Original research article

A brief version of this protocol appeared in:
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水稻叶绿素含量的测定   

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实验原理:根据叶绿素提取液对可见光谱的吸收,利用分光光度计在特定波长下测定叶绿素提取物的吸光值,然后根据公式计算出样品中叶绿素的含量。
实验目的:可以检测水稻叶片中叶绿素a、叶绿素b及总叶绿素含量。

关键词: 叶绿素a, 叶绿素b, 总叶绿素含量

材料与试剂

  1. 2 ml离心管
  2. Parafilm
  3. 乙醇 (国药试剂) (分析纯)
  4. 丙酮 (国药试剂) (分析纯)
  5. 抽提Buffer (见溶液配方)

仪器设备

  1. 分析天平 (Adventurer, USA)
  2. 分光光度计DU640 (Backman, USA)
  3. 离心机 (Backman, USA)

实验步骤

  1. 取新鲜水稻叶片,擦干后去中脉,剪碎,混匀。
  2. 用分析天平称取0.1 g左右的叶片到2 ml离心管中,每个编号的样品分成三份作为三个技术重复,每管加入抽提Buffer 2 ml,parafilm封口,4 °C避光抽提12 h。
  3. 以抽提Buffer为空白对照,使用紫外分光光度计DU640在波长645 nm和 663 nm下测定叶绿素提取物的吸光值。

结果与分析

根据Arnon法 (1949) 的公式加以修正计算叶绿素含量:
叶绿素a = (12.72A663-2.59A645) × v/w × 1,000
叶绿素b = (22.88A645-4.67A663) × v/w × 1,000
叶绿素总含量 = (20.29A645+8.05A663) × v/w × 1,000

注意事项

  1. 为避免叶绿素见光分解,操作时尽量在弱光下进行,且操作速度要快。
  2. 样品不宜太多,否则加入抽提Buffer后,2 ml离心管装不下。
  3. 若4 °C避光抽提12 h后,叶片还没有完全变白,则延长抽提时间至叶片全部变白为止。
  4. 若叶绿素抽提物浑浊,则可以短暂离心后测定吸光值,否则会对试验结果造成误差。

溶液配方

  1. 抽提Buffer
    乙醇:丙酮:H2O = 4.5:4.5:1 (体积比)
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用格式:周勇, 范晓磊, 林拥军, 陈浩. (2018). 水稻叶绿素含量的测定. Bio-101: e1010147. DOI: 10.21769/BioProtoc.1010147.
How to cite: Zhou, Y., Fan, X. L., Lin, Y. J. and Chen, H. (2018). Determination of Chlorophyll Content in Rice. Bio-101: e1010147. DOI: 10.21769/BioProtoc.1010147.
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