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Plant Tissue Trypan Blue Staining During Phytopathogen Infection
植物病原体感染期间的植物组织台盼蓝染色   

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Abstract

In this protocol plant tissue is stained with trypan blue dye allowing the researcher to visualize cell death. Specifically this method avoids the use of the carcinogen compound chloral hydrate, making this classical method of staining safer and faster than ever. The protocol is applied specifically to detect cell death on Arabidopsis leaves during the course of infection with necrotrophic fungus Botrytis cinerea.

Keywords: Trypan Blue staining(台盼蓝染色), Plant cell death(植物细胞死亡), Botrytis cinerea(灰葡萄孢), Arabidopsis thaliana(拟南芥), Chloral hydrate(水合氯醛)

Background

One of the most common methods to detect dead plant tissue is trypan blue staining (Keogh et al., 1980). This diazo dye is also used in histology and medicine to measure tissue viability through allowing the visualization of cell death1 (Keogh et al., 1980; Cooksey, 2014). Most microscopic procedures involving trypan blue staining require a long subsequent clearing step using chloral hydrate (CHL), a small organic compound currently used such as a carcinogen, an anesthetic and an analgesic in laboratory animals (Keogh et al., 1980; Lu and Greco, 2006; Salmon et al., 1995). CHL is not approved by the FDA in the USA or the EMA in the European Union for any medical indication (http://www.accessdata.fda.gov/). Only 250 mg or 50 mg of choral hydrate are sufficient to produce adult or pediatric sedation respectively, and its toxicity has also been measured in neonatals (http://www.drugs.com, Salazar et al., 2009). The LD50 (median lethal dose) for an adult is estimated to be a 4-h exposure to 0,440 mg/L vapour concentration, which is also the duration currently recommended for de-staining of plant leaves at a concentration of 250 g/100 ml. Long-term use of chloral hydrate also results in a rapid development of tolerance to its effects and possible addiction, as well as adverse effects including rashes, gastric discomfort and severe kidney, heart, and liver failure (Gelder et al., 2005).

Through avoiding the use of CHL, this protocol allows researchers to stain for plant cell death with trypan blue more rapidly and safely, substantially reducing the risk to researchers. Here we demonstrate the utility of this method by monitoring the course of infection of Col-0 leaves with Botrytis cinerea (B.c), the second phytopathogen fungus on scientific/economic importance with a broad host range, and a high capacity to produce hydrogen peroxide in plants (Rolke et al., 2004; Dean et al., 2012; Lehmann et al., 2015). This protocol has been also applied successfully to other Arabidopsis accessions.
1Note: Trypan blue is a synthetic compound derived from toluidine, invented by Paul Ehrlich, winner of the Nobel prize in Physiology and Medicine, 1904 (http://www.pei.de/).

Materials and Reagents

  1. Conical tubes (50 ml) (Corning, Falcon®, catalog number: 352070 )
  2. Parafilm M laboratory film (Hach, catalog number: 251764 )
  3. Plastic Petri dishes with a transparent lid (JET BIOFIL cell and tissue culture plates 6 well)
  4. Tips  
  5. Miracloth (EMD Millipore, catalog number: 475855 )
  6. Labeling tape (Shamrock, catalog number: ST-12-20 )
  7. Paper towels
  8. Arabidopsis accessions: Columbia (Col-0)
  9. Ethanol > 99.8% (Sigma-Aldrich, catalog number: 02860 )
    Note: This product has been discontinued.
  10. Lactic acid 85% (w:w) (Sigma-Aldrich, catalog number: L1250 , DL-Lactic acid 11.3 M)
  11. Phenol (TE buffer equilibrated, pH 7.5-8.0) (Roti®-Phenol, Carl Roth, catalog number: 0038.1 )
  12. Glycerol ≥ 99% (Sigma-Aldrich, catalog number: G7757 )
  13. Sterile distilled water (EMD Millipore, milli-q a10 water purification system)
  14. Trypan blue (Sigma-Aldrich, catalog number: T6146 )
  15. Potato dextrose agar (PDA) (BD, DifcoTM, catalog number: 213400 )
  16. Potato dextrose broth (PDB) (BD, DifcoTM, catalog number: 254920 )
  17. Sodium hypochlorite solution (Sigma-Aldrich, catalog number: 239305 )
  18. Sodium dodecyl sulfate (SDS) 98% (Sigma-Aldrich, catalog number: 862010 )
    Note: This product has been discontinued.
  19. Tween 20 detergent (Sigma-Aldrich, catalog number: P1379 )
  20. Trypan blue staining solution (see Recipes)
  21. Potato dextrose agar (PDA) (see Recipes)
  22. Bortytis cinerea (B.c) inoculum (see Recipe)
  23. Potato dextrose broth (PDB) (see Recipes)
  24. Seed sterilization solution (see Recipes)

