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Leaf Disc Stress Tolerance Assay for Tobacco
烟草叶盘抗逆性分析   

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Abstract

Stress tolerance is a multigenic trait that depends on the coordinated action of several genes. Various physiological parameters, such as plant height and weight, total yield, chlorophyll content, photosynthesis rate, level of reactive oxygen species (ROS) and anti-oxidant activity could be correlated directly with the level of stress tolerance potential of any particular genotype. To evaluate the stress tolerance potential of a plant, leaf disc stress tolerance assay is a very rapid and widely acceptable experiment with minimum instrumentation facilities.

Keywords: Leaf disc(叶盘), Abiotic stress tolerance(非生物胁迫的耐受性), Plant(植物), Tobacco(烟草), Rapid(迅速的)

Materials and Reagents

  1. Young tobacco plants (to be tested)
  2. Distilled water
  3. Acetone (Sigma-Aldrich, catalog number: I10010 )
  4. Stress reagents (such as NaCl, Sigma-Aldrich, catalog number: S7653 ; for salinity stress)
  5. Liquid nitrogen
  6. Extraction buffer (see Recipes)

Equipment

  1. Leaf puncher/ cork borer (Sigma-Aldrich, catalog number: Z165220 )
  2. Petri plates (60 x 15 mm)
  3. Microcentrifuge tube
  4. Tissue paper
  5. Graduated cylinder
  6. Mortar and pestle (Sigma-Aldrich, catalog number: Z112496 )
  7. Glass cuvette (1 ml) (Sigma-Aldrich, catalog number: C8550-1EA )
  8. Electronic balance
  9. Spectrophotometer
  10. Benchtop refrigerated centrifuge (Eppendorf, catalog number: 5427 R )
  11. Vortexer (Sigma-Aldrich, catalog number: Z755613-1EA )

Procedure

  1. Grow tobacco seedlings in earthern pots filled with normal well-drained soil and keep them in the green house for 6 weeks at optimum condition (26 ± 2 °C, 75% relative humidity, and a photoperiod of 16/8 h light and dark).
  2. Select healthy plants of equal height and age for all the lines to be tested for leaf disc stress tolerance assay.
  3. Collect leaves (third to fifth leaf from the top) from each plant and gently wash with distilled water to remove any extraneous material associated with the tissues.
  4. Cut leaves using a leaf puncher or cork borer to obtain leaf discs of similar size (1 cm diameter) and surface area. Avoid taking discs from the major veins of leaf.
  5. Float equal number of leaf discs (7-8) from each line (with abaxial surface down) on sterile distilled water in triplicates as experimental control (Figure 1, 0 day).
  6. Float the same number of leaf discs from each line (with abaxial surface down) on various stress containing solution such 200 mM NaCl for salinity stress (Figure 1, 0 day, lower panel).
  7. The leaf discs floated on particular solutions as well as experimental controls should be kept in the same environment and visualized after every 12 h until a visual difference in their “greenness” is observed.
  8. After observing a clear difference between the leaf discs floated in control solution and stress solution, photographs should be taken (Figure 1, after 3-4 days). Leaf discs can be tested for other stresses, such as oxidative, osmotic and heavy metal stresses using same protocol (Kumar et al., 2012; Singh et al., 2012).
  9. Quantitate this visual observation by extracting the total chlorophyll of the leaf discs as described (Arnon, 1949) and quantify according equation as described (Porra, 2002).
  10. Measure fresh weight (in grams, gm) of the leaf discs from each conditions and lines.
    Note: Remove surface moisture from the discs with tissue paper before measuring weight.
  11. Grind leaf discs to fine powder in a mortar and pestle using liquid nitrogen.
    Add 1 ml of extraction buffer (80% acetone), mix throughly and immediately transfer the extract into a microcentrifuge tube (MCT) in a fume hood. Alternatively, add the tissue powder to a tube containing 1 ml of extraction buffer. Mix properly by vortexing for 1 min and adjust the volume with extraction buffer in all the tubes after mixing.
    Note: Adjust volume of the mixture to 1 ml (if neccessary) with extraction buffer before centrifugation as acetone is volatile.
  12. Centrifuge the suspension at 3,000 x g for 5 min under cold condition (4 °C).
  13. Transfer the supernatant from each tube to new MCT tube and use this extract for measuring absorbance.
  14. Dilute the supernatant five times by adding 200 µl of supernatant in 800 µl of extraction buffer. Absorbance of this mixture is measured in triplicates at 664 and 647 nm wavelength by taking extraction buffer (80% acetone) as blank using a spectrophotometer and 1 ml glass cuvette.
  15. Calculate the content of Chlorophyll A, Chlorophyll B and total Chlorophyll using the following equations as described by Porra (2002).
    Chlorophyll A= [{(12.25xA664)-(2.55xA647)}xDF]/TW µg/ml/gm fresh weight
    Chlorophyll B= [{(20.31xA647)-(4.91xA664)}xDF]/TW µg/ml/gm fresh weight
    Total Chlorophyll= (Chlorophyll A + Chlorophyll B) µg/ml/gm fresh weight
    Here, DF (Dilution factor)= (Total volume of solution for absorbance (1 ml)/volume of supernatant taken for dilution (200 µl)
    TW (Tissue Weight) = Weight of the tissue taken (in grams)
  16. Leaf discs from tolerant cultivars would be able to maintain their total chlorophyll level with minimum loss over time as compared to their control (0 day) value. However, sensitive leaves will show drastic reduction of their chlorophyll level (more stress induced chlorophyll bleaching) in response to stress.

