- Email: [email protected]
Chemical imaging of intact seeds with NIR focal plane array assists plant breeding
Vibrational Spectroscopy 42 (2006) 215–221 www.elsevier.com/locate/vibspec
Chemical imaging of intact seeds with NIR focal plane array assists plant breeding Virgil W. Smail a,b, Allan K. Fritz c, David L. Wetzel a,b,* a
Microbeam Molecular Spectroscopy Laboratory, Shellenberger Hall, Kansas State University, Manhattan, KS 66506, USA b Department of Grain Science and Industry, Kansas State University, Manhattan, KS 66506, USA c Department of Agronomy, Kansas State University, Manhattan, KS 66506, USA Available online 11 May 2006
Abstract Early generation identification of desirable or undesirable traits of kernels of grain is useful to the breeding program to assist in selection of the best seeds to carry forward and to eliminate the inferior ones. In the case of wheat, resistance to premature sprouting (germination) in the field, before harvest under high moisture conditions is desirable. Visual examination of individual kernels provides non-destructive identification of severities of the problem. The germination process must be so advanced before visual detection is possible that severe damage to starch in the kernel has already occurred from release of ?-amylase enzyme. Several tests exist for the viscosity of solutions or for alpha amylase directly that are done in bulk and are destructive to the individual kernel. Near-IR imaging with an InGaAs focal plane array provides early localized chemical evidence of the process occurring in multiple kernels in the field of view. A Go/No-Go decision is made on individual kernels by the instrument using a specimen mounting plate that defines the position of the diffusely reflecting kernel, while rejecting adjacent pixels as specular reflection away from the detector array. The chemical imaging reported detects sprouting considerably earlier than can be found by both visual inspection and alpha amylase (?-amylase) testing procedures. Images are presented to illustrate the contrast between normal and sprouted specimens. # 2006 Elsevier B.V. All rights reserved. Keywords: Chemical imaging; Near-IR spectroscopy; Wheat breeding
1. Introduction
2. Background
This study reports on the first efforts to use a non-destructive chemical imaging approach to detect early germination in intact kernels of different hard winter wheat varieties. Wheat cultivars that sprout in the field under moist conditions result in economic devastation. Plant breeders need a reliable nondestructive method to allow single kernel selection of germinated versus nongerminated kernels. A non-destructive system for detection of sprouting at early stages in individual kernels of experimental breeding lines is ideal. Focal plane array imaging based on the very near (short wave) infrared spectroscopic region enables subsurface polychromatic contrast to reveal embryo development under experimental conditions.
Preharvest germination or ‘‘sprouting’’, in wheat is a major problem in most wheat production areas with heavy precipitation or humidity just prior to harvest. The 2004 harvest season in Kansas was especially severe and, as reported by the press [1,2], caused an estimated 24% of the entire hard wheat crop to have sprout damage and be discounted to ‘‘feed’’ grade wheat, with a severe economic loss. More than 40% of the hard white winter (HWW) wheat crop was estimated to be sprout damaged. Kansas wheat breeders have recently successfully developed HWW wheat varieties that represent approximately 20% of Kansas acreage. Growers in 2004 had the impression that these varieties suffered more sprout damage than the hard red winter (HRW) wheat varieties. Earlier evidence from a HRW wheat versus HWW wheat comparison in 1999 showed that this may be the case [3]. Improved tolerance to moisture is necessary to protect producer interests and provide a consistent supply of high quality hard white wheat in years when conditions are conducive to
* Corresponding author. Tel.: +1 785 532 4094; fax: +1 785 532 7010. E-mail address: [email protected] (D.L. Wetzel). 0924-2031/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vibspec.2006.02.004
216
V.W. Smail et al. / Vibrational Spectroscopy 42 (2006) 215–221
sprout damage. Progress continues to be made on the genetic improvement of HWW wheat. One of the challenges, however, is efficient selection of more sprout resistant breeding stock. Although some DNA markers are available for this trait since 1993, [4] marker-assisted selection is still relatively expensive and will probably only be feasible to apply at one or two points in the selection process. The development of tools to rapidly separate sprouted and unsprouted kernels would be a tremendous benefit to the breeding process and could conceivably also have future benefit in the market channels. In existing methods, visual (naked eye) inspection is the only present non-destructive method applicable to single kernels. Destructive testing of pooled ground kernels includes viscosity of a wheat flour suspension that is damaged relative to alpha amylase counteraction or via direct determination of ?-amylase using a test kit. Sprout damage is typically measured destructively using a viscoelastic measure (Falling NumberTM) in a slurry of starch damaged flour caused by the action of amylase from germination in the still nonharvested spike of the wheat [5]. Alternatively recently available antibody tests have been developed to measure specific amylase levels in ground wheat kernels. This test uses antibodies specific for ?-amylase extracted from ground kernels. These destructive tests determine the starch damage caused by germination of wheat, and require 1–100 g of ground kernels. All bulk test methods have high standard errors due to significant within spike variation levels of sprouting and sampling errors within the bulk sample [6]. With the proposed imaging procedures, a Go/No-Go decision is made on individual kernels with the instrument using a specimen mounting plate that defines the position of the diffusely reflecting kernel, while rejecting adjacent pixels as specular reflection away from the detector array. The chemical imaging reported detects sprouting considerably earlier than can be found by both visual inspection and alpha amylase (?-amylase) testing procedures. Images are presented to illustrate the contrast between normal and sprouted specimens. 3. Experimental 3.1. Wheat specimens Six 100-g replicates of seed of each variety were germinated for 3, 6, 12, 24, 36, and 48 h on soaked blotter paper in Petri dishes. The kernels were frozen at 80 8C for 24 h to stop all germination processes. The kernels were then freeze dried until no weight change could be detected (48 h) to remove all moisture and stabilize the kernels. The kernels have shown no germination or changes for the past several months. Kernels were then manually placed on the grid sample plates for imaging. In addition to the imaging specimens, 10 g of each replicate wheat specimen were ground in preparation for ?-amylase testing using the WheatRite1 Immune Scanner (C-Qentec Diagnostics, Epping, Australia) antibody side flow test kit. One kilogram samples of six HRW and HWW wheat varieties grown in
