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Use of Attenuated Total Reflection Fourier Transform Infrared (ATR FT-IR) Spectroscopy in Direct, Nondestructive, and Rapid Assessment of Developmental Cotton Fibers Grown In Planta and in Culture Yongliang Liu,a,* Hee-Jin Kimb a USDA, ARS, Cotton Structure and Quality Research Unit, Southern Regional Research Center (SRRC), New Orleans, LA 70124 USA b USDA, ARS, Cotton Fiber Bioscience Research Unit, Southern Regional Research Center (SRRC), New Orleans, LA 70124 USA

Cotton fibers are routinely harvested from cotton plants (in planta), and their end-use qualities depend on their development stages. Cotton fibers are also cultured in controlled laboratory environments, so that cotton researchers can investigate many aspects of experimental protocols in cotton breeding programs at reduced expenses. In this work, attenuated total reflection Fourier transform infrared (ATR FT-IR) spectra of cotton fibers grown in planta and in culture were collected to explore the potential of FT-IR technique as a simple, rapid, and direct method for characterizing the fiber development. Complementary to visual inspection of spectral variations, principal component analysis (PCA) of ATR FT-IR spectra revealed the occurrence of phase transition from primary to secondary cell wall synthesis and also the difference of starting the phase transition between two types of fibers. Like PCA observation, three simple algorithms were capable of monitoring the secondary cell wall formation effectively. Interestingly and uniquely, simple algorithms were able to detect the subtle discrepancies in fibers older than 25 days post-anthesis, which was not apparent from PCA results. The observation indicated the feasibility of FT-IR technique in rapid, routine, nondestructive, and direct assessment of fiber development for cotton physiology and breeding applications. Index Headings: Fourier transform infrared spectroscopy; FT-IR; Attenuated total reflection; ATR; Cellulose; Phase transition; Fiber secondary wall biosynthesis; Principal component analysis; PCA.

INTRODUCTION Cellulose I (b 1!4 linked glucose residues) is a major compositional component in mature cotton fibers. Its Received 14 January 2015; accepted 26 February 2015. * Author to whom correspondence should be sent. E-mail: yongliang. [email protected]. DOI: 10.1366/15-07876

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quantity affects the end-use qualities for yarn and fabric products. In general, cotton fiber cellulose development includes at least four overlapping but distinctive phases: initiation, primary cell wall formation for fiber elongation, secondary cell wall thickening for cellulose biosynthesis, and maturation.1 The day of flowering is referred to as anthesis, and the term ‘‘days post-anthesis’’ (DPA) is commonly used to describe the cotton fiber growth. The fiber cells initiate at 0 DPA and then elongate to reach a fiber length of 22–35 mm within 20–25 DPA. The secondary cell wall synthesis starts around 15–22 DPA and continues for an additional 30–40 days until the maturation phase, when the fibers dehydrate and collapse into flattened and twisted ribbons. Mature fibers exhibit thickened secondary walls composed of mainly cellulose component, which is primarily responsible for their physical properties. Such diverse developmental phases suggest a number of essential changes in fiber chemical compositions and structures while cotton fibers grow in the field. Despite the fibers usually becoming more mature with increasing DPA under similar growth conditions, the chronological classification of cotton fibers according to the DPA cannot be used as a parameter to reflect the degree of fiber maturation.2 As fibers develop in planta, they can also grow in Petri dishes in culture conditions.3 The cotton ovule culture methods were optimized and used as a powerful tool for numerous cotton studies.4 A comparison of fibers grown between in planta and in culture is illustrated in Fig. 1. Cotton ovule culture methods provide a number of advantages over field growth when unraveling many aspects of experimental protocols, including inhibitors, radiolabeled precursors, or controlled environmental conditions.4 The cultured fibers show substantial similarity in fiber development and chemical composition to the fibers grown in planta. However, the fibers grown in culture differ from those grown in planta in some attributes, such as fiber length, cellulose content, the degree of branching of carbohydrate polymers, and protein profiles.4 Compositional and structural differences of developing cotton fibers in distinct developmental phases with various DPA, together with their physical properties and end-use qualities, have been investigated considerably over the years using diversified and comprehensive techniques, including wet chemistry, microscopy, X-ray diffractometry, molecular spectroscopy, and well-defined fiber testing methods in the cotton industry.1,5–14 Given the obvious distinctions in compositions and structures between immature fibers with younger DPA and mature fibers with older DPA, appropriate optical and physical means are very successful in reflecting the differences of targeted properties between two types of fibers. Among the techniques, attenuated total reflection (ATR) sampling device based Fourier transform infrared (ATR FT-IR)

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of PCA pattern to verify the subjective assignment of either immature or mature fibers prior to two-dimensional correlation analysis on their ATR spectral intensity variations. The objectives of this study were to characterize the unique IR bands of cotton fibers grown in planta and in culture and also to compare the phase transition between two types of fibers with the aid of PCA and simple algorithms. The ultimate goal is to develop this ATR FT-IR procedure as an effective diagnostic tool in monitoring fiber cellulose biosynthesis for cotton physiology and breeding applications.

