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Arabidopsis Seed Germination Assay with Gibberellic Acid
GA处理条件下的拟南芥种子萌发实验   

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Abstract

This assay analyzes Arabidopsis seed germination in response to gibberellic acid (GA). During seed imbibition, visible physiological changes allow precise determination of germination rate. This protocol utilizes a stereoscopic microscope to improve characterization of seed germination process.

Background

Seed germination is a critical process of the plant life cycle controlled by phytohormones, such as GA and abscisic acid (ABA), and environmental factors. Seed germination comprises two physiological processes, including seed coat (testa) and endosperm ruptures. Usually, penetration of endosperm by the radicle indicates that germination is complete. Previous studies generally use endosperm rupture to calculate germination rate. However, seed coat rupture also measures the progression of seed germination. This protocol utilizes a stereoscopic microscope to provide a visible and precise calculation of seed germination, including the rates of both seed coat and endosperm rupture (Zhong et al., 2015).

Materials and Reagents

  1. 1.5 ml Eppendorf tubes (Eppendorf)
  2. 950 x 150 mm Petri dish (Shanhai Wuyi Glass Factory, catalog number: 1771 )
  3. Sterile pipette tips (Corning, Axygen®)
  4. Parafilm (Bemis, catalog number: PM-996 )
  5. Aluminum foil (GLAD, catalog number: F5M )
  6. 0.2 µm syringe filters (Pall, Acrodisc®, catalog number: 4554 )
  7. Arabidopsis seeds
  8. Murashige and Skoog basal medium (Sigma-Aldrich, catalog number: M5519 )
  9. Agar (MBCHEM, catalog number: 170837 , agented by WHIGA in China)
  10. Sucrose, purity: AR (Tianjin Damao Chemical Reagent Factory, catalog number: 57-50-1 )
  11. Gibberellic acid (GA3) (Sigma-Aldrich, catalog number: G7645 )
  12. Ethanol, purity: AR (Tianjin Damao Chemical Reagent Factory, catalog number: 64-17-5 )
  13. Potassium hydroxide (KOH), purity: AR (Chengdu Institute of Chemical Reagents, catalog number: 1310-73-2 )
  14. MS solid medium (see Recipes)
  15. 10 mM GA3 stock (see Recipes)
  16. 70% ethanol (see Recipes)
  17. 1% sodium hypochlorite (see Recipes)
  18. Autoclaved distilled water (see Recipes)

Equipment

  1. Aquapro water purification system (Aquapro, model: AQ06062001 )
    Note: This product has been discontinued.
  2. Refrigerator (Haier, model: BCD-648WDBE )
  3. Autoclave (HIRAYAMA, model: HVE-50 )
  4. 1 ml pipette (Eppendorf, catalog number: 3120620.001 )
  5. Drying oven (ZenithLabo, model: DHG 9070A )
  6. 100 ml flasks
  7. Microwave oven (Galanz, model: G70F20N2L-DG )
  8. Bechtop (Suzhou Antai Air-tech, model: SW-CJ-1FD )
  9. Stereoscopic microscope (Nikon, model: SMZ1500 )
  10. Digital camera (10 megapixels) (Nikon, model: COOLPIX4500 )
  11. Thermometer (WUqiang, China)

