Salinity and Drought Treatment Assays in Kenaf (Hibiscus cannabinus L.)

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Dec 2015



Salinity and drought are the two main factors that cause fiber yield and quality losses in kenaf. It is reported that salinity and drought can affect more than 10% of arable land and cause a global decline in the average yields of major crops by more than 50%. Therefore, understanding plant tolerance of drought and salinity is of fundamental importance and has become the focus of intensive research. This protocol describes a simple and reproducible protocol to imitate natural salinity and drought stress under soil conditions. Even though the water-culture method is most frequently used for salinity and drought treatments, the soil-culture method in this study was more applicable to studying natural stress conditions.

Keywords: Salinity (盐度), Drought (干旱), Kenaf (Hibiscus cannabinus L.) (Kenaf(红麻))

Materials and Reagents

  1. Sterile Petri dishes (JET, catalog number: TCD010090 )
  2. Sterile filter paper (Sigma-Aldrich, catalog number: Z241091 )
  3. Seeds: kenaf (cv Fuhong 992) [conserved in the Key Laboratory for Genetics, Breeding and Multiple Utilization of Crops of Fujian Agriculture and Forestry University (China)].
  4. Sterile water (sterilized for 20 min at 121 °C/1 atm using an autoclave)
  5. Sterile soil (Fafard® growing mix 1-PV/C-1PV, Sun Gro Horticulture Canada Ltd.) (sterilized for 30 min at 121 °C/1 atm using an autoclave)
  6. Sodium hypochlorite (NaClO) solution (15%) (Sinopharm Chemical Reagent, catalog number: 7681-52-9 )
  7. Potassium nitrate (KNO3) (Sinopharm Chemical Reagent, catalog number: 7757-79-1 )
  8. Ammonium dihydric phosphate (NH4H2PO4) (Sinopharm Chemical Reagent, catalog number: 7722-76-1 )
  9. Ammonium nitrate (NH4NO3) (Sinopharm Chemical Reagent, catalog number: 6484-52-2 )
  10. Calcium nitrate [Ca(NO3)2·4H2O] (Sinopharm Chemical Reagent, catalog number: 13477-34-4 )
  11. Magnesium sulphate (MgSO4·7H2O) (Sinopharm Chemical Reagent, catalog number: 10034-99-8 )
  12. Ethylene diamine-N,N bis (2 hydroxyphenylacetic acid) Ferric sodium complex (Fe-EDDHA) (Sinopharm Chemical Reagent, catalog number: 16455-61-1 )
  13. Boric acid (H3BO3) (Sinopharm Chemical Reagent, catalog number: 10043-35-3 )
  14. Manganese chloride (MnCl2·4H2O) (Sinopharm Chemical Reagent, catalog number: 7773-01-5 )
  15. Zinc sulphate (ZnSO4·7H2O) (Sinopharm Chemical Reagent, catalog number: 7446-20-0 )
  16. Copper sulphate (CuSO4·5H2O) (Sinopharm Chemical Reagent, catalog number: 7758-98-7 )
  17. Sodium molybdate (Na2MoO4·2H2O) (Sinopharm Chemical Reagent, catalog number: 10102-40-6 )
  18. Sodium chloride (NaCl) (Sinopharm Chemical Reagent, catalog number: 7647-14-5 )
  19. PEG6000 (Sigma-Aldrich, catalog number: 81260 )
  20. 5% (v/v) sodium hypochlorite(see Recipes)
  21. Hoagland solution (see Recipes)
  22. 200 mM sodium chloride (NaCl) solution (see Recipes)
  23. 20% (w/v) PEG6000 solution (see Recipes)


  1. Plastic pots (10 cm diameter and 8 cm depth)
  2. Clean bench (Suzhou AirTech , AirTech®, model: SW-CJ-1FD )
  3. Growth chamber (Yihengyiqi Shanghai, Blue pard®, model: MGC-300A )
  4. Autoclave (TOMY, model: SX-500 )
  5. Precision balance (± 0.001)
  6. Timer control


  1. Seed germination and culture
    1. Rinse the seeds under running water for 10 min, and then sterilize the seeds with 5% sodium hypochlorite for 10 min, and subsequently wash the seeds three times with sterile water (Figure 1A).
    2. Place the sterilized seeds in Petri dishes on filter paper saturated with sterile water, and maintain the seeds in complete darkness at 28 °C until germination (about 3 days) (Figure 1B and 1C).
    3. Transfer 3-5 days old seedlings to separate sterilized soil pots.
    4. Maintain the plants in growth chambers at 28 °C for 16 h with light and 26 °C for 8 h without light, 60% relative humidity, ~450 μmol m-2 s-1 light intensity, and supplement 100 ml 1/4 Hoagland solution to soil once a day (Figure 1D).
    5. Allow plants to grow up to 3-10 leaves stage (about 4 weeks) for salinity and drought treatments assays.

