Protocol

 

Authors

A.Anandan, A.Mahender, P.Sanghamitra, S.K.Pradhan

Author Affliations

National Rice Research Institute, Cuttack, Odisha, India

Overview

Grain colour is an important grain quality parameter concerned from the level of scientist to consumers. Seed/grain colour draws attention, affects the quality, nutrition and also related to dormancy. Therefore, high throughput technique has been formalized to measure the colour intensity of grain/seed by using digital image and ImageJ software.

Background

Scaling of grain colour by visual method is not accurate and creates soreness to eyes, likelihood of inconsistent estimation. Determination of colour by quantification is relatively low throughput, labor intensive and requires high cost analytical equipment. Therefore, accurate, high throughput selection of optimal genotypes and interpretable way with simple procedure is essential for plant breeders/ biologist during handling of large number of samples. Techniques will only be feasible when traits involved should favours in identification of traits and should be scored efficiently. Image analysis is getting popular in the field of agriculture as it is related to phenomics. Here an approach has been explained to measure the grain colour by using open source ImageJ software.

Materials/Equipment

– Digital camera and computer
– X-ray illuminator box with required power connection
– ImageJ software (open source: http://rsbweb.nih.gov/ij/download.html)
– Grains of different varieties
– Forceps
– Two fluorescent table lamps with good light intensity

Procedure

Step 1:
– Place the X-ray illuminator box over the convenient platform or table.

Step 2:
– Arrange the 10 – 15 numbers of grains/dehusked kernels of a genotype in equidistance over the X-ray illuminator box in linear fashion with help of forceps.
– Arrange the next genotype in similar fashion by leaving 1.5 to 2.0 cm space between two varieties.
– Similarly, arrange other genotypes in the above-mentioned manner. All the varieties should have uniform number of grains with length of seed arrangement.
– Place two fluorescent table lamps near the X-ray illuminator box to get uniform light around the grain (Under sufficient natural light, lamps can be avoided).
– Now switch on the X-ray illuminator box to get light source from bottom to avoid shade effect of grains from fluorescent table lamps during imaging.
– Next, image the X-ray illuminator box by using the camera as a single image covering all the grains. Then, download image from camera and save to computer for further process.

Step 3:
– Open ImgaeJ software and select open the desired photograph.
– Select the Analyze menu, “Gels” and “Gel analyzer options”. Then check Uncalibrated OD, Label with percentages and Invert peaks.
– Use the rectangular selection tool to outline the grains (10-15 nos.) of first genotype that arranged in linear fashion.
– Then select the Analyze menu, “Gels” and “Select first lane”. The first genotype with rectangular box will be highlighted with number 1.
– Next, move the rectangular selection box right to the next genotype by holding the mouse. Then, select “Select next lane” from Analyze menu, “Gels”.
– Repeat the pervious step for remaining genotypes. After completing the required genotypes, select the Analyze menu, “Gels” and “Plot lanes” to generate the graphical output (plots with peaks).

Step 4:
– Now by using the straight line selection tool, draw base line to close the each peak to define the closed area.
– To access the remaining genotypes, the graphical peak may be scrolled down by using mouse/scrolling tool.
– To measure the size of each peak, click the peak with the Wand tool.
– Now label the peaks in percent value by selecting “Label peaks” from Analyze menu, “Gels”.
– New window opens with results corresponding to the individual grains of all genotypes in “Area” and “Percent”.
– Output related to colour intensity appears as percent. Grains (10-15 nos. used per genotype) used to measure may be averaged to arrive the colour intensity of each genotype.
– This protocol would be more useful for grain/kernels in dark colour such as red, purple, black etc.

Notes and troubleshooting tips

Maintain uniform light throughout the experiment.