Equipment

  1. Micro scissors NOYE (Auxilab, catalog number: 62101111 )
  2. Dissecting forceps, Toothless 125 mm (Auxilab, catalog number: 61302012 )
  3. Growth chamber (Vötsch Industrietechnik, model: HERAEUS VB1514 )
  4. Biological safety cabinet (Germfree, model: BBF-2SSCH )
  5. Micropipets P1000, P200 and P20 from Rainin Instruments
  6. Weight analysis 440 (KERN & SOHN)
  7. Stereomicroscope (Leica Microsystems, model: MZ9 ) coupled to CCD camera (Leica Microsystems, model: DC 280 )
  8. Conductimeter (HACHLANGE SPAIN, CRISON, model: BASIC30 )
  9. Autoclave (JP SELECTA, model: MED 20 , catalog number: 4001757)

Software

  1. ImageJ software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997-2016)
  2. Statgraphics Centurion XVI.II software (StatPoint Technologies, Inc. www.statgraphics.com)

Procedure

  1. Growing Arabidopsis plants
    1. Sterilize Arabidopsis seeds with the seed sterilization solution (see Recipes) for 20 min, then wash with sterile water four times.
    2. Stratify seeds in 4 °C chamber for 2 days in darkness.
    3. Germinate seeds in a mix of 3:1 (soil:vermiculite) in the growth chamber, for 21 days under 10 h light/14 h darkness photoperiod, with a light intensity of 120 lux at 22 °C and 60% humidity.

  2. Inoculation with Botrytis cinerea
    1. Sterilize all the material in an autoclave at 120 °C for 20 min.
    2. Put the plants into a biological safety cabinet chamber and inoculate them with one drop of 5 μl of B.c fungal inoculum (see Recipes, Figure 1A) per leave.
    3. Cover the plants with Parafilm (Figure 1B) to avoid dispersion of conidiospores and to maintain high humidity (90-100%) for optimal growth of the fungus. Return them to the growth chamber and make small holes in the Parafilm (> 10) with a yellow tip to avoid ethylene accumulation within the tray, which would activate plant defense response and stop fungal growth.
    4. Incubate the B.c-inoculated plants in a growth chamber at 25 °C under a 10 h photoperiod until the day of sampling, usually 2 to 7 days post-inoculation (Figure 1C).
    5. At each time-point of the experiment, harvest inoculated leaves with scissors and put them directly into plates containing 5 ml of fresh trypan blue solution (see Recipes, Figure 1D). At this point be sure that the leaves are completely immersed in the liquid; use a blue tip to submerge them if necessary. Cover the plate and wait for at least 30 min. A blue color will then be visible in the locations of dead tissue. Total staining time should not exceed 1 h. Avoid the use of boiling methods, vacuum infiltration methods or microwave treatments which will impede the later de-staining process.
      Note: Timing of sampling might change depending on Arabidopsis/accession/mutant/line-B.c interaction and fungal virulence.
    6. Remove the trypan solution and re-fill the plates immediately with 98-100% ethanol. Seal the plates with Parafilm in order to avoid drying that might break the leaves or fix them to the plastic plate. Leave tissue in the ethanol solution at least overnight, then using a 5 ml pipet; replace the ethanol solution with fresh ethanol, repeatedly, until green tissue has become completely colorless (Figure 1E). Remove the ethanol and cover the leaves with 60% glycerol solution. At this point, the leaves are ready to be transferred to a glass surface to be visualized with a microscope (Figure 1F).
    7. If necessary, cell death measurements might be validated by ion leakage losses using a conductometer according to Govrin and Levine (2000)* or measuring leaf death area with ImageJ software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997-2016).
      *Note: To measure ion leakage we followed the protocol of Govrin and Levine (2000). We used 5 inoculated or control leaves per biological replicate, and four replicates per genotype. Leaves were immersed into water for 3 h at room temperature and later conductivity was measured following manufacturer’s instructions. Total (100%) ion leakage was obtained from same samples maintained o/n at -80 °C. Data analyses were performed as is indicated below.


