PhenoPhyte: a flexible affordable method to quantify 2D phenotypes from imagery

BackgroundAccurate characterization of complex plant phenotypes is critical to assigning biological functions to genes through forward or reverse genetics. It can also be vital in determining the effect of a treatment, genotype, or environmental condition on plant growth or susceptibility to insects or pathogens. Although techniques for characterizing complex phenotypes have been developed, most are not cost effective or are too imprecise or subjective to reliably differentiate subtler differences in complex traits like growth, color change, or disease resistance.ResultsWe designed an inexpensive imaging protocol that facilitates automatic quantification of two-dimensional visual phenotypes using computer vision and image processing algorithms applied to standard digital images. The protocol allows for non-destructive imaging of plants in the laboratory and field and can be used in suboptimal imaging conditions due to automated color and scale normalization. We designed the web-based tool PhenoPhyte for processing images adhering to this protocol and demonstrate its ability to measure a variety of two-dimensional traits (such as growth, leaf area, and herbivory) using images from several species (Arabidopsis thaliana and Brassica rapa). We then provide a more complicated example for measuring disease resistance of Zea mays to Southern Leaf Blight.ConclusionsPhenoPhyte is a new cost-effective web-application for semi-automated quantification of two-dimensional traits from digital imagery using an easy imaging protocol. This tool’s usefulness is demonstrated for a variety of traits in multiple species. We show that digital phenotyping can reduce human subjectivity in trait quantification, thereby increasing accuracy and improving precision, which are crucial for differentiating and quantifying subtle phenotypic variation and understanding gene function and/or treatment effects.

[1]  P. Benfey,et al.  Imaging and Analysis Platform for Automatic Phenotyping and Trait Ranking of Plant Root Systems1[W][OA] , 2010, Plant Physiology.

[2]  K. Hiratsu,et al.  Dominant repression of target genes by chimeric repressors that include the EAR motif, a repression domain, in Arabidopsis. , 2003, The Plant journal : for cell and molecular biology.

[3]  Michael D. Abràmoff,et al.  Image processing with ImageJ , 2004 .

[4]  T. Mitchell-Olds,et al.  Erratum: Induced plant defense responses against chewing insects. Ethylene signaling reduces resistance of arabidopsis against Egyptian cotton worm but not diamondback moth (Plant Physiology (2000) 124 (1007-1017)) , 2001 .

[5]  P. Ryser,et al.  Consequences of phenotypic plasticity vs. interspecific differences in leaf and root traits for acquisition of aboveground and belowground resources. , 2000, American journal of botany.

[6]  Eric J. W. Visser,et al.  Abramoff MD, Magalhaes PJ, Ram SJ. 2004. Image Processing with ImageJ. Biophotonics , 2012 .

[7]  B. Mueller‐Roeber,et al.  A growth phenotyping pipeline for Arabidopsis thaliana integrating image analysis and rosette area modeling for robust quantification of genotype effects. , 2011, The New phytologist.

[8]  E. Kombrink,et al.  The Hypersensitive Response and its Role in Local and Systemic Disease Resistance , 2004, European Journal of Plant Pathology.

[9]  Cynthia Weinig,et al.  Constraints on the evolution of adaptive plasticity: costs of plasticity to density are expressed in segregating progenies. , 2007, The New phytologist.

[10]  Daniel Parnham,et al.  LeafAnalyser: a computational method for rapid and large-scale analyses of leaf shape variation. , 2008, The Plant journal : for cell and molecular biology.

[11]  Peter J. Bradbury,et al.  Genome-wide association study of quantitative resistance to southern leaf blight in the maize nested association mapping population , 2011, Nature Genetics.

[12]  T. Mitchell-Olds,et al.  Induced plant defense responses against chewing insects. Ethylene signaling reduces resistance of Arabidopsis against Egyptian cotton worm but not diamondback moth. , 2000, Plant physiology.

[13]  M. Nishimura,et al.  EDR2 negatively regulates salicylic acid-based defenses and cell death during powdery mildew infections of Arabidopsis thaliana , 2007, BMC Plant Biology.

[14]  M. Malnoy,et al.  Evaluation of Transgenic ‘Chardonnay’ (Vitis vinifera) Containing Magainin Genes for Resistance to Crown Gall and Powdery Mildew , 2006, Transgenic Research.

[15]  K. Chenu,et al.  PHENOPSIS, an automated platform for reproducible phenotyping of plant responses to soil water deficit in Arabidopsis thaliana permitted the identification of an accession with low sensitivity to soil water deficit. , 2006, The New phytologist.

[16]  L. Lamari Assess 2.0 , 2008 .

[17]  E. Dubois,et al.  Digital picture processing , 1985, Proceedings of the IEEE.

[18]  Mark A. Matthews,et al.  Water deficits accelerate ripening and induce changes in gene expression regulating flavonoid biosynthesis in grape berries , 2007, Planta.

[19]  L. Fraser,et al.  Adaptive phenotypic plasticity of Pseudoroegneria spicata: response of stomatal density, leaf area and biomass to changes in water supply and increased temperature. , 2009, Annals of botany.

[20]  U. Melcher Symptoms of Cauliflower Mosaic Virus Infection in Arabidopsis thaliana and Turnip , 1989, Botanical Gazette.

[21]  G. Loake,et al.  The developmental selector AS1 is an evolutionarily conserved regulator of the plant immune response , 2007, Proceedings of the National Academy of Sciences.

[22]  J. J. Grant,et al.  Targeted activation tagging of the Arabidopsis NBS-LRR gene, ADR1, conveys resistance to virulent pathogens. , 2003, Molecular plant-microbe interactions : MPMI.

[23]  Guido Sanguinetti,et al.  LEAFPROCESSOR: a new leaf phenotyping tool using contour bending energy and shape cluster analysis. , 2010, The New phytologist.

[24]  M. Tester,et al.  Phenomics--technologies to relieve the phenotyping bottleneck. , 2011, Trends in plant science.

[25]  Dan Nettleton,et al.  Spatial analysis of arabidopsis thaliana gene expression in response to Turnip mosaic virus infection. , 2007, Molecular plant-microbe interactions : MPMI.

[26]  J. Trygg,et al.  LAMINA: a tool for rapid quantification of leaf size and shape parameters , 2008, BMC Plant Biology.

[27]  M. Grant,et al.  RIN13 Is a Positive Regulator of the Plant Disease Resistance Protein RPM1w⃞ , 2005, The Plant Cell Online.

[28]  N. Otsu A threshold selection method from gray level histograms , 1979 .

[29]  M. M. Christ,et al.  Dynamics of seedling growth acclimation towards altered light conditions can be quantified via GROWSCREEN: a setup and procedure designed for rapid optical phenotyping of different plant species. , 2007, The New phytologist.