【作者】 杜金华；

【导师】 郭勇；

【作者基本信息】 华南理工大学 ， 发酵工程， 1997， 博士

【摘要】 最初诱导出的玫瑰茄（Hibiscus sabdariffa L.）愈伤组织为黄色，不产花青素。调整B₅培养基中的某些成分，使其含1mg／L2，4—D、0.2mg／LKT，于25℃、16h／d光照培养愈伤组织。经过数代继代培养、黄色愈伤组织内出现红色“色素区”，适宜于高产花青素玫瑰茄细胞系的筛选。在光学显微镜下观察，细胞中的色素颗粒（anthocyanoplasts）呈现桔黄、桔红、粉红、红及紫等颜色，这些色素颗粒既存在于细胞质中也存在于液泡中。而且在非着色细胞的液泡中发现了紫色的小色素颗粒。玫瑰茄愈伤组织中的花青素可能在这些特定的细胞器（anthocyanoplasts）中生产、积累，然后释放到液泡中。 用平板培养及小细胞团培养两种方法筛选高产花青素的玫瑰茄细胞系。悬浮培养8天左右的玫瑰茄细胞最适宜于单细胞分离；pH5.84，CaCl₂·2H₂O为750mg／L的B₅培养基中植板率最高。植板细胞密度低于6×10³ cell／ml时，植板率急剧下降。于玫瑰茄愈伤组织的高色素区选择色深且颜色均一的小细胞团，置于B₅培养基（pH5.84，CaCl₂·2H₂ O:750mg／L）上培养。成活的细胞系与小细胞团继代培养10次，高产细胞系花青素含量为2.33％（g／g dw.），比筛选前提高了14.5倍。 用B₅培养基悬浮培养产花青素的玫瑰茄愈伤组织。培养13天时，花青素产量达0.25g／L。培养基中添加终浓度为10^-6 mol／L的外源L—Phe能显著地增加悬浮细胞花青素的积累量。浓度为10^-7 mol／L的槲皮素，可使悬浮培养的玫瑰茄细胞花青素产量2.3倍。无论是L-Phe还是槲皮素均不能启动不产花青素的细胞系积累花青素。 金属离子能提高培养细胞花青素的产量。改变B₅培养基中钙、锰、铜、铁等离子的浓度，悬浮培养玫瑰茄细胞。增加铁离子的浓度使花青素产量下降。其它三种离子对花青素的生产均有促进作用。其中锰离子最为有效，浓度为6×10^-4mol／L时，花青素产量为0.34g／L，提高了1.82倍。当培养基中铜含量超过10^-4mol／L时，培养细胞生长受到抑制或死亡。高浓度的钙使培养细胞结团现象严重。 在B₅培养基中添加L—抗坏血酸，悬浮培养的玫瑰茄细胞花青素产量下降。更多还原

【Abstract】 Callus of roselle （Hibiscus sabdariffa L.） in its initial stage was yellow and did not produced anthocyanin. At 25℃, 27.3 umol/s ? m² 、 16h/d light regime, the callus was cultivated on B₅ medium （pH5.8） containing lmg/L 2,4 — D and 0.2mg/L KT. Afer sveral generations, red"pigment areas" which were suitable for selecting were appear. Observation of Hibiscus sabdariffa cells under an optical microscope revealed that the occurence of pigment vesicles （anthocyanoplasts） which were orange 、 reddish orange 、 pink 、 red and purple both in the cytoplasm and in the vacuole. In some cases, anthocyanoplasts of purple color even observed in cells with nonpigment vacuoles, thus supporting the thesis according to which anthocyanoplasts would be the site of anthocyanin sythesis, then anthocyanin released into the vacuole.High production anthocyanin roselle cell lines were selected by both single cell clone plate culture and small-cell-aggregate culture. The cell suspension cultures for 8days were most favourable for the formation of cell clones. When PH value was 5.84 as well as CaCl₂ ? 2H₂ O concentration was 750mg/L in B5 medium, the platingefficiency （PE） was intensively increased.There was a lower PE when the cell plating density was no more than 6 ×10 cell/ml. Homogenous and dark color small-cell-aggregates were selected in high-content pigment areas of roselle callus and cultivated in B₅ medium （pH 5.84; CaCl₂ ? 2H₂ O: 750mg/L）. Both red cell lines and cellaggregates selected from cultures were subcultured successively for 10 generations. The anthocyanin content in high production cell line was 2.33 % （g/g dw）, as high as15.5 times of that of control.When pigment cells of roselle suspension cultures were grown in B₅ medium for 13 days, best production of anthocyanin which as high as 0.25g/L was obtained. The anthocyanin yield of pigmented suspension cultured cells was signifficantly increased if adding 10^-6 mol/L exogenous L-Phe into B₅ medium. When 10^-7 mol/L quercetin was supplied into the medium, the pigment yield was increased 1-3 fold of that of control. Either L-Phe or querectin feeding did not initate anthocyanin accumulation of yellow roselle callus.更多还原