      Figure 1. Procedure of Arabidopsis leaf inoculation with B.c. A and B. Leaf surfaces of Arabidopsis lines were inoculated with 5 µl of fungus (105-108 spores/ml), and plants were transferred to a plastic tray with transparent lid to allow fungal growth. C. The progress of infection was followed until plant symptoms were observed. D. Leaves were harvested on plates and imbibed with trypan blue staining solution for no more than 1 h. E. Leaves were immersed in ethanol overnight until they were translucent. F. Leaves were transferred to 60% glycerol for microscopy purposes.


      Figure 2. Arabidopsis leaves with different degrees of infection. A-F. Photos taken immediately after cutting leaves from plants grown on soil. G-L. Photos of representative leaves stained with trypan blue, exhibiting levels of infection similar to the leaves shown above. Bar = 0.5 cm.

Data analysis

For all experiments at least ten leaves per ecotype are sampled to be stained. Experiments are repeated at least four times. When necessary, the stained leaf surfaces were measured with the software ImageJ (Particle Analysis, Surface Plot, and Measure Tools, following the user guide of ImageJ, https://imagej.nih.gov/ij/docs/pdfs/examples.pdf). Leaf ion leakage data were analyzed with the Statgraphics Centurion XVI.II software (StatPoint Technologies, Inc. www.statgraphics.com). Unifactorial analyses of variance (ANOVA) tests were performed, calculating the mean and standard error as well as Duncan’s comparison of means for a confidence level of 0.05%.

Recipes

  1. Trypan blue staining solution
    10 ml lactic acid (85% w:w)
    10 ml phenol (TE buffer equilibrated, pH 7.5-8.0)
    10 ml glycerol (≥ 99%)
    10 ml of distilled water
    40 mg of trypan blue (final concentration of 10 mg/ml)
    Note: Store the solution at room temperature into a ventilation hood and always use fresh solutions.
  2. Potato dextrose agar (PDA)
    4 g/L PDA in distilled water
  3. Botrytis cinerea (B.c) inoculum*
    The Botrytis strain was grown at 28 °C on PDA medium plates (potato dextrose agar, Difco, Detroit, MI , USA) for eight days under darkness, and spores were collected in sterile water and stored in 20% glycerol at -80 °C until use.
    *Note: The stock of the fungi should be preserved at -80 °C in 20% glycerol in order to avoid virulence loss caused by repeated growing of the fungus on plates. The final concentration of the fungus will depend on the virulence of the fungal isolate usually between 105-107 spores/ml. Fungal concentration (conidiospores per ml) is measured using a hemocytometer (http://www.hemocytometer.org/). When the B.c isolate has low virulence, or when high levels or rapid fungal growth are required, the inoculation may be performed in presence of potato dextrose broth medium (PDB) using the maximum dilution of PDB possible, adding the same concentration of PDB to control inoculated leaves.
  4. Potato dextrose broth (PDB)
    4 g/L PDB in distilled water
  5. Seed sterilization solution
    Hypochlorite solution (30%, v:v)
    Sodium dodecyl sulfate (0.1%, v:v)

Acknowledgments

This protocol was modified from Keogh and collaborators (1980). We acknowledge M. Wilkinson from CGBP for his contribution. The research leading to these results received funding from No. AL13-P(I+D)-05, No. AL14- PID-26 to Domínguez J.A, and Berrocal-Lobo M, from RTA2013-00027-00-00 to Castellano M. M, and Berrocal-Lobo M.

