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Rice Science, 2015, 22(5): 245249

Copyright 2015, China National Rice Research Institute


Hosting by Elsevier B.V. All rights reserved
DOI: 10.1016/S1672-6308(14)60291-2

Discrimination of Transgenic Rice Based on Near Infrared


Reflectance Spectroscopy and Partial Least Squares
Regression Discriminant Analysis

ZHANG Long1, 3, WANG Shan-shan2, DING Yan-fei2, PAN Jia-rong2, ZHU Cheng2, 3
(1College of Ecology, Lishui University, Lishui 323000, China; 2College of Life Sciences, China Jiliang University, Hangzhou
310018, China; 3College of Life Sciences, Zhejiang University, Hangzhou 310058, China)

Abstract: Near infrared reflectance spectroscopy (NIRS), a non-destructive measurement technique, was
combined with partial least squares regression discrimiant analysis (PLS-DA) to discriminate the transgenic
(TCTP and mi166) and wild type (Zhonghua 11) rice. Furthermore, rice lines transformed with protein gene
(OsTCTP) and regulation gene (Osmi166) were also discriminated by the NIRS method. The performances of
-1 -1
PLS-DA in spectral ranges of 4 0008 000 cm and 4 00010 000 cm were compared to obtain the optimal
spectral range. As a result, the transgenic and wild type rice were distinguished from each other in the range of
-1
4 00010 000 cm , and the correct classification rate was 100.0% in the validation test. The transgenic rice
-1
TCTP and mi166 were also distinguished from each other in the range of 4 00010 000 cm , and the correct
classification rate was also 100.0%. In conclusion, NIRS combined with PLS-DA can be used for the
discrimination of transgenic rice.
Key words: near infrared reflectance spectroscopy; genetically-modified food; regulation gene; protein gene;
partial least squares regression discrimiant analysis

Genetically-modified foods (GMOs) are inserted with foreign liquid chromatography and gas chromatography are also useful
genes which increase resistance to diseases, pests and technologies (Alishahi et al, 2010). However, these technologies
herbicides or improve nutritional contents which never occur had many shortcomings, such as high cost, difficult to use,
naturally (Anklam et al, 2002). Since it is suspected with the special need, and long duration.
safety about environmental hazards, human health risks, and Near infrared reflectance spectroscopy (NIRS) is sensitive to
economic concerns, the public are scared of consuming GMOs, organic compounds with vibration overtones of CH, OH and
and the GMOs are severely rejected in many regions of the NH, which is rapid, lower cost and non-destructive. The
world. In order to regulate the introduction and production of content of modified DNA is in amount of ultra trace, so
GMOs, many regulations and legislations are promulgated with detection of the modified DNA is difficult to identify the
the Ministry of Health, the Ministry of Agriculture, the General GMOs, while detection of the products resulting from the
Administration of Quality Supervision, and the Inspection and genetic modification (particular proteins) or larger structural
Quarantine in China. Thus, the fast reliable detection methods changes (phenotype) are feasible (Munck et al, 2001; van Duijn
must be developed to support the regulations and legislations et al, 2002; Xie et al, 2007a, b; Alishahi et al, 2010). In
above. previous studies, NIRS has successfully used in detection of
There are many detection methods for transgenic food. transgenic tomato (Xie et al, 2007a), barley (Hurburgh et al,
Western blot (Lipton et al, 2000; Sambrook and Russel, 2000), 2000) and corn (Rossel et al, 2001) or mutant of barley (Munck
enzyme linked immunosorbent assays (Yates, 1999; Margarit et al, 2004) and corn (Campbell et al, 2000).
et al, 2006) and lateral flow strip (Fagan et al, 2001) are protein- Rice is relatively easy to consult transgenic. There are rarely
based methods. Southern blot (Ross et al, 1999; Stull, 2001), studies conducted on the detection of transgenic rice to our
qualitative polymerase chain reaction (qPCR) (van Hoef et al, knowledge (Jiao et al, 2010). Translationally-controlled tumor
1998; Lipp et al, 1999; Singh et al, 2007), microarray (Miraglia protein (TCTP) is most likely controlled by a cell housekeeping
et al, 2004) and real time PCR (Heid et al, 1996; Ahmed, 2000; gene which presents as a multifunctional protein and essential
Mde et al, 2006; Akiyama et al, 2007; Grohmann and Mde, in the development of mammals, higher plants and
2009) are DNA-based methods. Additionally, high performance Saccharomyces cerevisiae. It was recently discovered that
OsTCTP works on mercury resistance. As we all known,
Received: 11 November 2014; Accepted: 26 May 2015 microRNAs (miRNAs) are a class of small non-coding RNAs
Corresponding author: ZHU Cheng (pzhch@zju.edu.cn) which negatively regulate specific target mRNAs at the
246 Rice Science, Vol. 22, No. 5, 2015

