Study On The Accumulation Of Alkaloids Such As Galanthamine In Lycoris Chinensis

Because Amaryllidaceae plants have many alkaloids which have immunobiologic ctivity, scientists have paid their attention to them for a long time. Among these alkaloids galanthamine is the most interested one. Galanthamine is an isoquinoline alkaloid acetylcholinesterase inhibito and an important agent used all around the world for the symptomatic treatment of senile dementia of the Alzheimer's type.There are two galanthamine sources for medical applications. One is the total synthesis. The other one is the natural sources from bulbs of some Amaryllidaceae plants. Because the approach of total synthesis is complex and expensive, some Amaryllidaceae plants still are the sources of some alkaloids such as galanthamine.Nevertheless it is worth to note that the galanthamine contents in Amaryllidaceae plants is low, at the same time it is facing the growing demand of the pharmaceutical market and the natural source would be insufficient and will at least lead to the disappearance of these plants from their natural habitats and destroy the environments. It is a hot research problem for a long time that how to improve the contents of galanthamine in Amaryllidaceae plants.In the present work we generalized the researches of galanthamine production using in vitro cultures all over the world and introduced a species of Lycoris chinensis which was specially distributed in china. The characteristics of seed storage and germination of Lycoris chinensis and galanthamine, lycorine and lycoramine figure changes in Lycoris chinensis during development. The effect of callus and shoot clumps which come from different explants on the galanthamine, lycorine and lycoramine contents and the effect of biotic and abiotic elicitors on the accumulation of galanthamine, lycorine and lycoramine and the relationship of an important enzyme Catechol 3-O-methyltransferase with the content of galanthamine are studied. The results of studies were showed as follows:1. Mature seeds of Lycoris chinensis were placed in-80℃plastic bags;-20℃plastic bags;-4℃plastic bags; wet sand in the room temperature; silica gel in the room temperature for 30 days separately, afterwards the germinating rate and seedling percentage were measured. Result indicated that the seed of Lycoris chinensis was not suitable to be stored at the pure low temperature such as-80℃and-20℃or at silica gel drying station. Beacuse its germinating ability would lose very quickly after hoarding a month. On the other hand it can keep viability after storing in wet sand at room temperature (10℃-30℃) or-4℃for a month. Present study also proved that capsule of the seed restrain germinating and the seed without capsule germinated quickly and germinating rate and seedling percentage were higher than the seed with capsule. Our experiment showed that the seed of Lycoris chinensis need short time dormancy before germinating. Low temperature or GA3 all could break out the dormancy and improve the germinating rate and seedling percentage and shorten the germinating time. Under illumination and dark condition, Lycoris chinensis seed germination rate and seedling percentage didn't have distinct differences. Lycoris chinensis seed belonged to the sensitive type of dehydration. With the decline of moisture content of seed, the viability of the seed dropped also. The viability of seed declined to 33% when seed moisture content went down to 51.10%. Lycoris chinensis seed belonged to recalcitrant-type seed.2. The galanthamine, lycorine and lycoramine content of Lycoris chinensis were researched during development from young to old plants. The contents of alkaloids in seed,10-day seedling,3-month seedling,1-year seedling, perennial seedling were determined by HPLC. It was notable that the level of galanthamine, lycorine and lycoramine reduced to its lowest contents at seed germinating of 10 days. Then the accumulation of galanthamine tends to increase with ages, reaching the higher value at perennial years. The production rules of lycorine, lycoramine in these different development phases exhibited similarity with galanthamine. The contents of these alkaloids are the highest in seedlings. Different plant organs were evaluated revealing that the seed kernel, seed capsule, and root were the main repository site with galanthamine, lycorine and lycoramine. It was notable that mature seed has the highest content of galanthamine (671.33μg/g DW). The leaves were the least productive organs.3. In vitro cultures we induced shoot clumps initiated from bubs, inflorescence and seed of Lycoris chinensis. We studied the effect of different times of sterilization, the different time of getting the explants, the different combination of hormone, and the different concentration of sugar on the inducement rate of Lycoris chinensis. The results showed that after the explants of buds was sterilization