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Seed Coat Permeability Test: Tetrazolium Penetration Assay
种皮渗透性测试:四唑四氮唑蓝渗透分析   

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Abstract

Seed coat permeability is important to study as it plays significant roles in seed dormancy, germination, and protection from pathogens. Here we describe a commonly used seed coat permeability test known as the tetrazolium penetration assay with a method to quantify the levels of permeability. Tetrazolium red is a cationic dye that is widely used in seed viability testing. Tetrazolium salts are amphipathic cations, which, after penetrating the dead cells of the seed coat, are reduced to red-colored insoluble precipitates made up of formazans by active dehydrogenases (NADH-dependent reductases) in the embryo of seeds (Berridge et al., 1996). The intensity of red coloration is directly proportional to the permeability of the seeds. The quantification involves extraction of formazans from the incubated seeds and spectrophotometric determination of absorbance of formazan extracts at 485 nm.
Note: This protocol is optimized for testing Arabidopsis thaliana seeds.

Keywords: Seed coat(种子涂层), Permeability(透性), Tetrazolium(四氮唑), Formazan(甲臜), Arabidopsis(拟南芥)

Materials and Reagents

  1. Seeds (50 mg)
  2. 2,3,5-triphenyltetrazolium chloride (tetrazolium red) (Sigma-Aldrich, catalog number: T8877 )
  3. 95% ethanol (spectrophotometric grade)
  4. Distilled or ultra-pure deionized water

Equipment

  1. Brown bottle
  2. 1.5 ml microcentrifuge tubes
  3. Microcentrifuge tube rack
  4. Aluminum foil
  5. Air incubator (set to 30 °C)
  6. 1 ml Pipette (e.g., Gilson)
  7. Pestle and mortar
  8. Disposable pasteur pipettes (glass)
  9. Spectrophotometer
  10. Spectrophotometer cuvettes (polystyrene, 1.5 ml, path length 1 cm)
  11. Microcentrifuge
  12. Stereomicroscope with color camera

Procedure

  1. Prepare 1% (w/v) aqueous solution of tetrazolium red and store in a brown bottle. Set the incubator temperature at 30 °C.
  2. Weigh 50 ± 1 mg of dried seeds for each sample and place them in a 1.5 ml microcentrifuge tube. There should be at least three replicates for each sample. To measure the permeability over a time course, choose at least 5 time points (such as 4 h, 24 h, 48 h, 72 h, 96 h) and distribute the seed samples (with replicates) accordingly.
  3. Add 1 ml of 1% tetrazolium red solution to each tube containing seeds. Place these tubes in a microfuge tube rack and cover them with aluminium foil (incubation must be done in darkness). Place them in the air incubator at 30 °C for the specific time periods chosen. A negative control using water instead of tetrazolium red should be performed when doing the assay for the first time.  
  4. After completion of incubation for each time period, remove the tubes with seeds for observation. Observe the seeds change in color (look for formation of red color) and image some seeds using a stereomicroscope (Figure 1). The seeds can be kept at room temperature for microscope imaging but they should be imaged within 1 h. If longer, keep seeds at 4 °C.
  5. For the extraction of formazans, transfer the tube contents (seeds and the 1% tetrazolium red solution) into a mortar using a 1 ml pipette.
    Notes:
    1. Use cut tips to avoid clogging tip with seeds.
    2. The extractions should be performed immediately after each time point (do not store time points and do all at once).
  6. Remove the 1% tetrazolium red solution from the mortar using a glass pasteur pipette and discard. Wash the seeds with distilled water twice by rinsing using the pasteur pipette. Remove the water from the mortar and discard.
  7. Add 1 ml of 95% ethanol to the seeds in the mortar and finely grind the seeds using a pestle. Transfer the whole ground seed material in ethanol solution to a fresh microfuge tube using the pasteur pipette and adjust the final volume to 1.5 ml with 95% ethanol.
  8. Immediately centrifuge the tubes at 15,000 x g for 3 min. Collect the supernatant into a spectrophotometer cuvette (Figure 2).
    Note: Steps 7 and 8 must be performed quickly to avoid reaction of tetrazolium salts with the embryo cells after seed disruption.
  9. Using a spectrophotometer, measure the absorbance of the formazan extracts at 485 nm (Figure 2) and plot the readings. Use 95% ethanol as the reference (blank).

Representative data



Figure 1. Example of staining patterns prior to extraction of formazans. Staining pattern in seeds of Col-WT and seed coat permeability mutants incubated in 1% tetrazolium red at 30 °C and imaged using a stereomicroscope at the end of 48 h. The gpat5 mutant is severely affected and far1 far4 far5 triple mutant is moderately affected (Vishwanath et al., 2013).