References

  1. Cooksey, C. J. (2014). Quirks of dye nomenclature. 3. Trypan blue. Biotech Histochem 89(8): 564-567.
  2. Dean, R., Van Kan, J. A., Pretorius, Z. A., Hammond-Kosack, K. E., Di Pietro, A., Spanu, P. D., Rudd, J. J., Dickman, M., Kahmann, R., Ellis, J. and Foster, G. D. (2012). The Top 10 fungal pathogens in molecular plant pathology. Mol Plant Pathol 13(4): 414-430.
  3. Gelder, M., Mayou, R. and Geddes, J. (2005). Psychiatry (3rd). Oxford pp: 238.
  4. Govrin, E. M, and Levine, A. (2000). The hypersensitive response facilitates plant infection by the necrotrophic pathogen Botrytis cinerea. Curr Biol 10(13): 751-757.
  5. Keogh, R.C., Deverall, B.J. and Mcleod, S. (1980). Comparison of histological and physiological responses to Phakopsora pachyrhizi in resistant and susceptible soybean. Transactions of the British Mycological Society 74(2):329-333
  6. Lehmann, S., Serrano, M., L'Haridon, F., Tjamos, S. E. and Metraux, J. P. (2015). Reactive oxygen species and plant resistance to fungal pathogens. Phytochemistry 112: 54-62.
  7. Lu, J. and Greco, M. A. (2006). Sleep circuitry and the hypnotic mechanism of GABA drugs. J Clin Sleep Med 2(2): S19-26.
  8. Rolke, Y., Liu, S., Quidde, T., Williamson, B., Schouten, A., Weltring, K. M., Siewers, V., Tenberge, K. B., Tudzynski, B. and Tudzynski, P. (2004). Functional analysis of H(2)O(2)-generating systems in Botrytis cinerea: the major Cu-Zn-superoxide dismutase (BCSOD1) contributes to virulence on French bean, whereas a glucose oxidase (BCGOD1) is dispensable. Mol Plant Pathol 5(1): 17-27.
  9. Salazar M, Peralta C, Pastor F. J. (2009). Tratado de Psicofarmacología. 2 Ed. Panamericana.
  10. Salmon, A. G., Kizer, K. W., Zeise, L., Jackson, R. J. and Smith, M. T. (1995). Potential carcinogenicity of chloral hydrate--a review. J Toxicol Clin Toxicol 33(2): 115-121.
  11. Silverman, J, and Muir, W. W., 3rd (1993). A review of laboratory animal anesthesia with chloral hydrate and chloralose. Lab Anim Sci 43(3): 210-216.

简介

在该方案中,植物组织用台盼蓝染料染色,使研究人员可视化细胞死亡。具体来说,这种方法避免了使用致癌物水合氯醛,使得这种经典染色方法的染色比以往更加安全和快速。该方案专门用于在坏死性真菌Botrytis cinerea感染过程中检测拟南芥叶片上的细胞死亡。

【背景】 检测死植物组织的最常见的方法之一是台盼蓝染色(Keogh等人,1980)。该重氮染料也用于组织学和药物中,以通过允许细胞死亡的观察来测量组织存活力(Keogh等人,1980; Cooksey,2014)。涉及台盼蓝染色的大多数微观方法需要长时间的清除步骤,使用水合氯醛(CHL),一种目前使用的小型有机化合物,如实验动物中的致癌物,麻醉剂和止痛剂(Keogh等, 1980年; Lu和Greco,2006; Salmon等人,1995)。 CHL未经美国FDA批准或欧盟EMA在任何医疗指征( http://www.accessdata.fda.gov/ )。只有250mg或50mg的合唱水合物足以分别产生成人或儿科镇静剂,其毒性也在新生儿中测定( http://www.drugs.com ,Salazar等人,2009)。估计成人的LD50(中位数致死剂量)为4,4小时暴露于0.440mg / L蒸汽浓度,这也是目前推荐用于以250g / 100ml的浓度去除植物叶子的持续时间。长期使用水合氯醛也可以快速发展其对其作用和可能的成瘾的耐受性,以及不良反应,包括皮疹,胃不适和严重的肾脏,心脏和肝衰竭(Gelder等人,2005)。
 通过避免CHL的使用,该协议允许研究人员更迅速和安全地用台盼蓝染色植物细胞死亡,大大降低了研究人员的风险。在这里,我们通过监测Col-0叶片与灰葡萄孢()(> Bc )的感染过程来演示该方法的实用性,第二种植物病原真菌具有科学/经济重要性广泛的宿主范围,以及在植物中产生过氧化氢的高产能(Rolke等人,2004; Dean等人,2012; Lehmann等人,2015)。该协议也被成功应用于其他拟南芥种质。
注意:台盼蓝是来自甲苯胺的合成化合物,由Paul Ehrlich发明,诺贝尔生理与医学奖获得者,1904年( http://www.pei.de/)。