MATERIALS AND METHODS

FIG. 1. Schematic of fiber growth at 0, 12, and 20 DPA grown (a) in planta and (b) in culture.

spectroscopy could be a potential consideration, because it requires minimal sample preparation, permits routine analysis at both laboratory and on-field environments, and is easy to operate.10,11,13,14 To extract useful information from FT-IR spectra, a number of strategies have been explored, including the estimation of band intensities, the calculation of two- or three-band ratios, and the adoption of principal component analysis (PCA). Abidi et al.10,11 examined the DPAdependent integrated intensities of such vibration modes observed at 3335, 3286, 2918, 2850, 1738, 1639, 1543, 1236, 1161, 897, 710, and 667 cm1, and then concluded the potential of these bands as useful indicators of describing the deposition of secondary wall cellulose. As an alternative approach to estimating cellulose crystallinity from IR measurement, Nelson and O’Connor15 calculated the intensity ratio of the 1372 cm1 band against the 2900 cm1 vibration, Kra¨ssig16 proposed the use of intensity ratio of the 1429 cm1 band against the 897 cm1 vibration, and Liu et al.13,14 considered the relative amount of Ib to Ia crystal forms that are represented using the respective IR bands at 708 and 730 cm1. Principal component analysis is a very effective variable reduction technique for spectroscopic data from n variables (624 in this study from the 1800 to 600 cm1 IR region with a 1.949 cm1 interval) to a fewer number of dimensions.17 It decomposes a set of spectra into mathematical spectra (called loading vectors, factors, principal components, etc.) that represent the most common variations to all spectral data. The correlations among samples (or spectra) are indicated by their scores (or projections) on new principal components (PCs). Abidi et al.10,11 applied PCA to analyze the ATR FT-IR spectra of fibers as a function of developmental DPAs and observed that the PC1 scores, in general, change with DPA. Although the PC1 scores were not linear with DPA, they identified two groups of spectra representing immature fibers at the earlier developmental stage and mature fibers at the later developmental stage using their positive or negative PC1 scores alone, and further distinguished the transition phase for the respective cotton varieties. Liu et al.18 reported the use

Fibers Grown In Planta. Cotton plants (Gossypium hirsutum L. TM-1) were grown in a United States Department of Agriculture–Agricultural Research Service (USDA-ARS) field in New Orleans, LA, during the 2011 crop year. Cotton flowers were tagged at day of anthesis. Two biological replicates of cotton bolls from TM-1 variety were harvested at 10, 17, 24, 28, 33, and 37 DPA. The fibers at each DPA were collected, manually ginned, and dried in 40 8C incubator, leading to six samples representing the developing cotton fibers of TM-1 variety grown in planta at the respective DPAs of 10, 17, 24, 28, 33, and 37. The soil type was Aquent dredged over alluvium in an elevated location to provide adequate drainage. Fibers Grown in Culture. At flowering day (0 DPA), unfertilized cotton ovules from TM-1 variety harvested from the cotton field were cultured on Beasley and Ting (BT) medium containing 0.5 lM gibberellic acid and 5.0 lM 1-naphthaleneacetic acid.3 The cultures were kept in a dark environment at 30 8C in a 5% CO2 atmosphere. The cultured ovules at different developmental stages (16, 20, 24, 28, and 38 DPA) were harvested, manually ginned, and dried for further analyses. Under this experimental setup, five samples were used to reflect the developmental fibers of TM-1 variety grown in culture at the respective DPAs of 16, 20, 24, 28, and 38. From the accumulated knowledge on fiber growth, fiber collection in culture was delayed approximately one week subjectively compared with that in planta. Attenuated Total Reflection Fourier Transform Infrared (ATR FT-IR) Spectral Collection and Data Analysis. All spectra were collected with a Model 3000MX FT-IR spectrometer (Varian Instruments, Randolph, MA) equipped with a ceramic source, KBr beam splitter, and deuterated triglycine sulfate detector. The ATR sampling device used a DuraSamplIR single-pass diamond-coated internal reflection accessory (Smiths Detection, Danbury, CT), and a consistent contact pressure was applied using a stainless steel rod and an electronic load display. At least five measurements at different locations for individual sample were collected over the range of 4000–600 cm1 at 4 cm1 and 16 coadded scans. All spectra were given in absorbance units, and no ATR correction was applied. Following the import to Grams IQ application in Grams/AI (Version 9.1, Thermo Fisher Scientific, Waltham, MA), mean spectrum was taken for each sample and then was smoothed with a Savitzky–Golay function

