Whole‐Plant Manual and Image‐Based Phenotyping in Controlled Environments

Erica Agnew1, Adam Bray1, Eric Floro1, Nate Ellis1, John Gierer1, César Lizárraga1, Darren O'Brien1, Madeline Wiechert1, Todd C. Mockler1, Nadia Shakoor1, Christopher N. Topp1

1 Donald Danforth Plant Science Center, St. Louis, Missouri
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20044
Online Posting Date:  March, 2017
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Abstract

Phenotypic measurements and images of crops grown under controlled‐environment conditions can be analyzed to compare plant growth and other phenotypes from diverse varieties. Those demonstrating the most favorable phenotypic traits can then be used for crop improvement strategies. This article details a protocol for image‐based root and shoot phenotyping of plants grown in the greenhouse to compare traits among different varieties. Diverse maize lines were grown in the greenhouse in large 8‐gallon treepots in a clay granule substrate. Replicates of each line were harvested at 4 weeks, 6 weeks, and 8 weeks after planting to capture developmental information. Whole‐plant phenotypes include biomass accumulation, ontogeny, architecture, and photosynthetic efficiency of leaves. Image analysis was used to measure leaf surface area and tassel size and to extract shape variance information from complex 3D root architectures. Notably, this framework is extensible to any number of above‐ or below‐ground phenotypes, both morphological and physiological. © 2017 by John Wiley & Sons, Inc.

Keywords: whole‐plant; phenotypes; image‐based analysis; shoots; roots

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Planting
  • Basic Protocol 2: Preharvest (Non‐Destructive) Shoot Phenotyping
  • Basic Protocol 3: Destructive Harvesting and Phenotyping
  • Basic Protocol 4: Imaging Shoots and Roots
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Planting

  Materials
  • Seeds
  • Turface
  • Tempered water (i.e., water fixed at a moderate temperature [∼80°F])
  • Fertilizer water (e.g., Jack's Professional LX 15‐5‐15 Calcium + Magnesium Water‐Soluble Fertilizer diluted to 174 ppm elemental nitrogen)
  • Plug trays
  • 8‐gallon treepots
  • Greenhouse
  • Aluminum mesh window screen (12 in. × 12 in.) for bottom of treepot
  • Pot liners (optional)
  • Stakes (for plant support)
  • Barcoded identification labels (or similar)
  • Non‐adhesive, grip‐type shelf liner
  • Drip irrigation system
  • Irrigation picks

Basic Protocol 2: Preharvest (Non‐Destructive) Shoot Phenotyping

  Materials
  • Plant of interest
  • Ladder or step stool
  • Calipers
  • Meter stick or measuring tape
  • Chlorophyll meter (e.g., CCM‐200 chlorophyll content meter)
  • FluorPen (e.g., Photon System Instruments)
  • Porometer (e.g., Decagon SC‐1 Leaf Porometer)

Basic Protocol 3: Destructive Harvesting and Phenotyping

  Materials
  • Plant of interest
  • Shears/clippers to cut plant stalks
  • Labels and push pins (to affix labels to roots)
  • Brown paper bags (to dry down and store plant material)
  • Screen‐bottomed bin to wash root systems
  • Water hose and spray nozzles
  • Sturdy greenhouse bench to support and raise screen‐bottomed bin and allow flow through of water
  • Garbage bin on wheels to remove Turface removed from treepots
  • Pre‐printed data sheet or tablet to record data
  • Scale
  • Large weigh boats
  • Mesh bags for root storage

Basic Protocol 4: Imaging Shoots and Roots

  Materials
  • Shoots and roots of interest
  • Table for imaging surface to bring plants up to comfortable working height
  • Black muslin cloth for backdrop
  • Extendable tripod (e.g., Oben AC‐2361 3‐Section Aluminum Tripod Legs) with adjustable arm (e.g., Photek TRI‐X‐2500 Tripod Extender)
  • Digital camera (with ability to manually change focus and light settings; e.g., Canon EOS Rebel SL1 DSLR Camera)
  • Lens with ∼18 to 55 mm zoom (e.g., Canon EF‐S 17‐85mm f/4‐5.6 IS USM Lens)
  • Lights with flash and sync capability (e.g., Impact Two Monolight Kit)
  • Computer or tablet capable of running the native camera software of camera choice
  • Storage media: memory cards for camera and external hard drive for backup of images
  • Color and size markers: color card and white poker chip
  • Water bin (e.g., water in a large, black concrete mixing tub) or fabric for imaging roots
  • Prop to hold label when imaging roots (e.g., elevated, black placeholder)
  • Plant phenotyping software (e.g., PlantCV available at http://plantcv.danforthcenter.org/)
  • Post‐imaging and analysis software (e.g., DIRT available at dirt.iplantcollaborative.org)
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Figures

Videos

Literature Cited

Literature Cited
  Das, A., Schneider, H., Burridge, J., Ascanio, A.K.M., Wojciechowski, T., Topp, C.N., Lynch, J.P., Weitz, J.S., and Bucksch, A. 2015 Digital imaging of root traits (DIRT): A high‐throughput computing and collaboration platform for field‐based root phenomics. Plant Methods 11:51‐51. doi: 10.1186/s13007‐015‐0093‐3.
  Fahlgren, N., Feldman, M., Gehan, M.A., Wilson, M.S., Shyu, C., Bryant, D.W., Hill, S.T., McEntee, C.J., Warnasooriya, S.N., Kumar, I., Ficor, T., Turnipseed, S., Gilbert, K.B., Brutnell, T.P., Carrington, J.C., Mockler, T.C., and Baxter, I. 2015. A versatile phenotyping system and analytics platform reveals diverse temporal responses to water availability in setaria. Mol. Plant 8:1520‐1535. doi: 10.1016/j.molp.2015.06.005.
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