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一种优化的 Rhizobox 协议, 用于可视化根系生长和对局部营养素的响应

Published: October 22, 2018
doi:
10.3791/58674
, ,
1Department of Plant Sciences,University of California at Davis, 2Department of Natural Resources and the Environment,University of New Hampshire

Summary

在原位可视化和测量根系生长是极具挑战性的。我们提出了一种可自定义的 rhizobox 方法, 可根据营养丰富的时间跟踪根系发育和增殖。该方法用于分析玉米根系可塑性对有机氮源反应的基因型差异。

Abstract

根是众所周知的难以研究。土壤既是一种视觉和机械屏障, 在没有破坏性收获或昂贵设备的情况下, 很难在原地跟踪根。我们提出了一种可自定义且经济实惠的 rhizobox 方法, 可在一段时间内实现根系生长的无损可视化, 特别适合研究本地化资源修补程序的根塑性。通过对含有15个 n-标记豆科植物残渣的斑块的塑性响应进行了评价, 验证了玉米基因型的变化。方法描述了随着时间的推移获得代表性的发育测量值, 测量含资源和控制斑块中的根长度密度, 计算根系生长速率, 并通过植物根和芽来确定15N 的回收率。本文还讨论了该方法的优点、注意事项和潜在的未来应用。尽管必须注意确保实验条件不偏向根增长数据, 但在这里提出的 rhizobox 协议如果进行得足够注意细节, 就会产生可靠的结果。

Introduction

根系在植物养分采集中起着至关重要的作用, 但与地面上的同类相比往往被忽视。鉴于根系建造和维护的大量碳成本, 植物在觅食是值得投资的地方, 进化出了形成根系的机制。因此, 根系可以通过在热点中增殖、上调吸收率和快速转运养分向韧皮部进一步输送1, 从而高效、动态地挖掘资源斑块。可塑性反应在植物种类或基因型2、3和根据所涉及的养分的化学形式中有很大差异4,5。应进一步探讨根系可塑性的变化, 因为了解不同土壤资源的复杂根系反应, 可以告知育种和管理策略, 以提高农业的养分利用效率。

尽管它对了解植物系统的必要性和相关性, 但在相关的尺度上可视化和量化根塑性会带来技术挑战。从土壤中挖掘根冠 (“shovelomics”6) 是一种常见的方法, 但细根利用土壤骨料之间的小孔隙, 而开挖不可避免地导致这些脆弱的根的某种程度的损失。此外, 破坏性的收获使得不可能随着时间的推移在一个根系统中发生变化。原位成像方法, 如 X 射线计算机断层扫描允许直接可视化的根和土壤资源在高空间分辨率7, 但昂贵, 需要专门的设备。水培试验避免了从土壤中提取根的约束, 但与土壤的机械约束和生物物理复杂性相比, 在水性介质中根系形态和结构的差异8、9。最后, 在这些人工介质中, 根际过程和功能不能与发育可塑性相结合。

我们提出了一个用于构造和使用 rhizoboxes (窄的、透明的矩形容器) 的协议, 作为一种低成本、可定制的方法来表征土壤中的根系生长。特别设计的框架鼓励根在后面板上优先增长, 由于安歇, 提高根长度测量的准确性。Rhizoboxes 通常用于研究根系生长和根际相互作用10,11,12, 但这里提出的方法提供了简单的优势, 其单室设计和廉价材料, 旨在研究对局部营养素的根系反应。然而, 该方法也可用于研究一系列其他根和根际过程, 如内/种间竞争, 化合物的空间分布, 微生物, 或酶活性。在本文中, 我们研究了玉米杂交种对15个 n-标记豆科植物残留斑块的基因型差异, 并突出了代表性结果, 验证了 rhizobox 方法。

Protocol

1. 前、后面板和垫片的准备工作 准备前面板和背板。 将两块透明0.635 厘米厚的亚克力切成40.5 厘米宽61厘米每盒或购买预切割件 (见材料表)。 使用专为亚克力设计的钻头孔, 从顶部的2.5、19、38和53.3 厘米的侧面边缘0.635 厘米直径1.3 厘米的钻孔。钻孔1.3 厘米从底部边缘在 2.5, 20.3 和38厘米从左侧 (图 1)。注意: 一次使用钻床进行六到十页的?…