Software

  1. Photoshop CS5 software
  2. IBM SPSS Statistics 22 software
  3. Origin8.0 software or Microsoft Excel

Procedure

  1. Preparation: All the reagents used in this protocol should be sterile (see Recipes for detailed information). Sterilize 1.5 ml Eppendorf tubes, Petri dishes, and 1 ml pipette tips by autoclaving (121 °C, 20 min). Dry sterilized materials overnight in a drying oven at 37 °C.
  2. Prepare the solid MS medium plates with or without GA3: Thoroughly melt solid MS medium (two flasks with 100 ml each) using a microwave oven and then cool flasks down to 50-60 °C. In one flask make a 1 µM GA3 solution (stock solution of 10 mM GA3), thoroughly but gently mix media, and then pour into four plates. Without adding GA3 pour the other flask of medium into another four plates. After cooling, seal all the plates with Parafilm and store at 4 °C before use.
    Notes:
    1. ½ MS medium is optional in this step.
    2. The medium must be cooled before adding GA3 stock solution.
    3. The medium can be prepared and kept at 4 °C before conducting experiments. However, the medium with GA3 should be kept in the dark and used within a week.
    4. The concentration of GA3 can be adjusted according to experimental needs.
  3. Collect approximately 200 mature seeds into a 1.5 ml Eppendorf tube.
  4. Sterilize seeds at the clean bench: Add 500 μl 70% (v/v) ethanol to the tube containing the seeds and vigorously invert it several times. After 3 min, discard the ethanol, add 1 ml 1% (v/v) sodium hypochlorite, and invert several times over the course of 10 min. Finally, rinse seeds four or five times with sterile distilled water.
    Note: Make sure the time for sterilizing seeds is no longer than 3 min with ethanol and 10 min with sodium hypochlorite.
  5. Sowing seeds: Resuspend seeds in approximately 100 μl of water and then transfer to media by using a pipette. When transferring, make sure seeds are spaced apart and not touching each other. At least 80 seeds should be sowed for each treatment. Leave plates open long enough so excess water around seeds and on media surface can evaporate. Wrap plates in aluminum foil to maintain darkness.
  6. Stratification: Keep all plates together at 4 °C for 2 or 3 days.
    Note: Ensure the temperature of stratification to be 4 °C. Higher temperature will accelerate seed germination before the exposure to light.
  7. After stratification, put the medium plates into a culture room (22 °C with a 16 h light/8 h dark photoperiod, the light supplied by cool and warm white fluorescent bulbs, a light intensity of approximately 100 μmol m-2 s-1 on the shelf surface, and 75%-80% relative humidity).
    Notes:
    1. Make sure that the culture temperature is 22 °C, and a thermometer should be kept on the shelf surface. Higher temperature will affect seed germination.
    2. Make sure that all media plates are applied with the same intensity of light. Due to the variation of light intensity of single bulbs, plates should be kept in a narrow scope.
  8. Data recording: Mark 0 h when seeds are exposed to light. Using a stereoscopic microscope (5x magnification) take photos of each plate every 2 h until all seeds have germinated (see Figure 1). Data should be calculated based on at least three independent experiments with similar results.
    Note: It is optional to count seed germination after the radical tip emergence (see Figure 1, right panel), which is defined as the first sign of seed germination. Seeds are scored every 2 h until seed germination rate reaches over 98%.


    Figure 1. Seed germination process of Arabidopsis thaliana. Images show the status of seeds during seed coat and endosperm rupture under the normal condition. Photographs were taken before (left) or after seed coat rupture (middle) or after endosperm rupture (right). Seed coat, endosperm, and radicle are indicated by black arrows.

Data analysis

Using Photoshop CS5 software, count the number of the seeds with a ruptured testa or endosperm at each time point. The germination rate is indicated by the percentage of seeds with a ruptured endosperm compared to the total number of seeds. Additionally, the rate of testa rupture is indicated by the percentage of seeds with a ruptured testa compared to the total number of seeds (optional). Analyze the germination and testa rupture rates by using the IBM SPSS Statistics 22 software. Significant differences in comparison with control seeds (*P < 0.05, **P < 0.01), are analyzed by one-way analysis of variance (ANOVA). The line diagram of seed germination is made by using Origin8.0 software or Microsoft Excel.

Notes

  1. From the step of sterilizing seeds, all the operation must be done at a clean bench.

Recipes

  1. 100 ml MS solid medium
    0.44 g Murashige and Skoog basal medium powder
    1 g sucrose
    Dissolve in 90 ml ddH2O and adjust pH to 5.8-5.9 with KOH
    Bring volume to 100 ml with ddH2O
    Add 0.8 g agar powder and autoclave at 121 °C for 20 min
  2. 10 ml 10 mM GA3 stock
    34.638 mg GA3 powder
    Dissolve in 10 ml absolute ethanol and filter with a 0.2 µm filter at a clean bench
    Aliquot into sterile 1.5 ml Eppendorf tubes
    Cover the tubes with aluminum foil to protect from light, store at -20 °C
  3. 100 ml 70% ethanol
    70 ml absolute ethanol
    Bring volume to 100 ml with ddH2O
  4. 7 ml 1% sodium hypochlorite
    1 ml 7% sodium hypochlorite
    Bring volume to 7 ml with ddH2O
  5. Sterilized distilled water
    Autoclave distilled water at 121 °C, 20 min

Acknowledgments

This protocol was adapted from the Xiaojing Wang Lab at the South China Normal University. This work was supported by the National Natural Science Foundation of China (grant No. 90917011 to X.W.). We thank Dr. craig schluttenhofer (University of Kentucky) for proofreading the article.

References

  1. Zhong, C., Xu, H., Ye, S., Wang, S., Li, L., Zhang, S. and Wang, X. (2015). Gibberellic acid-stimulated Arabidopsis6 serves as an integrator of gibberellin, abscisic acid, and glucose signaling during seed germination in Arabidopsis. Plant Physiol 169(3): 2288-2303.

