X-ray, multispectral and chlorophyll fluorescence images: innovative methods for evaluating the physiological potential of rice seeds

Image analysis techniques are expanding in agriculture and, for being fast, simple, and not destroying samples, they are interesting alternatives in the detection of immature rice seeds. Therefore, the aim of this study was to evaluate the quality of rice seeds using X-ray, multispectral and chlorophyll fluorescence image analysis techniques. Initially, with the seeds identified and numbered, radiographic images were obtained to determine the free space (area between the endosperm + embryo and the glumes). Subsequently, with the same seeds used in the X-ray, multispectral and chlorophyll fluorescence images were acquired, and then the computerized seedling analysis was performed with the SVIS® software. It was concluded that rice seeds that do not germinate or originate abnormal seedlings have free space equal to or greater than 18.68%, higher reflectance in the spectral bands with wavelength from 590 nm to 780 nm (41.46% to 64.21%, respectively) and in the band of 970 nm (75.65%), in addition to having chlorophyll fluorescence values equal to or greater than 40.58 and 112.92 at excitation/emission energies of 630/700 nm and 645/700 nm, respectively.

Index terms:
free space; immature seeds; Oryza sativa L.; seed vigor; spectral reflectance

Authorship

Artur Sousa Silva ^**Corresponding author E-mail: artursilva.agro@gmail.com

Universidade de São Paulo (USP), Escola Superior de Agricultura “Luiz de Queiroz” - 13418-900, Piracicaba, São Paulo, Brasil.Universidade de São PauloBrazilPiracicaba, São Paulo, BrazilUniversidade de São Paulo (USP), Escola Superior de Agricultura “Luiz de Queiroz” - 13418-900, Piracicaba, São Paulo, Brasil.

https://orcid.org/0000-0001-6961-5781

Silvio Moure Cicero

https://orcid.org/0000-0002-0676-4007

Fabiano França da Silva

https://orcid.org/0000-0002-3805-0084

Francisco Guilhien Gomes-Junior

https://orcid.org/0000-0001-9620-6270

*Corresponding author
E-mail: artursilva.agro@gmail.com

SCIMAGO INSTITUTIONS RANKINGS

Figures

Figures (9)

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Figure 1
Flowchart of the analyses performed: positioning of the seeds of each replication in Petri dishes (1); acquisition of X-ray images (2) and multispectral and chlorophyll fluorescence images with VideometerLab instrument (3); acquisition of chlorophyll ‘a’ fluorescence images with the SeedReporter^® instrument (4); computerized analysis of seedling images (SVIS^®) (5).

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Figure 2
Variables obtained with X-ray image analysis: free space (A), seed area (B) and relative density (C); with chlorophyll fluorescence images of 620/730 nm - excitation/emission energy: chlorophyll fluorescence (D), chlorophyll index (E) and anthocyanin index (F), of seeds from six lots of the rice cultivar BRS Catiana. Different letters above each column, for each evaluation, indicate that the lots belong to different groups by the Scott-Knott test (p < 0.05). Bars represent the standard deviation.

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Figure 3
Multispectral analyses, corresponding to 22 spectral bands (365, 405, 430, 450, 470, 490, 515, 540, 570, 590, 630, 645, 660, 690, 780, 850, 880, 940, 970, 630/700, 645/700, 660/700 nm, respectively) with individual and color charts (CIELab L, CIELab a* and CIELab b*) of six lots of BRS Catiana rice seeds. Different letters above the columns, for each evaluation, indicate that the lots belong to different groups by the Scott-Knott test (p < 0.05). Bars represent the standard deviation.

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Figure 4
Germination (A), first germination count (B), accelerated aging (C), electrical conductivity (D), seedling emergence in sand (E), seedling length (F), vigor index (G), development uniformity index (H) of seeds from six lots of the rice cultivar BRS Catiana. Different letters above the columns, for each evaluation, indicate that the lots belong to different groups by the Scott-Knott test (p < 0.05). Bars represent the standard deviation.

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Figure 5
A: Biplot of principal component analysis obtained from the variables related to X-ray, multispectral, chlorophyll fluorescence and physiological analyses of seeds of the rice cultivar BRS Catiana. PC1 - Principal Component 1; PC2 - Principal Component 2. B: Estimates of Pearson’s correlations between the multiple characteristics of X-ray, multispectral, chlorophyll fluorescence and physiological analyses of six lots under study. FGC - first germination count; AA - accelerated aging; EC - electrical conductivity; Emergence - seedling emergence in sand; SL - seedling length; Uniformity - development uniformity index; Vigor - vigor index.

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Figure 6
Images of seeds of the rice cultivar BRS Catiana with similar mean values of chlorophyll ‘a’ fluorescence at 620/730 nm (1a, 2a and 3a), chlorophyll ‘a’ fluorescence at 645/700 nm (1b, 2b and 3b) and chlorophyll ‘b’ fluorescence at 660/700 nm (1c, 2c and 3c), originating dead seed (1d) and seedlings with different lengths (2d and 3d).

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Figure 7
Mean spectral intensity of 19 wavelengths (nm) for seeds that did not germinate or originated abnormal seedlings and seeds that generated normal seedlings of the rice cultivar BRS Catiana. ** indicates significant difference between non-germinated and germinated seeds by the Scott-Knott test (p < 0.05).

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Figure 8
Differences between seeds that did not germinate or originated abnormal seedlings and seeds that generated normal seedlings of the rice cultivar BRS Catiana, in chlorophyll fluorescence of the red region - 630/700 nm (A), chlorophyll ‘a’ fluorescence - 620/730 nm (D) and percentage of free space in the internal cavity (E). ** indicates a significant difference between seeds not germinated and germinated by the F test (p < 0.05). NS indicates that there is no significant difference. Green line represents the mean. The bars represent the standard deviation.