Kansas Agricultural Experiment Station plots in 2004 were supplied as follows: Variety
Class
Pedigree
Jagger Danby Trego Betty KS2174 KS89180B-2-1-2
HRW HWW HRW HWW HWW HRW
KS82W418/Stephens Trego/Betty ‘S’ KS87H325/Rio Blanco – IL71-5662/’’PL145’ (PI 00840)//21665 KS8010-73/KS8010-1-4-2/3/107349/ KS8181252//Karl 92
3.2. Instrumentation The Spectral Dimensions MatrixNIRTM Chemical Imaging system (Olney, MD, USA) was used [7,8]. It consisted of a liquid crystal tunable filter [9], a 320 256 pixel indium gallium arsenide (InGaAs) focal plane array detection system, and appropriate optics to preferentially capture diffusely reflected light from each specimen while, minimizing background specular reflection off of the polished mounting plate. Four tungsten source lamps illuminate the imaging target field. ISysTM Chemical Imaging Software was provided by Spectral Dimensions that enables simultaneous manipulation of spectra of 92,000 pixels from a single field of view. Sample mounts were designed and locally custom made for pre-positioning 24 or 30 kernels in the field of view of the imaging system. This device permitted us to scan 15 million spectra in 1 day, thus data acquisition was relatively efficient in comparison to sample preparation and data handling as described in other sections. A dedicated colorimeter (Immune Scanner) with direct readout in ?-amylase units was part of the WheatRite1 test kit. 3.3. Data manipulation Image contrast was maximized by pretreating the raw image response of the focal plane array to remove the specular reflection background, followed by selection of the wavelength or the principle component analysis factor that best accentuated the contrast required to distinguish embryo development within the germ area of each individual kernel. Data pretreatment in all instances involved rejection of pixels of specularly reflected radiation off of the polished stainless steel plate on which wheat seeds had been mounted with the germ up. Typically a 19 MB data cube resulting from spectra of all pixels in the full array was reduced to ca. 15 MB. The remaining pixels of diffusely reflected radiation constituted the database for chemical imaging of the whole kernels. In our wheat research application of this imaging technique post run data processing is acceptable. Image processing and a Go/No-Go census within each image is readily attained. Penetration of near-IR rays in the 1100–1700 nm range and their capture and detection by the InGaAs focal plane array enabled subsurface internal spectroscopic probing of the developing embryo and its surrounding scutellum within the germ. With visible light, a fully germinated seed produces a sprout tube that can be seen by the naked eye, however, the visible rays do not penetrate below the surface of the germ to show the
V.W. Smail et al. / Vibrational Spectroscopy 42 (2006) 215–221
217
biological development underneath the surface during the process that precedes emergence of the actual sprout. 4. Results Fig. 1 shows a series of log 1/R peak area images from a pair of model kernels representing an unsprouted (left) and a fully sprouted kernel (right) of the Jagger variety of HRW wheat from experimental plots of the Kansas Agricultural Experimental Station. These images are of kernels from the same lot that were subjected to different treatments described in Section 3. The series of images on the left are those of a representative unsprouted kernel of Jagger wheat that had been treated with moisture for only 3 h and that showed no evidence whatsoever of sprouting. Images on the right are from a representative kernel of Jagger wheat that was treated for 36 h under moist conditions and verified as definitely having sprouted. The nonsprouting and sprouting of the respective kernels were established by subjecting kernels from the same experimental treatment to ?-amylase testing as well as to careful microscopic examination. The first pair of images (Fig. 1a) are from broad spectral absorption, expressed as log 1/R, in the region of 1400– 1700 nm. The actual images displayed represent the peak area response at a wavelength of essentially 1680 nm. Subsequent pairs of images (Fig. 1b) were produced by a statistical treatment, pixel by pixel; of principle component analysis (PCA) applied to reflection intensity resulting in the images displayed. Factor 1 is responsible for 99% of the variance within the individual image. Successive images are of PCA factors 2–7. PCA loadings for each factor are shown in graphical form (Fig. 1c). From the images presented in Fig. 1a and b there are obvious differences in the images of the unsprouted kernel on the left and the sprouted kernel on the right. In each case the individual kernels were mounted with the germ up and the germ on the right of the mounting plate used to collect data. (The single exception to this is one member of the quartet of images on the Jagger plate that was exposed for 36 h. This was an experimental oversight.) Images produced with the germ end on the right and the germ facing up all showed evidence of advanced embryo formation in those images of the sprouted kernel. This was true not only for the first image, representing the direct spectroscopic response, but also for the images of factors 1–4, and 6. In all of those the area occupied by the germ was exhibited with a great deal of contrast, compared to the rest of the kernel. Furthermore, the intensity of the false color image contrast gave evidence of the density of that part of the kernel occupied by a highly developed embryo. The exact opposite case was observed for the germ region on the right side of the unsprouted kernel. There was no evidence of a developed germ from the simple spectroscopic response at 1680 nm. Similarly with the images produced from statistical contrast of reflection intensity, none of the PCA factors from 1 to 7 showed evidence of subsurface development of an embryo. The bar graph in Fig. 2 illustrates the results of a discriminant analysis scheme applied to spectra extracted
Fig. 1. (a) Broad band log 1/R spectroscopic images of unsprouted (left) and sprouted (right) single Jagger wheat kernels revealing the developed embryo below the surface at the germ end (right) of the sprouted kernel. [False color deep blue in the online version indicates pixels of higher absorption]. (b) False color contrasting images of unsprouted (left) and sprouted (right) kernels based on PCA factor 1 values assigned to individual pixel reflection intensities showing the size, location, and density of the developed embryo in the sprouted kernel. The first factor represents 99% of the variance within the single kernel image. Images contrast PCA factors 2–7. Note the clear revelation of the developed embryo in the images of the sprouted kernel (right) in factors 2–4, and 6. (c) PCA loadings (factors 1–7). [For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.]