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TABLE I. Characteristic ATR FT-IR spectral bands of shorter and longer DPA fibers grown in planta.a 10 DPA fiber 33 DPA fiber 1740 (s) 1620 (s) 1545 1425 1405 1365 1335 1315 1236 1200 1158 1104 1055 1028 985 895 662 FIG. 2. Representative of normalized ATR FT-IR spectra of cotton fibers grown in planta at 10 (dotted line), 17 (solid line), and 33 (dashed line) DPA.

(polynomial = 2 and points = 11). The spectral set was loaded into Microsoft Excel 2000 to execute simple algorithm analysis. Meanwhile, the spectra were normalized by dividing the average intensities in the 1800– 600 cm1 region, and subsequent PCA characterization was performed in the 1800–600 cm1 IR region with mean centering and Savitzky–Golay first-derivative (two degrees and 13 points) spectral pretreatment and also with the leave-one-out cross-validation method.

RESULTS AND DISCUSSION Attenuated Total Reflection Fourier Transform Infrared (ATR FT-IR) Spectral Characteristics of Developing Fibers Grown in Planta and in Cultures. Figure 2 shows the representative ATR FT-IR spectra in the 1800–600 cm1 region of developing cotton fibers of TM-1 variety grown in planta. Band assignments for these natural fibers have been studied in some detail.1,10,11,18 Commonly, the vibration at 1740 cm1 is attributed to the C=O stretching mode of carbonyl groups, and a broadband centered at 1630 cm1 mainly originates from the OH bending mode of adsorbed water. Bands in the region of 1500–1200 cm1 are mixtures of CH2 deformations and C–O–H bending vibrations, and a number of bands in the 1200–900 cm1 region are assignable to coupling modes of C–O and C–C vibrations. The bands between 800 and 700 cm1 are likely attributable to two crystal forms (Ia and Ib) of cotton cellulose. In addition, there are intense absorptions between the 3600 and 2750 cm1 region that are assignable to the O–H and C–H stretching vibrations (not shown). Along with the progressing DPA, apparent spectral intensity variations are expected. For example, intensities of the bands at 1740, 1620, 1545, 1455, 1405, 1236, and 1147 cm1 as well as those below 850 cm1 decrease, while those at 1425, 1365, 1335, 1315, 1200, 1158, 1104, 1055, and 1028 cm1 as well as those in the

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(s) (w) (s) (w) (w) (w) (s) (w) (w) (w) (w) (w) (w) (w) (w)

1740 (w) 1620 1545 1425 1405 1365 1335 1315 1236 1200 1158 1104 1055 1028 985 895 662

(w) (w) (s) (w) (s) (s) (s) (w) (s) (s) (s) (s) (s) (s) (s) (s)

Band assignment C=O stretching HOH bending of adsorbed water þ amide I HOH bending of adsorbed water Amide II CH2 scissoring O–H deformation C–H bending CH2 wagging CH2 wagging O–H deformation or NH deformation C–O stretching C–O–C stretching C–O stretching C–O stretching C–O stretching C–O stretching b-glycosidic linkage O–H out-of-plane bending

a Parentheses indicate the relative ATR FT-IR spectral intensities between 10 and 33 DPA fibers: s = strong, w = weak.

