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Salinity Assay in Arabidopsis
拟南芥耐盐实验   

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Abstract

Salinity is an important environmental constraint to crop productivity in arid and semi-arid regions of the world. The evaluation of the responses to salinity of different Arabidopsis ecotypes or transgenic lines is important to identify and investigate the role of different key genes. These new characterized genes involved in the response to salinity stress are of great interest to be incorporated in crops breeding programs. Here we provide a reproducible method to evaluate the performance of Arabidopsis lines to salinity stress by analysing primary and lateral root growth and fresh weight of plants grown under in vitro conditions in growth chambers. Even though NaCl is the most frequent used salinity tests, other salts (e.g. KCl, MgCl2) can be also evaluated by this method. Arabidopsis plants can be maintained for 15-20 days in these conditions, although effects on growth and biomass can be observed, depending on the used salt and concentration, within the first 10 days.

Keywords: Salinity tolerance(耐盐性), Arabidopsis(拟南芥), Plate assay(平板法), Root length(根长)

Materials and Reagents

  1. Arabidopsis thaliana ecotype Columbia (Col-0) and transgenic Arabidopsis lines 35S::SlCDF1 and 35S::SlCDF3 (background Col-0) seeds
  2. 70% ethanol
  3. Sodium hypochlorite (NaClO)
  4. Sodium dodecylsulphate (SDS)
  5. Sterile water
  6. Murashige & Skoog (MS) mediun including B5 vitamins (Duchefa Biochemie BV, catalog number: M0231 )
  7. MES (Duchefa Biochemie BV, catalog number: M1503 )
  8. Sucrose
  9. NaCl
  10. Agar (Duchefa Biochemie BV, catalog number: P1001 )
  11. Medium composition (see Recipes)

Equipment

  1. Square (120 x 120 mm) petri dishes polystyrene sterile
  2. Growth chamber
  3. Autoclave
  4. Shaker
  5. Image capturing device
  6. Precision balance (± 0.0001)

Software

  1. Image J software (http://rsb.info.nih.gov/ij) (Abrámoff et al., 2004)

Procedure

  1. Seed germination
    1. Seeds are surface sterilized by washing in 70% ethanol for 2 min, then in sterilization solution (1% sodium hypochlorite with 1% SDS in sterile water) for 12 min with shaking and finally washed five times with sterilized water.
    2. The seeds are stratified for 2 days at 4 °C in the dark.

  2. Growth conditions
    1. Sterilized seeds are placed in Petri dishes (approx. 70 seeds per Petri dish) on MS medium and grown for 6 days in vertical position, in a culture chamber at 22 °C under standard long-day conditions (16/8 h light/dark photoperiod).
    2. After six days of growth, seedlings exhibiting similar root length (7-10 mm) are transferred to square plates containing MS medium (control) or MS medium supplemented with 80 mM NaCl. About 20 seedlings are used per replicate and three replicates are made for each treatment.
    3. The initial position of the root tip is marked in the plate (Figure 1A).


      Figure 1. Example of primary and lateral root measurements with the Image J software. A) Root tip marked; B) Image of seedlings grown on MS medium supplemented with 80 mM NaCl after 10 days of treatment; C) Scale setting (e.g. 30 mm); D-E) Length measurement of the primary root from the marked point; and F) Length measurement of the lateral roots.

    4. The plants are grown in vertical position under standard  long day conditions (22 °C, 16/8 h light/dark) for 10 days, and then pictures are taken of each plate (Figure 1B). Primary and lateral root length and plant weight are then scored.