218
V.W. Smail et al. / Vibrational Spectroscopy 42 (2006) 215–221
Fig. 1. (Continued ).
from pixels in the germ region of both the sprouted and unsprouted images previously shown (Fig. 1b). The one group of 60 spectra (Fig. 2a) was obtained from three sprouted kernels in which spectra were extracted from 20 pixels at the center of the embryo location. The second group of 80 spectra (Fig. 2b) was from 20 pixels extracted at the center of the location of the undeveloped embryos of four unsprouted kernels. The bar graph (Fig. 2) shows the Mahalanobis distance of each of the 60 pixel spectra from the germ of sprouted wheat kernels from the mean of the 80 spectra representing the summation of pixels from the germ area of unsprouted kernels. It is evident from this graph that discriminant analysis shows promise. Fig. 3 exhibits spectra from four pixels extracted from the dead center of the Jagger 36 image shown in Fig. 1b. These spectra were extracted from the most highly contrasted pixels of the principle component factor 1 image. Although the spectroscopic features are not bold in the part of the spectrum shown, they do stand in contrast to the relatively featureless spectra from other pixels extracted elsewhere from the same
kernel. Although no attempt has been made to interpret these features, plots of second derivative spectra accentuate the features that are present. Having established that it is possible to recognize fully developed embryos beneath the surface in germinated kernels and the absence of these embryos in ungerminated kernels, we tested the relative sensitivity of the subsurface polychromatic contrast approach on groups of kernels of the same cultivar treated 3, 6, 12, 24, 36, and 48 h to moisture on the saturated blotter paper in a Petri dish. In the overall experiment, three replicates of six different moisture exposure times of six different cultivars were tested and imaged with the near-IR focal plane array. In this paper only the results from the composite plates (Fig. 4), containing quartets of six different exposure times of one variety, are shown. Among the six exposure times the contrast between the quartets of unsprouted kernels in the upper left corner for each cultivar, compared with those images of the kernels definitely established as having been sprouted after the 36 h treatment in the second group on
V.W. Smail et al. / Vibrational Spectroscopy 42 (2006) 215–221
219
Fig. 3. Spectra in the 1400–1700 nm region, extracted from pixels within the developed embryo region of the germ shown in Fig. 1a. Note the contrast of these for spectra with the relatively featureless spectrum extracted from a pixel at the outer portion of the germ of the same kernel.
Fig. 2. (a) Sixty spectra extracted from highest absorbing pixels within the germ of three kernels of Jagger wheat sprouted for 36 h. (b) Eighty spectra extracted from highest absorbing pixels within the germ of four unsprouted Jagger wheat kernels. (c) Discriminant analysis (Mahalanobis distances) of 60 pixels of the sprouted kernels from the cluster center of data representing 80 pixels from the image of corresponding unsprouted kernels.
