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Stress tolerance of plants is a complex phenomenon that depends on the inter-related action of several morphological, physiological and biochemical parameters. Although stress affects normal physiological growth of a plant irrespective of its developmental stage, seed germination and seed setting are considered to be the most sensitive two. Therefore, to evaluate the stress tolerance potential of a particular plant species or variety, rate of seed germination in presence of stress is an important agronomic trait. This will provide a clear indication about the stress tolerance potential with minimum instrumentation facilities. The method is very simple, effective and highly reproducible that would provide quick and reliable results to the researchers.

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Stress Tolerance Assay at the Seed Germination Stage for Tobacco
烟草种子萌芽阶段的抗逆性分析

植物科学 > 植物生理学 > 植物生长
作者: Ajit Ghosh
Ajit GhoshAffiliation: Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India
Bio-protocol author page: a2116
Ashwani Pareek
Ashwani PareekAffiliation: Stress Physiology and Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India
Bio-protocol author page: a2117
 and Sneh L Singla-Pareek
Sneh L Singla-PareekAffiliation: Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India
For correspondence: sneh@icgeb.res.in
Bio-protocol author page: a2118
Vol 5, Iss 7, 4/5/2015, 2771 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1441

[Abstract] Stress tolerance of plants is a complex phenomenon that depends on the inter-related action of several morphological, physiological and biochemical parameters. Although stress affects normal physiological growth of a plant irrespective of its developmental stage, seed germination and seed setting are considered to be the most sensitive two. Therefore, to evaluate the stress tolerance potential of a particular plant species or variety, rate of seed germination in presence of stress is an important agronomic trait. This will provide a clear indication about the stress tolerance potential with minimum instrumentation facilities. The method is very simple, effective and highly reproducible that would provide quick and reliable results to the researchers.
Keywords: Seed germination(种子萌发), Stress tolerance(胁迫的耐受性), Tobacco(烟草), Simple(简单的), Plant(植物)

[Abstract]

Materials and Reagents

  1. Tobacco seeds (Nicotiana tabacum L. cv. Petit Havana, wild type and transgenic)
  2. Sterile distilled H2O
  3. Ethanol
  4. Para film
  5. Murashige and Skoog medium salt (Caisson Laboratories, catalog number: MSP09-1lt )
  6. Sucrose (Sigma-Aldrich, catalog number: S0389 )
  7. Agar (Plant tissue culture grade, Sigma-Aldrich, catalog number: A7921 )
  8. Stress reagents (such as NaCl for salinity stress, and H2O2 for oxidative stress)
  9. Germination media (see Recipes)

Equipment

  1. Microcentrifuge tubes
  2. Growth chamber
  3. Laminar flow cabinet
  4. Petri plates (100 x 20 mm)
  5. Whatman filter paper
  6. Autoclaved forceps
  7. Electronic balance

