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Salinity Assay in Tomato
番茄耐盐实验   

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参见作者原研究论文

本实验方案简略版
Journal of Experimental Botany
Mar 2014

Abstract

Tomato is one of the most important horticultural crops worldwide, and is cultivated in semi-arid regions in which soil and groundwater salinity is an increasing limitation to yield. The assessment of the responses of new cultivars to salt and the comparisons among cultivars and wild species are of great interest in tomato breeding. This assay provides a reproducible and reliable method for screening tomato responses to NaCl salinity under hydroponic conditions in growth chambers. Although NaCl is the most commonly used salt in salinity studies, other salts such as Na2SO4, MgCl2 or MgSO4, usually found in saline soils, can also be assayed (Nebauer et al., 2013). Plants can be maintained for 30-45 days under the described conditions, although significant effects on growth can be observed after 10 days, depending on the salt and concentration used.

Keywords: Tomato (番茄), Salinity assay (盐度的测定), Hydroponic (水培), Growth (生长)

Materials and Reagents

  1. Solanum lycopersicum seeds
  2. Agar
  3. Sodium hypochlorite (NaClO)
  4. Potassium nitrate (KNO3)
  5. Ammonium nitrate (NH4NO3)
  6. Calcium nitrate [Ca(NO3)2.4H2O]
  7. Magnesium sulphate (MgSO4.7H2O)
  8. Ethylene diamine-N, N bis (2hydroxyphenylacetic acid) Ferric sodium complex (Fe-EDDHA)
  9. Boric acid (H3BO3)
  10. Manganese chloride (MnCl2.4H2O)
  11. Zinc sulphate (ZnSO4.7H2O )
  12. Copper sulphate (CuSO4.5H2O)
  13. Sodium molybdate (Na2MoO4.2H2O)
  14. Sodium chloride (NaCl)
  15. Sodium sulphate (Na2SO4)*
  16. Magnesium chloride (MgCl2)*
  17. Magnesium sulphate (MgSO4)*
    *Note: These salts are necessary only if they have to be assayed. The standard assay is performed with NaCl.
  18. Non-saline nutrient solution (see Recipes)
  19. Salt stock solutions (see Recipes)

Equipment

  1. Eppendorf-type tubes (1.5 ml)
    Note: Caps are removed and the tube end is cut with scissors (Figure 1A). Tubes are placed in a tube rack (with a sealed bottom, Figure 1B) and filled with 0.6% agar in tap water (melted in a microwave) using a 50 ml syringe (Figure 1C-D).


    Figure 1. Preparation of the tubes to hold plantlets. A) Cut tube, B) Racks with a sealed bottom, C) Filling tubes with agar in the rack and D) Tube filled with agar.

  2. Opaque 10 L containers with cover
    Polyethylene containers (40 cm long x 30 cm wide x 12 cm high) are used. Covers are bored with a drill to allow the placement of the Eppendorf-type tubes (Figure 2).


    Figure 2. Example of a container with a bored cover to hold Eppendorf-type tubes

  3. Petri dishes
  4. 50 ml syringe
  5. Growth chamber
  6. ‘Aquarium’-type air pumps
  7. Timer control
  8. Microwave oven
  9. Pasteur pipettes
  10. Racks for Eppendorf-type tubes with a sealed bottom
  11. Plastic trays with humidity domes
  12. Precision balance (± 0.001)

Procedure

  1. Seed germination
    1. Seeds are surface-sterilised in a sodium hypochlorite solution (2.5%) with 0.1% Tween 20 for 15 min and subsequently washed three times in sterile distilled water.
    2. Seeds are placed in 9 cm-diameter Petri dishes (20-50 seeds per Petri dish) on top of three layers of moistened blotting paper (Figure 3A) and maintained in the dark at 25 °C until germination (3-6 days depending on the genotype) (Figure 3B).


      Figure 3. Details of seed preparation and germination. A) Seeds in Petri dishes, B) Germinated seeds showing radicle emergence and C) Germinated seeds in Eppendorf-type tubes filled with agar.

    3. Place homogenously germinated seeds (3-6 mm root length) in Eppendorf-type tubes with the cut end filled with 0.6% agar (Figure 3C). Moisten the agar surface with a water drop using a Pasteur pipette. The racks holding the tubes are placed in a tray and covered with a humidity dome to maintain high air moisture (Figure 4A). Put some water (50-100 ml) into the tray to guarantee high air humidity within the tray. Maintain plantlets in growth chambers at 25/18 °C in a 16/8 h light/dark photoperiod.
    4. Plants (seedlings at fully expanded cotyledon stage, Figure 4B) are progressively exposed to ambient atmosphere by slightly opening (1-2 cm) the humidity dome (Figure 4C) and after one week transferred to containers.