the right column. Note the same image evidence for the corresponding seeds on the other five cultivars shown in Fig. 4. (The exception is the one seed in the 36 h Jagger set in which the germ was face down.) With the exception of that one instance, all of the other 23 seeds exposed to moisture 36 h showed obvious embryo development. It was reported in 2003 that selective debranning and degerming of sprouted wheat, prior to milling, reduced the levels of active ?-amylase in wheat flour [10]. This supports our observation that much of the sprouted activity changes relating to early germination occurred in the germ and aleurone layers. Further examination of individual quartets (Fig. 4) showed that embryo germination development was evident for all groups of 48 h, all groups of 36 h, and all groups of 24 h. Examination of seeds in the groups exposed for 12 h showed response of two or three of the four kernels. Even the groups
exposed only 6 h showed evidence within usually two of the four kernels present. Pagano et al. [11] showed that germination onset occurs in the embryo first with the release of ?-amylase and then other degradative enzymes. The results of subsurface polychromatic imaging are consistent with analytically determined alpha amylase values performed on groups of kernels ground and subjected to a ?-amylase test kit. Note ?-amylase values from Table 1. Relative precision is lacking at low levels, however, any value below 20 indicates no sprouting and any values above 20 indicate progressively higher ?-amylase activity and the presence of sprouting. Further note that in sample at the 36 h mark, there was sufficient ?-amylase in the kernels to show a sharp rise in the ?-amylase number. In contrast, not only did the imaging affirm a fully sprouted state at 36 h but it readily showed germination at 24 h and some evidence of germination at the 12- and 6-h time of the experiment. 5. Discussion The ability to see embryonic development below the surface provides important analytical assistance to the plant breeding program. Those current experimental breeding lines, subjected to the different times of moisture exposure, that show evidence in early stages of exposure, can be discarded as being more susceptible to sprouting. Those that show evidence of embryo germination only at the very later stages are assumed to be somewhat more resistant to premature sprouting. By identifying resistance at early stages of the breeding program, we can readily use the spectroscopic imaging as a selection guide for future cultivars that, besides the many other agronomic desirable characteristics, will also have better resistance to sprouting in the field and avoid the economic disaster that accompanies such a situation. Table 2 summarizes the relative
220
V.W. Smail et al. / Vibrational Spectroscopy 42 (2006) 215–221
Fig. 4. Image of six quartets of kernels assembled in columns according to the time of exposure of each group of kernels to a moist condition that enables sprouting. Left column (top to bottom) 03, 06, and 12 h; right column 24, 36, and 48 h. The 03 h group was independently determined to be unsprouted and the sprouted condition of the 36 h group was confirmed by two independent means. The composite images of this figure show the results of experiments with six hard wheat cultivars including Jagger HRW, Danby HWW, Trego HRW, Betty HWW, KS 2174 HWW, and KS 89180B-2-1-2 HRW. The numbered cultivars are experimental breeding lines. The named specimens are of released varieties. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
V.W. Smail et al. / Vibrational Spectroscopy 42 (2006) 215–221
221
Table 1 ?-Amylase test results (arbitrary units) from wheat kernels of six cultivars that have been exposed to sprouting conditions for six different periods of time Germination time (h)
Jagger HRW
Danby HWW
Trego HRW
Betty HWW
KS 2174 HWW
KS 89180B 2-1-2 HRW
3 6 12 24 36 48
11 12 10 19 91 229
16 30 17 14 173 166
16 10 10 13 76 444
21 9.2 6.1 11 39.4 434
7.8 6.2 5.4 14.6 154 501
10 12 11 10 26 191
Values listed are the average of triplicate determinations. Table 2 Relative sensitivity of non-destructive focal plane array imaging to visual and destructive testing for sprouting Non-destructive
Destructive
Time
Imaging
Visual
Alpha amylase
Viscosity
48 36 24 12 6
+ + + + +
+ +
+ +
+ +
sensitivity of near infrared focal plane array imaging, the visual inspection non-destructive test, and the destructive ?-amylase or bulk viscosity tests currently used. 6. Summary Subsurface kernel non-destructive imaging reveals sprouting for simultaneous single kernels with sensitivity greater than that of the human eye. Also, the sensitivity is greater than the destructive viscosity testing of aqueous slurries of ground wheat. Results of the test provide an informed breeding selection criterion indicator. This approach has future potential for automated inspection and sorting. Sequential testing of the same kernels is possible after non-destructive imaging. The techniques developed in this study have the potential for future study of biological process in living tissues.
Acknowledgements The assistance and suggestions from Joseph Schoepelrei and Neil Lewis of Spectral Dimension were a great help and the authors thank Hicran Koc, in particular, for assistance in data processing and graphics in preparation of the manuscript. This study was supported in part by the Kansas Agricultural Experiment Station and the Kansas State University Microbeam Molecular Spectroscopy Laboratory. Contribution no. 06-65-J Kansas Agricultural Experiment Station, Manhattan. References [1] Kansas Association of Wheat Growers, July 21, 2004, http://www. wheatonline.com, (sproutedwheat072104.doc). [2] R. Hegeman, San Diego Union Tribune, July 20, 2004. [3] K.L. Roozeboom, P.J. McCluskey, J.P. Shroyer, G.M. Paulsen, Kansas, Kansas State University Agricultural Experiment Station and Cooperative Extension Service, SRL 124, November 1999. [4] J.A. Anderson, M.E. Sorrells, S.D. Tanksley, Crop Sci. 33 (1993) 453– 459. [5] R. Olered, Cereal Res. Commun. 4 (1975) 195–199. [6] A.D. Evers, S. Ferguson, Cereal Res. Commun. 8 (1) (1980) 69–75. [7] E.N. Lewis, US Patent #5,5281,368. [8] E.N. Lewis, J. Schoppelrei, E. Lee, Spectroscopy 19 (4) (2004) 26–36. [9] D.L. Wetzel, A.J. Eilert, J.A. Sweat, in: J.M. Chalmers, P.R. Griffiths (Eds.), Handbook of Vibrational Spectroscopy, Wiley, Chichester, UK, 2002, 436–452. [10] G.A. Hareland, Cereal Chem. 80 (2) (2003) 232–237. [11] E.A. Pagano, R.L. Benech-Arnold, M. Wawrzkiewicz, H.S. Steinbach, Ann. Bot. (Oxford, UK) 79 (1997) 13–17.