post-transcriptional level. Thus, cd-responsive gene Osmi166 and 1 in the PLS-DA model, respectively. The predicted
were selected (Ding et al, 2011). values below 0 were considered to the category of -1, and
In the present study, heavy metal responsive genes OsTCTP the values above 0 were considered to the category of 1.
and Osmi166 transgenic rice were employed to discriminate the Leave-one-out cross-validation was used for the calibration,
transgenic rice (Wang, 2010; Ding et al, 2011). The aim of the which involved in using a single observation from the original
present study was to discriminate the transgenic and wild type sample as the validation data and the remaining observations as
rice, and the transgenic rice transformed with protein gene and the training data.
regulation gene. Additionally, the partial least squares
regression discrimiant analysis (PLS-DA) was employed as a Model evaluation
discriminant model.
The PLS-DA model was evaluated with the parameters of
root-mean-square error (RMSE) and correlation coefficient (r2).
MATERIALS AND METHODS RMSE used for calibration was designated as root mean square
error in calibration (RMSEC) and for validation or prediction
Transgenic rice as root mean square error in prediction (RMSEV). r2 used for
calibration was designated as r2c and for validation as r2v.
Wild type rice Zhonghua 11 (Oryza sativa L. subsp. japonica,
ZH11) was used as the transgenic material. Two single-copy
transgenic rice lines were developed in the authors laboratory RESULTS AND DISCUSSION
by respectively introducing OsTCTP and Osmi166 genes into
ZH11 using an Agrobacterium-mediated transformation method. Diffuse transmittance spectra of rice grains
The rice seed used were all T3 homozygous lines and
designated as TCTP and mi166, respectively. OsTCTP and Spectra of transgenic rice and wild types ZH11 in the region of
Osmi166 genes expressed in rice plants and the expression 4 00010 000 cm-1 are shown in Fig. 1. It is obviously that the
levels were stable. The rice lines were all planted and harvested shapes of the spectra of TCTP, mi166 and ZH11 were
under natural condition and dried in the sun in the same field. overlapped and quite homogeneous and cannot be identified by
naked eyes, but there were some variants in the 4 00010 000
NIRS analysis cm-1 region. The noise of spectra in the 8 00010 000 cm-1
region was a little more than that in the 4 0008 000 cm-1
The near infrared reflectance spectra of rice grains were region. Therefore, in the following calculation, the performance
scanned on a Nicolet Nexus 870 FT-IR spectrometer (Thermo of the model was compared between the 4 0008 000 cm-1 and
Corporation, USA) with the mode of diffuse transmission. The 4 00010 000 cm-1 regions.
spectra of 192 rice grain samples were measured at 8 cm-1
resolution with 32 scans above the detection window. And both PCA of rice grain spectra
the obverse and the reverse of the grains (three grains for each
sample) were scanned. The spectra were collected with the To examine the spectral variability of the selected samples,
OMNIC 6.0 software in range of 4 00010 000 cm-1 in the PCA was performed on the raw spectra across the spectral
absorbance mode. Each spectrum was structured in 3040 data range 4 00010 000 cm-1. The first two principal components
with the format of ASCII which are easily combined with the explained 62.90% of the variations in the raw spectra of rice
Matlab software version 6.5. grain samples (PC1, 54.51%; PC2, 8.39%) (Fig. 2). The score
plot of the first two PCs showed that the TCTP, mi166 and
PLS-DA
2
PLS is a well-established multivariate regression model which
constructs a mathematic relationship between descriptors and 1
dependent variables (Kleinbaum et al, 1988). In the PLS model,
Absorbance

principal component analysis (PCA) was first processed, and 0


then the scores of principal components (PCs) obtained were
considered as new eigenvectors of the original spectra. The -1
performance of PLS model is affected by the number of PCs
(Marengo et al, 2008). In order to discriminate the transgenic -2

and wild type rice, PLS was used as discrimination by designed


the values of different category. The values of transgenic -3
4 000 6 000 8 000 10 000
(TCTP and mi166) and wild type (ZH11) rice were designated
Wavenumber (cm-1)
as -1 and 1, respectively, in the PLS-DA model. To identify
rice transformed with protein gene and regulation gene, TCTP 1 grains.
Fig. 1. Standard normal variance pretreated spectra of rice
and mi166 were used and the values were designated as -1 ZH11, Zhonghua 11.
ZHANG Long, et al. Near Infrared Reflectance Spectroscopy for Discrimination of Transgenic Rice Seeds 247

ZH11 sets were overlapped in the PC1 and PC2 spaces (Fig. 3),
and the three kinds of rice cannot be discriminated from each 80
other clearly.

