Pot Level Drought Stress Tolerance Assay in Tobacco through Plant Phenotyping and Antioxidant Assay

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Journal of Plant Physiology
Oct 2013



Drought is an important abiotic factor which has a huge detrimental impact on crop productivity. Study of plant responses towards drought stress and investigating the mechanism of drought tolerance is crucial for achieving the target of developing drought-tolerant plants. Phenotyping is a cost effective approach which can be adopted to evaluate the severity of drought stress in a plant. Next to phenotyping parameters, biochemical parameters such as the study of antioxidant enzyme activity play significant roles in assessing the extent of drought stress caused injury in a plant. Among the antioxidant enzymes, ascorbate peroxidase is an enzyme which plays a crucial role in drought tolerance in plants. It has been well established that the activity of this enzyme increases under drought stress. Here, we present a simple and reproducible protocol to investigate the response of tobacco plants towards drought stress through measurement of phenotypic parameters and antioxidant enzyme activity. Though, these experiments have been conducted with tobacco plants, this protocol could be adopted for other crop species.

Keywords: Drought (干旱), Tolerance (容忍), Antioxidant (抗氧化剂), Ascorbate peroxidase (抗坏血酸过氧化物酶), Phenotyping (表型)

Materials and Reagents

  1. Pipette (BrandTech Scientific, Transferpette, catalog numbers:  704770 , 704778 , 704780 )
  2. Centrifuge tube (1.5 ml) (Corning, Axygen®, catalog number: MCT-150-C-S )
  3. Tobacco seed samples under study: Nicotiana tabacum L. cv. Petit Havana, wild type and transgenic lines
    Note: The complete open reading frame of histone-gene binding protein of rice (OsHBP1b) was cloned at BglII and SpeI sites in the plant expression vector (pCAMBIA1304) and used for transformation of Agrobacterium strain (LBA 4404). Fifteen days old tobacco seedlings were used for transformation through tissue culture method. The putative transgenic T0 plants were transferred to the greenhouse for the purpose of seeds multiplication. The seeds of T2 transgenic lines were used for experimental purpose.
  4. Distilled H2O (IndiaMART InterMESH Ltd, Mars Scientific Instruments Co., catalog number: BASIC/pH & XL )
  5. Agro peat (Agro peat super) (AswiniAgrotech)
  6. Vermiculite (Manidharma Biotech Private Limited)
  7. Sucrose (Sigma-Aldrich, catalog number: S0389 )
  8. Stress reagent (Polyethylene glycol 6000 or PEG 6000) (Sigma-Aldrich, catalog number: 81260 )
  9. Ascorbate (Sigma-Aldrich, catalog number: A4034 )
  10. Liquid Nitrogen (Advanced Instrumentation Research Facility, Jawaharlal Nehru University, New Delhi)
  11. Hydrogen peroxide (H2O2) (Sigma-Aldrich, catalog number: 216763 )
  12. Bovine serum albumin (Sigma-Aldrich, catalog number: A2153 )
  13. Murashige & Skoog (MS) with vitamins (Caisson Laboratories, catalog number: MSP09-1lt )
  14. K2HPO4 (Sigma-Aldrich, catalog number: 1551128 )
  15. KH2PO4 (Sigma-Aldrich, catalog number: 1551139 )
  16. Coomassie Brilliant Blue G-250-100 mg (Sigma-Aldrich, catalog number: 27815 )
  17. Methanol (100%) (Sigma-Aldrich, catalog number: 34860 )
  18. Phosphoric acid (85%) (Sigma-Aldrich, catalog number: W290017 )
  19. Phosphate buffer (see Recipes)
  20. Bradford reagent (see Recipes)
  21. MS media (see Recipes)


  1. Plastic pots (12 cm diameter and 13 cm depth) (Garden Aids, India)
  2. Forceps (ACE Surgical Co, catalog number: 10)
  3. Growth chamber (Daihan LabTech India Pvt. Ltd., model: LGC-S201 )
  4. Oven (Hicon India)
  5. pH meter (Control Dynamic Instrumentation Pvt. Ltd., model: APX175E )
    Note: Currently, it is “CD Hightech Pvt. Ltd., model: APX175E ”.
  6. Plastic ice tray
  7. Liquid nitrogen container (Kailash Gases, Cryocan, model: BA-11 )
  8. Motor and pestle (local supplier, New Delhi)
  9. Cuvette (both plastic and quartz) (Sigma-Aldrich, catalog number: Z276677 )
  10. Spectrophotometer (Cary 300 UV-Vis)
  11. Image capturing device (SONY CORPORATION OF AMERICA, model: DSC-HX300 )
  12. Weighing machine (Sartorius, model: BSA224S-CW )
  13. Scale (30 cm plastic scale)