Representative data



Figure 1. Response of tobacco leaf discs in presence of 200 mM NaCl for 4 days. Leaf discs are floated onto water to serve as experimental control and 200 mM NaCl for salinity stress. There is no change in the ‘greenness’ of discs under control condition, while discs are bleached and turned into yellow in the presence of 200 mM NaCl.

Notes

  1. The size and age of the leaf is an important factor for the assay. So make sure that the leaves are collected from the same position (third to fifth leaf from the top) and almost similar in size to avoid initial variance among different lines.
  2. All solutions should be made in sterile water and work under clean bench to avoid any type of bacterial or fungal infection.
  3. As the response could vary based on the plant species, age of plant, type and degree of imposed stress, experiment should be monitored regularly for at least seven days (not strictly 4 days). If there is no change within seven days time period, fresh experiment should be set up with increased degree of stress.
  4. As acetone could degrade disposable plastic cuvettes, glass cuvette should be used.

Recipes

  1. Extraction buffer (100 ml)
    Acetone: 80 ml
    H2O: 20 ml
    Prepare in a tightly sealed glass brown bottle and prepare fresh every time

Acknowledgments

Authors thank International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India for funding research.

References

  1. Arnon, D. I. (1949). Copper enzymes in isolated chloroplasts. polyphenoloxidase in Beta Vulgaris. Plant Physiol 24(1): 1-15.
  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. Porra, R. J. (2002). The chequered history of the development and use of simultaneous equations for the accurate determination of chlorophylls a and b. Photosynth Res 73(1-3): 149-156.
  4. Singh, A. K., Kumar, R., Pareek, A., Sopory, S. K. and Singla-Pareek, S. L. (2012). Overexpression of rice CBS domain containing protein improves salinity, oxidative, and heavy metal tolerance in transgenic tobacco. Mol Biotechnol 52(3): 205-216.

简介

胁迫耐受性是一种多基因性状,其取决于几个基因的协同作用。 各种生理参数,例如植物高度和重量,总产量,叶绿素含量,光合速率,活性氧(ROS)水平和抗氧化活性可以与任何特定基因型的胁迫耐受性水平的水平直接相关。 为了评价植物的胁迫耐受性潜力,叶盘胁迫耐受性测定是使用最小仪器设备的非常快速和广泛可接受的实验。

关键字:叶盘, 非生物胁迫的耐受性, 植物, 烟草, 迅速的

材料和试剂

  1. 年轻的烟草植物(待测)
  2. 蒸馏水
  3. 丙酮(Sigma-Aldrich,目录号:I10010)
  4. 应力试剂(如NaCl,Sigma-Aldrich,目录号:S7653;盐度胁迫)
  5. 液氮
  6. 提取缓冲液(参见配方)

设备

  1. 叶冲床/软木钻(Sigma-Aldrich,目录号:Z165220)
  2. 培养皿(60×15mm)
  3. 微量离心管
  4. 纸巾
  5. 量筒
  6. 砂浆和杵(Sigma-Aldrich,目录号:Z112496)
  7. 玻璃比色杯(1ml)(Sigma-Aldrich,目录号:C8550-1EA)
  8. 电子天平
  9. 分光光度计
  10. 台式冷冻离心机(Eppendorf,目录号:5427R)
  11. Vortexer(Sigma-Aldrich,目录号:Z755613-1EA)

