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Mar 2013
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Seed Germination and Viability Test in Tetrazolium (TZ) Assay
Tz法测定种子发芽率及种子活力   

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Abstract

Tetrazolium (TZ) assay is the fast evaluation for seed viability and alternative quick method for seed’s germinability (Porter et al., 1947; Wharton, 1955). All respiring tissues are capable of converting a colourless compound, TZ (2,3,5 triphenyl tetrazolium chloride) to a carmine red coloured water-insoluble formazan by hydrogen transfer reaction catalysed by the cellular dehydrogenases. TZ enters both living and dead cells but only living cells catalyse the formation of formazan which being non-diffusible stains the viable seeds red whereas the absence of respiration prevents formazan production making the dead seeds (aged tissue) remain unstained.

The brief description of this protocol has been reported in Verma et al., 2013.

Materials and Reagents

  1. Arabidopsis thaliana (Columbia-0) seeds were used in our study
  2. 2,3,5 triphenyl tetrazolium chloride (Sigma-Aldrich, catalog number: T8877 )
  3. Commercial bleach (Sodium hypochlorite)
  4. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  5. Autoclaved distilled water
  6. Lactic acid
  7. Phenol
  8. Glycerine
  9. Whatman no.1 filter paper
  10. 1% Tetrazolium (TZ) solution (see Recipes)
  11. Scarification solution (see Recipes)
  12. Clearing agent (lactophenol solution) (see Recipes)

Equipment

  1. 30 °C incubator
  2. Shaker at RT
  3. Weighing balance
  4. pH meter
  5. Stereo Microscope
  6. Culture room (22 °C ± 2, 16 h light 200 μmol/m2/s/8 h dark)
    (any instrument which provides above mentioned conditions will work)

Procedure

  1. Scarify the Arabidopsis seeds by soaking approximately 100 seeds (in three replicates) in 1 ml scarification solution for 15 min under shaking conditions at RT. Wash at least five times with distilled water to remove the bleach.
    Note: Perform steps 1-3 under sterile conditions.
  2. After scarification, remove excess water and incubate the seeds with 1% TZ solution at 30 °C for 24 to 48 h in dark (observe the seeds after 24 h and proceed with step 3 if stain appears). Take heat killed (100 °C, 1 h) seeds as negative control.
  3. After staining, wash the seeds 2-3 times with distilled water.
  4. Immerse the stained seeds in clearing agent for 1-2 h.
    Follow step 4, if the pigment within the seed coat prevents clear vision after staining.
  5. Observe the seeds under stereo microscope.
  6. Evaluate the seeds on the basis of staining pattern and colour intensity. Among stained seeds, seeds with bright red staining are completely viable while partially stained seeds may produce either normal or abnormal seedlings. Pink or greyish red stain indicates dead tissue. Completely unstained seeds are non-viable. Figure 1 shows TZ staining pattern in viable (normal), abnormal and dead or non-viable seeds.


    Figure 1. Different pattern of TZ staining showing visable, abnormal and dead or non-viable seeds

  7. In parallel, place about 100 seeds without scarification on two layers of Whatman no.1 filter paper soaked with distilled water in culture room for germination assay (perform the germination assay in triplicate). Score the seed germination by considering radicle protrusion beyond testa as germinated seeds. To score the germination, count the no. of seeds with radicle protrusion as germinated seeds and calculate the percentage germination against total no. of seeds.

Recipes

  1. 1% Tetrazolium (TZ) solution
    Add 1 g 2,3,5 triphenyl tetrazolium chloride in 100 ml autoclaved distilled water in amber colour bottle.
    Mix well and store in dark at 4 °C (can be kept for several months under such conditions).
  2. Scarification solution
    20 ml commercial bleach and 100 μl Triton X-100 in 100 ml autoclaved distilled water. Mix well and store under sterilized conditions (prepare fresh).
  3. Clearing agent (lactophenol solution)
    Mix lactic acid: phenol: glycerine: water in a ratio of 1:1:2:1. Use if the pigment within the seed coat prevents clear vision after staining (prepare fresh).