  1. GraphPad InStat3 software


  1. Seed germination
    1. Imbibe wild type (WT) and transgenic tobacco seeds in distilled water for overnight.
    2. Allow twenty to twenty five seeds of tobacco to germinate on soil (equal quantity of agro peat and vermiculite should be mixed to prepare the soil) filled pots inside a growth chamber at 28 ± 1 °C with PPFD of 100 μmol/m2/s, a relative humidity of 75-80%, and a photoperiod of 12/12 h light/dark. Supplement 50 ml of MS media every day in the soil-filed pots to provide nutrition to the plants.
    3. Transfer two-week-old seedlings individually to separate soil-filled pots.
    4. Allow the WT and transgenic plants to grow up to 25 days for drought tolerance assay at pot level (Lakra et al., 2015).

  2. Drought treatment and recovery
    1. For drought treatment, prepare 5% PEG solution in MS media and supplement 100 ml of this solution to the pots individually, on a regular basis (once a day), for 5- or 10- days (Kumar et al., 2012; Lakra et al., 2015).
      Note: Polyethylene glycol or PEG is a flexible, water-soluble polymer, it can be used to create high osmotic pressures. These properties make PEG one of the most useful molecules for applying osmotic pressure in plants to induce water deficit stress.
    2. After 5 or 10 days of drought treatment, allow the plants to recover by supplementing 50 ml of MS media for 7 days and subsequently, carry out drought tolerance assay.

  3. Assessment of drought stress response after recovery
    Evaluation of drought stress in WT and transgenic tobacco can be done by analysis of phenotypic and biochemical parameters.

  4. Phenotypic assessment
    1. As phenotypic parameters, number of yellow or green leaf, root length, shoot length, shoot fresh weight, root fresh weight, shoot dry weight and root dry weight of WT and transgenic tobacco plants can be considered (Lakra et al., 2015). As representative data, phenotype of WT and transgenic tobacco plants after drought stress has been shown here (Figure 1A).
    2. Measure the shoot and root lengths with the help of a scale.
    3. For shoot and root dry weight, cut shoot and root samples from the plant and dry them in an oven for 72 h. Subsequently, measure the dry weight of the samples through a weighing machine.
      Note: Use at least three individual lines from each type of plants for each experimental analysis. Analyse data for variance (ANOVA) by GraphPad InStat3 software.

