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Qualitative Analysis of Lipid Peroxidation in Plants under Multiple Stress Through Schiff’s Reagent: A Histochemical Approach
植物多重应激下脂质过氧化作用的希夫试剂定性分析:组织化学方法   

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PLOS ONE
Apr 2017

Abstract

Lipid peroxidation is a physiological indicator of both biotic and abiotic stress responses, hence is often used as a biomarker to assess stress-induced cell damage or death. Here we demonstrate an easy, quick and cheap staining method to assess lipid peroxidation in plant tissues. In this methodology, Schiff’s reagent, is used to assay for membrane degradation. Histochemical detection of lipid peroxidation is performed in this protocol. In brief, Schiff’s reagent detects aldehydes that originate from lipid peroxides in stressful condition. Schiff’s reagent is prepared and applied to plants tissue. After the reaction, plant tissue samples are rinsed with a sulfite solution to retain the staining color. From this analysis, qualitative visualization of lipid peroxidation in plant tissue is observed in the form of magenta coloration. This reagent is useful for visualization of stress induced lipid peroxidation in plants. In this protocol, Indica rice root, Assam tea root and Indian mustard seedlings are used for demonstration.

Keywords: Schiff’s reagent (希夫试剂), Rice (水稻), Tea (茶叶), Mustard (芥末), Membrane degradation (膜降解), Lipid peroxidation (脂质过氧化)

Background

Oxidative stress induced by both biotic and abiotic stress factors leads to lipid peroxidation. The peroxidation of unsaturated lipids in membrane is the most apparent symptom of oxidative stress. Lipid peroxidation is a deleterious process in plants, which affects membrane properties, causes protein degradation and limits the capacity of ionic transport, ultimately triggering the cell death process (Yamamoto et al., 2001). Malondialdehyde (MDA) content, which is a byproduct of lipid peroxidation process was found to be enhanced in rice (Ma et al., 2012; Awasthi et al., 2017), Salvinia (Mandal et al., 2013), pea (Yamamoto et al., 2001), Soyabean (Cakmak and Horst, 1991; Du et al., 2010), Indian mustard (Saha et al., 2016) on exposure to stress. The reactive oxygen species (ROS) associated with oxidative stress acts on membrane lipids to decrease membrane stability. Thus making the study of lipid peroxidation an important parameter and our protocol offers a very rapid method for the study. Schiff’s reagent reacts with aldehyde functional group of MDA to give the magenta coloration, thus acting as a key determinant of lipid peroxidation in plant tissues.

