rate of stem is the highest, the ratio of callus inducing reach 91.67%. The secondly is phyllophor, and the lowest is leaf. Comprehensive analysis was followed by the conclusion-phyllophor is the best explant.3.0 mg·L-12,4-D+1.0 mg·L-16-BA was best for callus induction of M officinalis.The callus was induced more than 90%.2. Choosing some calluses with well and uniform growth to subculture culture.the purpose is to study the influence of concentration of NAA and 6-BA on the growth of callus. The effect of proliferation of callus was the best when callus on B5+2.0 mg·L-1 NAA+1.0 mg·L-1 6-BA, and it’s proliferation times reach 2.865. It could cause the inhibition of callus to subculture culture when concentration of NAA is more than 3.0 mg·L-1. While the concentration of 6-BA is 2.0 mg·L-1, the average proliferation of callus was only 1.202 times, and with poor growth condition, dark color and lax tissue.3. Cell suspension cultures were established by calluses of phyllophores.Study on sucrose concentration,shaking speed,cell density and the volume of liquid for the effects of the growth of4. suspension cells.Research results have shown that suspension cells had largest contents of growth under the condition of sucrose concentration was 30 g·L-1,shaking speed was 120 r/min, cell density was 1.0:20, the volume of liquid was 30 mL.4. The experiment has been studied on concentration and composition of the hormone, pH,2 kinds of precursors and light quality for the effects of the growth of suspension cells and the total phenol contents. It has been demonstrated that NAA 0.5 mg·L/1+2,4-D 0.5 mg·L-1+6-BA 0.5 mg·L-1+KT 1.5 mg·L-1 was the best composition for the growth of M. officinalis cells and the production of secondary metabolite, most suitable pH for culturing M. officinalis cells and its secondary metabolite was pH 6.5. It could highly promote the growth of cells and accumulation of its secondary metabolite when concentration and composition of precursors was D,L, β-phenylalanine 0.1 mg·L-1+L-phenylalanine 0.5 mg·L-1. Yellow light was best for the growth of suspension cells, but total phenol contents in cells were low. Under the blue light, the cell dry weight increasing rate was second to the yellow-light’s. And it had maximum contents of total phenol, been its contrast 3.19 times. After comprehensively analyzed, the conclusion was followed-the optimum light quality for culturing M. officinalis suspension cells was blue light. In hormone, pH, precursor and light quality these four influencing factors, the influence of light quality in secondary metabolism of M. officinalis cells was more grave than other three. The higtest total phenol content in cells of first three factors was about 0.200%. But it could reach 0.423% in the light quality test.5. The growth curve of M. officinalis cells appeared as a "S" shape. The growth period of cell cultures was around 24 d with highest biomass on day 18 and biggest total phenol contents on day 21.The best harvest of cell is on the eighteenth day. |