  5. Biochemical assessment
    1. As biochemical parameter, ascorbate peroxidase (APX) activity can be measured through spectrophotometric method. As representative data, comparison of APX activity in WT and transgenic tobacco before and after drought stress has been shown (Figure 1B-C).
      Note: Under control (before drought stress) conditions no significant difference in APX activity was observed between WT and transgenic plants.
    2. For this purpose, weigh 100 mg of WT and transgenic tobacco leaf tissue and grind these separately using chilled motors and pestles (Figure 2A).
      Note: One may take young or old leaves, but, consistency should be maintained to take same aged leaves from each sample type.
    3. Supplement liquid nitrogen for fine crushing of tissue.
    4. Add 1 ml of potassium phosphate buffer (prepare fresh and keep at room temperature) immediately and then add 0.2 mM of PMSF to avoid protein degradation.
    5. Then transfer the homogenate (Figure 2B) to microcentrifuge tubes (Figure 2C) and centrifuge at 10,000 x g for 15 min. Maintain the temperature of the centrifuge machine at 4 °C.
    6. After centrifugation, the pellet and the clear supernatant will be visible (Figure 2D). Subsequently, transfer the supernatant to fresh microcentrifuge tubes (Figure 2E) which will serve as crude protein/enzyme extract and will be used for APX activity assay.
    7. Measure the concentration of crude protein by using Bradford method (Bradford, 1976). Use BSA standard curve for protein quantification.
      Note: Previously prepared standard curve can be used here.
    8. For APX activity assay, use double beam spectrophotometer and quartz cuvette (Figure 3A and B).
    9. Prepare 1 ml of reaction mixture (sample) which will contain 50 mM K-PO4 buffer (pH 7.0), 10 µg crude enzyme extract and 0.35 mM ascorbate. Initiate the reaction by adding 5 µl of 10 mM H2O2.
      Note: H2O2 and ascorbate should be prepared in 50 mM K-PO4 buffer to maintain the buffer molarity.
      Reaction formula: The reaction initiates immediately upon addition of H2O2 where H2O2-dependent oxidation of ascorbate starts. The gradual decrease in absorbance can be visualized on the computer screen after initiation of the reaction.
    10. Use the reaction mixture without H2O2 as reference.
    11. Follow H2O2-dependent oxidation of ascorbate spectrophotometrically by recording the decrease in absorbance at 290 nm (€ = 2.8 mM-1 cm-1).
      Note: The reaction time is 120 sec and the reaction temperature is 20 °C.
    12. Recollect that Beer's law is expressed as A = €CL where, ‘A’ is absorbance, ‘€’ is absorption coefficient (a constant that reflects the efficiency or the extent of absorption at selected wavelengths), ‘C’ is concentration of the substance and ‘L’ is the path length or the thickness of the cuvette. To find the concentration for a solution, you will first need to find the slope of the best-fit line. From the slope of the best-fit line together with the absorbance, you can now calculate the concentration for that solution (i.e., Concentration = Absorbance/Slope)
    13. Here, slope value of absorbance in 290 nm (Δ OD s-1) from first 0.6 min is considered for rate calculation (Figure 4).
      Note: The slope of the best-fit line in this case is actually the product of the molar absorptivity constant and the path length (1.00 cm).Cary 300 UV-Vis instrument has the option to show the slope value of the graph.
    14. The activity of APX can be represented by the unit ‘µmol/min/mg protein’ or ‘µmol/min/g protein’.

      Figure 1. Plant Morphology and APX activity of WT and transgenic tobacco leaves following drought stress treatment and subsequent recovery. (A) Shows yellow and green leaves in WT and transgenic tobacco after drought stress. (B) APX activity in WT and transgenic tobacco leaves before drought stress treatment. (C) APX activity in WT and transgenic tobacco leaves after drought stress treatment.

      Figure 2. Protein extraction from tobacco leaves for measuring APX activity. (A) Set up for tissue crushing. Red, yellow, black and blue arrows show liquid N2 container, motor, pestle and ice container respectively. (B) Tissue homogenate after crushing. (C) Tissue homogenate after transferring to microcentrifuge tube. (D) Supernatant (Red arrow) and Pellet (Blue arrow) after centrifugation. (E) Supernatant containing the crude protein/enzyme after transferring to a fresh microcentrifuge tube.

      Figure 3. View of double beam spectrophotometer and cuvette. (A) Double beam UV-Vis spectrophotometer. Red arrow shows cuvette holder for reference and blue arrow shows cuvette holder for sample. (B) Quartz cuvettes used to read reference and sample absorbance.

      Figure 4. APX activity graph showing decline in enzyme activity due to depletion of H2O2 in unit time. Black line indicates the slope and blue arrow indicates the slope value.


Precautions to be followed:

  1. Proper maintenance of nutrient medium is required for plant growth in pots.
  2. Light, temperature and humidity should be maintained properly.
  3. If algal growth is observed on the soil surface, it can be removed by removing the algal layer along with the thin upper layer of soil with the help of a spatula. If it appears that soil surface is reducing after removing the algal layer along with a layer of soil, additional soil can be added to the pot carefully without harming the plant.
  4. Cuvette should be cleaned properly before use.


  1. Phosphate buffer
    Make 10 ml of 50 mM K2HPO4
    Make 10 ml of 50 mM KH2PO4
    Place 50mM of K2HPO4 under pH meter
    Add required amount of KH2PO4 to get 7.0
  2. Bradford reagent
    Take Coomassie Brilliant Blue G-250 100 mg
    Add 47 ml methanol (100%) to it
    Add 100 ml phosphoric acid (85%) to it
    Add required distilled water to get final volume of 1 L
  3. MS media
    Weigh 4.4 mg of readily available MS powder
    Dissolve 4.4 mg of MS powder in 1 L of distilled water
    Add 30 g of sucrose to 1 L of MS solution
    Adjust pH 5.8


Authors are thankful to Department of Science and Technology (Indo-Sri Lanka Program) and University Grants Commission, Govt. of India for the financial support. Authors acknowledge previous work published by Lakra et al. (2015), from which this protocol is adopted.


