胡萝卜及其愈伤组织细胞质Ca~(2 )水平分析的研究

为测定植物细胞质内Ca~(2 )]_i,对胡萝卜(Daucus carota var.sativa DC.)原生质体制备介质做了改进,并在正常生理条件下,用温和的、非损伤性的方法将Ca~(2 )荧光指示剂indo-1 K~ 和fura-2 K~ 导入该原生质体,能很好地标记细胞质内的游离Ca~(2 )。在此基础上,用显微荧光光度单波法测定被标记原生质体单个细胞胞质Ca~(2 )]_i。结果表明:被indo-1 K~ 标记的胡萝卜及其愈伤组织的原生质体Ca~(2 )]_i分别为88.3nmol/L和263.0nmol/L;fura-2 K~ 标记的分别为99.9nmol/L和255.5nmol/L。由此可见,脱分化的、处在细胞周期中的愈伤组织细胞质中Ca~(2 )]_i远高于分化了的、处于静息态的胡萝卜细胞。此外,为了确认测量的可靠性,对两种Ca~(2 )荧光指示剂分别做了体外校正,证明其线性相关。

ANALYSIS OF CYTOPLASMIC Ca~(2 ) LEVELS IN CARROT AND ITS CALLUS

Measurement of cytoplasmic Ca2 ]; in Daitcus carota var. saliva DC. was improved first modifying the preparative medium of, protoplast, then, under normal physiological condition, introducing fluorescent Ca indicators indo-1 K and fura-2 K into the protoplasts with gentle and non-invasiva loading procedure. The cytoplasmic free Ca2 could be well labeled. Cytoplasmic calcium levels of individual cells were measured via single-wave microfluorometiy. That Ca2 ]; of protoplasts of carrot and its callus labeled with indo-1 K and with fura-2 K were 88.3 nmol/L, 263.0 nmol/L and 99.9 nmol/L, 255.5 nmol/L, respectively. It was shown that cytoplasmic Ca2 ]ij of carrot callus in the state of dedifferentiation in cell cycle was much higher than carrot root cells in the differentiated resting cells. In addition, the authors performed the in vitro calibration of the two fluorescent Ca2 indicators respectively to determine their linear relationship between calcium ion concentration and the two indicators in order to ensure the reliability of measurements.