简介

该测定分析响应于赤霉酸(GA)的拟南芥种子萌发。在种子吸水期间,可见的生理变化允许精确测定发芽率。该协议利用立体显微镜来改善种子发芽过程的表征。

[背景] 种子萌发是由植物激素(如GA和脱落酸(ABA))和环境因素控制的植物生命周期的关键过程。种子萌发包括两个生理过程,包括种皮(睾丸)和胚乳破裂。通常,胚乳通过胚根的渗透表明萌发是完全的。以前的研究通常使用胚乳破裂来计算发芽率。然而,种皮破裂也测量种子发芽的进展。该协议利用立体显微镜提供种子发芽的可见和精确计算,包括种皮和胚乳破裂的速率(Zhong等人,2015)。

材料和试剂

  1. 1.5ml Eppendorf管(Eppendorf)
  2. 950×150mm培养皿(山海五一玻璃厂,目录号:1771)
  3. 无菌移液器吸头(Corning,Axygen ®
  4. Parafilm(Bemis,目录号:PM-996)
  5. 铝箔(GLAD,目录号:F5M)
  6. 0.2微米注射器过滤器(Pall,Acrodisc ,目录号:4554)
  7. 拟南芥种子
  8. Murashige和Skoog基础培养基(Sigma-Aldrich,目录号:M5519)
  9. 琼脂(MBCHEM,目录号:170837,由WHIGA在中国代理)
  10. 蔗糖,纯度:AR(天津大美化学试剂厂,目录号:57-50-1)
  11. 赤霉酸(GA 3)(Sigma-Aldrich,目录号:G7645)
  12. 乙醇,纯度:AR(天津大高化学试剂厂,目录号:64-17-5)
  13. 氢氧化钾(KOH),纯度:AR(成都化学试剂研究所,目录号:1310-73-2)
  14. MS固体培养基(参见配方)
  15. 10mM GA 3 原料(见配方)
  16. 70%乙醇(见配方)
  17. 1%次氯酸钠(参见配方)
  18. 高压蒸馏水(见配方)

设备

  1. Aquapro水净化系统(Aquapro,型号:AQ06062001)
    注意:此产品已停产。
  2. 冰箱(海尔,型号:BCD-648WDBE)
  3. 高压灭菌器(HIRAYAMA,型号:HVE-50)
  4. 1ml移液管(Eppendorf,目录号:3120620.001)
  5. 干燥炉(ZenithLabo,型号:DHG 9070A)
  6. 100ml烧瓶中
  7. 微波炉(Galanz,型号:G70F20N2L-DG)
  8. Bechtop(苏州安泰空气技术,型号:SW-CJ-1FD)
  9. 立体显微镜(尼康,型号:SMZ1500)
  10. 数码相机(10兆像素)(尼康,型号:COOLPIX4500)
  11. 温度计(中国吴强)

软件

  1. Photoshop CS5软件
  2. IBM SPSS Statistics 22软件
  3. Origin8.0软件或Microsoft Excel

程序

  1. 准备:本协议中使用的所有试剂均应无菌(参见配方中的详细信息)。通过高压灭菌(121℃,20分钟)灭菌1.5ml Eppendorf管,培养皿和1ml移液管吸头。将干燥的无菌材料在37℃的干燥炉中过夜。
  2. 制备具有或不具有GA 3的固体MS培养基平板:使用微波炉彻底熔化固体MS培养基(两个烧瓶,每个具有100ml),然后将烧瓶冷却至50-60℃。在一个烧瓶中制备1μMGA 3溶液(10mM GA 3的储备溶液),彻底但轻轻地混合培养基,然后倒入四个板中。没有添加GA 3将另一瓶培养基倒入另外四个板中。冷却后,使用Parafilm密封所有的板,使用前保存在4°C 注意:
    1. 在此步骤中,半介质是可选的。
    2. 在添加GA 3 储备溶液之前,必须冷却介质。
    3. 在进行实验之前,可以制备培养基并保持在4℃。但是,GA 3 的培养基应保存在黑暗中,并在一周内使用。
    4. 的浓度可以根据实验需要进行调整。
  3. 收集大约200个成熟的种子放入1.5毫升的Eppendorf管。
  4. 在净化台消毒种子:向含有种子的管中加入500μl70%(v/v)乙醇,并剧烈反转几次。 3分钟后,弃去乙醇,加入1ml 1%(v/v)次氯酸钠,在10分钟内倒置几次。最后,用无菌蒸馏水冲洗种子四或五次。
    注意:请确保灭菌种子的时间不超过3分钟乙醇和10分钟次氯酸钠。
  5. 播种:将种子重悬于约100μl水中,然后使用移液管转移至培养基中。转移时,确保种子分开,不要相互接触。每次处理至少播种80粒种子。让板开得足够长,这样种子周围和介质表面上的过量水可以蒸发。包装板在铝箔保持黑暗。
  6. 分层:将所有板在4℃保持2或3天。
    注意:确保分层温度为4°C。较高的温度会加速种子在暴露于光之前的发芽。
  7. 分层后,将培养基平板放入培养室(22℃,16小时光照/8小时黑暗光周期,由冷和暖白色荧光灯提供的光,光强度约100μmol/m 2 s -1 ,相对湿度为75%-80%)。
    注意:
    1. 确保培养温度为22°C,并且应在架子表面上保存温度计。高温会影响种子发芽。
    2. 确保所有介质板都应用相同的光强度。由于单个灯泡的光强度变化,板材应保持在较窄的范围内。
  8. 数据记录:当种子暴露于光时标记0小时。使用立体显微镜(5x放大)每2小时拍摄每个板的照片,直到所有种子都发芽(参见图1)。数据应根据至少三次独立实验计算,结果类似。
    注意:在根尖提示后计数种子萌发是可选的(参见图1,右图),其被定义为种子萌芽的第一个标志。每2小时对种子进行评分,直到种子发芽率达到98%以上

