M.Sc course seminar On the topic

Supervisor : Prof. J. P Shahi Name : Kumari ShikhaI.D. NO. G-15096 M.Sc (Ag) ,final year

Department of Genetics and plant breedingBanasars Hindu University

Varanasi-2210055/24/2017 1

CONTENTS Introduction

Importance

Imaging technology

Gloss of modern technologies

Traits phenotyped

Relevance

Data management

conclusion

3-D ImagingFar –infrared imagingNIR ImagingFluorescence imagingAutomated watering and sprayingHyperspectral imagingVisible imaging

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Plant High-throughput Phenotyping

• Phenomics term given by Gerlai, 2002.

• The term phenomic refers to sum total of phenotypes

at various levels ranging from molecules to organs

and the whole organism.

• Study of plant growth ,architecture, performance and

composition using high throughput methods of data

acquisition and analysis.

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Continue..

• Phenotyping is essential for –

functional analysis of specific genes

forward and reverse genetic analysis

production of elite plants

• High throughput is essential for phenotyping in

different growth conditions of many different lines.

mutant populations

mapping population

breeding population

germplasm collection 5/24/2017 4

Forward phenomics vs Reverse phenomics

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Why phenotyping needed?

Need more food

By 2050, 9.1 billion people Efficient breeding technique required

Tilman, et al5/24/2017 6

Accelerating Plant Phenomics

High-throughput screens

Multiple camera units

Non-destructive measurements

Quantitative analysis

Monitor gowth dynamics

Stress assessment

Link to genomics

Opening new prospects

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Automated Facilities for HTP

Australian plant phenotypingnetworking

ICAR-IIHR5/24/2017 8

Imaging Technique

3-D imaging

Near infrared imaging

Far infrared imaging

Flourescence imaging

Visible light scanning

Automated spraying and watering

High throughput Plant Phenotyping1486203666711

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3-Dimensional imaging

SIDE VIEW SIDE VIEW TOP VIEW

After 90 degree rotation

Digital photos of the top and side view of a plant combined into 3D image.

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Measurements taken using 3-D

• Shoot mass

• Leaf number, shape and angle

• Leaf colour

• Leaf health

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Far-infrared Imaging

• Measure temperature difference between leaves and plants.

• Temperature ranges between 15-1000 micrometer.

• Cooler plants absorb more water

• Can be used for single plant to whole plant

• Temperature differences used to measure:-

Photosynthetic activity

Salinity tolerance

Effective water use efficiency

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Near- infrared imaging

• More the presence of chlorophyll more will be

reflectance in NIR range.

• Facilitates estimation of

Water content and movement within leaves and soil

Carbohydrate content of leaves

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Spongy layer present backside of leaves that reflects lot of light in NIR range,after dehydration or stress condition this spongy layer get collapsed hencereflect less NIR light but same visible range. Thus , we can differentiate healthyplant from sickly plant.

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Fluorescence imaging

Measure photosynthesis ratebiotic and abiotic stress responsesChlorophyll content.

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Visible light scanning (VIS)

• Colour information give estimation of the degreeof senescence.

• Senescence of older leaves during drought suggestan escape or avoidance.

• Genotypes with stay-green type can be identifiedthat would be able to continue photosynthesisunder water stress

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Continued..

Measure aspects of plant architecture such as

• Image-based projected biomass, leaf area, colour, growth dynamics, seedling vigour, seed morphology,

• Root architecture, leaf disease severity assessments, yield, and fruit number and distribution.

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Leaf growth decreases in response to drought even before a decline in stomatal conductance or photosynthesis.

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Hyperspectral Imaging

• Spectral reflectance is the fraction of light reflected

by non transparent surface.

• Researchers use this spectral reflectance fordetection of plants stressed by saline soil ordrought, well before it can be eye.

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Positron emission tomography(PET)

• Nuclear imaging system

• Produces 3-D image of a functional process

• While consumption of co2 , transport of

radiolabelled carbon imaged in 3-D by PET.

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Root Phenotyping

• Different procedure-

Visualization of excavated root system.

Analysis by camera systems which are introduced into

soil through small tubes made up of Plexiglass (changes in

electrical properties of soil due to water uptake by soil).

2-D and 3-D analysis

Phenotyping platforms using aeroponic or hydroponic

culture systems for direct visualization and imaging of roots.

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Steps used in the analysis ofroot morphology.Step 1: soil coring;

Step 2: dividing soil samples

Step 3: washing of roots

Step 4: separating roots fromsoil

Step 5: preserving roots in 25%ethanolStep 6: scanning roots using rootscanner Epson Perfection

step 7 analyzing the scannedroot images using Winrhizosoftware.

