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Vanillin Assay of Arabidopsis Seeds for Proanthocyanidins

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The Plant Journal
Mar 2014



Proanthocyanidins (PAs) are colorless flavonoid polymers and deposit in Arabidopsis seed coat specifically. Oxidation of PAs gives rise to brown color of mature seeds. PA accumulation can be affected by a number of growth conditions, such as temperature and sun light. PAs, which are converted from anthocyanidins, can protect seeds from outer environment and have a positive effect in seed longevity (Debeaujon, 2003). Vanillin turns red upon binding to leucoanthoanthocyanidins, catechins and monomers and terminal subunits of PAs (Butler et al., 1982; Deshpande et al., 1986). Based on this principle, PA deposition in Arabidopsis seed coat can be visualized.

Keywords: Seed coat color (种皮颜色), Vanillin assay (香草醛), Arabidopsis (拟南芥), Proanthocyanidins (原花青素)

Materials and Reagents

  1. Arabidopsis seeds (Col-0 and tt mutants)
  2. 6 M HCl
  3. Sterile distilled water
  4. Dye solution (for coloration and reaction with PAs) containing 1% w/v vanillin (Sangon Biotech, catalog number: VT0974- 100g) and 6 M HCl stored in brown bottle
    Note: The dye solution should be used after preparation as soon as possible (not longer than half an hour).
  5. Vanillin reagent (see Recipes)


  1. Brown bottle (or glass bottle covered with aluminum foil)
  2. 1.5 ml microcentrifuge tubes
  3. Glass slides (25.4 mm x 76.2 mm) and coverslips (20 mm x 20 mm)
  4. Dissecting needle and tweezer
  5. An SZ61-zoom stereomicroscope (OLYMPUS, model: SZ61)
  6. A compound light microscope (OLYMPUS, model: BX61 )


  1. Intact seeds should be dipped into dye solution at room temperature.
  2. For mature seeds, the incubation should last for approximately 1 h, whereas for immature seeds, the incubation time can be up to 10 min, depending on the degree of immaturity. Figure 2 indicates silliques at different developmental stages.
  3. After the reaction with vanillin, seeds can be gently separated into embryos and seed coats with dissecting needle and tweezers under an SZ61-zoom stereomicroscope on glass slides.
  4. Cover slides with coverslips. A little bit extra vanillin reagent should be left to avoid drying (water and glycerol will fade this red color).
  5. Stained seed coats were observed and photographed with a compound light microscope.

Representative data

Figure 1. Comparison of the darkness of PA staining in seed coats among various test transparent mutants. Scale bars = 100 µm. (Wang et al., 2014)

Figure 2. Comparison of Arabidopsis siliques between different developmental stages. Stage 1: Young siliques: less than or about one week after pollination. 10 min is enough for reaction between PAs and vanillin. Stage 2: Immature siliques: 1-3 weeks after pollination. 20 to 40 min are required for the reaction. Stage 3: Mature silique: 3 weeks or more after pollination, color of siliques appears pale yellow. 1 h is necessary for the reaction.


  1. Reaction between vanillin and PAs is affected by temperature. Make sure stain all the seeds at the same condition.
  2. Do not try to rinse seeds, especially young seeds, with water after dying with vanillin. Red color can be rinsed away by water.
  3. Make sure all the seeds were dipped into vanillin reagent at the same condition, and a quick centrifuge may help. For seed quantity, just cover the bottom of a 1.5 ml microcentrifuge tube (one or two layers).


  1. Vanillin reagent
    The vanillin reagent should be fresh and 1 ml is quite enough for seed staining. Make sure vanillin reagent volume and seed number are the same in wildtype and mutant microcentrifuge tubes. Stained red color continues getting dark along with time duration. It is very important to photograph seed coats at the same light condition. Light intensity can affect visual effects.


The work was sponsored by the Natural Science Foundation of China (Grant nos. 31171463). Our protocol is an improvement based on a method described by Hagerman (2002) with sorghum grains.


  1. Butler, L. G., Price, M. L. and Brotherton, J. E. (1982). Vanillin assay for proanthocyanidins (condensed tannins): modification of the solvent for estimation of the degree of polymerization. J Agr Food Chem 30(6): 1087-1089.
  2. Debeaujon, I., Nesi, N., Perez, P., Devic, M., Grandjean, O., Caboche, M. and Lepiniec, L. (2003). Proanthocyanidin-accumulating cells in Arabidopsis testa: regulation of differentiation and role in seed development. Plant Cell 15(11): 2514-2531.
  3. Deshpande, S. S., Cheryan, M. and Salunkhe, D. K. (1986). Tannin analysis of food products. Crit Rev Food Sci Nutr 24(4): 401-449.
  4. Hagerman, A. E. (2002). Vanillin assay. The Tannin Handbook.
  5. Wang, Z., Chen, M., Chen, T., Xuan, L., Li, Z., Du, X., Zhou, L., Zhang, G. and Jiang, L. (2014). TRANSPARENT TESTA2 regulates embryonic fatty acid biosynthesis by targeting FUSCA3 during the early developmental stage of Arabidopsis seeds. Plant J 77(5): 757-769.