Representative Results

像预期的那样, 根在盒子后面的生长优先。盒背面的总追踪根长度介于400到 1956 cm 之间, 与盒前93-758 厘米相比。在扫描的根长度和追踪的根长度之间, 在盒子的前部、盒子的背面, 计算出成对皮尔逊相关系数, 并使用前后的总和来确定跟踪是否准确地反映了总根长度 (n = 23, 如在一个盒子里的植物在实验中死了)。扫描的总根长度与盒背面的追踪根长度显著相关 (<strong class="xfig…

Discussion

本协议中描述的 rhizoboxes 可用于回答根和根际科学中的各种问题, 并在别处发现了不同用途10、20、21、22、23,24,25. 其他研究人员捕获了 rhizoboxes21、25、26</sup…

Declarações

The authors have nothing to disclose.

Acknowledgements

作者希望感谢匿名审阅者对他们的反馈意见, 以及 J.C. 卡希尔和谭宝在开发 rhizobox 协议方面的初步指导。资金由粮食和农业研究基金会、美国农业部 (USDA) 国家粮食和农业研究所、农业试验站项目 CA-D-PLS-2332-H、司法部和加州大学戴维斯分校提供。科学通过 J.S. 的奖学金

Materials

1.27 cm diameter PVC pipe JM Eagle 530048 305 cm per box, cut into lengths as specified in the protocol
PVC side elbows Lasco 315498 2 per box
PVC 90-degree elbows Charlotte PVC 02300 0600 4 per box
PVC T joints Charlotte PVC 02402 0600 4 per box
Extruded acrylic panes TAP Plastics N/A 2 per box, 0.64 cm thick x 40.5 cm wide x 61 cm long
HDPE spacers (sides) TAP Plastics N/A 2 per box, 0.64 cm thick x 2.5 cm wide x 57 cm long
HDPE spacers (bottom) TAP Plastics N/A 1 per box, 0.64 cm thick x 2.5 cm wide x 40.5 cm long
HDPE spacers (patch) TAP Plastics N/A 2 per box, 0.64 cm thick x 3.8 cm wide x 28 cm long
Polyester batting Fairfield #A-X90 2.5 cm x 40.5 cm strip per box
20-thread screws N/A N/A 3.2 cm long, 0.64 cm diameter
Washers N/A N/A 0.64 cm internal diameter
Hex nuts N/A N/A sized to fit the screws
Light deprivation fabric Americover, Inc. Bold 8WB26.5 1 piece 95 cm wide and 69 cm long per box
Sand Quikrete No. 1113
Field soil N/A N/A
Transparencies for tracing FXN FXNT1319100S One per side of the box to be traced
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Referências