Chemical imaging of intact seeds with NIR focal plane array assists plant breeding Virgil W. Smail a,b, Allan K. Fritz c, David L. Wetzel a,b,* a
Microbeam Molecular Spectroscopy Laboratory, Shellenberger Hall, Kansas State University, Manhattan, KS 66506, USA b Department of Grain Science and Industry, Kansas State University, Manhattan, KS 66506, USA c Department of Agronomy, Kansas State University, Manhattan, KS 66506, USA Available online 11 May 2006
Abstract Early generation identification of desirable or undesirable traits of kernels of grain is useful to the breeding program to assist in selection of the best seeds to carry forward and to eliminate the inferior ones. In the case of wheat, resistance to premature sprouting (germination) in the field, before harvest under high moisture conditions is desirable. Visual examination of individual kernels provides non-destructive identification of severities of the problem. The germination process must be so advanced before visual detection is possible that severe damage to starch in the kernel has already occurred from release of ?-amylase enzyme. Several tests exist for the viscosity of solutions or for alpha amylase directly that are done in bulk and are destructive to the individual kernel. Near-IR imaging with an InGaAs focal plane array provides early localized chemical evidence of the process occurring in multiple kernels in the field of view. A Go/No-Go decision is made on individual kernels by the instrument using a specimen mounting plate that defines the position of the diffusely reflecting kernel, while rejecting adjacent pixels as specular reflection away from the detector array. The chemical imaging reported detects sprouting considerably earlier than can be found by both visual inspection and alpha amylase (?-amylase) testing procedures. Images are presented to illustrate the contrast between normal and sprouted specimens. # 2006 Elsevier B.V. All rights reserved. Keywords: Chemical imaging; Near-IR spectroscopy; Wheat breeding
1. Introduction
2. Background
This study reports on the first efforts to use a non-destructive chemical imaging approach to detect early germination in intact kernels of different hard winter wheat varieties. Wheat cultivars that sprout in the field under moist conditions result in economic devastation. Plant breeders need a reliable nondestructive method to allow single kernel selection of germinated versus nongerminated kernels. A non-destructive system for detection of sprouting at early stages in individual kernels of experimental breeding lines is ideal. Focal plane array imaging based on the very near (short wave) infrared spectroscopic region enables subsurface polychromatic contrast to reveal embryo development under experimental conditions.
Preharvest germination or ‘‘sprouting’’, in wheat is a major problem in most wheat production areas with heavy precipitation or humidity just prior to harvest. The 2004 harvest season in Kansas was especially severe and, as reported by the press [1,2], caused an estimated 24% of the entire hard wheat crop to have sprout damage and be discounted to ‘‘feed’’ grade wheat, with a severe economic loss. More than 40% of the hard white winter (HWW) wheat crop was estimated to be sprout damaged. Kansas wheat breeders have recently successfully developed HWW wheat varieties that represent approximately 20% of Kansas acreage. Growers in 2004 had the impression that these varieties suffered more sprout damage than the hard red winter (HRW) wheat varieties. Earlier evidence from a HRW wheat versus HWW wheat comparison in 1999 showed that this may be the case [3]. Improved tolerance to moisture is necessary to protect producer interests and provide a consistent supply of high quality hard white wheat in years when conditions are conducive to
* Corresponding author. Tel.: +1 785 532 4094; fax: +1 785 532 7010. E-mail address: [email protected] (D.L. Wetzel). 0924-2031/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vibspec.2006.02.004
216
V.W. Smail et al. / Vibrational Spectroscopy 42 (2006) 215–221
sprout damage. Progress continues to be made on the genetic improvement of HWW wheat. One of the challenges, however, is efficient selection of more sprout resistant breeding stock. Although some DNA markers are available for this trait since 1993, [4] marker-assisted selection is still relatively expensive and will probably only be feasible to apply at one or two points in the selection process. The development of tools to rapidly separate sprouted and unsprouted kernels would be a tremendous benefit to the breeding process and could conceivably also have future benefit in the market channels. In existing methods, visual (naked eye) inspection is the only present non-destructive method applicable to single kernels. Destructive testing of pooled ground kernels includes viscosity of a wheat flour suspension that is damaged relative to alpha amylase counteraction or via direct determination of ?-amylase using a test kit. Sprout damage is typically measured destructively using a viscoelastic measure (Falling NumberTM) in a slurry of starch damaged flour caused by the action of amylase from germination in the still nonharvested spike of the wheat [5]. Alternatively recently available antibody tests have been developed to measure specific amylase levels in ground wheat kernels. This test uses antibodies specific for ?-amylase extracted from ground kernels. These destructive tests determine the starch damage caused by germination of wheat, and require 1–100 g of ground kernels. All bulk test methods have high standard errors due to significant within spike variation levels of sprouting and sampling errors within the bulk sample [6]. With the proposed imaging procedures, a Go/No-Go decision is made on individual kernels with the instrument using a specimen mounting plate that defines the position of the diffusely reflecting kernel, while rejecting adjacent pixels as specular reflection away from the detector array. The chemical imaging reported detects sprouting considerably earlier than can be found by both visual inspection and alpha amylase (?-amylase) testing procedures. Images are presented to illustrate the contrast between normal and sprouted specimens. 3. Experimental 3.1. Wheat specimens Six 100-g replicates of seed of each variety were germinated for 3, 6, 12, 24, 36, and 48 h on soaked blotter paper in Petri dishes. The kernels were frozen at 80 8C for 24 h to stop all germination processes. The kernels were then freeze dried until no weight change could be detected (48 h) to remove all moisture and stabilize the kernels. The kernels have shown no germination or changes for the past several months. Kernels were then manually placed on the grid sample plates for imaging. In addition to the imaging specimens, 10 g of each replicate wheat specimen were ground in preparation for ?-amylase testing using the WheatRite1 Immune Scanner (C-Qentec Diagnostics, Epping, Australia) antibody side flow test kit. One kilogram samples of six HRW and HWW wheat varieties grown in