  3. Measurements root length
    The measurement of primary and lateral root length is performed by analysis of the pictures with the Image J software.
    1. First, set the scale using the ImageJ software: > draw a straight line of known distance > click on Analyze > set scale > known distance (e.g. 30 mm) > unit of length (mm) > global > ok (Figure 1C).
    2. Measure the length of primary root from the marked start position to the tip using the segmented line option of the software: Press Control + M to measure the length of the root (Figure 1D-E).
    3. Measure the length of each lateral root following the same procedure (Figure 1F).
    4. To evaluate growth differences between control and saline stress, measured lengths of the primary and lateral roots are represented as the percentage of root growth reduction relative to control conditions (MS medium) (for representative data see Figure 2).
    5. Statistical analyses are carried out by one-way analysis of variance (ANOVA) followed by a Student-Newman-Keuls test (P<0.01) (Figure 2).

  4. Measurement of plant fresh weight
    Fresh weight measurements are estimated using a precision balance.
    1. Whole 10 day-old-plants grown in MS medium or MS medium supplemented with 80 mM NaCl are weighed using a precision balance (Figure 3A-C).  
    2. Statistical analyses are carried out by one-way analysis of variance (ANOVA) followed by a Student–Newman–Keuls test (P<0.01). The percentage of growth reduction by salinity relative to control conditions is compared between the studied genotypes.


      Figure 2. View of plant fresh weight measurements. A) Plantlets are carefully removed from the medium and B-C) weighed with a precision balance.

Representative data

  1. Example of results primary and lateral root elongation measurements under salinity stress after 10 days of treatment using ImageJ software. The measurement are performed as described above (item C: 4 and 5).


    Figure 3. Example of measured effects of salt stress on Arabidopsis root growth in lines overexpressing SlCDF1 and SlCDF3 genes (Corrales et al., 2014). A) Salt stress tolerance estimated by determining the reduction of primary root and lateral root growth of 35S::SlCDF1 lines 1.2, 1.4 and 2.6, 35S::SlCDF3 lines 2.10, 10.4 and 10.7 and wild type Col-0 plants after 10 days in MS supplemented with 80 mM NaCl. The percentage of reduction relative to control conditions is represented. Data are expressed as means ± standard errors of three independent experiments with at least 20 plants each. Asterisks indicate significant differences between Col-0 and 35S::SlCDF1 or 35S::SlCDF3 overexpressing lines (P<0.05; ANOVA Student-Newman-Keuls tests. B) Representative images of WT, 35S::SlCDF1 line 2.6 and 35S::SlCDF3 line 2.10 after the treatments.

Recipes

  1. Medium composition
    1. For 1 L of MS control medium
      2.2 g of Murashige & Skoog (MS) medium including B5 vitamins
      5 g of sucrose
      5 g MES
      Adjust pH to 5.7 with KOH
      Add 7 g agar
      Sterilized for 20 min at 121 °C/1 atm using an autoclave
    2. For 1 L the medium MS supplemented 80 mM with NaCl
      4.67 g NaCl is added to the medium

Acknowledgments

We gratefully acknowledge funding through grants from Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA; project numbers: 2009-0004-C01, 2012-0008-C01) and the Spanish Ministry of Science and Innovation (project number: BIO2010-14871 and ERA-NET GEN2006-27772-C2-2). Additionally, we are grateful for the protocol described by Lakhssassi et al. (2012).

References

  1. Abràmoff, M. D., Magalhães, P. J. and Ram, S. J. (2004). Image processing with ImageJ. Biophotonics Inter 11(7): 36-43.
  2. Corrales, A. R., Nebauer, S. G., Carrillo, L., Fernandez-Nohales, P., Marques, J., Renau-Morata, B., Granell, A., Pollmann, S., Vicente-Carbajosa, J., Molina, R. V. and Medina, J. (2014). Characterization of tomato Cycling Dof Factors reveals conserved and new functions in the control of flowering time and abiotic stress responses. J Exp Bot 65(4): 995-1012.
  3. Lakhssassi, N., Doblas, V. G., Rosado, A., del Valle, A. E., Posé, D., Jimenez, A. J., Castillo, A. G., Valpuesta, V., Borsani, O. and Botella, M. A. (2012). The Arabidopsis tetratricopeptide thioredoxin-like gene family is required for osmotic stress tolerance and male sporogenesis. Plant Physiol 158(3): 1252-1266.

