Primary Studies On Accumulation Of Triterpenoids In Culture Medium Of Betula Platyphylla Suk.

In the thesis,barks of Betula platyphylla Suk.were taken as material to analysis the difference of the betulin content in different parts and provenances in birch.Then,tissuecultured seedings of Betula platyphylla Suk.were taken as culture material to induce callus,for solid culture and suspension culture,and culture technique of metabolite regulation of triterpenoid content in callus was studied.Primary studies the connection between callus growth and triterpenoid accumulation,this studies would provide theory and technology basis for producing total triterpenoid by cell engineering in birch.The results were as follows:1,The betulin content variation in different parts and provenances in Betula platyphylla Suk.Betulin content in the same birch bark through the four extraction methods,Saxhlet apparatus,supersonic wave was 240.25mg/g,239.57 mg/g respectively.In the comparison with other methods,the method of supersonic wave had more advantages,So the method with supersonic wave was suggested to extract betulin in birch.The content of betulin in different parts of birch was:bark＞branch skin＞inside skin＞bud＞leaf＞root＞pollen.Among of them,the tallest betulin content was in the bark,and the lowest was in pollen.Betulin contents in the bark of eight-year old birches of 13 provenances from Xinjiang,Gansu,Ningxia, Neimeng and the Northeast region were detected,and their range was from 132.45mg/g to 257.11mg/g.The higher betulin content was 257.11 mg/g and 240.36 mg/g in the birches of the two provenances of Beihu and Liangshui in north-east areas,respectively;the lower betulin content was 139.24 mg/g,136.26 mg/g and 132.45 mg/g in those of Liaoning,Neimeng and Ningxia,respectively.2,The optimization of callus solid culture system and studies on metabolic regulation of trierpenoid from Betula platyphylla Suk.The system for triterpenoid accumulation in callus was established:The highest yield of trierpenoid was obtained when callus cultured in the IS medium and add 0.8mg/L6-BA and 0.6 mg/L NAA with sucrose concentration 30 g/L,strong light and blue light.Compared with MS,NT,IS,WPM,B₅ medium,WPM medium was best suitable for callus growth,the fresh weight of callus yield achieved finally6.6g(FW)/bottle;IS medium was ideal to the triterpenoid accumulation,the highest yield achieved 2.3670 mg/g(DW).On combinations of all the plant hormones,6-BA and TDZ composition was suitable for callus growth,but 0.8 mg/L6-BA +0.6 mg/LNAA was suitable for accumulation of triterpenoid in callus,yield achieved 3.5319 mg/g(DW).Under different carbon sources conditions,30g/L sucrose was ideal to the triterpenoid accumulation,the conten of triterpenoid was 4.040 mg/g(DW).On different light conditions,blue light was suitable for accumulation of triterpenoid in callus,the highest yield achieved 8.413 mg/g(DW).3,Primary establishment of callus suspension culture system and studies on metabolic regulation of trierpenoid from Betula platyphylla Suk.The system for triterpenoid accumulation in callus suspension culture was established: The highest yield of trierpenoid was obtained when callus suspension cultured in the B₅ medium and add 0.4mg/L6-BA and 0.2 mg/L TDZ,the inoculation weight was 10～20g/L,deal with light.During all the suspension culture period,the accumulation of fresh weight of birch callus showed an upward trend,the content of triterpenoid was achieved the ninth day,the content was14.3075 mg/g(DW).Compared with MS,NT,IS,WPM,B₅,1/2MS medium, B₅ medium was best suitable for callus suspension cultured growth,the fresh weight of callus yield achieved finally13.72g(FW) /bottle;NT medium was ideal to the triterpenoid accumulation,the highest yield achieved 11.9671mg/g(DW).The plant hormones were 0.4mg/L 6-BA and 0.2mg/L TDZ was suitable for accumulation of triterpenoid in callus suspension,yield achieved 14.2264mg/g(DW).The inoculation weight was 10g/L was suitable for accumulation of triterpenoid in callus suspension,yield achieved 14.3638mg/g(DW).Light was not only to be suitable for callus suspension cultured growth,but also was ideal to the triterpenoid accumulation.4,All of the test show that suspension culture was better suitable for triterpenoid accumulation in callus then solid culture,the best culture condtion was:suspension cultured with B₅ medium and add 0.4mg/L6-BA and 0.2 mg/L TDZ,the inoculation weight was 10 g/L (FW),deal with light.