1000–875 and 700–600 cm1 region increase. A comparison of intensity increasing or decreasing of these bands between 10 and 33 DPA fibers is tabulated in Table I. These intensity distinctions suggest chemical, compositional, and structural changes during cotton fiber cellulose development, and subsequently these unique bands have proved to be effective in monitoring the increasing dominance of secondary cell wall cellulose.10,11,13,14 Cotton fibers cultured from ovule medium in Fig. 3 exhibit quite similar intensity fluctuations as those in Fig. 2, which is not unexpected. Like those grown in planta, fiber growth in culture medium results in the dominant production of the major common chemical component in cotton fibers, cellulose. With the cellulose deposition in fibers being continuous, there are anticipated spectral intensity differences of the developing fibers grown between in planta and in culture with similar DPA (Fig. 4). For the young fibers at the early developmental stages that included 10 DPA fibers in planta and 16 DPA fibers in culture, three bands at 1565, 1525, and 1405 cm1 are distinguished owing to the difference in fiber growing environments (in culture versus in planta), with two intense bands (1565 and 1405 cm1) in the 10 DPA fibers in planta and one intense band (1525 cm1) in the 16 DPA fibers in culture. Among old fibers at the later developmental stage, including the 33 DPA fibers grown in planta and the 38 DPA fibers grown in culture, the spectral intensity changes are not apparent with respect to two types of fibers. On the basis of relative ATR FT-IR intensities and band positions, cotton fiber from the plant developed faster than those from the cultures at earlier stages, which is consistent with visual observation of fiber growth during the experiments. However, subjective interpretation of spectra cannot be applied for comparing or assessing the degree of fiber secondary wall biosynthesis in a semiquantitative way.

FIG. 3. Representative of normalized ATR FT-IR spectra of cotton fibers grown in culture at 16 (dotted line), 24 (solid line), and 38 (dashed line) DPA.

Principal Component Analysis (PCA) Characterization of Attenuated Total Reflection Fourier Transform Infrared (ATR FT-IR) Spectra. To understand the similarity or dissimilarity of samples (or spectra) in Figs. 2 and 3, all spectra were subjected to PCA characterization in the 1800–600 cm1 region. The first two PCs accounted for 95.2% of the total variation, with PC1 explaining 90.3% of the spectral variation. The plot of PC1 score versus DPA in Fig. 5 provides a good visualization of sample distribution between two sets of fibers. When fibers are grown in planta, PC1 increases

FIG. 4. Representative of normalized ATR FT-IR spectra of (bottom) shorter DPA and (top) longer DPA fibers; the former included the 10 DPA fibers in planta and 16 DPA fibers in culture, while the latter included the 33 DPA fibers in planta and 38 DPA fibers in culture. Spectra of longer DPA cotton fibers were shifted 0.7 units vertically for direct comparison. Among each spectral cluster, solid and dotted lines represent the fibers grown in culture and in planta, respectively.

FIG. 5. Plot of PC1 scores against DPA from normalized ATR FT-IR spectra of fibers collected in planta (*) and in culture ( ).



rapidly between 10 and 24 DPA before reaching the nearly constant PC1 score. The observation implies the occurrence of phase transition from primary to secondary cell wall synthesis between 10 and 24 DPA, and it is consistent with the earlier results showing that elongating fibers at 10 DPA contain no secondary walls, whereas thickening fibers at 24 DPA are composed of relatively more content of secondary wall cellulose.9–11 For fibers grown in culture, PC1 increases significantly from 20 through 27 DPA. Briefly, secondary cell wall synthesis of developing fibers grown in planta started between 10 and 17 DPA, and that grown in culture started between 20 and 27 DPA. In other words, the phase transition of cultured fibers from primary to secondary wall fiber biosynthesis is about 10 days delay compared with the fibers grown in planta, and this result is in line with expectations. Notably, PC1 scores for both fiber sets are nearly independent of fiber DPA when DPA is older than 25 days; the spectral intensity changes at this period of fiber maturation are probably insignificant. Meanwhile, there are unclear disparities in PC1 score between two types of fibers with DPA greater than 25 days. Figure 6 shows the PC1 loadings plot, which features a nearly equal number of negative and positive peaks. The moderate and intense peaks, defined as absolute loading values greater than or equal to 0.05, occur mainly in the 1200–900 cm1 region assignable to the C–O stretching modes in fiber cellulose. Large positive loadings in Fig. 6 contribute largely to positive PC1 scores in Fig. 5, and the same is true between negative loadings in Fig. 6 and negative PC1 scores in Fig. 5. The highest negative and positive loadings at 1062 and 976 cm1 might originate from the C–O vibrations in noncellulosic species or amorphous celluloses and the C–O modes in crystalline celluloses, respectively.18 Algorithms for Analyzing Attenuated Total Reflection Fourier Transform Infrared (ATR FT-IR) Spectra of Cotton Fibers. Based on a spectral intensity

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FIG. 6. PC1 loadings plot from the PCA of 11 normalized ATR FT-IR spectra of fibers collected in planta and in culture.