简介

盐度是世界干旱和半干旱地区作物生产力的重要环境约束。对不同拟南芥生态型或转基因株系的盐度的响应的评价对于鉴定和研究不同关键基因的作用是重要的。这些新的表征基因参与对盐分胁迫的反应是非常感兴趣的被纳入作物育种计划。在这里我们提供了一种可再现的方法,通过分析在生长室中的体外条件下生长的植物的初级和侧根生长和鲜重来评估拟南芥株系对盐度胁迫的性能。 。即使NaCl是最常用的盐度测试,也可以通过该方法评价其它盐(例如KCl,MgCl 2)。拟南芥植物在这些条件下可以维持15-20天,但是在前10天内可以观察到对生长和生物量的影响,这取决于所使用的盐和浓度。

关键字:耐盐性, 拟南芥, 平板法, 根长

材料和试剂

  1. 拟南芥生态型哥伦比亚( Col-0 )和转基因拟南芥系35S :: S1CDF1 和<35> :: SlCDF3 (背景 Col-0 )种子
  2. 70%乙醇
  3. 次氯酸钠(NaClO)
  4. 十二烷基硫酸钠(SDS)
  5. 无菌水
  6. Murashige& 包括B5维生素的Skoog(MS)介质(Duchefa Biochemie BV,目录号:M0231)
  7. MES(Duchefa Biochemie BV,目录号:M1503)
  8. 蔗糖
  9. NaCl
  10. 琼脂(Duchefa Biochemie BV,目录号:P1001)
  11. 中等组成(见配方)

设备

  1. 方形(120×120mm)培养皿聚苯乙烯无菌
  2. 生长室
  3. 高压灭菌器
  4. 振动器
  5. 图像捕获设备
  6. 精密平衡(±0.0001)

软件

  1. Image J软件( http://rsb.info.nih.gov/ij )(Abrámoff< et al。,2004)

程序

  1. 种子萌发
    1. 种子通过在70%乙醇中洗涤2分钟,然后在灭菌溶液(1%次氯酸钠与1%SDS的无菌水溶液)中振荡12分钟进行表面灭菌,最后用无菌水洗涤5次。
    2. 将种子在4℃下在黑暗中分层2天。

  2. 生长条件
    1. 将灭菌的种子置于培养皿中(约70个种子/培养皿)在MS培养基上并在22℃的标准长日条件(16/8小时光照/黑暗)下在培养室中在垂直位置生长6天 光周期)。
    2. 生长6天后,将显示相似根长度(7-10mm)的幼苗转移到含有MS培养基(对照)或补充有80mM NaCl的MS培养基的方板上。 约20棵幼苗 每次重复使用,每次处理重复三次
    3. 根尖的初始位置在板中标记(图1A)。


      图1.使用Image J软件进行原始和侧根测量的示例。A)根尖标记; B)处理10天后在补充有80mM NaCl的MS培养基上生长的幼苗的图像; C)比例设置(例如 30mm); D-E)标记点的主根长度测量;和F)侧根的长度测量。

    4. 植物在垂直位置下生长,长日照条件(22℃,16/8h光/暗)10天,然后拍摄每个平板的照片(图1B)。然后计算原始和侧根长度和植物重量

  3. 测量根长
    通过使用Image J软件分析图像来进行原始和侧根长度的测量。
    1. 首先,使用ImageJ软件设置刻度:>绘制已知距离的直线>点击分析>设置比例>已知距离(例如30mm)>长度单位(mm)>全局> ok(图1C)。
    2. 使用软件的分段线选项从标记的起始位置到尖端测量主根的长度:按Control + M测量根的长度(图1D-E)。
    3. 按照相同的程序测量每个侧根的长度(图1F)。
    4. 为了评价对照和盐胁迫之间的生长差异,将原始和侧根的测量长度表示为相对于对照条件(MS培养基)的根生长减少的百分比(代表性数据参见图2)。
    5. 通过单向方差分析(ANOVA),然后进行Student-Newman-Keuls试验(P <0.01)(图2)进行统计学分析。