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Figure 9
Images of seeds of the rice cultivar BRS Catiana: seed 1 - with low percentage of free space in the internal cavity, measured from radiographic image (A) and low mean values of chlorophyll ‘a’ fluorescence at 620/730 nm (B), chlorophyll ‘a’ fluorescence at 645/700 nm (C) and chlorophyll ‘b’ fluorescence at 660/700 nm (D), and 19 spectral bands using the ‘Inverse jet’ filter (VideometerLab4® software), with the respective histogram of reflectance (E), originating a normal seedling (F); seed 2 - with high percentage of free space in the internal cavity, measured from radiographic image (G) and high mean values of chlorophyll ‘a’ fluorescence at 620/730 nm (H), chlorophyll ‘a’ fluorescence at 645/700 nm (I) and chlorophyll ‘b’ fluorescence at 660/700 nm (J), and 19 spectral bands using the ‘Inverse jet’ filter (VideometerLab4^® software), with the respective histogram of reflectance (K), originating a dead seed (L).

Figure 1 Flowchart of the analyses performed: positioning of the seeds of each replication in Petri dishes (1); acquisition of X-ray images (2) and multispectral and chlorophyll fluorescence images with VideometerLab instrument (3); acquisition of chlorophyll ‘a’ fluorescence images with the SeedReporter^® instrument (4); computerized analysis of seedling images (SVIS^®) (5).

Figure 2 Variables obtained with X-ray image analysis: free space (A), seed area (B) and relative density (C); with chlorophyll fluorescence images of 620/730 nm - excitation/emission energy: chlorophyll fluorescence (D), chlorophyll index (E) and anthocyanin index (F), of seeds from six lots of the rice cultivar BRS Catiana. Different letters above each column, for each evaluation, indicate that the lots belong to different groups by the Scott-Knott test (p < 0.05). Bars represent the standard deviation.

Figure 3 Multispectral analyses, corresponding to 22 spectral bands (365, 405, 430, 450, 470, 490, 515, 540, 570, 590, 630, 645, 660, 690, 780, 850, 880, 940, 970, 630/700, 645/700, 660/700 nm, respectively) with individual and color charts (CIELab L, CIELab a* and CIELab b*) of six lots of BRS Catiana rice seeds. Different letters above the columns, for each evaluation, indicate that the lots belong to different groups by the Scott-Knott test (p < 0.05). Bars represent the standard deviation.

Figure 4 Germination (A), first germination count (B), accelerated aging (C), electrical conductivity (D), seedling emergence in sand (E), seedling length (F), vigor index (G), development uniformity index (H) of seeds from six lots of the rice cultivar BRS Catiana. Different letters above the columns, for each evaluation, indicate that the lots belong to different groups by the Scott-Knott test (p < 0.05). Bars represent the standard deviation.

Figure 5 A: Biplot of principal component analysis obtained from the variables related to X-ray, multispectral, chlorophyll fluorescence and physiological analyses of seeds of the rice cultivar BRS Catiana. PC1 - Principal Component 1; PC2 - Principal Component 2. B: Estimates of Pearson’s correlations between the multiple characteristics of X-ray, multispectral, chlorophyll fluorescence and physiological analyses of six lots under study. FGC - first germination count; AA - accelerated aging; EC - electrical conductivity; Emergence - seedling emergence in sand; SL - seedling length; Uniformity - development uniformity index; Vigor - vigor index.

Figure 6 Images of seeds of the rice cultivar BRS Catiana with similar mean values of chlorophyll ‘a’ fluorescence at 620/730 nm (1a, 2a and 3a), chlorophyll ‘a’ fluorescence at 645/700 nm (1b, 2b and 3b) and chlorophyll ‘b’ fluorescence at 660/700 nm (1c, 2c and 3c), originating dead seed (1d) and seedlings with different lengths (2d and 3d).

Figure 7 Mean spectral intensity of 19 wavelengths (nm) for seeds that did not germinate or originated abnormal seedlings and seeds that generated normal seedlings of the rice cultivar BRS Catiana. ** indicates significant difference between non-germinated and germinated seeds by the Scott-Knott test (p < 0.05).

Figure 8 Differences between seeds that did not germinate or originated abnormal seedlings and seeds that generated normal seedlings of the rice cultivar BRS Catiana, in chlorophyll fluorescence of the red region - 630/700 nm (A), chlorophyll ‘a’ fluorescence - 620/730 nm (D) and percentage of free space in the internal cavity (E). ** indicates a significant difference between seeds not germinated and germinated by the F test (p < 0.05). NS indicates that there is no significant difference. Green line represents the mean. The bars represent the standard deviation.

Figure 9 Images of seeds of the rice cultivar BRS Catiana: seed 1 - with low percentage of free space in the internal cavity, measured from radiographic image (A) and low mean values of chlorophyll ‘a’ fluorescence at 620/730 nm (B), chlorophyll ‘a’ fluorescence at 645/700 nm (C) and chlorophyll ‘b’ fluorescence at 660/700 nm (D), and 19 spectral bands using the ‘Inverse jet’ filter (VideometerLab4® software), with the respective histogram of reflectance (E), originating a normal seedling (F); seed 2 - with high percentage of free space in the internal cavity, measured from radiographic image (G) and high mean values of chlorophyll ‘a’ fluorescence at 620/730 nm (H), chlorophyll ‘a’ fluorescence at 645/700 nm (I) and chlorophyll ‘b’ fluorescence at 660/700 nm (J), and 19 spectral bands using the ‘Inverse jet’ filter (VideometerLab4^® software), with the respective histogram of reflectance (K), originating a dead seed (L).

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