关键字:台盼蓝染色, 植物细胞死亡, 灰葡萄孢, 拟南芥, 水合氯醛

材料和试剂

  1. 锥形管(50ml)(Corning,Falcon ®,目录号:352070)
  2. Parafilm M实验室电影(Hach,目录号:251764)
  3. 具有透明盖的塑料培养皿(JET BIOFIL细胞和组织培养板6孔)
  4. 提示
  5. Miracloth(EMD Millipore,目录号码:475855)
  6. 标签胶带(三叶草,目录号:ST-12-20)
  7. 纸巾
  8. 加利福尼亚加拿大:哥伦比亚(Col-0)
  9. 乙醇> 99.8%(Sigma-Aldrich,目录号:02860)
    注意:本产品已停产。
  10. 乳酸85%(w:w)(Sigma-Aldrich,目录号:L1250,DL-乳酸11.3M)
  11. 苯酚(TE缓冲液平衡,pH 7.5-8.0)(Roti - 苯酚,Carl Roth,目录号:0038.1)
  12. 甘油≥99%(Sigma-Aldrich,目录号:G7757)
  13. 无菌蒸馏水(EMD Millipore,milli-q a10水净化系统)
  14. 台盼蓝(Sigma-Aldrich,目录号:T6146)
  15. 马铃薯葡萄糖琼脂(PDA)(BD,Difco TM,目录号:213400)
  16. 马铃薯葡萄糖肉汤(PDB)(BD,Difco TM,目录号:254920)
  17. 次氯酸钠溶液(Sigma-Aldrich,目录号:239305)
  18. 十二烷基硫酸钠(SDS)98%(Sigma-Aldrich,目录号:862010)
    注意:本产品已停产。
  19. 吐温20洗涤剂(Sigma-Aldrich,目录号:P1379)
  20. 台盼蓝染色溶液(见配方)
  21. 马铃薯葡萄糖琼脂(PDA)(见食谱)
  22. (B.c )接种物(见配方)
  23. 马铃薯葡萄糖汤(PDB)(见食谱)
  24. 种子灭菌方案(见食谱)

设备

  1. 微型剪刀NOYE(Auxilab,目录号:62101111)
  2. 解剖镊子,无齿125毫米(Auxilab,目录号:61302012)
  3. 生长室(VötschIndustrietechnik,型号:HERAEUS VB1514)
  4. 生物安全柜(无菌,型号:BBF-2SSCH)
  5. 来自Rainin Instruments的Micropipets P1000,P200和P20
  6. 重量分析440(KERN& SOHN)
  7. 耦合到CCD相机(Leica Microsystems,型号:DC 280)的立体显微镜(Leica Microsystems,型号:MZ9)
  8. 电导率计(HACHLANGE SPAIN,CRISON,型号:BASIC30)
  9. 高压釜(JP SELECTA,型号:MED 20,目录号:4001757)

软件

  1. ImageJ软件(Rasband,WS,ImageJ,美国国立卫生研究院,Bethesda,马里兰州,美国, http://imagej.nih.gov/ij/,1997-2016)
  2. Statgraphics Centurion XVI.II软件(StatPoint Technologies,Inc. www.statgraphics.com

程序

  1. 生长拟南芥植物
    1. 用种子灭菌溶液杀灭拟南芥种子(见食谱)20分钟,然后用无菌水洗涤四次。
    2. 在黑暗中将种子在4°C室中分层2天。
    3. 在生长室中以3:1(土壤:蛭石)的混合物种植种子,在10小时光/14小时黑暗光周期下持续21天,在22℃和60%湿度下光强度为120勒克斯。 />
  2. 与灰葡萄孢接种
    1. 将高压釜中的所有材料在120℃下灭菌20分钟
    2. 将植物放入生物安全柜中,每次一次接种一滴5μl的B.c真菌接种物(见食谱,图1A)。
    3. 用Parafilm覆盖植物(图1B),以避免分生孢子的分散,并保持高湿度(90-100%)以获得真菌的最佳生长。将它们返回到生长室,并在黄色末端的Parafilm(> 10)中形成小孔,以避免乙烯在托盘内积聚,这将激活植物防御反应并停止真菌生长。
    4. 在25℃,10小时光照条件下,在生长室中孵育 C.c 接种植物,直到取样日期,通常在接种后2至7天(图1C)。
    5. 在实验的每个时间点,用剪刀收获接种的叶子,并将其直接放入含有5ml新鲜的台盼蓝溶液的平板(参见食谱,图1D)。此时应确保叶子完全浸入液体中;如有需要,请使用蓝色的烙铁头将其淹没。盖板,等待至少30分钟。然后在死组织的位置看到蓝色。总染色时间不应超过1小时。避免使用沸腾方法,真空渗透方法或微波处理,这将阻碍以后的脱色过程。
      注意:取样的时间可能会根据拟南芥/加入/突变体/线-Bc相互作用和真菌毒力而改变。
    6. 取出锥虫溶液,立即用98-100%乙醇重新填充板。用Parafilm密封板,以避免可能会破坏叶子或将其固定在塑料板上的干燥。将组织置于乙醇溶液中至少过夜,然后使用5ml移液管;用新鲜的乙醇代替乙醇溶液,重复,直到绿色组织变得完全无色(图1E)。取出乙醇并用60%甘油溶液覆盖叶子。此时,叶片准备转移到玻璃表面,用显微镜可视化(图1F)。
    7. 如果需要,可以使用根据Govrin和Levine(2000)*的电导率的离子泄漏损失验证细胞死亡测量,或者使用ImageJ软件测量叶死亡面积(Rasband,WS,ImageJ,美国国立卫生研究院,Bethesda,Maryland,美国, http://imagej.nih.gov/ij/, 1997-2016) 注意:为了测量离子泄漏,我们遵循Govrin和Levine的方案(2000)。我们每个生物重复使用5个接种或对照叶,每个基因型有4个重复。将叶子在室温下浸入水中3小时,然后按照制造商的说明书测量电导率。在-80℃下保持o/n的相同样品获得了离子泄漏总量(100%)。数据分析如下所示进行。