2-D Phenotyping of root

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3-D PHENOTYPING

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Rhizoponics is an innovative design of hydroponic rhizotronsadapted to Arabidopsis thaliana. The setup allows tosimultaneously characterize the RSA and shoot development fromseedling to adult stages, i.e. from seed to seed. This system offersthe advantages of hydroponics such as control of rootenvironment and easy access to the roots for measurements orsampling. Being completely movable and low cost, it can be usedin controlled cabinets.

Hydroponic

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Measurement of leaf area

• Morphometric method

• Optical flow method

• Particle /marker tracking method

Plant biomass estimation

• 3-digital imaging technique

• Hyperspectral imaging

• Non-optical method (electrical determination of water content of plant , portable nuclear magnetic resonance device)

Seed and fruit phenotyping

• 3-D Laser –scanning technology

• Visual imaging

• NIR Spectroscopy5/24/2017 25

Analysis of root system

• Rootreader 2D

• Smartreader

• Rootreader 3D

Analysis of shoot system

• Hyperspectral imaging

• Visible imaging

Analysis of chemical content

• Mass spectrometry and Gas chromatography (amino acids present in fresh plant material )

• Liquid chromatography

• Flow cytometry

• NIR Spectroscopy5/24/2017 26

Analysis of physiological parameters

• SPAD chlorophyll meter

• Fluorescent imaging (chlorophyll fluorescence show negative co-relation with photosynthetic activity)

Assessment of water use

• CID (carbon isotope discrimination technique used in wheat)

• Leaf and canopy temperature ( higher in case of decreased transpiration rate)

• SPAD chlorophyll meter

Assessment of soil water content

• Mobile NIR

• Visual spectrophotometer

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Relevance of HTP

• Identification of stress.

• Rapid and efficient screening for mutants.

• Detection and monitoring of disease epidemics in field.

• Detection of root attack by pathogens.

• Facilitate screening of germplasm.

• Study of various physiological processes.

• Modelling of biomass production.

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continued..

Facilitate selection of superior genotypes from breeding population.

Allow huge genomic information to be reliably related to specific phenotypes.

Permit systematic study of pleiotropic effects of the genes.

Crop improvement .

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Data management

Phenomic data management involves three critical components:

ALGORITHM AND PROGRAM

PHENOTYPIC INFORMATION

SENSORY DATA

MODEL DEVELOPMENT

GENOTYPE AND PHENOTYPE

INTERACTIONSUNDERSTAND

MANAGEMENT DATABASES

RESOURCE DEVELOPMENT AND RESOURCE SHARING

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Continued

Some suggestion :-

Creating complete and accurate metadata

Deposition of data into primary repository

Easily Accessible to every researchers

Development of open source community database

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Software companies

• Some of these companies encourage co-development (system customization, softwaredevelopment for computation) as a process ofimproving their current product and productutilization such as Lemnashare and Lemnalauncher etc.

PHENOSPEX5/24/2017 32

Case study

• GiNA platform developed for phenotyping of horticultural crops. For small fruits such as grapes, cranberries, or cherries a picture of 40 fruits can be taken every minute (or less). Therefore, in an hour, at least 20 different parameters for 2400 fruits can be accurately measured from 60 images.

LENGTH , WIDTH , PERIMETER AREA , COLOR ESTIMATED

POTATO AND CHERRY FRUIT5/24/2017 33

Access genotypic adaptation to water stress

Thermography

Leaf temperature detection byinfrared thermometers hasbeen used to detect waterstress, which results instomatal closure and anincrease in temperaturethrough decreased adiabaticcooling

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a): Excavated maize root stocks which were split lengthwiseb): imaged root stock under constant illuminationc): root top angled): gap size distribution e): cluster thickness distribution Colombi et al.2015

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This high-throughput phenotypingsystem developed at the USDA Arid-Land Agricultural Research Center inMaricopa, AZ is being used to collectplant height, canopy temperature, andcanopy reflectance data from cottonplants.

Researchers at CSIRO using aremote-controlled gas-poweredmodel helicopter called the“phenocopter” to measure plantheight, canopy cover, lodging, andtemperature throughout a day.

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Future of HTP

• Current phenotyping is largely extensive hence need for intensive approach.

• Available methods are not satisfactory , hence urgent need to develop suitable statistical models.

• Software developed for statistical analysis should permit automated data analysis.

• Integration of all phenomic related research as did for genomic efforts.

• Phenomics teams must be transdisciplinary.

Bilder et al. 2009

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Phenotyping

Conclusion

Identifying differences among genotypes andinterpretation of detected differences.

(Fiorani and Schurr, 2013; Klukaset al., 2014)

Analytical approach

Management

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Thankyou5/24/2017 39