Notes

  1. The pH of the TZ staining solution should be 7. Solution with pH > 8 or pH < 4 would result in either intense staining or would not stain even viable seed tissues. If water is out of neutral range then use phosphate buffer with pH 7 to dissolve TZ.
  2. TZ assay can be used for seeds of legume, cotton and grasses. The incubation time varies with seed type and morphology. Remove the seed coats of larger seeds (like legume seeds) before examination.
  3. The dicot seeds can be germinated further as the stained seeds are not damaged.
  4. When performed appropriately, the percentage of viable seeds obtained by tetrazolium assay is very close to the percentage of seed germination expected under most favourable conditions.

Acknowledgments

This protocol was adapted from Porter et al. (1947) and Wharton (1955). This work was supported by the Department of Biotechnology (grant no. BT/PR10262/GBD/27/77/2007) and National Institute of Plant Genome Research, Government of India.

References

  1. Porter, R., Durrell, M. and Romm, H. (1947). The use of 2, 3, 5-triphenyl-tetrazoliumchloride as a measure of seed germinability. Plant Physiol 22(2): 149.
  2. Verma, P., Kaur, H., Petla, B. P., Rao, V., Saxena, S. C. and Majee, M. (2013). PROTEIN L-ISOASPARTYL METHYLTRANSFERASE2 is differentially expressed in chickpea and enhances seed vigor and longevity by reducing abnormal isoaspartyl accumulation predominantly in seed nuclear proteins. Plant Physiol 161(3): 1141-1157.
  3. Wharton, M. J. (1955). The use of tetrazolium test for determining the viability of seeds of the genus Brassica. Proc Int Seed Test Assoc 20: 81-88. 

简介

四唑(TZ)测定是种子生活力的快速评估和种子发芽的替代快速方法(Porter等人,1947; Wharton,1955)。 所有呼吸组织能够通过由细胞脱氢酶催化的氢转移反应将无色化合物TZ(2,3,5-三苯基四唑氯化物)转化为胭脂红红色的水不溶性甲。。 TZ进入活细胞和死细胞,但只有活细胞催化形成甲酸,其是非扩散性的,将活的种子染成红色,而没有呼吸阻止甲酸生产使死种子(老化组织)保持未染色。 ;  此协议的简要说明已在Verma 等人,2013年中报告。

材料和试剂

  1. 在我们的研究中使用拟南芥(Arabidopsis thaliana)(哥伦比亚-0)种子
  2. 2,3,5-三苯基四唑氯化物(Sigma-Aldrich,目录号:T8877)
  3. 商业漂白剂(次氯酸钠)
  4. Triton X-100(Sigma-Aldrich,目录号:T8787)
  5. 高压蒸馏水
  6. 乳酸
  7. 苯酚
  8. 甘油
  9. Whatman 1号滤纸
  10. 1%四唑(TZ)溶液(参见配方)
  11. 澄清解决方案(参见配方)
  12. 清除剂(乳糖醇溶液)(参见配方)

设备

  1. 30℃培养箱
  2. 室温下振荡器
  3. 称重余额
  4. pH计
  5. 立体显微镜
  6. 培养室(22℃±2,16h光200μmol/m 2/s /8h黑暗)
    (提供上述条件的任何仪器都可以工作)

程序

  1. 通过在室温下在摇动条件下在1ml除草溶液中浸泡约100粒种子(三次重复)来稀释拟南芥种子15分钟。 用蒸馏水洗至少五次,以去除漂白剂。
    注意:在无菌条件下执行步骤1-3。
  2. 划痕后,去除多余的水,种子与1%TZ溶液在30℃暗处24至48小时孵育种子(24小时后观察种子,如果出现染色,继续步骤3)。 取热杀死(100℃,1小时) 种子作为阴性对照
  3. 染色后,用蒸馏水将种子洗2-3次
  4. 将染色的种子浸泡在清洁剂中1-2小时。
    按照步骤4,如果种皮内的颜料在染色后阻碍清晰的视觉
  5. 在立体显微镜下观察种子。
  6. 根据染色模式和颜色强度评估种子。在染色的种子中,具有亮红色染色的种子是完全存活的,而部分染色的种子可以产生正常或异常幼苗。粉红色或灰色红色指示死亡组织。完全未染色的种子是不存活的。图1显示了存活(正常),异常和死亡或非存活种子的TZ染色模式