Materials and Reagents

  1. Glass Petri plates of 10 cm diameter (Scott Duran)
  2. Absorbent cotton
  3. Whatman 1 filter paper
  4. Razor blade (Glassvan, No. 23, Scalpel blade)
  5. 15 ml disposable centrifuge tubes (Tarsons)
  6. Slide
  7. Needle (Dispovan, sterile needle, 0.45 x 13 mm)
  8. Brush (Camel, Size 0)
  9. Gas mask
  10. Plant tissue (Leaf, root or whole seedling) collected freshly after stress treatment
  11. Distilled water
  12. Mercuric chloride (HgCl2) (SRL Sisco Research Laboratories, catalog number: 25699 )
  13. Schiff’s reagents (HiMedia Laboratories, catalog number: S074-500ML )
  14. Calcium nitrate tetrahydrate (Ca(NO3)2·4H2O) (HiMedia Laboratories, catalog number: GRM3903-500G )
  15. Potassium nitrate (KNO3) (HiMedia Laboratories, catalog number: GRM1401-500G )
  16. Potassium phosphate monobasic (KH2PO4) (HiMedia Laboratories, catalog number: RM3943-500G )
  17. Magnesium sulfate heptahydrate (MgSO4·7H2O) (HiMedia Laboratories, catalog number: RM684-5KG )
  18. Boric acid (H3BO3) (HiMedia Laboratories, catalog number: MB007-1KG )
  19. Manganese(II) chloride tetrahydrate (MnCl2·4H2O) (HiMedia Laboratories, catalog number: RM685-500G )
  20. Zinc sulfate heptahydrate (ZnSO4·7H2O) (HiMedia Laboratories, catalog number: PCT0118-1KG )
  21. Sodium molybdate dihydrate (Na2MoO4·2H2O) (HiMedia Laboratories, catalog number: GRM415-100G )
  22. Copper(II) sulfate pentahydrate (CuSO4·5H2O) (HiMedia Laboratories, catalog number: RM630-500G )
  23. Ferric chloride (FeCl3) (HiMedia Laboratories, catalog number: RM1178-1KG )
  24. EDTA (HiMedia Laboratories, catalog number: RM678-100G )
  25. Ammonium sulfate ((NH4)2SO4) (HiMedia Laboratories, catalog number: MB004-250G )
  26. Potassium sulfate (K2SO4) (HiMedia Laboratories, catalog number: GRM404-500G )
  27. Manganese sulfate monohydrate (MnSO4·H2O) (SRL Sisco Research Laboratories, catalog number: 1347151 , 500G)
  28. Sodium Meta-bisulphate (Na2S2O5) (Sigma-Aldrich, catalog number: 255556-100G )
  29. Calcium chloride dihydrate (CaCl2·2H2O) (HiMedia Laboratories, catalog number: PCT0004-500G )
  30. Aluminum chloride (AlCl3) (Merck, catalog number: 8010810100 )
  31. Cadmium chloride (CdCl2) (Thermo Fisher Scientific, catalog number: Q17584 )
  32. Zinc chloride (ZnCl2) (Thermo Fisher Scientific, catalog number: Q28785 )
  33. Ethanol (Diluent for DNA Extraction, HiMedia Laboratories, catalog number: MB228-500ML )
  34. Glycerol (Sigma-Aldrich, catalog number: V800196-500ML )
  35. Acetic acid (Thermo Fisher Scientific, catalog number: Q21057 )
  36. Hydrochloric acid (HCl) (HiMedia Laboratories, catalog number: AS004-2.5L )
  37. Schiff’s reagent staining solution (see Recipe 1)
  38. Hoagland solution (see Recipe 2)
  39. Modified nutrient solution (see Recipe 3)
  40. Treatment solutions (AlCl3, CdCl2, ZnCl2) (see Recipe 4)
  41. Sulfite solution (see Recipe 5)
  42. Bleaching solution (see Recipe 6)
  43. Storage solution (see Recipe 7)

Equipment

  1. Pipettes (Gilson, PIPETMAN, 2-20,20-200 and 100-1,000 µl)
  2. pH meter (pH Tutor, Eutech Instruments)
  3. Magnetic stirrer cum hot plate (Tarsons)
  4. Weighing balance (Sartorius, 0.1 mg-220 g)
  5. Water bath (Equitron unstirred water bath, Medica Instruments)
  6. Light microscope (Olympus, 10x objective)
  7. Digital camera (Nikon, model: COOLPIX P100 , 10.3 megapixel, 26x zoom)