Procedure

  1. Preparation of media
    Prepare half strength MS medium as described in the recipe. There is no need to add anything externally to the medium for control plates, and autoclave directly. To mimic stress condition during germination, add stress inducing agents such as 200 mM NaCl for salinity stress to the medium before autoclaving. But for oxidative stress, add 5 mM H2O2 to the medium after autoclaving. After autoclaving, allow the medium to cool down to around 40 to 50 °C temperature and then pour into sterilized petridishes (100 x 20 mm). After that, let the medium to solidify and seal the petridishes properly with para film if not to be used immediately.
  2. Seeds sterilization
    As the seeds could be contaminated by fungi or bacteria during maturation or harvesting or storage, they need to be surface sterilized properly before germinating in a nutrient rich medium. The steps of seed sterilization are described below:
    1. Put certain amount of seeds (around 200) into a 1.5 ml Eppendorf tube.
    2. Add 1 ml of 70% ethanol.
    3. Briefly shake for less than 1 min (strictly not more than that).
    4. Pour off ethanol.
    5. Rinse with sterile distilled water for 3 to 5 times.
  3. Germination of seeds
    Keep the plates with media open in a laminar flow cabinet for some time to remove the surface moisture (if any). Then place the sterile seeds (around 20) on the media in three separate plates (for biological replicates) with autoclaved sharp end forceps. After putting seeds, seal the plates properly with para film and keep in the growth chamber under dark (Figure 1b). The plates should be kept in dark for 3 to 4 days and then keep under normal long day condition (16/8 h light/dark cycle) at 26 ± 2 °C for 15 days. Monitor the growth of seedlings every day (Figure 1c-d).
  4. Rate of seed germination calculation
    Allow the seedlings to germinate and grow for certain period (10 to 15 days) under both control and stress condition. Count the number of germinated seedlings for each line in all the three biological replicates of both control and stress condition. Rate of seed germination for each line was calculated using the following equation.
    Germination rate (%) = (Average number of seeds germinated under stress condition/ Average number of seeds germinated under control condition) x 100%
  5. Stress tolerance index (STI) calculation
    Measure the fresh weight of 10 seedlings in triplicates for each line under both control and stress conditions and calculate the stress tolerance index (STI) using the following equation (Mustafiz et al., 2014).
    STI (%) = (Average fresh weight of 10 stressed seedlings/Average fresh weight of 10 control seedlings) x 100%

Representative data



Figure 1. Seed germination assay for stress tolerance. (A) Pictorial depiction of the position of various type of tobacco seeds WT (wild type; non-transgenic) and three different transgenic lines; line-1, line-2 and line-3) used in the study. (B) Inoculation of seeds in the MS medium alone (experimental control) or MS medium supplemented with 200 mM NaCl for salinity stress. Germination of seeds after 15 days under control condition (C) and stress condition (D). Transgenic seeds showed higher germination rate and better seedling growth as compared to the WT under stress condition.

Notes

  1. Seed sterilization with 70% ethanol for more than one minute will lead to complete inhibition of germination. So do not exceed the time.
  2. As germination of seeds may vary based on plant species, seed storage conditions, type and degree of imposed stress, experiment should be monitored regularly (not strictly 15 days) for distinguishable results.

Recipes

  1. Germination media (1 L)
    1/2 Murashige and Skoog medium salt (2.215 gm)
    30 g sucrose
    Adjust pH to 5.7 with 1 M NaOH
    Add 0.8% agar
    Then autoclave for 15 min

Acknowledgments

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

References

  1. Mustafiz, A., Ghosh, A., Tripathi, A. K., Kaur, C., Ganguly, A. K., Bhavesh, N. S., Tripathi, J. K., Pareek, A., Sopory, S. K. and Singla-Pareek, S. L. (2014). A unique Ni2+ -dependent and methylglyoxal-inducible rice glyoxalase I possesses a single active site and functions in abiotic stress response. Plant J 78(6): 951-963.

材料和试剂

  1. 烟草种子( Nicotiana tabacum L.cv。Petit Havana,野生型和转基因)
  2. 无菌蒸馏H 2 O 2 /
  3. 乙醇
  4. 参与影片
  5. Murashige和Skoog中间盐(Caisson Laboratories,目录号:MSP09-11tt)
  6. 蔗糖(Sigma-Aldrich,目录号:SO389)
  7. 琼脂(植物组织培养级,Sigma-Aldrich,目录号:A7921)
  8. 应力试剂(例如NaCl用于盐度胁迫,H 2 O 2用于氧化应激)
  9. 萌发培养基(参见配方)