  1. Hodge, A. Roots: The Acquisition of Water and Nutrients from the Heterogeneous Soil Environment. Progress in Botany 71. , 307-337 (2010).
  2. Grossman, J. D., Rice, K. J. Evolution of root plasticity responses to variation in soil nutrient distribution and concentration. Evolutionary Applications. 5 (8), 850-857 (2012).
  3. Zhang, H., Forde, B. G. An Arabidopsis MADS box gene that controls nutrient-induced changes in root architecture. Science. 279 (5349), 407-409 (1998).
  4. Hodge, A., Stewart, J., Robinson, D., Griffiths, B. S., Fitter, A. H. Competition between roots and soil micro-organisms for nutrients from nitrogen-rich patches of varying complexity. Journal of Ecology. 88 (1), 150-164 (2000).
  5. Trachsel, S., Kaeppler, S. M., Brown, K. M., Lynch, J. P. Shovelomics: high throughput phenotyping of maize (Zea mays L.) root architecture in the field. Plant and Soil. 341 (1-2), 75-87 (2011).
  6. Rogers, E. D., Monaenkova, D., Mijar, M., Nori, A., Goldman, D. I., Benfey, P. N. X-ray computed tomography reveals the response of root system architecture to soil texture. Plant Physiology. , (2016).
  7. Groleau-Renaud, V., Plantureux, S., Guckert, A. Effect of mechanical constraint on nodal and seminal root system of maize plants. Comptes Rendus De L Academie Des Sciences Serie Iii-Sciences De La Vie-Life Sciences. 321 (1), 63-71 (1998).
  8. Lin, Y., Allen, H. E., Di Toro, D. M. Barley root hair growth and morphology in soil, sand, and water solution media and relationship with nickel toxicity. Environmental Toxicology and Chemistry. 35 (8), 2125-2133 (2016).
  9. Wenzel, W. W., Wieshammer, G., Fitz, W. J., Puschenreiter, M. Novel rhizobox design to assess rhizosphere characteristics at high spatial resolution. Plant and Soil. 237 (1), 37-45 (2001).
  10. Spohn, M., Carminati, A., Kuzyakov, Y. Soil zymography – A novel in situ method for mapping distribution of enzyme activity in soil. Soil Biology and Biochemistry. 58, 275-280 (2013).
  11. Vollsnes, A. V., Futsaether, C. M., Bengough, A. G. Quantifying rhizosphere particle movement around mutant maize roots using time-lapse imaging and particle image velocimetry. European Journal of Soil Science. 61 (6), 926-939 (2010).
  12. Hewitt, E. J. . Sand and Water Culture Methods Used in the Study of Plant Nutrition. , (1966).
  13. Choudhary, M. I., Shalaby, A. A., Al-Omran, A. M. Water holding capacity and evaporation of calcareous soils as affected by four synthetic polymers. Communications in Soil Science and Plant Analysis. 26 (13-14), 2205-2215 (1995).
  14. Bakker, P. A. H. M., Berendsen, R. L., Doornbos, R. F., Wintermans, P. C. A., Pieterse, C. M. J. The rhizosphere revisited: root microbiomics. Frontiers in Plant Science. 4, 2013 (2013).
  15. McNear, D. H. The Rhizosphere – Roots, Soil, and Everything In Between. Nature Education Knowledge. 4 (3), 1 (2013).
  16. Ortas, I. Determination of the extent of rhizosphere soil. Communications in Soil Science and Plant Analysis. 28 (19-20), 1767-1776 (1997).
  17. . Carbon (13C) and Nitrogen (15N) Sample Preparation Available from: https://stableisotopefacility.ucdavis.edu/13cand15nsamplepreparation.html (2018)
  18. Barraclough, D. 15N isotope dilution techniques to study soil nitrogen transformations and plant uptake. Fertilizer research. 42 (1-3), 185-192 (1995).
  19. Belter, P. R., Cahill, J. F. Disentangling root system responses to neighbours: identification of novel root behavioural strategies. AoB PLANTS. 7, (2015).
  20. Nagel, K. A., et al. GROWSCREEN-Rhizo is a novel phenotyping robot enabling simultaneous measurements of root and shoot growth for plants grown in soil-filled rhizotrons. Functional Plant Biology. 39 (11), 891-904 (2012).
  21. Adu, M. O., Yawson, D. O., Bennett, M. J., Broadley, M. R., Dupuy, L. X., White, P. J. A scanner-based rhizobox system enabling the quantification of root system development and response of Brassica rapa seedlings to external P availability. Plant Root. 11, 16-32 (2017).