Procedure

  1. Prepare Schiff’s reagent solution (Recipe 1).
  2. Grow plants hydroponically.
    1. Indica rice: Germinate surface sterilized (with 0.01% mercuric chloride) rice seeds (undehusked) in Petri plates with cotton bed (absorbent cotton covered with Whatman 1 filter paper) soaked in Hoagland nutrient solution (Recipe 2) at 28 ± 2 °C in dark condition. Grow the germinated seedlings hydroponically on Hoagland nutrient solution (Recipe 2) at 26 ± 2 °C over a photoperiod of 14 h with a photon flux density of 220 μmol m-2 sec-1 (PAR) for 5 days and then pretreat with 500 µM CaCl2 solution for 24 h. Expose the pretreated seedlings to 100 µM AlCl3 solution (dilute from the stock solution, Recipe 4) containing 500 µM CaCl2 at pH 4.5 for 48 h and harvest root tips for histochemical assay.
      Note: Because of toxic nature of mercuric chloride (HgCl2), use of precautionary clothing is highly recommended.
    2. Assam tea: Plant the cuttings procured from local tea gardens, after pulling out the polythene sleeves and then acclimatize for 30-35 days in soil at 26 ± 2 °C over a photoperiod of 14 h with photon flux density of 220 μmol m-2 sec-1 (PAR). After acclimatization, grow tea plantlets hydroponically in pots with the nutrient solution (Recipe 3). Pre-culture the plants for a week in the nutrient solution (unsterile) before the application of Al3+ in the form of AlCl3, at a concentration of 250 µM. The pH is maintained at 4.2. After 3 days, harvest the root segments for histochemical assay.
    3. Indian mustard: Surface sterilize healthy seeds of Brassica juncea (L.) Czern & Coss with 0.01% HgCl2 and set for germination. Impart 1 mM CdCl2 and 2.5 mM ZnCl2 treatments (dilute from the stock solutions, Recipe 4) to seeds by placing in Petri plates with cotton bed soaked in the stress solutions when kept for germination. After 7th day, harvest the seedlings for histochemical study.
      Note: Since no carbohydrate source is added to the nutrient solution, so no bacterial contamination occurs.
  3. In another set of experiments, keep control samples apart from stress and allow to grow under normal unstressed condition (plants not treated with any of the stress factors).
  4. Excise (using a razor blade) root tissues for Indica rice and Assam tea. Transfer root tissues and whole seedlings of Indian mustard to 15 ml centrifuge tubes and add 5 ml of Schiff’s reagent solution to each tube.
    Note: Tissues should be completely immersed in the solution.
  5. Vacuum infiltrate the tissue samples dipped in Schiff’s reagent for 1-2 h and then rinsed with a sulfite solution for 10 min (Recipe 5).
    Note: The time for vacuum infiltration varies with tissue and plant age, should be kept until and unless a contrasting visual difference in Magenta coloration is obtained. A representative image that shows the extent of color change indicative of sufficient infiltration has been provided (Figure 1).


    Figure 1. Representative image that shows the color change indicative of sufficient infiltration/treatment. Schiff’s reagent in Indica rice root (A) control, (B) stressed, scale bar = 1 cm; Assam tea roots (C) control, scale bar = 1 cm, (D) stressed, scale bar = 1 cm; and in Indian mustard seedling (E) control, scale bar = 1 cm, (F) 1 mM Cd treated seedling, scale bar = 1 cm, (G) 2.5 mM Zn treated seedling, scale bar = 1 cm.

  6. Wash thoroughly with distilled water to remove the extra stain and then bleach the tissue samples with bleaching solution (Recipe 6) for 5-10 min at 100 °C in a water bath.
    Note: Chlorophyll should be completely removed from tissue samples.
  7. After complete bleaching is achieved, carefully mount samples on slide (for rice and tea roots using needle and brush) and take photographs under a microscope at 10x magnification (Figure 2). Whereas for mustard, take direct photographs with a digital camera for seedlings (Figure 2). If required, store the tissue samples by completely dipping in storage solution at 4 °C (Recipe 7) for future photography sessions. 


    Figure 2. Histochemical determination of lipid peroxidation in different plant species by Schiff’s reagent. Detection of lipid peroxidation using Schiff’s reagents in Indica rice root (A) control, scale bar = 100 μm; (B) stressed, scale bar = 100 μm; Assam tea roots (C) control, scale bar = 100 μm, (D) stressed, scale bar = 100 μm; and in Indian mustard seedling (E) control, scale bar = 5 mm, (F) 1 mM Cd treated seedling, scale bar = 5 mm, (G) 2.5 mM Zn treated seedling, scale bar = 5 mm.
    Note: Root in Figure 2C looks brownish because of various phenolic compounds secreted by tea plant. 

Data analysis

Since this is a qualitative assay, no data analysis is performed. The stained plant tissue samples are simply documented by taking photographs with a digital camera. A similar analysis is presented in the following paper: Awasthi et al. (2017).