    图1. TZ染色的不同模式显示可见,异常和死亡或非活性种子

  7. 平行地,将约100个种子无划痕地放置在用蒸馏水浸泡在培养室中的两层Whatman 1号滤纸上以用于发芽测定(进行一式三份的发芽测定)。通过考虑超过睾丸的胚根突起作为发芽的种子来评分种子发芽。要得分萌发,计数不。的种子与胚根突起作为发芽的种子,并计算发芽百分比对总数。的种子

食谱

  1. 1%四唑(TZ)溶液 加入1克2,3,5-三苯基四唑氯化物在100ml高压灭菌蒸馏水的琥珀色瓶中。
    混合均匀,在4℃避光保存(可在这样的条件下保存几个月)
  2. 划痕解决方案
    20ml商业漂白剂和100μlTriton X-100装入100ml高压灭菌的蒸馏水中。混合均匀,在无菌条件下储存(新鲜制备)。
  3. 清除剂(乳酚溶液)
    混合乳酸:苯酚:甘油:水的比例为1:1:2:1。 如果种皮内的颜料在染色后(清新制备)阻碍清晰的视觉,则使用

笔记

  1. TZ染色溶液的pH应为7。 8或pH < 4将导致强染色或甚至不会染色即使存活的种子组织。 如果水超出中性范围,则使用pH为7的磷酸盐缓冲液溶解TZ
  2. TZ测定可用于豆科植物,棉花和草的种子。 孵育时间随种子类型和形态而变化。 在检查之前,取出较大种子(如豆类种子)的种皮
  3. 双子叶植物种子可以进一步发芽,因为染色的种子没有被损坏
  4. 当适当地进行时,通过四唑测定获得的活的种子的百分比非常接近在最有利条件下预期的种子发芽的百分比。

致谢

该协议改编自Porter等人(1947)和Wharton(1955)。 这项工作得到生物技术部(授权号BT/PR10262/GBD/27/77/2007)和印度政府植物基因组研究国家研究所的支持。

参考文献

  1. Porter,R.,Durrell,M。和Romm,H。(1947)。 使用2,3,5-三苯基四唑氯化物作为种子发芽率的量度。 a> Plant Physiol 22(2):149
  2. Verma,P.,Kaur,H.,Petla,B.P.,Rao,V.,Saxena,S.C.and Majee,M。(2013)。 蛋白L-ISOASPARTYL METHYLTRANSFERASE2 在鹰嘴豆中差异表达,并增强种子活力 和通过减少异常的异天冬氨酸积累主要在种子核蛋白中的长寿。植物生理学161(3):1141-1157。
  3. Wharton,M.J。(1955)。 使用四唑测试来确定芸苔属种子的存活力。 Proc Int Seed Test Assoc 20:81-88。 
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Copyright: © 2013 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. Verma, P. and Majee, M. (2013). Seed Germination and Viability Test in Tetrazolium (TZ) Assay. Bio-protocol 3(17): e884. DOI: 10.21769/BioProtoc.884.
  2. Verma, P., Kaur, H., Petla, B. P., Rao, V., Saxena, S. C. and Majee, M. (2013). PROTEIN L-ISOASPARTYL METHYLTRANSFERASE2 is differentially expressed in chickpea and enhances seed vigor and longevity by reducing abnormal isoaspartyl accumulation predominantly in seed nuclear proteins. Plant Physiol 161(3): 1141-1157.
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Ignacio Lescano
Centro de Investigaciones en Química Biológica de Córdoba
Dear Pooja and Manoj,
Nice protocol! It worked perfectly for Arabidopsis Col-0 (Columbia), it is fairly easy to distinguish between viable and non-viable seeds, and few seeds have intermediate staining.
However, other accessions such as Ws (Wassilewskija), show high heterogeneity with many partially stained seeds, even for controls, and it becomes very difficult to distinguish between viable and non-viable seeds. This has already been described in some publications since Ws seed coat seems to be slightly permeable for tetrazolium.
I have tried to improve this staining by washing with a scarification solution (20 ml commercial bleach and 100 µl Triton X-100 in 100 ml autoclaved distilled water), or increasing incubation time, but was unsuccessful. Any recommendations to improve this assay? Thank you very much for reading me.
2021/4/28 7:44:10 回复
Hera Flarountzou
Food Industry
Dear Pooja,