  1. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.
  2. Kumar, G., Kushwaha, H. R., Panjabi-Sabharwal, V., Kumari, S., Joshi, R., Karan, R., Mittal, S., Pareek, S. L. and Pareek, A. (2012). Clustered metallothionein genes are co-regulated in rice and ectopic expression of OsMT1e-P confers multiple abiotic stress tolerance in tobacco via ROS scavenging. BMC Plant Biol 12: 107.
  3. Lakra, N., Nutan, K. K., Das, P., Anwar, K., Singla-Pareek, S. L. and Pareek, A. (2015). A nuclear-localized histone-gene binding protein from rice (OsHBP1b) functions in salinity and drought stress tolerance by maintaining chlorophyll content and improving the antioxidant machinery. J Plant Physiol 176: 36-46.



关键字:干旱, 容忍, 抗氧化剂, 抗坏血酸过氧化物酶, 表型


  1. 移液管(BrandTech Scientific,Transferpette,目录号:704770,704770,704778,704780)
  2. 离心管(1.5ml)(Corning,Axygen ,目录号:MCT-150-C-S)
  3. 所研究的烟草种子样品:烟草(Nicotiana tabacum)L.cv。 Petit Havana,野生型和转基因系
    注意:将水稻组织蛋白基因结合蛋白(OsHBP1b)的完全开放阅读框架克隆到植物表达载体(pCAMBIA1304)中的BglII和SpeI位点,并用于农杆菌菌株(LBA 4404)的转化。使用15天龄的烟草幼苗通过组织培养方法进行转化。为了种子繁殖的目的,将推定的转基因T 0植物转移到温室中。将T sub2转基因株系的种子用于实验目的。
  4. 蒸馏H 2 O(IndiaMART InterMESH Ltd,Mars Scientific Instruments Co.,目录号:BASIC/pH& XL)
  5. 农业泥炭(Agro peat super)(AswiniAgrotech)
  6. 蛭石(Manidharma Biotech Private Limited)
  7. 蔗糖(Sigma-Aldrich,目录号:SO389)
  8. 应力试剂(聚乙二醇6000或PEG 6000)(Sigma-Aldrich,目录号:81260)
  9. 抗坏血酸盐(Sigma-Aldrich,目录号:A4034)
  10. 液氮(Advanced Instrumentation Research Facility,Jawaharlal Nehru University,New Delhi)
  11. 过氧化氢(H 2 O 2)(Sigma-Aldrich,目录号:216763)
  12. 牛血清白蛋白(Sigma-Aldrich,目录号:A2153)
  13. Murashige&含维生素的Skoog(MS)(Caisson Laboratories,目录号:MSP09-11t)
  14. (Sigma-Aldrich,目录号:1551128)。
  15. KH sub 2 PO 4(Sigma-Aldrich,目录号:1551139)
  16. 考马斯亮蓝G-250-100mg(Sigma-Aldrich,目录号:27815)
  17. 甲醇(100%)(Sigma-Aldrich,目录号:34860)
  18. 磷酸(85%)(Sigma-Aldrich,目录号:W290017)
  19. 磷酸盐缓冲液(参见配方)
  20. Bradford试剂(见配方)
  21. MS介质(参见配方)


  1. 塑料盆(直径12厘米,深13厘米)(印度的Garden Aids)
  2. 镊子(ACE Surgical Co,目录号:10)
  3. 生长室(Daihan LabTech India Pvt.Ltd。,型号:LGC-S201)
  4. 烤箱(Hicon India)
  5. pH计(Control Dynamic Instrumentation Pvt。Ltd.,型号:APX175E) 注意:目前,它是"CD Hightech Pvt。 Ltd.,型号:APX175E"。
  6. 塑料冰盘
  7. 液氮容器(Kailash Gases,Cryocan,型号:BA-11)
  8. 汽车和杵(本地供应商,新德里)
  9. 比色杯(塑料和石英)(Sigma-Aldrich,目录号:Z276677)
  10. 分光光度计(Cary 300 UV-Vis)
  12. 称重机(Sartorius,型号:BSA224S-CW)
  13. 刻度(30厘米塑料刻度)