Recipes

  1. Schiff’s reagent staining solution (Jensen, 1962)
    Prepare 10% (v/v) Schiff’s reagent solution in distilled water and mixed well
    Note: Schiff’s reagent commercially available is used. Schiff’s reagent staining solution is prepared freshly each time.
  2. Hoagland solution (Table 1)

    Table 1. Composition of Hoagland solution


    Prepare stock solutions separately and mix in appropriate proportions to obtain working solution. Finally adjust the pH to 6.0-6.2 with 1 N NaOH
  3. Modified nutrient solution (Ghanati et al., 2005) (Table 2)

    Table 2. Composition of modified nutrient solution


    Prepare stock solutions separately and mix in appropriate proportions to obtain a working solution. Finally adequate nutrient solution and adjust the pH to 6.0-6.2 with 1 N NaOH
  4. Treatment solutions
    AlCl3, CdCl2, ZnCl2: Prepare 10 mM stock and store separately at 4 °C for future use
    Note: When preparing AlCl3 solution noxious fumes of HCl are formed. So it should be prepared with proper precaution like in a fume hood, use of gas mask etc. CdCl2 is highly carcinogenic, so use of precautionary clothing is highly recommended.
  5. Sulfite solution
    0.5% (w/v) Na2S2O5 in 0.05 M HCl
  6. Bleaching solution
    Acetic acid:glycerol:ethanol (1:1:3, v/v/v) solution
  7. Storage solution
    Glycerol:ethanol (1:4, v/v) solution

Acknowledgments

This protocol is adapted from Pompella et al. (1987) and Yamamoto et al. (2001). We sincerely acknowledge the financial support from UGC, India (UGC NON-NET fellowship). Authors declare no conflicts of interest or competing interests.

References

  1. Awasthi, J. P., Saha, B., Regon, P., Sahoo, S., Chowra, U., Pradhan, A., Roy, A. and Panda, S. K. (2017). Morpho-physiological analysis of tolerance to aluminum toxicity in rice varieties of North East India. PLoS One 12(4): e0176357.
  2. Cakmak, I. and Horst, W. J. (1991). Effect of aluminum on lipid peroxidation, superoxide dismutase, catalase, and peroxidase activities in root tips of soybean (Glycine max). Physiologia Plantarum 83: 463-468.
  3. Du, B., Nian, H., Zhang, Z. and Yang, C. (2010). Effects of aluminum on superoxide dismutase and peroxidase activities, and lipid peroxidation in the roots and calluses of soybeans differing in aluminum tolerance. Acta Physiologiae Plantarum 32: 883-890.
  4. Ghanati, F., Morita, A. and Yokota, H. (2005). Effects of aluminum on the growth of tea plant and activation of antioxidant system. Plant Soil 276: 133-141.
  5. Jensen, W. A. (1962). Botanical Histochemistry: Principles and Practice. W. H. Freeman and Company, San Francisco.
  6. Ma, B., Gao, L., Zhang, H., Cui, J. and Shen, Z. (2012). Aluminum-induced oxidative stress and changes in antioxidant defenses in the roots of rice varieties differing in Al tolerance. Plant Cell Rep 31(4): 687-696.
  7. Mandal, C., Ghosh, N., Maiti, S., Das, K., Gupta, S., Dey, N. and Adak, M. K. (2013). Antioxidative responses of Salvinia (Salvinia natans Linn.) to aluminium stress and it's modulation by polyamine. Physiol Mol Biol Plants 19(1): 91-103.
  8. Pompella, A., Maellaro, E., Casini, A. F. and Comporti, M. (1987). Histochemical detection of lipid peroxidation in the liver of bromobenzene-poisoned mice. Am J Pathol 129(2): 295-301.
  9. Saha, B., Mishra, S., Awasthi, J. P., Sahoo, L., and Panda, S. K. (2016). Enhanced drought and salinity tolerance in transgenic mustard [Brassica juncea (L.) Czern & Coss.] overexpressing Arabidopsis group 4 late embryogenesis abundant gene (AtLEA4-1). Environ Exp Bot 128: 99-111.
  10. Yamamoto, Y., Kobayashi, Y. and Matsumoto, H. (2001). Lipid peroxidation is an early symptom triggered by aluminum, but not the primary cause of elongation inhibition in pea roots. Plant Physiol 125(1): 199-208.