程序

  1. 在充满正常排水良好的土壤的土盆中种植烟草幼苗,并在最佳条件(26±2℃,75%相对湿度,16/8h光照和黑暗的光周期)下将它们保持在温室中6周, 。
  2. 选择相同高度和年龄的健康植物用于所有要测试叶片胁迫耐受性测定的品系
  3. 从每个植物收集叶子(从顶部第三至第五叶),轻轻地用蒸馏水洗涤以去除与组织相关的任何外来物质。
  4. 使用叶片打孔器或软木钻孔器切割叶以获得相似大小(1cm直径)和表面积的叶盘。避免从叶子的主静脉采集碟片。
  5. 将无菌蒸馏水上的每一行(具有背面表面向下)的叶盘(7-8)浮动成等份作为实验对照(图1,0天)。
  6. 在各种应力​​含有溶液(例如200mM NaCl)上,对于盐度胁迫(图1,0天,下图),从每条线(具有背面表面向下)漂浮相同数目的叶盘。
  7. 浮在特定溶液上的叶盘和实验对照应保持在相同的环境中,并且每12小时后可视化,直到观察到它们的"绿色"的视觉差异。
  8. 观察浮在对照溶液中的叶盘和应激溶液之间的明显差异,应拍摄照片(图1,3-4天后)。可以使用相同的方案(Kumar等人,2012; Singh等人,2012)测试叶盘的其它应力,例如氧化,渗透和重金属应力。 。
  9. 通过如(Arnon,1949)所述提取叶圆片的总叶绿素并通过如所述的方法(Porra,2002)定量来定量该目视观察。
  10. 测量来自每个条件和品系的叶圆片的鲜重(克,gm)。
    注意:在测量体重之前,用棉纸从盘上除去表面水分。
  11. 用研钵将叶片磨成细粉,用液氮研磨杵 加入1ml提取缓冲液(80%丙酮),充分混合,立即将提取物转移到通风橱中的微量离心管(MCT)中。或者,将组织粉末添加到含有1ml提取缓冲液的管中。通过涡旋混合适当地混合1分钟,并在混合后在所有管中用提取缓冲液调节体积。
    注意:在离心前用丙酮挥发,用萃取缓冲液将混合物的体积调节至1ml(如果必要)。
  12. 在寒冷条件(4℃)下将悬浮液以3,000×g离心5分钟。
  13. 将每个管的上清液转移到新的MCT管中,并使用该提取物测量吸光度
  14. 通过在800μl提取缓冲液中加入200μl上清液来稀释上清液五次。通过使用分光光度计和1ml玻璃试管将提取缓冲液(80%丙酮)作为空白,在664和647nm波长下一式三份测量该混合物的吸光度。
  15. 使用Porra(2002)描述的以下等式计算叶绿素A,叶绿素B和总叶绿素的含量。
    叶绿素A = [{(12.25×A664) - (2.55×A647)}×DF]/TWμg/ml/g鲜重
    叶绿素B = [{(20.31×A647) - (4.91×A664)}×DF] /TWμg/ml/g鲜重
    总叶绿素=(叶绿素A +叶绿素B)μg/ml/g鲜重
    这里,DF(稀释因子)=(吸光度溶液总体积(1ml)/稀释用上清液体积(200μl)
    TW(组织重量)=所用组织的重量(克)
  16. 与其对照(0天)值相比,来自耐受品种的叶片将能够保持其总叶绿素水平,同时随时间具有最小损失。然而,敏感的叶片将显示它们的叶绿素水平(更多的应力诱导叶绿素漂白)响应于应激的急剧减少。

代表数据



图1.烟叶叶片在200mM NaCl存在下4天的应答。将叶片浮在水上作为实验对照,200mM NaCl用于盐胁迫。在控制条件下盘的"绿色度"没有变化,而盘在200mM NaCl的存在下漂白并变成黄色。

笔记

  1. 叶的大小和年龄是测定的重要因素。因此,请确保叶子是从相同的位置(从顶部第三到第五叶)收集,并且尺寸几乎相似,以避免不同行之间的初始差异。
  2. 所有溶液应在无菌水中制成,并在清洁工作台下工作,以避免任何类型的细菌或真菌感染
  3. 由于响应可以根据植物物种,植物年龄,施加的胁迫的类型和程度而变化,应定期监测实验至少7天(不严格地为4天)。如果在七天的时间内没有变化,新的实验应该设置有更大的压力
  4. 由于丙酮会降低一次性塑料比色杯,因此应使用玻璃比色杯

食谱

  1. 提取缓冲液(100ml)
    丙酮:80 ml
    H sub 2 O:20ml
    准备在密封的玻璃棕色瓶中,每次准备新鲜

致谢

作者感谢国际遗传工程和生物技术中心(ICGEB),新德里,印度资助研究。

参考文献

  1. Arnon,D.I。(1949)。 分离的叶绿体中的铜酶。多酚氧化酶在植物生理中 24(1):1-15。
  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
  3. Porra,R.J。(2002)。 用于准确测定叶绿素的联立方程的开发和使用的方格历史a 73(1-3):149-156。
  4. Singh,A.K.,Kumar,R.,Pareek,A.,Sopory,S.K.and Singla-Pareek,S.L。(2012)。 含有CBS结构域的蛋白的过表达 盐度,氧化性和重金属耐受性。转基因烟草中的抗氧化性,氧化性和重金属耐受性。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Ghosh, A., Pareek, A. and Singla-Pareek, S. L. (2015). Leaf Disc Stress Tolerance Assay for Tobacco . Bio-protocol 5(7): e1440. DOI: 10.21769/BioProtoc.1440.
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