      Figure 4. Plantlets growing in covered trays. A) Trays with humidity domes for seedling culture, B) Detail of seedlings at fully expanded cotyledon stage and C) Dome opened to allow acclimatisation (indicated by the arrow).

  2. Culture
    1. Containers are filled with nutrient medium and aerated regularly (for 10 min every half hour with an ‘Aquarium’-type air pump). Nutrient solutions (see Recipes) are renewed every 4 days (the old solution has to be completely removed).
    2. Insert the Eppendorf-type tubes with homogeneous plantlets (at fully expanded cotyledon stage with active root growth) into the cover holes allowing the contact of the cut end with the nutrient solution (Figure 5).


      Figure 5. Detail of plantlets (12-16 days old) growing in the containers

    3. Maintain plants in growth chambers at 25/18 °C in a 16/8 h light/dark photoperiod (200 μmol photon m-2 s-1) during the experiment.
    4. After 3-4 weeks, when plants have three-four leaves (Figure 6), an aliquot of the salt stock solution (see Recipes) is added to the nutrient solution to obtain the desired salt concentration (see Representative data). The plants cultured in the containers with non-saline nutrient solution are used as the controls. Eight to 12 plants are used for each condition and genotype.
    5. Plant biomass (dry and fresh weight) can be measured after 10-15 days to determine the effect of salinity. Roots and shoots are separated and fresh weights are weighed on a precision balance. Dry weights are recorded after keeping the material for 48 h at 60 °C.


      Figure 6. Tomato plantlets (30-40 days old) growing in the containers. View of A) shoots and B) roots.

Representative data

  1. It has been described that 75-100 mM NaCl significantly reduces growth in several tomato cultivars (Nebauer et al., 2013 and references herein; Corrales et al., 2014). In Figure 7, the representative data obtained in the RAF tomato cultivar are shown. Smaller amounts (50-75 mM) of MgCl2, MgSO4 and Na2SO4 accomplished similar reductions, and higher toxicity was found with magnesium.


    Figure 7. Effect of different salts and concentration on the total fresh weight of RAF tomato measured after 4, 8 and 12 days of salt exposure. ○: control; ●: 25 mM; ▲: 50 mM; ◆: 100 mM (Nebauer et al. 2013)

Notes

  1. A representative video of the protocol is also available.

    Video 1. Salinity assay in tomato

Recipes

  1. Non-saline nutrient solution (control medium)
    Non-saline nutrient solution (control medium) is based on Hoagland no.2 solution (Hoagland and Arnon, 1950).
    Macronutrients
    Stock solution (g per 1 L)
    ml stock per L medium
    1 M KNO3
    101.11 g
    3
    1 M NH4H2PO4
    115.03 g
    0.5
    1 M Ca(NO3)2.4H2O
    236.15 g
    2
    1 M MgSO4.7H2O
    246.48 g
    1
    0.5% (w/v) Fe-EDDHA
    5 g
    0.5
    Micronutrients


    H3BO3
    2.86 g
    1
    MnCl2.4H20
    1.81 g
    1
    ZnSO4.7H20
    0.22 g
    1
    CuSO4.5H20
    0.051 g
    1
    Na2MoO4.2H2O
    0.09 g
    1

  2. Salt stock solutions
    Salt stock solutions (in distilled water):
    5 M NaCl (292.2 g NaCl per litre stock solution)
    1 M Na2SO4 (142.04 g Na2SO4 per litre stock solution)
    2.5 M MgCl2 (508.25 g MgCl2.6H2O per litre stock solution)
    2 M MgSO4 (492.96 g MgSO4.7H2O per litre stock solution)
    An aliquot of the salt stock solutions is added to the control media to prepare saline media.

Acknowledgments

We gratefully acknowledge funding through grants from the 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 numbers: BIO2010-14871 and ERA-NET GEN2006-27772-C2-2).

References

  1. 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.
  2. Hoagland, D. R. and Arnon, D. I. (1950). The water-culture method for growing plants without soil. Circular. California Agricultural Experiment Station 347 (2nd edit).
  3. Nebauer, S. G., Sanchez, M., Martinez, L., Lluch, Y., Renau-Morata, B. and Molina, R. V. (2013). Differences in photosynthetic performance and its correlation with growth among tomato cultivars in response to different salts. Plant Physiol Biochem 63: 61-69.