简介

脂质过氧化是生物和非生物胁迫反应的生理指标,因此常被用作评估应激诱导的细胞损伤或死亡的生物标志物。在这里,我们展示了一种简单,快速和便宜的染色方法来评估植物组织中的脂质过氧化。在这种方法中,Schiff's试剂用于测定膜降解。在该方案中进行脂质过氧化的组织化学检测。简而言之,席夫试剂检测压力条件下源自脂质过氧化物的醛。希夫氏试剂准备并应用于植物组织。反应后,用亚硫酸盐溶液漂洗植物组织样品以保持染色的颜色。从该分析中,观察到品红着色形式的植物组织中脂质过氧化的定性可视化。该试剂可用于植物中胁迫诱导的脂质过氧化的可视化。在这个协议中,使用籼稻根,阿萨姆茶根和印度芥菜幼苗来示范。

【背景】生物和非生物胁迫因子诱导的氧化应激导致脂质过氧化。膜中不饱和脂质的过氧化是氧化应激最明显的症状。脂质过氧化是植物中的有害过程,其影响膜性质,引起蛋白质降解并限制离子转运能力,最终引发细胞死亡过程(Yamamoto等人,2001)。发现在水稻中脂质过氧化过程的副产物丙二醛(MDA)含量增加(Ma等人,2012; Awasthi等人,2017) ,Salvinia(Mandal等人,2013),豌豆(Yamamoto等人,2001),Soyabean(Cakmak和Horst,1991; Du等人, ,2010),印度芥末(Saha et。,2016)暴露于压力。与氧化应激相关的活性氧(ROS)作用于膜脂质以降低膜稳定性。因此,使脂质过氧化的研究是一个重要的参数,我们的方案提供了一个非常快速的研究方法。 Schiff试剂与MDA的醛官能团反应产生品红着色,从而作为植物组织中脂质过氧化的关键决定因素。

关键字:希夫试剂, 水稻, 茶叶, 芥末, 膜降解, 脂质过氧化

材料和试剂

  1. 直径10厘米的玻璃培养皿(斯科特杜兰)
  2. 吸水棉
  3. Whatman 1滤纸
  4. 剃刀刀片(Glassvan,23号刀片刀片)

