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[Bio101] Pollen Fertility/viability Assay Using FDA Staining
[Bio101] 利用FDA染色测定花粉的育性和活力   

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Abstract

Pollen grains can be fertile or sterile by nature. This method stains pollen grains for an enzyme as the vital indicator of membrane integrity. Only fertile grains fluoresce under microscopic examination.

Keywords: Pollen(花粉), Fertility(生育), Vital staining(活体染色), Fluorescein diacetate(荧光素二乙酸酯), Pollen grain(花粉粒)

Materials and Reagents
  1. Fluorescein diacetate (FDA)
  2. Excision: At the time of anthesis
  3. Stain: Stock solution of FDA 2 mg FDA/ml of acetone (stored at -20 °C in an Eppendorf tube)
  4. BK buffer S15 MOPS (see Recipes)
  5. BK buffer S15 (see Recipes)

Equipment

  1. Fluorescence microscope
  2. Eppendorf tube

Procedure

  1. Take 1 μl of the stock solution of FDA and add to 1 ml of the BK buffer S15 MOPS (pH 7.5).
    Note: The stock solution of FDA is very volatile-the FDA-buffer mixture will not keep for more than 2 h.
  2. Mounting: Place a drop of the FDA-buffer mixture on a slide cleaned with alcohol and put a few pollen grains on the drop. Place a coverslip on top.
  3. Observation: Observe under optical microscope in blue light (wavelength = 495 nm). The viable pollen grains show fluorescence (FCR+).
    Remarks: The fluorescein diacetate, an apolar and non-fluorescent molecule, penetrates the pollen grain. Its hydrolysis by pollen esterases liberates fluorescein, a polar and fluorescent molecule. When the properties of membrane permeability are intact, the fluorescein accumulates inside the pollen grain, which appears fluorescent in blue light. The FCR test brings to light the esterase activity and the membrane integrity of the pollen grains.

Recipes

  1. BK buffer S15 MOPS (pH 7.5)
    Ca(NO3)2•4H2O (MW 236) 
    30 mg/L (0.127 mM)
    MgSO4•7H2O (MW 246.5)
    20 mg/L (0.081 mM)
    KNO3 (MW 101)
    10 mg/L (0.1 mM)
    Sucrose
    15%
    MOPS (MW209)
    10 mM (pH 7.5)
    Stored at -20 °C in an Eppendorf tube.
  2. BK buffer
    5 μl
    100 mM MOPS (pH 7.5)
    Sucrose 
    7.5 g
    Ca(NO3)2 (1 M)
    6.35 μl
    MgSO4 (1 M)
    4.05 μl
    KNO3 (1 M) 
    5 μl

References

  1. Heslop-Harrison, J. and Heslop-Harrison, Y. (1970). Evaluation of pollen viability by enzymatically induced fluorescence; intracellular hydrolysis of fluorescein diacetate. Stain Technol 45(3): 115-120.

简介

花粉粒可以是天然的能育的或无菌的。 该方法将酶的花粉颗粒染色为膜完整性的重要指标。 只有肥沃的谷物在显微镜检查下发出荧光。

关键字:花粉, 生育, 活体染色, 荧光素二乙酸酯, 花粉粒

材料和试剂
  1. 荧光素二乙酸酯(FDA)
  2. 切除:在开花时
  3. 染色:FDA的储备溶液2mg FDA/ml丙酮(在-20℃下在Eppendorf管中储存)
  4. BK缓冲器S15 MOPS(参见配方)
  5. BK缓冲区S15(见配方)

设备

  1. 荧光显微镜
  2. Eppendorf管

程序

  1. 取1μl的FDA储备溶液,并加入1 ml的BK缓冲液S15 MOPS(pH 7.5)。
    注意:FDA的储备溶液非常易挥发 - FDA缓冲液混合物不能保存超过2小时。
  2. 安装:将一滴FDA缓冲液混合物放在用酒精清洗过的载玻片上,并在滴上加几个花粉粒。将盖玻片放在上面。
  3. 观察:在光学显微镜下在蓝光(波长= 495nm)下观察。活花粉粒显示荧光(FCR +) 备注:荧光素二乙酸酯,非极性和非荧光分子,渗透花粉颗粒。其通过花粉酯酶的水解释放荧光素,极性和荧光分子。当膜通透性的性质完好时,荧光素积聚在花粉颗粒内部,其在蓝光中呈现荧光。 FCR测试揭示酯酶活性和花粉颗粒的膜完整性。

食谱

  1. BK缓冲液S15 MOPS(pH 7.5)
    Ca(NO 3)2·4H 2 O(MW 236)
    30mg/L(0.127mM)
    MgSO 4·7H 2 O(MW 246.5) 20mg/L(0.081mM)

    。KNO <3> (MW 101)
    10mg/L(0.1mM)
    蔗糖
    15%
    MOPS(MW209)
    10mM(pH7.5)
    储存在-20°C的Eppendorf管中
  2. BK缓冲区
    5微升
    100mM MOPS(pH7.5)
    蔗糖
    7.5克
    Ca(NO 3)2(1M)
    6.35微升
    MgSO 4(1M) 4.05微升
    KNO3(1 M) 
    5微升

参考文献

  1. Heslop-Harrison,J。和Heslop-Harrison,Y。(1970)。 通过酶诱导荧光评价花粉活力; 荧光素二乙酸酯的细胞内水解。染色技术 45(3):115-120。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Li, X. (2011). Pollen Fertility/viability Assay Using FDA Staining. Bio-protocol Bio101: e75. DOI: 10.21769/BioProtoc.75;
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Why pollen turns red?
11/13/2012 12:14:37 PM Reply
Xiyan Li
Department of Genetics, Stanford University, USA

Is it illuminated under blue light? Most likely it is the autoflurescence of the pollen wall.

11/17/2012 12:22:40 AM


why is it green in color is it due to the reaction of of an enzyme esterase?
8/9/2012 4:13:28 AM Reply
Xiyan Li
Department of Genetics, Stanford University, USA

Yes. FDA fluoresces only after being hydrolyzed by a cytosolic esterase.This enzyme is leaking out in dead pollen grains.

11/17/2012 12:23:44 AM