by 0.1% mercuric chloride for 12 minutes, the contamination rate declined to 32%, the survival rate was 96%, and the sterilization effect was the best. The research for different time of sampling showed that it was proper when the plant was in the first dormancy time (May-July). The research for different hormone combination showed that:MS+6-BA5.0 mg/L+NAA0.5 mg/L was the best inducement culture medium and the inducement rate was 100% and the buds induced was 8.5 every explant; the proper culture medium for rootage was 1/2 MS+6-BA0.15-0.18 mg/L+IBA 1.5-1.0 mg/L. The effect of sugar on the growth of shoot clumps showed that the diameter of little bud improved with the incensement of the concentration of sugar. When the sugar concentration was 90g/L, the diameter increased most, which was 2.7 times of control one (30g/L), at the same time the leaves of buds decreased.The explants of inflorescence was sterilization by 0.1% mercuric chloride for 13-14 minutes, the contamination rate declined to 57-42%, the survival rate was 66-54%, and the sterilization effect was the best. The research for different time of sampling showed that it was proper when the plant was flowering. The research for different hormone combination showed that:MS+6-BA5.0 mg/L+NAA0.5-0.7 mg/L was the best inducement culture medium.It was easier for the seed sterilization without capsule, when the explants of seed was sterilization by 0.1% mercuric chloride for 10-11 minutes, the contamination rate declined to 22-19%, the survival rate was 93-94%, so it was the proper sterilization time. The inducement rate was 98% after the germinating seeds were inoculated to inducement culture medium:MS+6-BA5.0 mg/L+NAA0.5 mg/L.4. Correlation between the state of differentiation and the content of galanthamine and related alkaloids were studied also. The best callus induceing culture was MS+2.0 mg/L 2, 4-D+0.5 mg/L BA and the callus inducement rate was 95.27% and the weight of callus was 1.72 g. The differentiation medium was MS+6-BA5.0 mg/L+NAA0.5 mg/L.The results showed that the differentiation have much relationship with the content of galanthamine and related alkaloids. Galanthamine was not been detected in the callus, but been detected in somatic embryogenesis and shoot clumps. The content of galanthamine, lycorine and lycoramine in the somatic embryogenesis and shoot clumps were less than in the 3-month-old seedlings and perennial plants also. The growth and content of alkaloids of shoot clumps induced from different explants were studied. The results showed that the sboot clumps came from different explants had different gowth state. The diameter of the bud was bigger in shoot clumps came from seeds than others, it was about 1.65 times than that came from buds. But the numbers of the little buds was the most in the shoot clumps came from buds; it was about 2.7 times of that of seed. The content of galanthamine, lycorine and lycoramine in the shoot clumps induced from different explants were less than in the 3-month-old seedlings and perennial plants also.5. Three-month-old seedlings of Lycoris chinensis were treated with biotic and abiotic elicitors. We have shown that the addition of MJ and NO promote the accumulation of galanthamine in these seedlings separately. The effect of these elicitors on the growth of the seedlings, as well as on the amount of the alkaloids accumulated in the seedlings was studied. The results showed that the elicitors had a negative effect on the growth of the seedlings when MJ was 50,100,250μM and SA was 0.25,0.5,1.0 mM, while the elicitors had a positive effect on the growth of the seedlings when YE was 0.05,0.10,0.15 g/L and NO was 50,100,500μM. It was remarkable that the addition of MJ, NO and YE can promote galanthamine accumulation in seedlings. The accumulation was higher in treatments at higher concentrations of NO (100μM), where the accumulation of galanthamine was 1.72-fold higher than the control by the 10th d of culture. The highest values of lycorine were also obtained in the seedlings treated with YE at a concentration of 0.01 g/L and by the 10th d of culture; the level was 1.38 times of control.6. A putative segment of Catechol O-methyltransferase (COMT) gene was obtained which has been proved to be related with biosynthesis of galanthamine. Semi-quantitative RT-PCR indicated that there was some correlation between COMT gene expression and the accumulation of galanthamine in flowers, flower stalks, bulbs and roots. In flowering stage, the expression of COMT was richer in flowers than in roots and bulbs, at the same time it has the highest content of galanthamine. On the contrary, the galanthamine content of flower stalks was the lowest; the expression of COMT in flower stalks was richer among all organs of Lycoris chinensis in flowering stage. Therefore further study was needed to elucidate the relationship between COMT and the biosynthesis of galanthamine.