  1. GraphPad InStat3软件


  1. 种子发芽
    1. 将Imbibe野生型(WT)和转基因烟草种子在蒸馏水中过夜
    2. 允许二十二十五颗烟草种子在土壤上发芽 (等量的农业泥炭和蛭石应混合准备 土壤)填充的盆在生长室内在28±1℃,PPFD为 100μmol/m 2/s,相对湿度为75-80%,光周期为12/12 ?h光/暗。在土壤中每天补充50ml的MS培养基 盆为植物提供营养
    3. 将两周龄的幼苗单独转移到分离的充满土壤的盆中。
    4. 允许WT和转基因植物生长达25天用于罐水平的耐旱测定(Lakra等人,2015)。

  2. 抗旱治疗和恢复
    1. 对于干旱处理,在MS培养基中制备5%PEG溶液, 补充100毫升这种溶液到花盆个别,在 定期(每天一次),5或10天(Kumar等人,2012; Lakra ,2015)。
      注意:聚乙二醇或PEG是a 柔性的水溶性聚合物,它可以用于创造高渗透性 压力。这些性质使PEG成为最有用的分子之一 用于在植物中施加渗透压以诱导水分缺乏应激。
    2. 在干旱处理5或10天后,允许植物恢复 ?通过补充50ml的MS培养基7天,随后,携带 耐旱测定。

  3. 恢复后的干旱应激反应评估

  4. 表型评估
    1. 作为表型参数,黄或绿叶数,根长, 苗长,苗鲜重,根鲜重,苗干重 和根干重可以是WT和转基因烟草植物 (Lakra等人,2015)。作为代表性数据,WT的表型 ?和干旱胁迫后的转基因烟草植物 (图1A)。
    2. 借助标尺测量枝条和根长。
    3. 对于苗和根干重,从茎中切下嫩枝和根样品 植物并在烘箱中干燥72小时。随后,测量干燥 通过称重机的样品重量 注意:至少使用 ?每个实验的每种类型的植物的三个单独的系 分析。通过GraphPad InStat3分析方差数据(ANOVA) 软件。

  5. 生化评估
    1. 作为生化参数,抗坏血酸 过氧化物酶(APX)活性可以通过分光光度法测量 方法。作为代表性数据,比较APX活性在WT和 已经显示干旱胁迫前后的转基因烟草(图1B-C) 注意:在控制下(干旱胁迫前)条件无显着性 在WT和转基因之间观察到APX活性的差异 植物。
    2. 为此目的,称重100mg的WT和转基因 烟叶组织,并使用冷冻马达分别研磨 杵(图2A) 注意:可以采取年轻或老的叶子,但是,应保持一致性,以从每个样品类型采集相同的老年叶。
    3. 补充液氮以细碎组织。
    4. 加入1毫升磷酸钾缓冲液(准备新鲜,保持在 室温),然后加入0.2mM PMSF以避免 蛋白质降解
    5. 然后转移匀浆(图2B) 微量离心管(图2C),并在10,000×g离心15分钟 min。保持离心机温度在4°C。
    6. 离心后,沉淀和澄清的上清液 可见(图2D)。随后,将上清液转移至新鲜 微离心管(图2E),其将用作粗制的 蛋白质/酶提取物,并将用于APX活性测定
    7.  使用Bradford法测量粗蛋白的浓度 (Bradford,1976)。使用BSA标准曲线进行蛋白质定量 注意:此处可以使用以前制作的标准曲线。
    8. 对于APX活性测定,使用双光束分光光度计和石英比色杯(图3A和B)
    9. 制备1ml含有50mM的反应混合物(样品) K-PO 4缓冲液(pH 7.0),10μg粗酶提取物和0.35mM抗坏血酸盐。 ?通过加入5μl10mM H 2 O 2 sub启动反应。
      注意:H >和抗坏血酸应在50mM K-PO 4 缓冲液中制备,以维持缓冲液摩尔浓度。 反应式:反应在加入时立即开始 em> 2 - 依赖氧化开始。渐进 在计算机屏幕上可以看到吸光度的降低 启动反应。
    10. 使用无H 2 O 2子分子的反应混合物作为参考
    11. 按照抗坏血酸分光光度法的H 2 O 2依赖性氧化 ?通过记录290nm处的吸光度的降低(€= 2.8mM -1 cm -1 )。 注意:反应时间为120秒,反应温度为20℃。
    12. 回顾啤酒定律表示为A =€CL其中,'A'是 吸光度,是吸收系数(反映吸光度的常数 效率或在选定波长处的吸收程度),"C"是 物质的浓度,"L"是路径长度或 比色杯的厚度。要找到解决方案的浓度,你 将首先需要找到最佳拟合线的斜率。从斜坡 的最佳拟合线与吸光度一起,您现在可以计算 ?该溶液的浓度(即浓度= 吸光度/斜率)
    13. 这里,考虑从开始0.6分钟起290nm处的吸光度的斜率值(ΔODs-1)用于速率计算(图4)。
      注意:在这种情况下,最佳拟合线的斜率实际上是 摩尔吸光系数常数和路径长度的乘积(1.00 cm).Cary 300 UV-Vis仪器具有显示斜率值的选项 ?图表。
    14. APX的活性可以由单位"μmol/min/mg蛋白质"或"μmol/min/g蛋白质"表示。

