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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。
In order to determine [Ca ~ (2 +)] _i in plant cytoplasm, the protoplast preparation of carrot (Daucus carota var.sativa DC.) Was improved and under normal physiological conditions, with a mild, non-invasive method The introduction of Ca 2+ fluorescent indicator indo-1 K ~ + and fura-2 K ~ + into the protoplast can well mark the intracellular cytoplasm free Ca ~ (2+). On this basis, the cytoplasmic [Ca ~ (2 +)] _i of single cells of the labeled protoplasts was determined by single-wave fluorescence spectrophotometry. The results showed that the protoplasts [Ca ~ (2 +)] _ i of carrot and callus marked by indo-1 K ~ + were 88.3nmol / L and 263.0nmol / L, respectively. The fura- Respectively 99.9nmol / L and 255.5nmol / L. Thus, [Ca ~ (2 +)] _ i in the dedifferentiated callus cytoplasm in the cell cycle is much higher than that of the differentiated, resting carrot cells. In addition, in order to confirm the reliability of the measurements, two Ca 2+ fluorescent indicators were calibrated in vitro, respectively, and proved to be linearly related.