  5. 15毫升一次性离心管(Tarsons)
  6. 幻灯片
  7. 针(Dispovan,无菌针,0.45 x 13毫米)
  8. 刷(骆驼,大小0)
  9. 防毒面具
  10. 植物组织(叶,根或整株幼苗)在压力处理后新鲜收集
  11. 蒸馏水
  12. 氯化汞(HgCl 2)(SRL Sisco Research Laboratories,目录号:25699)
  13. 希夫试剂(HiMedia Laboratories,目录号:S074-500ML)
  14. 硝酸钙四水合物(Ca(NO 3)2·4H 2 O)(HiMedia Laboratories,目录号:GRM3903-500G) >
  15. 硝酸钾(KNO 3)(HiMedia Laboratories,目录号:GRM1401-500G)
  16. 磷酸二氢钾(KH 2 PO 4)(HiMedia Laboratories,目录号:RM3943-500G)
  17. 硫酸镁七水合物(MgSO 4·7H 2 O)(HiMedia Laboratories,目录号:RM684-5KG)
  18. 硼酸(H 3 BO 3)(HiMedia Laboratories,目录号:MB007-1KG)
  19. 氯化锰(II)四水合物(MnCl 2·4H 2 O)(HiMedia Laboratories,目录号:RM685-500G)
  20. 硫酸锌七水合物(ZnSO 4·7H 2 O)(HiMedia Laboratories,目录号:PCT0118-1KG)
  21. 钼酸钠二水合物(Na 2 MoO 4·2H 2 O)(HiMedia Laboratories,目录号:GRM415-100G)
  22. 硫酸铜(II)五水合物(CuSO 4·5H 2 O)(HiMedia Laboratories,目录号:RM630-500G)
  23. 氯化铁(FeCl 3)(HiMedia Laboratories,目录号:RM1178-1KG)
  24. EDTA(HiMedia Laboratories,目录号:RM678-100G)
  25. 硫酸铵((NH 4)2 SO 4)(HiMedia Laboratories,目录号:MB004-250G)
  26. 硫酸钾(K 2 SO 4)(HiMedia Laboratories,目录号:GRM404-500G)
  27. 硫酸锰一水合物(MnSO 4·2H 2 O)(SRL Sisco Research Laboratories,目录号:1347151,500G)
  28. 偏硫酸氢钠(Na 2 S 2 O 5)(Sigma-Aldrich,目录号:255556-100G)
  29. 氯化钙二水合物(CaCl 2·2H 2 O)(HiMedia Laboratories,目录号:PCT0004-500G)
  30. 氯化铝(AlCl 3)(Merck,目录号:8010810100)
  31. 氯化镉(CdCl 2)(Thermo Fisher Scientific,目录号:Q17584)
  32. 氯化锌(ZnCl 2)(Thermo Fisher Scientific,目录号:Q28785)
  33. 乙醇(用于DNA提取的稀释剂,HiMedia Laboratories,目录号:MB228-500ML)
  34. 甘油(Sigma-Aldrich,目录号:V800196-500ML)
  35. 醋酸(Thermo Fisher Scientific,目录号:Q21057)
  36. 盐酸(HCl)(HiMedia Laboratories,目录号:AS004-2.5L)
  37. Schiff's试剂染色溶液(见配方1)
  38. Hoagland解决方案(见配方2)
  39. 改良的营养液(见配方3)
  40. 处理溶液(AlCl 3,CdCl 2,ZnCl 2)(见方案4)
  41. 亚硫酸盐溶液(见配方5)
  42. 漂白解决方案(见第6章)
  43. 存储解决方案(请参阅配方7)

设备

  1. 移液器(Gilson,PIPETMAN,2-20,20-200和100-1,000μl)
  2. pH计(pH Tutor,Eutech Instruments)
  3. 磁力搅拌器和热板(Tarsons)
  4. 称量天平(Sartorius,0.1 mg-220 g)
  5. 水浴(Equitron unstirred水浴,Medica Instruments)
  6. 光学显微镜(奥林巴斯,10倍物镜)
  7. 数码相机(尼康,型号:COOLPIX P100,1030万像素,26倍变焦)