      图1.用拟南芥进行拟南芥叶片接种的程序 .A和B.将拟南芥线条的叶表面接种用10μl真菌(10μg/ml)将植物转移到具有透明盖子的塑料托盘中以允许真菌生长。 C.进行感染进展,直到观察到植物症状。 D.在板上收获叶子,并用台盼蓝染色溶液吸收不超过1小时。 E.将叶子浸入乙醇中过夜,直到它们变得透明。 F.将叶转移至60%甘油进行显微镜检查。


      图2.拟南芥具有不同程度的感染。 A-F。切割叶子后立即拍摄照片,种植在土壤上的植物。 G-L。用台盼蓝染色的代表性叶片的照片,表现出类似于上述叶片的感染水平。 Bar = 0.5cm。

数据分析

对于所有实验,每个生态型至少10个叶被取样染色。实验重复至少四次。必要时,使用ImageJ(Particle Analysis,Surface Plot,and Measure Tools)软件,按照ImageJ的用户指南 https://imagej.nih.gov/ij/docs/pdfs/examples.pdf )。叶离子泄漏数据用Statgraphics Centurion XVI.II软件(StatPoint Technologies,Inc. www.statgraphics.com )。进行单因素方差分析(ANOVA)测试,计算平均值和标准误差以及Duncan的置信水平为0.05%的平均值。

食谱

  1. 台盼蓝染色液
    10ml乳酸(85%w:w)
    10毫升苯酚(TE缓冲液平衡,pH 7.5-8.0) 10ml甘油(≥99%)
    10ml蒸馏水
    40毫克台盼蓝(终浓度10毫克/毫升)
    注意:将溶液在室温下储存在通风罩中,并始终使用新鲜的溶液。
  2. 马铃薯葡萄糖琼脂(PDA)
    4 g/L PDA在蒸馏水中
  3. (B.c )接种物*
    将Botrytis菌株在28℃下在PDA培养基平板(马铃薯葡萄糖琼脂,Difco,Detroit,MI,USA)上生长8天,将孢子收集在无菌水中并储存在20℃ %甘油在-80°C直到使用。
    注意:真菌的存量应保存在-80℃的20%甘油中,以避免由于真菌重复生长而引起的毒力损失。真菌的最终浓度将取决于真菌分离物的毒力通常在105-107孢子/ml之间。使用血细胞计数器( http://www.hemocytometer)测量真菌浓度(每ml的分生孢子)。 org/)。当Bc分离株具有低毒力,或者当需要高水平或快速真菌生长时,可以使用可能的PDB的最大稀释度在马铃薯葡萄糖肉汤培养基(PDB)存在下进行接种,将相同浓度的PDB加入到对照接种叶。
  4. 马铃薯葡萄糖肉汤(PDB)
    4 g/L PDB在蒸馏水中
  5. 种子灭菌解决方案
    次氯酸盐溶液(30%,v:v)
    十二烷基硫酸钠(0.1%,v:v)

致谢

该协议由Keogh和合作者(1980)进行了修改。我们承认CGBP的Wilkinson先生的贡献。导致这些结果的研究从第AL13-P(I + D)-05号,第AL14-PID-26号至DomínguezJA号和Berrocal-Lobo M号,从RTA2013-00027-00-00至Castellano M M和Berrocal-Lobo M.