Figure 2. Example of formazan extracts collected in spectrophotometer cuvettes and ready for absorbance measurements. The extracts are from 48 h time point. The data below each cuvette is the mean value of three replicates. The gpat5 mutant is severely affected and far1 far4 far5 triple mutant is moderately affected (Vishwanath et al., 2013).

Notes

  1. It is important to use seed that is relatively freshly harvested (less than 6 months old). If comparing genotypes, the plants should be grown together and the seeds harvested at the same time.
  2. For each sample, seeds should be uniformly well ground.
  3. Avoid collecting any seed debris while transferring the supernatant into the spectrophotometer cuvette.
  4. Use fresh cuvettes for each sample reading.
  5. The absorbance values of all the samples should be measured as quickly as possible. There should not be a big time gap between reading samples.
  6. Dilutions of samples in 95% ethanol should be made for absorbance values higher than 0.8 to remain in the linear range of the spectrophotometer. Then calculate the absorbance in the original samples using the dilution factor.

Acknowledgments

This protocol was adapted from Molina et al. (2008). This work was supported by a Discovery grant to O.R. from the Natural Sciences and Engineering Research Council of Canada.

References

  1. Berridge, M. V., Tan, A. S., McCoy, K. D. and Wang, R. (1996). The biochemical and cellular basis of cell proliferation assays that use tetrazolium salts. Biochemica 4(1): 15-19.
  2. Molina, I., Ohlrogge, J. B. and Pollard, M. (2008). Deposition and localization of lipid polyester in developing seeds of Brassica napus and Arabidopsis thaliana. Plant J 53(3): 437-449.
  3. Vishwanath, S. J., Kosma, D. K., Pulsifer, I. P., Scandola, S., Pascal, S., Joubes, J., Dittrich-Domergue, F., Lessire, R., Rowland, O. and Domergue, F. (2013). Suberin-associated fatty alcohols in Arabidopsis: distributions in roots and contributions to seed coat barrier properties. Plant Physiol 163(3): 1118-1132.

简介

种子包衣渗透性对于研究是重要的,因为它在种子休眠,发芽和对病原体的保护中起重要作用。 在这里,我们描述了一种常用的种皮渗透性测试称为四唑渗透测定法与量化渗透性水平的方法。 四唑红是一种阳离子染料,广泛用于种子活力测试。 四唑盐是两亲性阳离子,其在穿透种皮的死细胞后,在种子胚中被活性脱氢酶(NADH依赖性还原酶)还原为由甲偶合物形成的红色不溶性沉淀物(Berridge等人 al。,1996)。 红色着色的强度与种子的渗透性成正比。 定量包括从孵育的种子中提取甲seeds,并在485nm下分光光度测定甲an提取物的吸光度。
注意:此协议已针对测试拟南芥种子进行了优化。

关键字:种子涂层, 透性, 四氮唑, 甲臜, 拟南芥

材料和试剂

  1. 种子(50mg)
  2. 2,3,5-三苯基四唑氯化物(四唑红)(Sigma-Aldrich,目录号:T8877)
  3. 95%乙醇(分光光度级)
  4. 蒸馏水或超纯去离子水

设备

  1. 棕色瓶
  2. 1.5 ml微量离心管
  3. 微量离心机管架
  4. 铝箔
  5. 空气培养箱(设定为30℃)
  6. 1 ml移液管(,例如,Gilson)
  7. 杵和臼
  8. 一次性巴斯德移液器(玻璃)
  9. 分光光度计
  10. 分光光度计比色杯(聚苯乙烯,1.5ml,路径长度1cm)
  11. 微量离心机
  12. 带有彩色摄像机的立体显微镜

程序

  1. 准备1%(w/v)的四唑红色水溶液,并存储在一个棕色的瓶子。将培养箱温度设置为30°C
  2. 每个样品称取50±1mg干燥种子,并将其放置在1.5ml微量离心管中。每个样品至少应有三次重复。为了测量时间过程中的渗透性,选择至少5个时间点(例如4小时,24小时,48小时,72小时,96小时)并相应地分配种子样品(重复)。
  3. 向每个含有种子的管中加入1ml的1%四唑红色溶液。将这些管放置在微量离心管架中,并用铝箔覆盖(孵育必须在黑暗中进行)。将它们在空气孵化箱中在30°C下选择的特定时间段。使用水代替四唑红的阴性对照应该在第一次进行测定时进行。  
  4. 在每个时间段的孵育完成后,取出具有种子的试管用于观察。观察种子颜色变化(寻找红色的形成),并使用立体显微镜(图1)对一些种子进行成像。种子可以保持在室温下显微镜成像,但他们应在1小时内成像。如果更长,保持种子在4℃。
  5. 对于甲的提取,使用1ml移液管将管内容物(种子和1%四唑红色溶液)转移到研钵中。
    注意:

    1. 提取应在每个时间点后立即执行(不要存储时间点,一次执行所有操作)。
  6. 使用玻璃巴斯德移液管从研钵中取出1%的四唑红色溶液并丢弃。 用蒸馏水洗涤种子两次,使用巴斯德吸管冲洗。 从砂浆中取出水并丢弃
  7. 在研钵中向种子中加入1ml的95%乙醇,并使用杵仔细研磨种子。 使用巴斯德移液管将整个磨碎的种子材料在乙醇溶液中转移到一个新的微量离心管,并调整 最终体积用95%乙醇至1.5ml
  8. 立即在15,000×g离心管离心3分钟。收集上清液到分光光度计比色皿(图2)。
    注意:步骤7和8必须迅速进行,以避免种子破坏后四唑盐与胚胎细胞的反应。
  9. 使用分光光度计,在485nm处测量甲an提取物的吸光度(图2)并绘制读数。使用95%乙醇作为参考(空白)。

代表数据



在Col-WT和种皮通透性突变体的种子中在30℃下在1%四唑红中孵育并在立体显微镜下在48小时结束。 gpat5 突变体严重受影响,并且far1 far4 far5 三重突变体受到中度影响(Vishwanath等人,2013)。


图2.在分光光度计比色杯中收集的formazan提取物的实例,并准备进行吸光度测量。 提取物来自48小时时间点。每个比色杯下面的数据是三次重复的平均值。 gpat5 突变体严重受影响,并且far1 far4 far5 三重突变体受到中度影响(Vishwanath等人,2013)。

笔记

  1. 重要的是使用相对新鲜收获的种子(少于6个月大)。如果比较基因型,植物应该一起种植,种子同时收获
  2. 对于每个样品,种子应均匀地研磨
  3. 避免收集任何种子碎片,同时将上清液转移到分光光度计比色皿
  4. 每次样品读数都使用新鲜的比色杯。
  5. 所有样品的吸光度值应尽可能快地测量。 阅读样本之间不应有大的时间间隔。
  6. 对于高于0.8的吸光度值,应在95%乙醇中稀释样品,以保持在分光光度计的线性范围内。 然后使用稀释因子计算原始样品的吸光度

致谢

该方案改编自Molina等人(2008)。 这项工作是由一个发现授予O.R. 来自加拿大自然科学和工程研究理事会。

参考文献

  1. Berridge,M.V.,Tan,A.S.,McCoy,K.D.and Wang,R。(1996)。 使用四唑盐的细胞增殖测定的生化和细胞基础。 4(1):15-19。
  2. Molina,I.,Ohlrogge,J.B.and Pollard,M。(2008)。 沉积和定位脂质聚酯在欧洲油菜的发育种子中和< em> Arabidopsis thaliana 。 Plant J 53(3):437-449。
  3. Vishwanath,SJ,Kosma,DK,Pulsifer,IP,Scandola,S.,Pascal,S.,Joubes,J.,Dittrich-Domergue,F.,Lessire,R.,Rowland,O。和Domergue, )。 拟南芥中的Suberin相关脂肪醇:根系中的分布和对种子外壳屏障性质。植物生理学 163(3):1118-1132。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Vishwanath, S. J., Domergue, F. and Rowland, O. (2014). Seed Coat Permeability Test: Tetrazolium Penetration Assay. Bio-protocol 4(13): e1173. DOI: 10.21769/BioProtoc.1173.
  2. Vishwanath, S. J., Kosma, D. K., Pulsifer, I. P., Scandola, S., Pascal, S., Joubes, J., Dittrich-Domergue, F., Lessire, R., Rowland, O. and Domergue, F. (2013). Suberin-associated fatty alcohols in Arabidopsis: distributions in roots and contributions to seed coat barrier properties. Plant Physiol 163(3): 1118-1132.
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Srinath Srinath Rao
Gulbarga University
How to overcome seed dormancy in Nothopoditis
7/12/2014 6:00:12 AM Reply
Sollapura Vishwanath
Agriculture and Agri-Food Canada

I guess you mean Nathopodytes foetida. The seed dormancy in Nathopodytes is due to excess amounts of phenolics in seeds. Also, the treatment differs between variety and population of the seeds. You can try scarification of seed coat and GA3 treatment. Hope this helps.

7/12/2014 8:53:46 AM