    图1.拟南芥的种子萌发过程。图像显示在正常条件下种子外壳和胚乳破裂期间种子的状态。在种皮破裂(中)或胚乳破裂(右)之前(左)或之后拍摄照片。种子衣,胚乳和胚根用黑色箭头表示

数据分析

使用Photoshop CS5软件,计数在每个时间点具有破裂的睾丸或胚乳的种子的数量。发芽率由具有破裂的胚乳的种子的百分比相比于种子的总数来指示。此外,睾丸破裂的速率由具有破裂的睾丸的种子的百分比与种子的总数相比来指示(任选的)。使用IBM SPSS Statistics 22软件分析发芽率和试验破裂率。通过单因素方差分析(ANOVA)分析与对照种子(* P <0.05,** P <0.01)比较的显着差异。种子发芽的线图通过使用Origin8.0软件或Microsoft Excel进行。

笔记

  1. 从种子灭菌的步骤,所有的操作必须在干净的工作台上进行。

食谱

  1. 100ml MS固体培养基
    0.44g Murashige和Skoog基础培养基粉末
    1克蔗糖
    溶于90ml ddH 2 O中并用KOH调节pH至5.8-5.9。 用ddH 2 O/b/b使体积为100ml 加入0.8g琼脂粉末并在121℃下高压灭菌20分钟
  2. 10ml 10mM GA 3原料
    34.638mg GA 3粉末
    溶于10ml无水乙醇中,用0.2μm过滤器在干净的平台上过滤 分装到无菌的1.5 ml Eppendorf管中 用铝箔覆盖管,以防止光照,存储在-20°C
  3. 100ml 70%乙醇
    70毫升无水乙醇 用ddH 2 O/b/b使体积为100ml
  4. 7ml 1%次氯酸钠
    1ml 7%次氯酸钠
    用ddH 2 O 2将体积调至7ml
  5. 灭菌蒸馏水
    121℃,20分钟高压灭菌蒸馏水

致谢

这个协议改编自华南师范大学的晓静王实验室。这项工作得到了中国国家自然科学基金会(授予No. 90917011,X.W.)的支持。我们感谢craig schluttenhofer博士(肯塔基大学)校对这篇文章。

参考文献

  1. 中国科技大学学报(自然科学版)refrefrefrefrefrefrefrefrefrefrefrefrefrefrefrefrefrefrefrefref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ="http://www.ncbi.nlm.nih.gov/pubmed/26400990"target ="_ blank">赤霉酸刺激的拟南芥6在种子发芽期间作为赤霉素,脱落酸和葡萄糖信号传导的整合子>拟南芥。植物生理学 169(3):2288-2303。
  • English
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 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. Zhong, C., Xu, H., Ye, S., Zhang, S. and Wang, X. (2016). Arabidopsis Seed Germination Assay with Gibberellic Acid. Bio-protocol 6(22): e2005. DOI: 10.21769/BioProtoc.2005.
  2. Zhong, C., Xu, H., Ye, S., Wang, S., Li, L., Zhang, S. and Wang, X. (2015). Gibberellic acid-stimulated Arabidopsis6 serves as an integrator of gibberellin, abscisic acid, and glucose signaling during seed germination in Arabidopsis. Plant Physiol 169(3): 2288-2303.
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