An image analysis for determination of genetic purity of different wheat varieties

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E. Nasrollahi, G. Tohidloo, S. Mobasser, M. Dehghanshoar, F. Khazaei, M. Mohammadpour [email protected] An image analysis for determination of genetic purity of different wheat varieties

Transcript of An image analysis for determination of genetic purity of different wheat varieties

E. Nasrollahi, G. Tohidloo, S. Mobasser, M. Dehghanshoar, F. Khazaei, M. [email protected]

An image analysis for determination of genetic purity of different wheat varieties

First: A view over the prevalent methods Second: Introducing a new method for genetic purity determination Finally: considering validation test of the new method

Seed quality

Seed quality factors are as follows:

Improved varieties

Genetic purity

Physical purity

Seed viability

Seed vigor

Seed health

Seed moisture

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Genetic PurityGenetic purity refers to the percentage of contamination by seeds or genetic material of other varieties or species.

seedquest.com4

Prevalent methods for determination of genetic purity are as follows: 1. Control plot method2. Phenol test 3. Molecular method

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Off-type

1 .Control plot method

Disadvantages Advantages

High cost Relatively precise

Time consuming No special equipment

Labor force

Expert

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Disadvantages Advantages

Low precision Rapid

Expert Low cost

More human errors No special equipment

2. Phenol test

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3 .Molecular method

Disadvantages Advantages

High cost Rapid

Complicated and difficult Precise More experience

Advanced equipment

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We are looking for a technique to:• Increase test precision• Decrease the costs • Reduce human errors• And save time

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Biochemical and Imaging Technique for Analysis (BITA)This technique is developed by the present research for determination of genetic purity

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Genetic purity testing procedures based on the BITA-technique

1- Seed sampling

BITA-technique procedure

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2- Washing and Soaking the seeds The seeds were washed and soaked in distilled water and kept at room temperature for 20 h.

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3- Seed replacement and adding Phenol Each seed was placed in 0.5mL tube. Then, 400 µL of 1% phenol solution was added to the tube and kept at room temperature

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16Color differentiation of different varieties posed to phenol

Biochemical principle of phenol reaction

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Intensity of color depends on amount of phenoloxidase enzyme

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4- Sampling of phenol solution 250µL of solution was added to Elisa testwells and aniline blue solution was used as index points.

index points19

5- Image capturing of the platesThe plates were placed on Hp Scanner (G4050) and images were captured.

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Image Processing and Analysis

BITA-Image Analyzer :

BITA developed in this study run under MATLAB

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6- Image processing:6-1- Automatically Rotating of the imagesThe images were rotated automatically and kept in correct position using Pattern of index points.

index point

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6-2- Identification of test wells center: Identification of the center of all test wells and making a circle with 200 pixel area for each center of test well

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6-3- Converting RGB to HSV mode:RGB color mode converted to HSV mode and the colors of pixels were averaged.

(RGB)RED, GREEN and BLUE

(HSV)HUE, SATURATION and VALUE

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Colors classification

classification of colors and

determination of their percentage

EXAMPLE FIGURE

7 -Image Analysis

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Validation ofBITA-TECHNIQUE and BITA-IMAGE ANALYZER

A B

A+B

AB1A=10%B=90%

AB2 A=15%B=85%

AB3 A=20%B=80%

R4R3R2R1R4R3R2R1R4R3R2R1

Validation of BITAValidation of the software and methodExperimental material: two samples of nucleus seed class of wheat were mixed in 10, 15 and 20% each with 4 replications

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AB110% impurity

AB215% impurity

AB320% impurity

R1 R2 R3 R4

R1 R2 R3 R4

R1 R2 R3 R4

Software input

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AB1 Software result

Expected 10%Observed 10.5%

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AB2 Software result

Expected 15%Observed 15%

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AB3 Software result

Expected 20%Observed 20%

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AB1/R4

This color shows impurity of a variety

Off types were also distinguished and recorded

Variety A

Variety B

unknown variety  

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BITA-techniquePros and cons

(advantages and disadvantages)

Disadvantages Advantages

Required Software Rapid(It takes 3 days)

Precise

Simplifies the performance and easy to use

Low cost

No special equipment

Low human errors

keeping data and retrieval of the test information

BITA-technique

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Rapid Precise Simplifies the performance

Easy to use cost special

equipmenthuman errors

keeping data

Required Software

Control plot method o

Phenol test

Molecular method o o

BITA + + + + + + + +

+ + +++++++

+ + +

+ :Positive point - : negative point O : Relative point

- - - -- -

--

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AdvantagesMethods

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• BITA software and method can be used for determination of genetic purity in wheat varieties

• The method has more advantages in comparison with prevalent methods

• BITA Software can keep the data and retrieval of the test information

• Despite the efficiency of this method, it should be validate by ISTA laboratories as well

Conclusion

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Validation needs ISTA member laboratories corporation

Thank you!