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目的优化喜树胚轴细胞悬浮培养体系生产喜树碱.方法将由喜树种子下胚轴诱导的愈伤组织进行悬浮培养,研究了基本培养基、激素和培养条件对培养物生长的影响,测定了培养细胞生长和喜树碱积累的动态变化.结果悬浮培养细胞在MS培养基中细胞生长良好,细胞分散性和喜树碱积累优于在其他培养基中.最佳激素组合为2,4-D 0.2mg/L+NAA 0.5 mg/L+6-BA 0.5mg/L,细胞继代周期以12d左右为宜,悬浮细胞适合在22~26℃,120r/min下生长.悬浮培养细胞在第4~20天为对数生长期,细胞倍增时间为t(d)=3.92d-1,细胞干重至第20天达到最大,为27.44g/L.在细胞对数生长期内,喜树碱积累与细胞生长呈线形正相关性,第20天喜树碱质量分数达到最大值9.319×10-5,喜树碱产量为2.56mg/L.结论MS基本培养基适合喜树胚轴细胞悬浮培养,细胞生长前20天内,培养体系中喜树碱积累与细胞生长呈正线形相关性.
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[Abstract]
Objective To optimize cell suspensing culture system of callus initiated from hypocotyls inCamptotheca acuminatato pro-duce camptothecin.Methods The callus initiated from hypocotyls ofC.acuminata seeds were suspensing-cultured,the effects of basic media,plant growth regulators and culture condition on cell growth were studied and the dynamic changes of cell growth and camptothecin accumulation were monitored.Results Suspension culture cells grew well in MS medium and cell homogeneity and eamp-tothecin content were superior to those in other media.The optimum plant growth regulators were 2,4-D 0.2mg/L+NAA 0.5mg/L+6-BA0.5mg/L, proper subculture time was about 21 and suspension cells developed well in 22-26℃ at 120r/min.The logarithm phase for cell growth was from 4th day to 20th day and during this time the double biomass accumulation time was 3.92 d.Meanwhile,during logarithm phase,campto-thecin was positively linear correlative to Cell growth.Consequently,at 20th day the maximum cell dry weight,camptothecin, and yield were 27.44 g/L,9.319×10-5,and 2.56 mg/L,respectively.Conclusion MS basic medium adapted to establish cell suspens-ion culture system of callus initiated from hypocotyls inC.acuminata and camptothecin is positively linear correlative to cell growth during the cell growth until the first day.
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