简介

番茄是世界上最重要的园艺作物之一,并且在半干旱地区栽培,其中土壤和地下水盐度对产量有越来越大的限制。 新品种对盐的反应的评估以及栽培品种和野生品种之间的比较在番茄育种中是非常有意义的。 该测定提供了在生长室中在水培条件下筛选番茄对NaCl盐度的反应的可重复和可靠的方法。 尽管NaCl是盐度研究中最常用的盐,但是其它盐如Na 2 SO 4,MgCl 2或MgSO 4 通常存在于盐水土壤中,也可以测定(Nebauer等人,2013)。 植物可以在所述条件下维持30-45天,但是在10天后可以观察到对生长的显着影响,这取决于所使用的盐和浓度。

关键字:番茄, 盐度的测定, 水培, 生长

材料和试剂

  1. Solanum lycopersicum 种子
  2. Agar
  3. 次氯酸钠(NaClO)
  4. 硝酸钾(KNO 3)
  5. 硝酸铵(NH 4 NO 3)
  6. 硝酸钙[Ca(NO 3)2] 2 SiO 4 [O]
  7. 硫酸镁(MgSO 4)7H 2 O 3)
  8. 乙二胺-N,N双(2-羟基苯基乙酸)铁钠复合物(Fe-EDDHA)
  9. 硼酸(H 3 BO 3)
  10. 氯化锰(MnCl 2→4H 2 O)
  11. 硫酸锌(ZnSO 4,7H 2 O,7H 2 O)
  12. 硫酸铜(CuSO 4钠)5 H 2 O 2)/
  13. 钼酸钠(Na 2 MoO 4)2/2H 2 O 2)。
  14. 氯化钠(NaCl)
  15. 硫酸钠(Na 2 SO 4)*
  16. 氯化镁(MgCl 2)*
  17. 硫酸镁(MgSO 4)*
    注意:这些盐只有在必须进行测定时才是必要的。 标准测定用NaCl进行。
  18. 无盐营养液(见配方)
  19. 盐储备溶液(见配方)

设备

  1. Eppendorf型管(1.5ml) 注意:取下盖子,用剪刀剪切管端(图1A)。 将管置于管架(具有密封底部,图1B)中,并使用50ml注射器(图1C-D)填充自来水中的0.6%琼脂(在微波中熔融)。


    A)切割管,B)具有密封底部的架子,C)在架子中填充琼脂,和D)填充琼脂的管子。

  2. 不透明10升带盖的容器
    使用聚乙烯容器(40cm长×30cm宽×12cm高)。 用钻子钻孔以允许放置Eppendorf型管(图2)。


    图2.带有钻孔盖以容纳Eppendorf型管的容器示例

  3. 培养皿
  4. 50 ml注射器
  5. 生长室
  6. 水族箱式空气泵
  7. 定时器控制
  8. 微波炉
  9. 巴斯德移液器
  10. 带有密封底部的Eppendorf型管的支架
  11. 塑料托盘,湿度圆顶
  12. 精密平衡(±0.001)

程序

  1. 种子萌发
    1. 将种子在具有0.1%吐温20的次氯酸钠溶液(2.5%)中表面灭菌15分钟,随后在无菌蒸馏水中洗涤三次。
    2. 将种子置于9cm直径的培养皿(每个培养皿中20-50粒种子)的三层湿润吸墨纸(图3A)的顶部,并在25℃下保持黑暗,直到发芽(3-6天,取决于 基因型)(图3B)

      图3.种子准备和发芽的细节 A)培养皿中的种子,B)显示胚根出芽的发芽种子和C)填充有琼脂的Eppendorf型管中的发芽种子。
    3. 将均质发芽的种子(3-6mm根长)放置在切割端填充有0.6%琼脂的Eppendorf型管中(图3C)。使用巴斯德吸管用水滴润湿琼脂表面。将保持管的架放置在托盘中并用湿度圆顶覆盖以保持高空气湿度(图4A)。将一些水(50-100毫升)放入托盘,以确保托盘内高的空气湿度。在25/18℃的生长室中在16/8小时光/暗光周期中保持苗
    4. 通过稍微打开(1-2cm)湿度圆顶(图4C)和在一周转移到容器之后,将植物(完全膨胀的子叶阶段的幼苗,图4B)逐渐暴露于环境大气。


      图4.在覆盖的托盘中生长的小植物。A)用于幼苗培养的具有湿度圆顶的托盘,B)在完全膨胀的子叶阶段的幼苗的细节,和C)打开圆顶以允许驯化(由箭头指示) 。

  2. 文化
    1. 容器中充满营养培养基并定期充气(每半小时用"水族箱"型空气泵10分钟)。营养溶液(见配方)每4天更新一次(旧溶液必须完全除去)
    2. 插入Eppendorf型管与同种植物(充分膨胀的子叶阶段有活性根生长)到盖孔,允许切口端与营养液接触(图5)。