Cumulative variance (%)


60
Distinction between transgenic and wild type and between
TCTP and mi166 with PLS-DA model 40

The number of PCs in the PLS-DA model is very important 20


because very few components will generate an under-fitted
model, i.e., fits loosely the data structure. However, using too 0
many components generates an over-fitted model, one which
fits parts of the noise of the calibration set, thus generating a 0 2 4 6 8 10

low RMSEC but performing poorly in the validation set (Broomhead Number of PCs

and Lowe, 1988). Different ranges of spectra contained the Fig. 2. Variation explained by principal component1 (PC) in
variant information of rice grains. In the present study, two principal component analysis.
regions of spectra 4 0008 000 cm-1 and 4 00010 000 cm-1
were compared to evaluate the optimal range spectra, which
could get the best discrimination result. In the experiment of mi166
discriminate, for transgenic and wild type rice, in the range of TCTP
Zhonghua 11
4 0008000 cm-1, the optimal numbers of PC were five in the
both calibration and validation tests, whereas in the range of

Score of PC2
4 00010 000 cm-1, the optimal numbers of PC were six in the
both calibration and validation tests (Table 1). In the experiment
of discriminating rice transformed with protein (TCTP) or
regulation genes (mi166), the optimal numbers of PC were all
five in the both calibration and validation tests for the two
wavenumber ranges (Table 2).
PLS-DA was constructed to distinguish rice from those with -7 0 7
transformed foreign genes. The performance of PLS-DA was Score of PC1
evaluated by the values of RMSE and r2 in the calibration and
1
Fig. 3. Plots of the first and second principal components (PCs).
validation tests. The spectral range which got the lowest values
of RMSE and the highest values of r2 showed the best
performance of model. It was obviously that spectra in the value of RMSEV and higher value of r2v, which were 0.2878
range of 4 00010 000 cm-1 showed lower value of RMSEC and 0.8979, respectively. Thus, the optimal bands responsible
and higher value of r2c, which were 0.2695 and 0.9183, for the discrimination of transgenic and wild type rice were in
respectively, in the discrimination of transgenic and wild type the range of 4 00010 000 cm-1. The correct classification of
rice. Similarly, the result of validation test showed the lower calibration test and validation test all achieved 100% perfectly

Table 1. Effects of different wavenumber range on classification results of partial least squares regression discrimiant analysis models
between transgenic and wild type rice.
Wavenumber Calibration Validation
(cm-1) No. of PCs RMSEC r2
c CRc (%) No. of PCs RMSEV r2v CRv (%)
4 0008 000 5 0.3555 0.8578 100 5 0.3543 0.8344 100
4 00010 000 6 0.2695 0.9183 100 6 0.2878 0.8979 100
PC, Principal component; RMSEC, Root mean square error in calibration; r2c, Correlation coefficient of calibration; CRc, Correct rate of
calibration; RMSEV, Root mean square error in validation; r2p, Correlation coefficient of validation; CRv, Correct rate of validation.

Table 2. Effects of different wavenumber range on classification results of partial least squares regression discrimiant analysis models
between TCTP and mi166.
Wavenumber Calibration Validation
(cm-1) No. of PCs RMSEC r2c CRc (%) No. of PCs RMSEV r2v CRv (%)
4 0008 000 5 0.3659 0.8661 97.5 5 0.3799 0.8123 98.3
4 00010 000 5 0.2945 0.9133 97.5 5 0.3873 0.7537 100.0
PC, Principal component; RMSEC, Root mean square error in calibration; r2c, Correlation coefficient of calibration; CRc, Correct rate of calibration;
RMSEV, Root mean square error in validation; r2v, Correlation coefficient of validation; CRv, Correct rate of validation.
248 Rice Science, Vol. 22, No. 5, 2015

transgenic rice TCTP and mi166 from the wild type rice, and
also to differentiate TCTP from mi166. The correct classification
1 rate of validation test all achieved 100.0% in the spectral range
of 4 00010 000 cm-1. Thus, it might be possible to apply the
Predicted value

non-destructive technology NIRS and PLS-DA model in the


0 identification of transgene and wild type rice as well as rice
transformed with different kinds of genes.
-1
ACKNOWLEDGEMENTS
-1 1
Designed value The research was supported by the projects under the
Innovation Team of the Safety Standards and Testing Technology
Fig. 4. Regression plot of designed and prediction 1category
variables of transgenic (TCTP and mi166) and wild type for Agricultural Products of Zhejiang Province, China (Grant
(Zhonghua 11) rice. No. 2010R50028), and the National Key Technologies R&D
Program of China during the 11th Five-Year Plan Period (Grant
No. 2006BAK02A18).

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