设备

  1. 微量离心管
  2. 生长室
  3. 层流柜
  4. 培养皿(100×20mm)
  5. Whatman过滤纸
  6. 高压钳
  7. 电子天平

程序

  1. 准备媒体
    按配方中所述制备半强度MS培养基。不需要向控制板的介质外部添加任何东西,并直接高压灭菌。为了模拟萌发期间的胁迫条件,在高压灭菌之前向培养基中加入胁迫诱导剂,例如200mM NaCl用于盐度胁迫。但对于氧化应激,在高压灭菌后向培养基中加入5mM H 2 O 2 O 2。高压灭菌后,使介质冷却至约40至50℃的温度,然后倒入灭菌的培养皿(100×20mm)中。然后,如果不立即使用,使介质固化并用对膜正确密封。
  2. 种子灭菌
    由于种子在成熟或收获或储存期间可能被真菌或细菌污染,所以它们需要在营养丰富的培养基中发芽之前进行适当的表面灭菌。种子灭菌的步骤描述如下:
    1. 将一定量的种子(约200)放入1.5ml的Eppendorf管中
    2. 加入1ml 70%乙醇
    3. 短暂摇动不到1分钟(严格不超过)。
    4. 倒出乙醇。
    5. 用无菌蒸馏水冲洗3〜5次。
  3. 种子发芽
    保持板在介质打开在层流柜一段时间,以消除表面水分(如果有)。 然后将无菌种子(约20)置于三个单独的平板(用于生物学重复)中的介质上,用高压灭菌的尖端镊子。 在种子之后,用对膜适当地密封板并在黑暗下保持在生长室中(图1b)。 将板在黑暗中保持3至4天,然后在正常长日照条件(16/8小时光/黑暗循环)下在26±2℃保持15天。 每天监测幼苗的生长(图1c-d)
  4. 种子发芽率计算
    允许幼苗在对照和胁迫条件下发芽和生长一定时期(10至15天)。在控制和胁迫条件的所有三个生物学重复中,计数每条线的发芽幼苗的数目。每行的种子发芽率使用下式计算 发芽率(%)=(在胁迫条件下发芽的种子的平均数/对照条件下发芽的种子的平均数)×100%
  5. 压力容忍指数(STI)计算
    在对照和胁迫条件下,在每个品系中测量10个幼苗的鲜重(一式三份),并使用以下方程式计算胁迫耐受指数(STI):(br/> STI(%)=(10株胁迫幼苗的平均鲜重/10株对照幼苗的平均鲜重)×100%

代表数据



图1.胁迫耐受性的种子萌发测定(A)各种类型的烟草种子WT(野生型;非转基因)和三种不同的转基因品系的位置的图示; line-1,line-2和line-3)。 (B)在单独的MS培养基(实验对照)或补充有200mM NaCl的MS培养基中接种种子用于盐胁迫。在对照条件(C)和胁迫条件(D)下15天后种子的发芽。与胁迫条件下的WT相比,转基因种子显示出更高的发芽率和更好的幼苗生长。

笔记

  1. 用70%乙醇种子灭菌超过一分钟将导致完全抑制萌发。所以不要超过时间。
  2. 由于种子的发芽可能根据植物种类,种子储存条件,施加的胁迫的类型和程度而不同,应定期(不严格地15天)监测实验以获得可区分的结果。

食谱

  1. 发芽培养基(1L)
    1/2 Murashige和Skoog中盐(2.215gm) 30克蔗糖 用1M NaOH调节pH至5.7 加入0.8%琼脂
    然后高压灭菌15分钟

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. Mustafiz,A.,Ghosh,A.,Tripathi,A.K.,Kaur,C.,Ganguly,A.K.,Bhavesh,N.S.,Tripathi,J.K.,Pareek,A.,Sopory,S.K.and Singla-Pareek, 独特的Ni 2+ 2+依赖性和甲基乙二醛诱导型水稻乙醛酸酶I 具有单一活性位点并在非生物应激反应中起作用。 Plant J 78(6):951-963。
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How to cite this protocol: Ghosh, A., Pareek, A. and Singla-Pareek, S. L. (2015). Stress Tolerance Assay at the Seed Germination Stage for Tobacco. Bio-protocol 5(7): e1441. DOI: 10.21769/BioProtoc.1441; Full Text



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