程序

  1. 准备希夫试剂溶液(配方1)。
  2. 水培植物。
    1. 籼稻:在用Hoagland营养液(方案2)浸泡的棉床(用Whatman 1滤纸覆盖的脱脂棉)的培养皿中发芽表面灭菌(具有0.01%氯化汞)水稻种子在28±2°C黑暗条件下。在光照时间为14小时,光子通量密度为220μmolm -2 s -1 -1的条件下,在26±2℃的Hoagland营养液(配方2)上水培生长发芽的幼苗, (PAR)5天,然后用500μMCaCl 2溶液预处理24小时。将经过预处理的幼苗暴露于pH 4.5的含有500μMCaCl 2 2的100μMAlCl 3溶液(稀释自储备溶液,配方4)48小时,并收获根尖用于组织化学测定。
      注:由于氯化汞(HgCl 2)的毒性,强烈建议使用预防性服装。
    2. 阿萨姆茶:从当地的茶园采集,然后拔出聚乙烯套管,然后在26±2°C的土壤中在14±2°C的光周期内适应环境适应30-35天,光子通量密度为220μmolm -2 -1(PAR)。适应后,用营养液在盆中水培茶苗(方案3)。在施用浓度为250μM的AlCl 3 3 Al 3+前,在营养液(未消毒)中将植物预培养一周。 pH保持在4.2。 3天后,收获根部进行组织化学分析。
    3. 印度芥末:表面消毒芥菜的健康种子(L.)Czern&具有0.01%HgCl 2的Coss并设定用于萌发。将1mM CdCl 2和2.5mM ZnCl 2处理(稀释自储备溶液,配方4)施用于种子,方法是放置在Petri平板中,棉花床浸泡在胁迫溶液中当保持发芽时。第7天后,收获幼苗进行组织化学研究。
      注意:由于没有碳水化合物来源添加到营养液中,所以不会发生细菌污染。
  3. 在另一组实验中,保持对照样本不受压力的影响,并允许在正常的非应激条件下生长(未用任何应激因子处理的植物)。
  4. 榨取(使用剃刀刀片)籼米和阿萨姆茶的根组织。将根部组织和印度芥菜的整个幼苗转移到15ml离心管中,并向每个管中加入5ml希夫氏试剂溶液。
    注意:组织应完全浸入溶液中。
  5. 真空渗入浸泡在希夫氏试剂中的组织样品1-2小时,然后用亚硫酸盐溶液冲洗10分钟(配方5)。
    注意:真空渗透的时间随组织和植物年龄而变化,应保持至少,并且除非获得品红色着色的对比视觉差异。已经提供了一个代表性的图像,显示颜色变化的程度,表明有足够的渗透(图1)。


    图1.代表性图像,显示颜色变化,指示足够的渗透/处理。籼米中的希夫试剂(A)对照,(B)应激,比例尺= 1厘米;阿萨姆茶根(C)对照,比例尺= 1厘米,(D)强调,比例尺= 1厘米; (E)对照,比例尺= 1cm,(F)1mM Cd处理的幼苗,比例尺= 1cm,(G)2.5mM Zn处理的幼苗,比例尺= 1厘米。

  6. 用蒸馏水彻底清洗以去除多余的污渍,然后用漂白溶液(配方6)将组织样品在100℃水浴中漂洗5-10分钟。
    注意:叶绿素应从组织样本中完全去除。
  7. 在完成漂白后,将样品小心地放在载玻片上(使用针头和刷子对大米和茶叶根部)并在10倍放大倍数的显微镜下拍照(图2)。而对于芥末,用数码相机直接拍摄幼苗(图2)。如果需要,将组织样本储存在4°C的储存溶液中(方法7),以备将来摄影时使用。 


    图2.用Schiff's试剂测定不同植物物种中脂质过氧化作用的组织化学测定使用Schiff's试剂在籼稻根中检测脂质过氧化(A)对照,比例尺= 100微米; (B)应力,比例尺=100μm;阿萨姆茶根(C)对照,比例尺=100μm,(D)应力,比例尺=100μm;和印度芥菜幼苗(E)对照,比例尺= 5mm,(F)1mM Cd处理的幼苗,比例尺= 5mm,(G)2.5mM Zn处理的幼苗,比例尺= 5毫米。
    注意:由于茶树分泌的各种酚类化合物,图2C中的根看起来呈褐色。 

数据分析

由于这是定性分析,因此不进行数据分析。通过用数码相机拍摄照片简单记录染色的植物组织样品。下面的文章介绍了类似的分析:Awasthi et al。(2017)。

食谱

  1. Schiff's试剂染色溶液(Jensen,1962)
    在蒸馏水中配制10%(v / v)希夫氏试剂溶液并充分混合
    使用市售的Schiff's试剂。每次新鲜制备Schiff's试剂染色溶液。
  2. Hoagland解决方案(表1)

    表1. Hoagland解决方案的组成


    分别制备储备溶液并按适当比例混合以获得工作溶液。最后用1 N NaOH将pH值调节至6.0-6.2
  3. 改良的营养液(加纳蒂等人,2005年)(表2)

