用GPC-HPLC法测定植物组织中的细胞分裂素

Gel permeation chromatography (GPC)-high performance liquid chromatographic (HPLC) determination of cytokinin in plant tissues

为了探讨和开发凝胶渗透色谱(GPC)--高压液相色谱(HPLC)法定量测定植物组织中的细胞分裂素,以群众杨叶片和烟草组培苗为试验材料,根据细胞分裂素标准品回收率的测定,得出最佳试验方法:80%甲醇研磨后4℃浸提24h,抽滤,滤液离心,上清液加2滴氨水,40℃浓缩至干,用含3%甲醇的CH2Cl2溶解样品,经0.22μm滤膜过滤后过GPC纯化,最后HPLC外标曲线法定量。用液相--质谱联用仪(LC-MS)对所检测的细胞分裂素定性。测试表明:群众杨叶片中玉米素鲜质量含量439.5018ng/g,烟草叶片中玉米素鲜质量含量为617.9957ng/g。在植物样品中检测到的激动素(鲜质量含量49.4739～124.7124ng/g)应为外源引入。研究结果表明,GPC纯化植物细胞分裂素后用HPLC定量是可行的。

Using leaves originated from Populus popularis cuttings and Nicotiana tabacum plantlets, the research aimed to develop a GPC-HPLC method to determine the concentration of cytokinin in plant tissues. According to the reclaim efficiency of standard cytokinin, the recommended procedures were as follows： plant samples were grinded with 80% methanol and extracted at 4℃ overnight. Crude extract was filtrated and then centrifugated. The liquid was added with two drops of ammonia, and then vacuum dried at 40℃. The samples were resolved by 3% methanol-CH2C12 and filtrated through 0.22 μm filter. The filtrate was purified through GPC （Gel permeation chromatography）. The GPC-purified samples were quantified with I-IPLC by means of external standard curves. The cytokinins detected in plant tissues were confirmed with LC-MS. The results showed that the contents of zeatin in poplar and tobacco leaves were 439. 501 8 and 617. 995 7 ng/g FW, respectively. Kinetin （49. 473 9 - 124. 712 4 ng/g FW） was found in plant tissues, presumably resulting from the uptake from tissue culture medium of tabacco and insects feeding （aphid） in poplar.