      图 ?WT和转基因烟草的植物形态和APX活性 干旱胁迫处理后随后恢复。(A) 在WT和转基因烟草中显示黄色和绿色叶片在干旱以后 ?强调。 (B)WT和转基因烟草叶中的APX活性 干旱胁迫处理。 (C)WT和转基因烟草中的APX活性 干旱胁迫处理后的叶子

      图2.蛋白质提取 从用于测量APX活性的烟叶。(A)设置组织 破碎。红色,黄色,黑色和蓝色箭头显示液体N 2容器, 马达,杵和冰容器。 (B)组织匀浆 破碎后。 (C)转移至微量后的组织匀浆 离心管。 (D)上清液(红色箭头)和颗粒(蓝色箭头) 离心后。 (E)含有粗产物的上清液 蛋白/酶转移到新鲜的微量离心管中。

      图3.双光束分光光度计和比色皿的视图。(A)双 ?光束UV-Vis分光光度计。红色箭头显示比色杯持有人 参考和蓝色箭头显示样品的比色杯架。 (B)石英 用于读取参考和样品吸光度的比色杯

      图4. APX ?活性图,其显示由于消耗的酶活性的下降 。黑线表示斜率和蓝色箭头 表示斜率值。



  1. 在花盆中植物生长需要适当维持营养培养基
  2. 光,温度和湿度应保持正常
  3. 如果在土壤表面观察到藻类生长,可以将其除去 通过去除藻类层与薄的上层土壤 ?一个铲的帮助。如果看起来土壤表面正在还原 在除去藻类层与一层土壤之后,另外 土壤可以小心地添加到锅中而不伤害植物
  4. 在使用前应正确清洁比色杯。


  1. 磷酸盐缓冲液
    制备10ml 50mM K 2 HPO 4
    制备10ml 50mM KH 2 PO 4/dub 在pH计
    下放置50mM K 2 HPO 4 添加所需量的KH 2 PO 4 即可获得7.0
  2. Bradford试剂
    以考马斯亮蓝G-250 100 mg
    向其中加入47ml甲醇(100%) 向其中加入100 ml磷酸(85%) 加入所需的蒸馏水,使最终体积为1 L
  3. MS媒体
    调整pH 5.8


作者感谢科学技术部(印度斯里兰卡计划)和大学教育资助委员会。的印度的财政支持。作者承认Lakra等人发表的以前的工作。 (2015),从中采用此协议。


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  2. Kumar,G.,Kushwaha,H. R.,Panjabi-Sabharwal,V.,Kumari,S.,Joshi,R.,Karan,R.,Mittal,S.,Pareek,S.L.和Pareek, 集群金属硫蛋白基因在水稻中共调节,OsMT1e-P的异位表达赋予多种非生物胁迫耐受性在烟草中通过ROS清除。 BMC植物生物学 12:107
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引用:Das, P., Lakra, N., Nutan, K. K., Singla-Pareek, S. L. and Pareek, A. (2015). Pot Level Drought Stress Tolerance Assay in Tobacco through Plant Phenotyping and Antioxidant Assay. Bio-protocol 5(19): e1605. DOI: 10.21769/BioProtoc.1605.