Kansas Agricultural Experiment Station plots in 2004 were supplied as follows: Variety
Class
Pedigree
Jagger Danby Trego Betty KS2174 KS89180B-2-1-2
HRW HWW HRW HWW HWW HRW
KS82W418/Stephens Trego/Betty ‘S’ KS87H325/Rio Blanco – IL71-5662/’’PL145’ (PI 00840)//21665 KS8010-73/KS8010-1-4-2/3/107349/ KS8181252//Karl 92
3.2. Instrumentation The Spectral Dimensions MatrixNIRTM Chemical Imaging system (Olney, MD, USA) was used [7,8]. It consisted of a liquid crystal tunable filter [9], a 320 256 pixel indium gallium arsenide (InGaAs) focal plane array detection system, and appropriate optics to preferentially capture diffusely reflected light from each specimen while, minimizing background specular reflection off of the polished mounting plate. Four tungsten source lamps illuminate the imaging target field. ISysTM Chemical Imaging Software was provided by Spectral Dimensions that enables simultaneous manipulation of spectra of 92,000 pixels from a single field of view. Sample mounts were designed and locally custom made for pre-positioning 24 or 30 kernels in the field of view of the imaging system. This device permitted us to scan 15 million spectra in 1 day, thus data acquisition was relatively efficient in comparison to sample preparation and data handling as described in other sections. A dedicated colorimeter (Immune Scanner) with direct readout in ?-amylase units was part of the WheatRite1 test kit. 3.3. Data manipulation Image contrast was maximized by pretreating the raw image response of the focal plane array to remove the specular reflection background, followed by selection of the wavelength or the principle component analysis factor that best accentuated the contrast required to distinguish embryo development within the germ area of each individual kernel. Data pretreatment in all instances involved rejection of pixels of specularly reflected radiation off of the polished stainless steel plate on which wheat seeds had been mounted with the germ up. Typically a 19 MB data cube resulting from spectra of all pixels in the full array was reduced to ca. 15 MB. The remaining pixels of diffusely reflected radiation constituted the database for chemical imaging of the whole kernels. In our wheat research application of this imaging technique post run data processing is acceptable. Image processing and a Go/No-Go census within each image is readily attained. Penetration of near-IR rays in the 1100–1700 nm range and their capture and detection by the InGaAs focal plane array enabled subsurface internal spectroscopic probing of the developing embryo and its surrounding scutellum within the germ. With visible light, a fully germinated seed produces a sprout tube that can be seen by the naked eye, however, the visible rays do not penetrate below the surface of the germ to show the
V.W. Smail et al. / Vibrational Spectroscopy 42 (2006) 215–221
217
biological development underneath the surface during the process that precedes emergence of the actual sprout. 4. Results Fig. 1 shows a series of log 1/R peak area images from a pair of model kernels representing an unsprouted (left) and a fully sprouted kernel (right) of the Jagger variety of HRW wheat from experimental plots of the Kansas Agricultural Experimental Station. These images are of kernels from the same lot that were subjected to different treatments described in Section 3. The series of images on the left are those of a representative unsprouted kernel of Jagger wheat that had been treated with moisture for only 3 h and that showed no evidence whatsoever of sprouting. Images on the right are from a representative kernel of Jagger wheat that was treated for 36 h under moist conditions and verified as definitely having sprouted. The nonsprouting and sprouting of the respective kernels were established by subjecting kernels from the same experimental treatment to ?-amylase testing as well as to careful microscopic examination. The first pair of images (Fig. 1a) are from broad spectral absorption, expressed as log 1/R, in the region of 1400– 1700 nm. The actual images displayed represent the peak area response at a wavelength of essentially 1680 nm. Subsequent pairs of images (Fig. 1b) were produced by a statistical treatment, pixel by pixel; of principle component analysis (PCA) applied to reflection intensity resulting in the images displayed. Factor 1 is responsible for 99% of the variance within the individual image. Successive images are of PCA factors 2–7. PCA loadings for each factor are shown in graphical form (Fig. 1c). From the images presented in Fig. 1a and b there are obvious differences in the images of the unsprouted kernel on the left and the sprouted kernel on the right. In each case the individual kernels were mounted with the germ up and the germ on the right of the mounting plate used to collect data. (The single exception to this is one member of the quartet of images on the Jagger plate that was exposed for 36 h. This was an experimental oversight.) Images produced with the germ end on the right and the germ facing up all showed evidence of advanced embryo formation in those images of the sprouted kernel. This was true not only for the first image, representing the direct spectroscopic response, but also for the images of factors 1–4, and 6. In all of those the area occupied by the germ was exhibited with a great deal of contrast, compared to the rest of the kernel. Furthermore, the intensity of the false color image contrast gave evidence of the density of that part of the kernel occupied by a highly developed embryo. The exact opposite case was observed for the germ region on the right side of the unsprouted kernel. There was no evidence of a developed germ from the simple spectroscopic response at 1680 nm. Similarly with the images produced from statistical contrast of reflection intensity, none of the PCA factors from 1 to 7 showed evidence of subsurface development of an embryo. The bar graph in Fig. 2 illustrates the results of a discriminant analysis scheme applied to spectra extracted
Fig. 1. (a) Broad band log 1/R spectroscopic images of unsprouted (left) and sprouted (right) single Jagger wheat kernels revealing the developed embryo below the surface at the germ end (right) of the sprouted kernel. [False color deep blue in the online version indicates pixels of higher absorption]. (b) False color contrasting images of unsprouted (left) and sprouted (right) kernels based on PCA factor 1 values assigned to individual pixel reflection intensities showing the size, location, and density of the developed embryo in the sprouted kernel. The first factor represents 99% of the variance within the single kernel image. Images contrast PCA factors 2–7. Note the clear revelation of the developed embryo in the images of the sprouted kernel (right) in factors 2–4, and 6. (c) PCA loadings (factors 1–7). [For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.]