  2. Salinity and drought treatments
    1. For salinity treatment, prepare 200 mM sodium chloride solution in Hoagland nutrient medium and supplement 100 ml of this solution to 10 pots individually once a day (Figure 1D).
    2. For drought treatment, prepare 20% (w/v) PEG6000 solution in Hoagland nutrient medium and supplement 100 ml of this solution to 10 pots individually once a day (Figure 1D).
    3. Harvest 3 treated leaves from each plant at different time points (e.g., 6, 12 and 24 h). Untreated samples were collected at the same interval as a control. Three biological replicates were obtained from each treatment (Figure 1E).

      Figure 1. Example of experimental procedure. A. Seeds rinsed under running water; B. Sterilized seeds on saturated filter paper in Petri dishes; C. Germinated seeds; D. Seedlings at cotyledon stage; E. Harvesting leaf samples (about 4 weeks) as pointed by red arrows after salinity and drought treatments.

Data analysis

RNA was isolated and used for selection reliable reference gene (such as ACT, 18S rRNA, EF1α) for Real-Time PCR (RT-qPCR) assay under salinity and drought stress, and this section was stated clearly in our paper (Niu et al., 2015).


  1. Soil is sterilized for at least 30 min at 121 °C/1 atm using an autoclave.
  2. Polyethylene glycol (PEG) is a flexible, water-soluble polymer, and it can be used to create high osmotic pressure. Therefore, it is used for applying osmotic pressure in plants to induce water deficit.
  3. The same aged leaves should be taken from each sample type. Also use at least three individual lines from each treatment for analysis.
  4. The volume of supplement is depended on the size of pots, watering frequency, and plant species. It is recommended that a series of assays should be done for a given plant species before stress treated.


  1. Hoagland solution
    Note: Hoagland solution is based on Hoagland No.2 solution (Hoagland et al., 1950) (Table 1).

    Table 1. Hoagland solution composition

  2. 5% (v/v) sodium hypochlorite
    Add 33 ml 15% NaClO solution in the distilled water to get a final volume of 100 ml
  3. 20% (w/v) PEG6000 solution
    Dissolve 20 g PEG6000 in distilled water
    Dilute with distilled water to get a final volume of 100 ml, and mix
  4. 200 mM sodium chloride solution
    Dissolve 11.7 g NaCl in 1 L distilled water


This work was supported by grants from the 948 project of the Agricultural Department of China (2013-Z70), the National bast fiber germplasm resources project of China (K47NI201A) and National bast fiber research system of China (CARS-19-E06). This protocol was adapted from our recent publications (Niu et al., 2015).


  1. Hoagland, D. R., Arnon, D. I. (1950). The water-culture method for growing plants without soil. California Agriculture Experiment Station 347: 34.
  2. Niu, X., Qi, J., Chen, M., Zhang, G., Tao, A., Fang, P., Xu, J., Onyedinma, S. A. and Su, J. (2015). Reference genes selection for transcript normalization in kenaf (Hibiscus cannabinus L.) under salinity and drought stress. Peer J 3: e1347.


盐度和干旱是导致红麻产量和质量损失的两个主要因素。 据报道,盐度和干旱可影响10%以上的耕地,导致主要作物平均单产下降50%以上。 因此,了解植物对干旱和盐分的耐受性具有根本重要性,已成为深入研究的重点。 该协议描述了在土壤条件下模拟天然盐度和干旱胁迫的简单和可重复的方案。 即使水培法最常用于盐分和干旱处理,本研究中的土壤培养方法更适用于研究自然胁迫条件。

关键字:盐度, 干旱, Kenaf(红麻)


  1. 无菌培养皿(JET,目录号:TCD010090)
  2. 无菌滤纸(Sigma-Aldrich,目录号:Z241091)
  3. 种子:槿麻(cv Fuhong 992)[保存于福建农林大学作物遗传育种与利用重点实验室(中国)]。
  4. 无菌水(使用高压灭菌器在121℃/1大气压下灭菌20分钟)
  5. 无菌土壤(Fafard生长混合物1-PV/C-1PV,Sun Gro Horticulture Canada Ltd.)(使用高压灭菌器在121℃/1大气压下灭菌30分钟)
  6. 次氯酸钠(NaClO)溶液(15%)(Sinopharm Chemical Reagent,目录号:7681-52-9)
  7. 硝酸钾(KNO 3)(Sinopharm Chemical Reagent,目录号:7757-79-1)
  8. 二氢磷酸铵(NH 4 H H 2 PO 4)(Sinopharm Chemical Reagent,目录号:7722-76-1)
  9. 硝酸铵(NH 4 NO 3)(Sinopharm Chemical Reagent,目录号:6484-52-2)
  10. 硝酸钙[Sinopharm Chemical Reagent,目录号:13477-34-4)]的制备在室温下, br />
  11. 硫酸镁(MgSO 4·7H 2 O)(Sinopharm Chemical Reagent,目录号:10034-99-8)
  12. 乙二胺-N,N双(2-羟基苯基乙酸)铁钠复合物(Fe-EDDHA)(Sinopharm Chemical Reagent,目录号:16455-61-1)
  13. 硼酸(H 3 BO 3)(Sinopharm Chemical Reagent,目录号:10043-35-3)
  14. 氯化锰(MnCl 2·4H 2 O)(Sinopharm Chemical Reagent,目录号:7773-01-5)
  15. 硫酸锌(ZnSO 4·7H 2 O)(Sinopharm Chemical Reagent,目录号:7446-20-0)
  16. 硫酸铜(CuSO 4·5H 2 O)(Sinopharm Chemical Reagent,目录号:7758-98-7)
  17. 钼酸钠(Na 2 SO 4·MoO 4·2H 2 O)(Sinopharm Chemical Reagent,目录号:10102-40-6)
  18. 氯化钠(NaCl)(Sinopharm Chemical Reagent,目录号:7647-14-5)
  19. PEG6000(Sigma-Aldrich,目录号:81260)
  20. 5%(v/v)次氯酸钠(参见配方)
  21. Hoagland解决方案(参见配方)
  22. 200 mM氯化钠(NaCl)溶液(参见配方)
  23. 20%(w/v)PEG6000溶液(参见配方)