参考文献

  1. Cooksey,C.J。(2014)。染料命名的怪癖。 3.台an蓝。 生物技术Histochem 89(8):564-567。
  2. Dean,R.,Van Kan,JA,Pretorius,ZA,Hammond-Kosack,KE,Di Pietro,A.,Spanu,PD,Rudd,JJ,Dickman,M.,Kahmann,R.,Ellis, ,GD(2012)。  十大真菌病原体分子植物病理学。 Mol Plant Pathol 13(4):414-430。
  3. Gelder,M.,Mayou,R。和Geddes,J。(2005)。精神病学(第3版)牛津版 pp:238.
  4. Govrin,E.M和Levine,A。(2000)。过敏反应促进了坏死性病原体灰霉病菌的植物感染。 10卷(13):751-757。
  5. Keogh,RC,Deverall,BJ和Mcleod,S。(1980)。  比较抗性和易感大豆中Phakopsora pachyrhizi的组织学和生理学反应。英国真菌学学会交易会(74)(2):329-333
  6. Lehmann,S.,Serrano,M.,L'Haridon,F.,Tjamos,SE和Metraux,JP(2015)。< a class ="ke-insertfile"href ="http://www.ncbi。 nlm.nih.gov/pubmed/25264341"target ="_ blank">反应性氧物种和植物对真菌病原体的抗性。植物化学 112:54-62。
  7. Lu,J.和Greco,MA(2006)。睡眠电路和GABA药物的催眠机制。
    J Clin Sleep Med 2(2):S19-26。
  8. Rolber,Y.,Liu,S.,Quidde,T.,Williamson,B.,Schouten,A.,Weltring,KM,Siewers,V.,Tenberge,KB,Tudzynski,B.and Tudzynski,P。(2004) 。  H(2)O(2)的功能分析主要的Cu-Zn超氧化物歧化酶(BCSOD1)对法国豆有毒,而葡萄糖氧化酶(BCGOD1)是不必要的。植物Pathol 5(1):17-27。
  9. Salazar M,Peralta C,Pastor FJ(2009)。  Tratado dePsicofarmacología。 2 Ed。 Panamericana
  10. Salmon,AG,Kizer,KW,Zeise,L.,Jackson,RJ和Smith,MT(1995)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih。 gov/pubmed/7897749"target ="_ blank">水合氯醛的潜在致癌性 - 综述。 Toxicol Clin Toxicol 33(2):115-121。
  11. Silverman,J和Muir,WW,3rd(1993)。  用水合氯醛和氯醛糖对实验室动物麻醉进行回顾实验动物科学 43(3):210-216。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Fernández-Bautista, N., Domínguez-Núñez, J. A., Moreno, M. C. and Berrocal-Lobo, M. (2016). Plant Tissue Trypan Blue Staining During Phytopathogen Infection. Bio-protocol 6(24): e2078. DOI: 10.21769/BioProtoc.2078.
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Jinping Zhao
Texas A&M University
Hi, I am doing trypan blue staining in Nicotiana benthamiana according to the protocol. In the Recipe of staining buffer it says that the final concentration of trypan blue is 10 mg/ml (1%), however 40 mg of trypan blue resolved in the liquid mixture makes a concentration of 1 mg/ml (0.1%). Which concentration should be used?
6/19/2017 10:54:43 AM Reply
Marta Berrocal-Lobo
Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM) – Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Parque Científico y Tecnológico, UPM, Campus de Montegancedo, Spain

Dear user,
You can try with 0,04g in 40ml of mix, this is the minimum for Arabidopsis leave staining, however with Nicotiana you might need more, the leaves are thicker.
Let me know your results. I will review the text to fix some error.
We observed with Nicotiana sometimes some whitering on leaves during ethanol step.
Good luck
Marta

8/23/2017 4:13:31 AM


xiao Zhang
Chinese Academy of Agricultural Sciences(CAAS)

您好,我叫张晓,请问你也在用台酚蓝染色的方法看烟草的cell death,请问可否分享一下这个方法的经验?

8/30/2017 4:54:55 PM