218
V.W. Smail et al. / Vibrational Spectroscopy 42 (2006) 215–221
Fig. 1. (Continued ).
from pixels in the germ region of both the sprouted and unsprouted images previously shown (Fig. 1b). The one group of 60 spectra (Fig. 2a) was obtained from three sprouted kernels in which spectra were extracted from 20 pixels at the center of the embryo location. The second group of 80 spectra (Fig. 2b) was from 20 pixels extracted at the center of the location of the undeveloped embryos of four unsprouted kernels. The bar graph (Fig. 2) shows the Mahalanobis distance of each of the 60 pixel spectra from the germ of sprouted wheat kernels from the mean of the 80 spectra representing the summation of pixels from the germ area of unsprouted kernels. It is evident from this graph that discriminant analysis shows promise. Fig. 3 exhibits spectra from four pixels extracted from the dead center of the Jagger 36 image shown in Fig. 1b. These spectra were extracted from the most highly contrasted pixels of the principle component factor 1 image. Although the spectroscopic features are not bold in the part of the spectrum shown, they do stand in contrast to the relatively featureless spectra from other pixels extracted elsewhere from the same
kernel. Although no attempt has been made to interpret these features, plots of second derivative spectra accentuate the features that are present. Having established that it is possible to recognize fully developed embryos beneath the surface in germinated kernels and the absence of these embryos in ungerminated kernels, we tested the relative sensitivity of the subsurface polychromatic contrast approach on groups of kernels of the same cultivar treated 3, 6, 12, 24, 36, and 48 h to moisture on the saturated blotter paper in a Petri dish. In the overall experiment, three replicates of six different moisture exposure times of six different cultivars were tested and imaged with the near-IR focal plane array. In this paper only the results from the composite plates (Fig. 4), containing quartets of six different exposure times of one variety, are shown. Among the six exposure times the contrast between the quartets of unsprouted kernels in the upper left corner for each cultivar, compared with those images of the kernels definitely established as having been sprouted after the 36 h treatment in the second group on
V.W. Smail et al. / Vibrational Spectroscopy 42 (2006) 215–221
219
Fig. 3. Spectra in the 1400–1700 nm region, extracted from pixels within the developed embryo region of the germ shown in Fig. 1a. Note the contrast of these for spectra with the relatively featureless spectrum extracted from a pixel at the outer portion of the germ of the same kernel.
Fig. 2. (a) Sixty spectra extracted from highest absorbing pixels within the germ of three kernels of Jagger wheat sprouted for 36 h. (b) Eighty spectra extracted from highest absorbing pixels within the germ of four unsprouted Jagger wheat kernels. (c) Discriminant analysis (Mahalanobis distances) of 60 pixels of the sprouted kernels from the cluster center of data representing 80 pixels from the image of corresponding unsprouted kernels.