  4. 植物鲜重的测量
    鲜重测量使用精密天平估计。
    1. 使用精密天平称量在MS培养基或补充有80mM NaCl的MS培养基中生长的全部10日龄植物(图3A-C)。  
    2. 通过单因素方差分析(ANOVA),随后的Student-Newman-Keuls试验(P <0.01)进行统计分析。 在研究的基因型之间比较相对于对照条件的盐度生长减少的百分比

      图2.植物鲜重测量视图 A)小心地从培养基中取出小植株,B-C)用精密天平称重。

代表数据

  1. 使用ImageJ软件进行10天处理后,在盐度胁迫下的初级和侧根伸长测量结果的实例。测量如上所述进行(项目C:4和5)

    图3.盐胁迫对拟南芥的根生长的测量效应的实例,所述根生长在过表达S1CDF1和scCDF3基因的细胞系中(Corrales ,2014)。 A)通过测定35S :: S1CDF1 品系1.2,1.4和2.6的初生根和侧根生长的减少来评估盐胁迫耐受性,在补充有80mM NaCl的MS中10天后,在野生型植物中,野生型Col-0植物,野生型 Col-0 35S :: SlCDF3系2.10,10.4和10.7。表示相对于对照条件的降低百分比。数据表示为三次独立实验的平均值±标准误差,每次至少有20株植物。星号表示Col-0和35S :: S1CDF1或35S :: S1CDF3过表达株系之间的显着差异(P <0.05; ANOVA Student-Newman-Keuls检验B)治疗后WT, 35S :: S1CDF1 系列2.6和 35S :: S1CDF3 系列2.10的图像。

食谱

  1. 中等组成
    1. 对于1 L的MS对照培养基
      2.2g Murashige& 包括B5维生素的Skoog(MS)培养基 5克蔗糖 5克MES
      用KOH调节pH至5.7 加入7克琼脂
      使用高压灭菌器在121℃/1atm下灭菌20分钟
    2. 对于1L培养基MS补充80mM用NaCl
      将4.67g NaCl加入到培养基中

致谢

我们衷心感谢通过国家农业技术研究所(INIA;项目编号:2009-0004-C01,2012-0008-C01)和西班牙科学和创新部(项目编号:BIO2010-14871和ERA-NET GEN2006-27772-C2-2)。此外,我们感谢Lakhssassi等人(2012年)描述的协议。

参考文献

  1. Abràmoff,M.D.,Magalhães,P.J.and Ram,S.J。(2004)。 使用ImageJ进行图像处理 生物光子学 11(7):36-43。
  2. Corrales,AR,Nebauer,SG,Carrillo,L.,Fernandez-Nohales,P.,Marques,J.,Renau-Morata,B.,Granell,A.,Pollmann,S.,Vicente-Carbajosa,J.,Molina ,RV和Medina,J。(2014)。 番茄循环因子的表征揭示了控制开花时间和非生物胁迫反应的保守和新功能。 65(4):995-1012。
  3. Lakhssassi,N.,Doblas,V. G.,Rosado,A.,del Valle,A.E.,Posé,D.,Jimenez,A.J.,Castillo,A.G.,Valpuesta,V.,Borsani,O.and Botella, 拟南芥三七肽硫氧还蛋白样基因家族是渗透性 胁迫耐受性和雄性孢子形成。植物生理学158(3):1252-1266。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Corrales, A. R., Carrillo, L., Nebauer, S. G., Renau-Morata, B., Sánchez-Perales, M., Fernández-Nohales, P., Marqués, J., Granell, A., Pollmann, S., Vicente-Carbajosa, J., Molina, R. V. and Medina, J. (2014). Salinity Assay in Arabidopsis. Bio-protocol 4(16): e1216. DOI: 10.21769/BioProtoc.1216.
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