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Non-invasive Imaging of Rice Roots in Non-compacted and Compacted Soil

非压实和压实土壤中水稻根系的无创成像

BP Bipin K. Pandey
JA Jonathan A. Atkinson
CS Craig J. Sturrock

发布: 2021年12月20日第11卷第24期 DOI: 10.21769/BioProtoc.4252 浏览次数: 5611

评审: Wenrong HeMin CaoAnonymous reviewer(s)

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Abstract

Roots are the prime organ for nutrient and water uptake and are therefore fundamental to the growth and development of plants. However, physical challenges of a heterogeneous environment and diverse edaphic stresses affect root growth in soil. Compacted soil is a serious global problem, causing inhibition of root elongation, which reduces surface area and impacts resource foraging. Visualisation and quantification of roots in soil is difficult due to this growth substrate’s opaque nature; however, non-destructive imaging technologies are now becoming more widely available to plant and soil scientists working to address this challenge. We have recently developed an integrated approach, combining X-ray Computed Tomography (X-ray CT) and confocal microscopy to image roots grown in compacted soil conditions from a plant to a cellular scale. The method is suited to visualize cellular responses of root tips grown in both non-compacted and compacted soils. This protocol presents a fully integrated workflow, including soil column preparation, creation of compaction conditions, plant growth, imaging, and quantification of root adaptive responses at a cellular scale.


Keywords: Soil (土壤) search Root (根) search Computed tomography (计算机断层扫描) search Soil compaction (土壤压实) search Rice (大米) search

Background

Soil compaction is an increasingly challenging problem for modern farming, as it deforms the physical, chemical, and biological properties of agricultural soil. Modern agriculture practices rely on the use of large and heavy machinery such as tractors and harvesters. These modern practices compress both top- and sub-soil (Keller et al., 2019). Hard (compacted) soils represent a major challenge facing modern agriculture that can reduce crop yields by over 50% through reducing root growth, causing significant losses annually. Soil compaction triggers a reduction in root penetration and uptake of water and nutrients. Despite its clear importance for agriculture and global food security, the mechanism underpinning root compaction responses has been unclear until now. Imaging root adaptive responses in natural soil conditions can provide invaluable and critical responses of rhizosphere biology. Previously, most soil compaction studies did not adopt an integrated approach to image the growing roots from cellular to plant organ scales. More generally, this integrated imaging approach can be applied to other root-soil related stress responses. This protocol is applicable to image the root tip responses of several crop species, including both monocot and dicotyledonous plants in mechanical impedance stress or other edaphic stresses. So far, we have tested this protocol on the roots of rice, wheat, barley, brachypodium, pearl millet, foxtail millet, maize, and tomato plants growing in soil.

Materials and Reagents

  1. Soil column preparation

    1. 3D-printed plastic column/polyvinyl chloride (PVC) or high-density polyethylene (HDPE) drainage pipe

    2. Double-sided adhesive tape (Radabar Double-Sided membrane tape 4000GA, 10 × 50 mm)

    3. Cloth-backed adhesive tape (Gaffa tape, Kitepackaging, UK)

    4. 20 l plastic storage box

    5. Air-dried and sieved (<2 mm) field soil (sandy loam and clay loam)

    6. Spatula (A2Z SciLab, 32 mm × 8 mm), 7-inch tapered end spatula

    7. Rice, Arabidopsis seeds


  2. Root anatomy imaging

    1. Slides (BS70011/2, 1.0-1.2 mm, Thermo Scientific) and coverslip (22 × 50 mm, No.1, SLS Ltd. UK)

    2. Calcofluor white (M2R, fluorescent brightener 28) (Polysciences, catalog number: 4359)

    3. 4% paraformaldehyde (Merck, CAS number: 30525-89-4)

    4. PFA power (Sigma-Aldrich, catalog number: P6148-1Kg)

    5. Agarose (Sigma-Aldrich, catalog number: A9539)

    6. Xylitol (Sigma-Aldrich, catalog number: W507930-1Kg)

    7. Sodium deoxycholate (Sigma-Aldrich, catalog number: SRE0046-500g)

    8. Urea (Sigma-Aldrich, catalog number: U5128-1Kg)

    9. 4% PFA (see Recipes)

    10. ClearSee (root clearing solution) (see Recipes)

Equipment

  1. Hand saw or pipe cutter (RS Pro 150 mm hacksaw, UK)

  2. Fine mesh (nylon dress net fabric, Fabric Land, UK)

  3. Stainless steel scissors (115 mm) (Spring Iris Scissors, SS, N828/01, SAMCO)

  4. Digital balance (HF4000, A&D Company limited)

  5. Microwave oven

  6. Fine paint brush

  7. Tweezers (FisherbrandTM Dumont #5 Fine Tip Tweezers, Z3) and forceps (BochemTM Stainless Steel Sharp Tip Forceps, 1133)

  8. GE Phoenix v|tome|x M 240 kV X-ray tomography system (GE Inspection Technologies, Wunstorf, Germany)

  9. Confocal microscope (Leica SP5/SP8)

  10. Leica vibratome (Leica Microsystems, model: VT1000s)

  11. RS PRO hot plate (700W) (RS Components, Corby, UK. RS Stock No: 490-1036)

  12. High performance workstation computer

  13. Recommended systems requirements: Intel Core i7 with 2.9 GHz and 32-64 GB of RAM (or higher)

Software

  1. Volume Graphics VGStudioMax 2.2 (Volume Graphics GmbH, Germany)

  2. Datos|REC software (GE Inspection Technologies, Wunstorf, Germany)

  3. Polyline tracing (VGStudioMAX V2.2 (Volume graphics GmbH, Germany)

  4. Leica LASX (Leica Microsystems)

  5. Fiji/ImageJ (NIH) (Schneider et al., 2012; Rueden et al., 2017), Version 1.53c

Procedure

English
中文翻译

文章信息

版权信息

© 2021 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

  1. Pandey, B. K., Atkinson, J. A. and Sturrock, C. J. (2021). Non-invasive Imaging of Rice Roots in Non-compacted and Compacted Soil. Bio-protocol 11(24): e4252. DOI: 10.21769/BioProtoc.4252.
  2. Pandey, B. K. and Huang, G. (2021). Plant roots sense soil compaction through restricted ethylene diffusion. Science 371(6526): 276-280.

    3. ris ris Download Citation in RIS Format

分类

植物科学 > 植物育种 > 栽培技术

细胞生物学 > 细胞成像 > 共聚焦显微镜

生物科学 > 生物技术

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