the right column. Note the same image evidence for the corresponding seeds on the other five cultivars shown in Fig. 4. (The exception is the one seed in the 36 h Jagger set in which the germ was face down.) With the exception of that one instance, all of the other 23 seeds exposed to moisture 36 h showed obvious embryo development. It was reported in 2003 that selective debranning and degerming of sprouted wheat, prior to milling, reduced the levels of active ?-amylase in wheat flour [10]. This supports our observation that much of the sprouted activity changes relating to early germination occurred in the germ and aleurone layers. Further examination of individual quartets (Fig. 4) showed that embryo germination development was evident for all groups of 48 h, all groups of 36 h, and all groups of 24 h. Examination of seeds in the groups exposed for 12 h showed response of two or three of the four kernels. Even the groups
exposed only 6 h showed evidence within usually two of the four kernels present. Pagano et al. [11] showed that germination onset occurs in the embryo first with the release of ?-amylase and then other degradative enzymes. The results of subsurface polychromatic imaging are consistent with analytically determined alpha amylase values performed on groups of kernels ground and subjected to a ?-amylase test kit. Note ?-amylase values from Table 1. Relative precision is lacking at low levels, however, any value below 20 indicates no sprouting and any values above 20 indicate progressively higher ?-amylase activity and the presence of sprouting. Further note that in sample at the 36 h mark, there was sufficient ?-amylase in the kernels to show a sharp rise in the ?-amylase number. In contrast, not only did the imaging affirm a fully sprouted state at 36 h but it readily showed germination at 24 h and some evidence of germination at the 12- and 6-h time of the experiment. 5. Discussion The ability to see embryonic development below the surface provides important analytical assistance to the plant breeding program. Those current experimental breeding lines, subjected to the different times of moisture exposure, that show evidence in early stages of exposure, can be discarded as being more susceptible to sprouting. Those that show evidence of embryo germination only at the very later stages are assumed to be somewhat more resistant to premature sprouting. By identifying resistance at early stages of the breeding program, we can readily use the spectroscopic imaging as a selection guide for future cultivars that, besides the many other agronomic desirable characteristics, will also have better resistance to sprouting in the field and avoid the economic disaster that accompanies such a situation. Table 2 summarizes the relative
220
V.W. Smail et al. / Vibrational Spectroscopy 42 (2006) 215–221
Fig. 4. Image of six quartets of kernels assembled in columns according to the time of exposure of each group of kernels to a moist condition that enables sprouting. Left column (top to bottom) 03, 06, and 12 h; right column 24, 36, and 48 h. The 03 h group was independently determined to be unsprouted and the sprouted condition of the 36 h group was confirmed by two independent means. The composite images of this figure show the results of experiments with six hard wheat cultivars including Jagger HRW, Danby HWW, Trego HRW, Betty HWW, KS 2174 HWW, and KS 89180B-2-1-2 HRW. The numbered cultivars are experimental breeding lines. The named specimens are of released varieties. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
V.W. Smail et al. / Vibrational Spectroscopy 42 (2006) 215–221
221
Table 1 ?-Amylase test results (arbitrary units) from wheat kernels of six cultivars that have been exposed to sprouting conditions for six different periods of time Germination time (h)
Jagger HRW
Danby HWW
Trego HRW
Betty HWW
KS 2174 HWW
KS 89180B 2-1-2 HRW
3 6 12 24 36 48
11 12 10 19 91 229
16 30 17 14 173 166
16 10 10 13 76 444
21 9.2 6.1 11 39.4 434
7.8 6.2 5.4 14.6 154 501
10 12 11 10 26 191
Values listed are the average of triplicate determinations. Table 2 Relative sensitivity of non-destructive focal plane array imaging to visual and destructive testing for sprouting Non-destructive
Destructive
Time
Imaging
Visual
Alpha amylase
Viscosity
48 36 24 12 6
+ + + + +
+ +
+ +
+ +
sensitivity of near infrared focal plane array imaging, the visual inspection non-destructive test, and the destructive ?-amylase or bulk viscosity tests currently used. 6. Summary Subsurface kernel non-destructive imaging reveals sprouting for simultaneous single kernels with sensitivity greater than that of the human eye. Also, the sensitivity is greater than the destructive viscosity testing of aqueous slurries of ground wheat. Results of the test provide an informed breeding selection criterion indicator. This approach has future potential for automated inspection and sorting. Sequential testing of the same kernels is possible after non-destructive imaging. The techniques developed in this study have the potential for future study of biological process in living tissues.
Acknowledgements The assistance and suggestions from Joseph Schoepelrei and Neil Lewis of Spectral Dimension were a great help and the authors thank Hicran Koc, in particular, for assistance in data processing and graphics in preparation of the manuscript. This study was supported in part by the Kansas Agricultural Experiment Station and the Kansas State University Microbeam Molecular Spectroscopy Laboratory. Contribution no. 06-65-J Kansas Agricultural Experiment Station, Manhattan. References [1] Kansas Association of Wheat Growers, July 21, 2004, http://www. wheatonline.com, (sproutedwheat072104.doc). [2] R. Hegeman, San Diego Union Tribune, July 20, 2004. [3] K.L. Roozeboom, P.J. McCluskey, J.P. Shroyer, G.M. Paulsen, Kansas, Kansas State University Agricultural Experiment Station and Cooperative Extension Service, SRL 124, November 1999. [4] J.A. Anderson, M.E. Sorrells, S.D. Tanksley, Crop Sci. 33 (1993) 453– 459. [5] R. Olered, Cereal Res. Commun. 4 (1975) 195–199. [6] A.D. Evers, S. Ferguson, Cereal Res. Commun. 8 (1) (1980) 69–75. [7] E.N. Lewis, US Patent #5,5281,368. [8] E.N. Lewis, J. Schoppelrei, E. Lee, Spectroscopy 19 (4) (2004) 26–36. [9] D.L. Wetzel, A.J. Eilert, J.A. Sweat, in: J.M. Chalmers, P.R. Griffiths (Eds.), Handbook of Vibrational Spectroscopy, Wiley, Chichester, UK, 2002, 436–452. [10] G.A. Hareland, Cereal Chem. 80 (2) (2003) 232–237. [11] E.A. Pagano, R.L. Benech-Arnold, M. Wawrzkiewicz, H.S. Steinbach, Ann. Bot. (Oxford, UK) 79 (1997) 13–17.