    表2.改良营养液的组成


    分别制备储备溶液并按适当比例混合以获得工作溶液。最后用足量的营养液,用1N NaOH调pH至6.0-6.2
  4. 治疗方案
    AlCl 3,CdCl 2,ZnCl 2:制备10mM储备液并在4℃下分开储存以备将来使用
    注:当制备AlCl 3溶液时,形成HCl有害烟雾。所以应该采取适当的预防措施,例如在通风橱里,使用防毒面具等。CdCl 2具有高致癌性,因此强烈建议使用预防性服装。
  5. 亚硫酸盐溶液
    在0.05M HCl中的0.5%(w / v)Na 2 S 2 O 5。
  6. 漂白解决方案
    乙酸:甘油:乙醇(1:1:3,v / v / v)溶液
  7. 存储解决方案
    甘油:乙醇(1:4,v / v)溶液

致谢

该协议改编自Pompella等人(1987)和Yamamoto等人(2001)。我们真诚地感谢印度UGC的经济支持(UGC NON-NET奖学金)。作者声明不存在利益冲突或利益冲突。

参考

  1. Awasthi,J. P.,Saha,B.,Regon,P.,Sahoo,S.,Chowra,U.,Pradhan,A.,Roy,A.and Panda,S.K。(2017)。 印度东北部水稻品种耐铝性的形态生理学分析 PLoS One 12(4):e0176357。
  2. Cakmak,I.和Horst,W.J。(1991)。 铝对脂质过氧化,超氧化物歧化酶,过氧化氢酶和过氧化物酶在大豆根尖中的活性( Physiologia Plantarum 83:463-468。
  3. Du,B。,Nian,H.,Zhang,Z。和Yang,C.(2010)。 铝对超氧化物歧化酶和过氧化物酶活性的影响以及根中的脂质过氧化和大豆在铝耐受性方面不同的愈伤组织。 Acta Physiologiae Plantarum 32:883-890。
  4. Ghanati,F.,Morita,A。和Yokota,H。(2005)。 铝对茶树生长和抗氧化系统激活的影响。 a> Plant Soil 276:133-141。
  5. Jensen,W.A。(1962)。 植物组织化学:原理与实践 W。 H.Freeman and Company ,旧金山。
  6. Ma,B.,Gao,L.,Zhang,H.,Cui,J。和Shen,Z。(2012)。 铝诱导的氧化胁迫以及耐铝性不同的水稻品种根部抗氧化防御能力的变化。 Plant Cell Rep 31(4):687-696。
  7. Mandal,C.,Ghosh,N.,Maiti,S.,Das,K.,Gupta,S.,Dey,N.and Adak,M.K。(2013)。 Salvinia(Salvenia natans Linn。)对铝胁迫的抗氧化反应和它被多胺调节。 Physiol Mol Biol Plants 19(1):91-103。
  8. Pompella,A.,Maellaro,E.,Casini,A.F和Comporti,M。(1987)。 溴苯中毒小鼠肝脏脂质过氧化的组织化学检测 Am J Pathol 129(2):295-301。
  9. Saha,B.,Mishra,S.,Awasthi,J. P.,Sahoo,L.和Panda,S. K.(2016)。 转基因芥菜中耐旱性和耐盐性的增强[ Brassica juncea (L. )Czern& Coss。]过表达拟南芥组4晚期胚胎发生丰富基因(AtLEA4-1)。
  10. Yamamoto,Y.,Kobayashi,Y。和Matsumoto,H。(2001)。 脂质过氧化是铝引发的早期症状,但不是豌豆根部延长抑制的主要原因。植物生理学 125(1):199-208。
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引用:Awasthi, J., Saha, B., Chowardhara, B., Devi, S., Borgohain, P. and Panda, S. (2018). Qualitative Analysis of Lipid Peroxidation in Plants under Multiple Stress Through Schiff’s Reagent: A Histochemical Approach. Bio-protocol 8(8): e2807. DOI: 10.21769/BioProtoc.2807.
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