原花青素(PA)是无色黄酮类聚合物,特异性地沉积在拟南芥种皮中。 PA的氧化产生成熟种子的棕色。 PA积累可能受到许多生长条件的影响,如温度和日光。 从花青素转化的PA可以保护种子免受外界环境影响,对种子寿命有正面影响(Debeaujon,2003)。 香兰素在结合到无色花青素,儿茶素和单体和PA的末端亚单位时变红(Butler等,1982; Deshpande等,1986)。 基于这一原理,拟南芥种皮中的PA沉积可以被可视化。

关键字:种皮颜色, 香草醛, 拟南芥, 原花青素


  1. 拟南芥种子(Col-0和 tt 突变体)
  2. 6 M HCl
  3. 无菌蒸馏水
  4. 含有1%w/v香草醛(Sangon Biotech,目录号:VT0974-100g)和储存在棕色瓶中的6M HCl的染料溶液(用于着色和与PA反应)
  5. 香兰素试剂(见配方)


  1. 棕色瓶(或用铝箔覆盖的玻璃瓶)
  2. 1.5 ml微量离心管
  3. 玻璃载玻片(25.4mm×76.2mm)和盖玻片(20mm×20mm)
  4. 解剖针和镊子
  5. SZ61变焦立体显微镜(OLYMPUS,型号:SZ61)
  6. 复合光学显微镜(OLYMPUS,型号:BX61)


  1. 完整的种子应在室温下浸入染料溶液中
  2. 对于成熟种子,孵育应持续约1小时,而对于未成熟种子,孵育时间可高达10分钟,这取决于不成熟的程度。 图2显示了在不同发育阶段的血清。
  3. 在与香草醛反应之后,可以在玻璃载玻片上的SZ61变焦立体显微镜下将种子轻轻地分离成胚和种皮,使用解剖针和镊子。
  4. 盖玻片与盖玻片。应留少许额外的香草醛试剂以避免干燥(水和甘油将褪去这种红色)
  5. 观察染色的种皮并用复合光学显微镜拍照


图1.比较各种测试透明突变体之间的种皮中PA染色的黑暗。比例尺=100μm。 (Wang等人,2014年)

图2.不同发育阶段之间的拟南芥长角果比较。 阶段1:幼小长角:在授粉后不到或大约一周。 10分钟足以使PA和香草醛之间的反应。阶段2:不成熟的长角果:授粉后1-3周。反应需要20至40分钟。阶段3:成熟长角果:授粉后3周或更长,长角果的颜色显现为浅黄色。 1小时是必要的 反应。


  1. 香草醛和PA之间的反应受温度的影响。 确保在相同条件下将所有种子染色。
  2. 在用香草醛染色后,不要尝试用水冲洗种子,特别是幼嫩的种子。 红色可以用水冲洗掉。
  3. 确保所有的种子浸在香草醛试剂在相同的条件,和快速离心可能有帮助。 对于种子数量,仅覆盖1.5ml微量离心管的底部(一层或两层)。


  1. 香兰素试剂
    香草醛试剂应该是新鲜的,1ml足够用于种子染色。 确保香草醛试剂体积和种子数在野生型和突变型微量离心管中相同。 染色的红色随着持续时间持续变暗。 在相同的光照条件下拍摄种皮是非常重要的。 光强度可以影响视觉效果。


这项工作由中国自然科学基金会资助(批准号31171463)。 我们的方案是基于Hagerman(2002)描述的方法用高粱粒子的改进。


  1. Butler,L.G.,Price,M.L。和Brotherton,J.E。(1982)。 原花色素(缩合单宁)的香兰素测定:对溶剂的改性 估计聚合度。 J Agr Food Chem 30(6):1087-1089。
  2. Debeaujon,I.,Nesi,N.,Perez,P.,Devic,M.,Grandjean,O.,Caboche,M.and Lepiniec,L。(2003)。 拟南芥中的原花色素 - 蓄积细胞:调节分化和在种子发育中的作用。植物细胞 15(11):2514-2531。
  3. Deshpande,S.S.,Cheryan,M。和Salunkhe,D.K。(1986)。 食品的单宁分析。 Crit Rev Food Sci Nutr 24(4):401-449。
  4. Hagerman,A.E。(2002)。香草醛测定。丹宁手册。
  5. Wang,Z.,Chen,M.,Chen,T.,Xuan,L.,Li,Z.,Du,X.,Zhou,L.,Zhang,G.and Jiang, TRANSPARENT TESTA2通过在拟南芥早期发育阶段期间靶向FUSCA3来调节胚胎脂肪酸生物合成,/em>种子。植物J 77(5):757-769。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Xuan, L., Wang, Z. and Jiang, L. (2014). Vanillin Assay of Arabidopsis Seeds for Proanthocyanidins. Bio-protocol 4(23): e1309. DOI: 10.21769/BioProtoc.1309.