| In recent years, the technology of palnt tissue culture has been widely employed in the production of plant secondary metobolites. Psammosilene tunicoides is a rare and endangered medicinal plant species, which was used to investigate the effect of the different culture conditions on the induction and proliferation of Psammosilene tunicoides callus and the pigment accumulation of secondary metabolites that would need the preliminary separation identification in this paper. On the one hand, we can provide a new idea for the protection and utilization of precious germplasm resources. On the other hand, we can also explicit the main pigment composition of Psammosilene tunicoides callus to provide the foundation for further exploitation of natural pigment. The main results and conclusions were as follows:1. The plant tissue culture technology was adopted to research the effect of the different culture conditions and disinfectants on the rate of seeds germination and the proliferation of Psammosilene tunicoides callus that were induced by different explant(stem, leaf and bud).The results showed that the method of Psammosilene tunicoides seeds germination after respectively putting them in 75% alcohol(time of 75 seconds), 0.1% potassium permanganate solution(time of 30 seconds) and 0.5% mercuric chloride solution(time of 15 minutes) was the better way to reduce the pollution of seeds than other methods. By using different explants and plant growth regulators to inducing callus, the results showed that the effect of buds inducing callus was better than that of stems and leaves inducing callus and the appropriate combination of plant growth regulators were 0.2 mg·L-1 2,4-D and 0.5 mg·L-1 6-BA. At the same time, the appropriate combination of plant growth regulators contributed to the growth of white callus and would provide enough experiment materials for further induction and accumulation of pigment.2. Comparing the effects of different culture conditions on Psammosilene tunicoides callus biomass and pigment content to select suitable culture condition that callus induced pigment fast. MS as the basic culture medium while individually adding plant growth regulator NAA and 6-BA that was better than the inductive effect of simultaneous addition.The specific content of NAA was 1.5 mg·L-1 and that of 6-BA was 0.5 mg·L-1. 2,4-D has a remarkable inhibitory effect on the accumulation of pigment. IBA had no obvious effect on the induction of pigment. The low concentration of IAA 1.5 mg·L-1 made for a small amountof pigment induction. The White medium that induced the accumulation effect of pigment was better than other medium, which has no significant difference comparing with MS. At the same time, the induction and accumulation effect of pigment was better under the conditions that were temperature of 25?, light of 2000 lx and pH of 5.8.3. To preliminary extract and identify the pigment that was induced by Psammosilene tunicoides callus. The extraction effects of pigment were different under different extraction conditions of pigment. The results showed that the better extraction method was that material-liquid ratio was 1 to 10(g:mL), time was 20 hours, temperature was 30 ? and solvent was 2% HCl-ethanol solution. By using paper chromatography to preliminary identify the composition of pigment. It isolated two colored tapes from the pigment by the paper chromatography, and one of the colored tapes was cyanidin-3-O-glucoside chloride. Besides,cyanidin-3-O-glucoside chloride was the main composition of pigment which was further identify by high performance liquid chromatography. The result of analysis of pigment stability showed that the pigment was fairly stability under the condition of temperature of4~60?, the existence of reducing agent and acid. While the metal ions(Cu2+and Fe3+) and light had significant damage for the stability of pigment. Therefore, the pigment of Psammosilene tunicoides should be kept the condition of dark and without the metal ions of Cu2+ and Fe3+. |
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