  1. 塑料盆(直径10厘米,深8厘米)
  2. 洁净台(苏州航空科技,AirTech ®,型号:SW-CJ-1FD)
  3. 生长室(上海益一益上海,Blue pard ,型号:MGC-300A)
  4. 高压灭菌器(TOMY,型号:SX-500)
  5. 精密平衡(±0.001)
  6. 定时器控制


  1. 种子发芽和培养
    1. 在流水下冲洗种子10分钟,然后用5%次氯酸钠消毒种子10分钟,然后用无菌水冲洗种子三次(图1A)。
    2. 将无菌种子放在用无菌水饱和的滤纸上的培养皿中,并在28℃下将种子保持在完全黑暗中,直到发芽(约3天)(图1B和1C)。
    3. 将3-5天的幼苗转移到无菌的土壤罐中
    4. 将植物在28℃的生长室中在光下和26℃下保持8小时,无光,60%相对湿度,〜450μmol·m < - > 1 光强度,并且每天向土壤中补充100ml的1/4 Hoagland溶液(图1D)。
    5. 允许植物长到3-10叶期(约4周),用于盐度和干旱处理测定
  2. 盐渍和干旱治理
    1. 对于盐度处理,在Hoagland营养培养基中制备200mM氯化钠溶液,并且每天一次将100μl该溶液补充至10个盆中(图1D)。
    2. 对于干旱处理,在Hoagland营养培养基中制备20%(w/v)PEG6000溶液,并且每天一次向10个盆中补充100ml该溶液(图1D)。
    3. 在不同的时间点(例如6,12和24小时)从每株植物收获3处理的叶子。以与对照相同的间隔收集未处理的样品。从每次处理获得三个生物学重复(图1E)

      图1.实验步骤示例:A.在流水下冲洗种子; B.在培养皿中的饱和滤纸上灭菌的种子;发芽的种子;子叶期幼苗; E.收获叶片样品(约4周),如盐渍和干旱处理后的红色箭头所示


分离RNA并用于实时PCR(RT-qPCR)的选择可靠参照基因(例如,ACT ,18S rRNA ,EF1α )测定在盐度和干旱胁迫下,并且该部分在我们的论文中清楚地描述(Niu等人,2015)。


  1. 使用高压灭菌器将土壤在121℃/1atm下灭菌至少30分钟
  2. 聚乙二醇(PEG)是柔性的水溶性聚合物,并且其可以用于产生高渗透压。 因此,它用于在植物中施加渗透压以引起水缺乏
  3. 应从每个样品类型中取相同的老化叶。 每次治疗至少使用三条独立的线进行分析
  4. 补充量取决于盆的大小,浇水频率和植物物种。 建议在胁迫处理前对一个给定的植物种进行一系列试验


  1. Hoagland解决方案
    注意:Hoagland解决方案基于Hoagland No.2解决方案(Hoagland等,1950)(表1)。

    表1. Hoagland解决方案组合

  2. 5%(v/v)次氯酸钠
    在蒸馏水中加入33ml 15%NaClO溶液,使最终体积为100ml
  3. 20%(w/v)PEG6000溶液
    将20g PEG6000溶于蒸馏水中
  4. 200mM氯化钠溶液
    将11.7g NaCl溶于1L蒸馏水中


这项工作得到了中国农业部948项目(2013-Z70),中国国家韧皮纤维种质资源项目(K47NI201A)和中国国家韧皮纤维研究系统(CARS-19-E06)的资助。 该协议改编自我们最近的出版物(Niu等人,2015)。


  1. Hoagland,DR,Arnon,DI(1950)。  水文化方法的成长 植物无土。 加利福尼亚农业实验站 347:34.
  2. Niu,X.,Qi,J.,Chen,M.,Zhang,G.,Tao,A.,Fang,P.,Xu,J.,Onyedinma,SAand Su,J.(2015) a参照基因选择用于槿麻中的转录物标准化( Hibiscus cannabinus ) /em> L.)在盐度和干旱胁迫下。 PeerJ 3:e1347。
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引用:Niu, X., Chen, M., Tao, A. and Qi, J. (2016). Salinity and Drought Treatment Assays in Kenaf (Hibiscus cannabinus L.). Bio-protocol 6(17): e1918. DOI: 10.21769/BioProtoc.1918.