      图5.生长在容器中的小植株(12-16天龄)的细节

    3. 在实验期间,在25/18℃下在16/8小时光/暗光周期(200μmol光子m -2 s -1 s -1 )中的生长室中维持植物。
    4. 3-4周后,当植物具有三叶(图6)时,将等份的盐储备溶液(参见Recipes)加入到营养液中以获得所需的盐浓度(参见代表性数据)。将在含有非盐水营养液的容器中培养的植物用作对照。每种条件和基因型使用8到12株植物
    5. 植物生物量(干重和鲜重)可以在10-15天后测量以确定盐度的影响。分离根和芽并在精密天平上称重鲜重。在将材料在60℃下保持48小时后记录干重

      图6.生长在容器中的番茄小植株(30-40天)。查看A)芽和B)根。

代表数据

  1. 已经描述了75-100mM NaCl显着降低了几种番茄品种中的生长(Nebauer等人,2013和本文中的参考文献; Corrales等人,2014)。在图7中,显示了在RAF番茄栽培品种中获得的代表性数据。较小量(50-75mM)的MgCl 2,MgSO 4和Na 2 SO 4,实现了类似的还原,并发现镁的毒性更高。


    图7.不同盐和浓度对在暴露4,8和12天后测量的RAF番茄的总鲜重的影响。○:对照; ●:25mM; ▲:50mM; ◆:100mM(Nebauer等人 2013)

笔记

      
  1. 还提供了协议的代表性视频。

    视频1.番茄中的盐度测定
                                                                                                                                                                                                                     <! - [if!IE]> - > <! - <![endif] - >                                                                                                                                                                                                                                                          

    要播放视频,您需要安装较新版本的Adobe Flash Player。

    获取Adobe Flash Player

    <! - [if!IE]> - >
    <! - <![endif] - >
                    
                                                                                                                                                                                                                                          <! - [if!IE]> - > <! - <![endif] - >                                                                                                                                                                                                                                                                                   

    要播放视频,您需要安装较新版本的Adobe Flash Player。

    获取Adobe Flash播放器

    <! - [if!IE]> - >
    <! - <![endif] - >
                       

食谱

  1. 非盐水营养液(对照培养基)
    非盐水营养液(对照培养基)基于Hoagland 2号溶液(Hoagland和Arnon,1950)。
    大量营养素
    储备溶液(g/1L)
    ml原液/L培养基
    1 M KNO 3
    101.11克
    3
    1 NH NH 4 H H 2 PO 4 4
    115.03克
    0.5
    1 M Ca(NO 3)2 sub 2 4H 2 O 236.15克
    2
    1 M MgSO 4。 7H 2 O 246.48克
    1
    0.5%(w/v)Fe-EDDHA
    5克
    0.5
    微量营养素


    H 3 BO 3
    2.86克
    1
    MnCl 2 4H 0
    1.81克
    1

    0.22克
    1
    CuSO 4 5H 2 0
    0.051克
    1
    Na 2 MoO 4 sub 。 2H O
    0.09克
    1

  2. 盐储备溶液
    盐储备溶液(在蒸馏水中):
    5 M NaCl(292.2 g NaCl /升储液)
    1 2M Na 2 SO 4(142.04g Na 2 SO 4 4)/升储备溶液)
    2.5M MgCl 2(508.25g MgCl 2·6H 2 O /升储备溶液)
    2M MgSO 4(492.96g MgSO 4)/7H 2 O 2 /升储备溶液)
    将等份的盐储备溶液加入对照培养基中以制备盐水培养基

致谢

我们衷心感谢来自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的拨款(项目编号:BIO2010-14871和ERA-NET GEN2006-27772-C2-2)。

参考文献

  1. 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。
  2. Hoagland,D.R。和Arnon,D.I。(1950)。 。 加利福尼亚农业实验站 347(第二次编辑)。
  3. Nebauer,S.G.,Sanchez,M.,Martinez,L.,Lluch,Y.,Renau-Morata,B.and Molina,R.V。(2013)。 光合性能的差异及其与番茄品种对不同盐响应的生长的相关性。 Plant Physiol Biochem 63:61-69
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Renau-Morata, B., Sánchez-Perales, M., Medina, J., Molina, R. V., Corrales, R., Carrillo, L., Fernández-Nohales, P., Marqués, J., Pollmann, S., Vicente-Carbajosa, J., Granell, A. and Nebauer, S. G. (2014). Salinity Assay in Tomato. Bio-protocol 4(16): e1215. DOI: 10.21769/BioProtoc.1215.
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Srinath Srinath Rao
Gulbarga University
Nice paper. Thanks for uploading.
Dr. Srinathrao
9/17/2014 12:06:52 PM Reply
Sergio Nebauer
Departamento de Producción Vegetal, Universitat Politècnica de València, España

Dear Dr. Srinathrao,
Thank you very much for yor comments and interest in our work.
Kind regards
SG Nebauer

9/18/2014 4:30:39 AM