  22. Neumann, G., George, T. S., Plassard, C. Strategies and methods for studying the rhizosphere-the plant science toolbox. Plant and Soil. 321 (1-2), 431-456 (2009).
  23. Bodner, G., Alsalem, M., Nakhforoosh, A., Arnold, T., Leitner, D. RGB and Spectral Root Imaging for Plant Phenotyping and Physiological Research: Experimental Setup and Imaging Protocols. JoVE (Journal of Visualized Experiments). (126), e56251-e56251 (2017).
  24. Kuchenbuch, R. O., Ingram, K. T. Image analysis for non-destructive and non-invasive quantification of root growth and soil water content in rhizotrons. Journal of Plant Nutrition and Soil Science. 165 (5), 573-581 (2002).
  25. Dresbøll, D. B., Thorup-Kristensen, K., McKenzie, B. M., Dupuy, L. X., Bengough, A. G. Timelapse scanning reveals spatial variation in tomato (Solanum lycopersicum L.) root elongation rates during partial waterlogging. Plant and Soil. 369 (1-2), 467-477 (2013).
  26. Wu, J., et al. RhizoChamber-Monitor: a robotic platform and software enabling characterization of root growth. Plant Methods. 14 (1), 44 (2018).
  27. Rogers, S. W., Moorman, T. B., Ong, S. K. Fluorescent In Situ Hybridization and Micro-autoradiography Applied to Ecophysiology in Soil. Soil Science Society of America Journal. 71 (2), 620-631 (2007).
  28. Eickhorst, T., Tippkötter, R. Detection of microorganisms in undisturbed soil by combining fluorescence in situ hybridization (FISH) and micropedological methods. Soil Biology and Biochemistry. 40 (6), 1284-1293 (2008).
  29. Spohn, M., Kuzyakov, Y. Distribution of microbial- and root-derived phosphatase activities in the rhizosphere depending on P availability and C allocation – Coupling soil zymography with 14C imaging. Soil Biology and Biochemistry. 67, 106-113 (2013).
  30. Lv, G., Kang, Y., Li, L., Wan, S. Effect of irrigation methods on root development and profile soil water uptake in winter wheat. Irrigation Science. 28 (5), 387-398 (2010).
  31. Asseng, S., Ritchie, J. T., Smucker, A. J. M., Robertson, M. J. Root growth and water uptake during water deficit and recovering in wheat. Plant and Soil. 201 (2), 265-273 (1998).
  32. Hernandez-Ramirez, G., et al. Root Responses to Alterations in Macroporosity and Penetrability in a Silt Loam Soil. Soil Science Society of America Journal. 78 (4), 1392-1403 (2014).
  33. Zhang, Y. L., Wang, Y. S. Soil enzyme activities with greenhouse subsurface irrigation. Pedosphere. 16 (4), 512-518 (2006).
  34. Robinson, D., Hodge, A., Griffiths, B. S., Fitter, A. H. Plant root proliferation in nitrogen-rich patches confers competitive advantage. Proceedings of the Royal Society of London B: Biological Sciences. 266 (1418), 431-435 (1999).
  35. Lobet, G., Draye, X. Novel scanning procedure enabling the vectorization of entire rhizotron-grown root systems. Plant Methods. 9, 1 (2013).
  36. Swarup, R., Wells, D. M., Bennett, M. J. Root Gravitropism. Plant Roots: The Hidden Half. , (2013).
  37. Smit, A. L., Bengough, A. G., Engels, C., van Noordwijk, M., Pellerin, S., van de Geijn, S. C. . Root Methods: A Handbook. , (2000).
  38. van Dusschoten, D., et al. Quantitative 3D Analysis of Plant Roots Growing in Soil Using Magnetic Resonance Imaging1[OPEN]. Plant Physiology. 170 (3), 1176-1188 (2016).
  39. Metzner, R., et al. Direct comparison of MRI and X-ray CT technologies for 3D imaging of root systems in soil: potential and challenges for root trait quantification. Plant Methods. 11, 17 (2015).

Tags

Keywords: RhizoboxRoot GrowthLocalized NutrientsRoot And Rhizosphere ScienceNon-invasive MeasurementsSoilAcrylic PanelsPolyester PaddingPolyethylene SheetsPVC FrameSoil-sand SubstrateNitrogen Source
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Schmidt, J. E., Lowry, C., Gaudin, A. C. An Optimized Rhizobox Protocol to Visualize Root Growth and Responsiveness to Localized Nutrients. J. Vis. Exp. (140), e58674, doi:10.3791/58674 (2018).
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