could you be so kind to tell me the commercial type of glycerine, phenol and NaOCl you use?
Thank you in advance!

Hera
2019/10/18 0:47:09 回复
Kamal Sharma
Jnkvv jablpur
Can TTC. Test substitute the germination test, explain with reasons?
2019/5/7 5:52:15 回复
Michael EZE
Michael EZE

While germination test takes a longer time, TTC test is a quick alternative with comparable result. However, both tests can still be performed.

2019/10/21 0:02:15 回复


Lee Chen
Bioriginal
Hi Pooja,

My borage seeds have thick and black seed coats that make the staining solution hard to get in contact with the endosperm or other tissues inside the seeds. What do you think can be done to improve the permeability of the staining solution through a thick seed coat?

Thanks!
2019/2/21 8:18:29 回复
Michael EZE
Michael EZE

An option is to bisect the seeds longitudinally through the centre of the embryo. In that case, use 0.25% to 0.5% TTC instead of 1% TTC solution.

2019/10/21 0:06:54 回复


YOGENDRA THAKUR
YOGENDRA THAKUR
Hi pooja,
I wanted to ask instead of phenol can we use other chemicals for preparation of clearing solution. we do not have pure phenol.(we have it in combination with other chemicals like chloroform)
2018/3/27 1:28:55 回复
Sanjoy Sadhukhan
Raiganj University
What is your positive control?
2018/3/1 21:16:37 回复
Pooja Verma
UDSC

Hi Sanjoy, The seeds with germination percentage of 95% or more were taken as a positive control for the TZ assay. Thanks.

2018/3/10 2:43:12 回复


Nwabisa Magengelele
Rhodes University
How long must the distilled water be autoclaved
2017/10/3 2:58:42 回复
Pooja Verma
UDSC

Hi Nwabisa, Distilled water should be autoclaved for 25-30 minutes at 121 degree Celsius (250 degree Fahrenheit) for sterilization. Thanks.

2017/10/10 4:29:35 回复


Raghu Tembe
MSc agricultural
Is there any alternative chemica quick test for viability other than TZ
2017/9/7 2:58:10 回复
Pooja Verma
UDSC

Hi Raghu, Another chemical test known for seed viability is Indigo Carmine (IC) test, it takes 2 to 30 hours for staining depending on variety of seeds. Here, only dead tissue takes the stain. IC staining has been reported for Pinus silvestris and Picea abies seed. Acid Fuchsin is another chemical for testing seed viability and it also takes 24 hrs for staining the non-viable tissue or embryos red (reported for orchid seeds). TZ has been widely accepted and reliable among chemical seed viability tests. Hope this helps you. Thanks.

2017/10/10 4:27:09 回复


Emily Bossard
Western Washinton University
Hi there, what percentage Triton-X do you recommend for the scarification solution?
2017/8/1 14:36:28 回复
Pooja Verma
UDSC

Hi Emily,

In this protocol, scarification solution with 0.1% of Triton X-100 was used. Recommended range for this surfactant is from 0.02% to 0.1%.

2017/8/1 23:01:33 回复