FIG. 7. Plot of MIR against DPA from ATR FT-IR spectra of fibers collected in planta (*) and in culture ( ).



differential between immature and mature fibers, Liu et al.13 previously identified the key wavelengths first and then developed two simple algorithms (R1 and R2) for their respective discrimination, noting that the R1 values increase with R2 values in a dataset consisting of 402 seed cotton bolls. Next, they proposed formulas to estimate the degree of cotton cellulose maturity (MIR) and crystallinity index (CIIR) by representing the R1 and R2 values. In this algorithm, either MIR values of 0.0 and 1.0 or CIIR values of 0.0% to 100.0% were assigned to the most immature and mature fibers in the dataset, respectively.13,14 Both algorithms were applied to spectral data in Fig. 5, and the plots of MIR and CIIR against DPA are shown in Figs. 7 and 8, respectively. There are obvious differing trends between two types of cotton fibers in Fig. 7, which is reasonable mostly due to their growing environments. The greater MIR at 10 DPA fibers in planta decreases to 17 DPA, then increases up to 37 DPA, whereas for cultured fibers, the turning point of MIR decreasing to increasing happens around 27 DPA. Regarding phase transition, Fig. 7 is in good agreement with PCA presentation in Fig. 5. On the other hand, MIR should increase gradually with raising DPA, hence caution should be taken when applying this algorithm to the younger and more immature fibers. Most likely, the dominant presence of sugars (sucrose, glucose, fructose, and galacturonic acid) in young or shorter DPA fibers contributes significantly to the bands (1032 and 956 cm1) used in this algorithm development, in which these sugars are essential building blocks for synthesizing cellulose, and their percentages decrease as the second cell wall is thickening at the older stage of developing fibers.9 Coincidently, none of the bands in the 1130–900 cm1 region was used to access the secondary cell wall formation.10,11 Overall, CIIR increases steadily for each fiber set as the fibers in planta or in culture grow with increasing DPA (Fig. 8). The CIIR values of in planta fibers matches

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well with the previous report on different cotton varieties.14 Although Fig. 8 resembles Fig. 5 more than Fig. 7 in general, it indicates a continuous augmentation of fiber crystalline fraction during the period of fiber maturation. Because of distinctive fiber growth environmental conditions, there is a reasonable lag in crystallinity development between two types of fibers, in which greater CIIR of the developing fibers grown in planta than those in culture is anticipated. Relative to PCA results, Fig. 8 apparently suggests a difference in CIIR among the fibers with 25 DPA and older, likely implying the ability of this algorithm in detecting the subtle difference within these fibers.

FIG. 8. Plot of CIIR against DPAs from ATR FT-IR spectra of fibers collected in planta (*) and in culture ( ).



FIG. 9. Plot of R values from ATR FT-IR spectra vs. fiber DPAs collected in planta (*) and in culture ( )



Compared with those in the 1180–600 cm1 region that were used to develop the R1 and R2 algorithms, significant intensity reduction or increment of the bands in the 1800–1180 cm1 spectral region could be informative as well (Figs. 2 and 3). From a spectral intensity difference, we developed a simple three-band algorithm (R) in Eq. 1. R ¼ ðA1315  A1800 Þ=ðA1236  A1800 Þ

ð1Þ

where R represents the intensity ratio, and A1800, A1315, and A1236 are each a multi-point average of the band intensities at respective range of 1802–1798, 1317–1313, and 1238–1234 cm1. The 1315 cm1 absorbance arises from the C–H characteristic group that is increasing in intensity with DPA, and by coupling with the intensity decrease of the 1236 cm1 band, the ratio R values can be used to monitor fiber growth. The 1800 cm1 band was selected to be zero in intensity subjectively. Undoubtedly, R values rise along with the elevation of DPA (Fig. 9). The pattern in Fig. 9 is identical to that in Fig. 8, and both are slightly different from Figs. 5 and 7.

CONCLUSION This study demonstrates the usefulness and effectiveness of the ATR FT-IR technique in rapid, nondestructive, and direct characterization of cotton fibers grown in planta and in culture. With DPA progressing, fibers undergo a number of significant chemical, physical, and structural changes. These changes can simply be monitored using unique ATR FT-IR spectral features, but it is not easy to acquire semiquantitative information on these fibers from the spectra directly. PCA results of these spectra indicate that the transition from primary to secondary cell wall biosyntheses of the fibers in culture occurred approximately 10 days later than that of the fibers in planta. As a comparison, three simple algorithms involving the characteristic bands in the 1500–

700 cm1 region were applied, and as with the PCA pattern, any algorithm can be used to describe the fiber secondary cell wall formation. Notably, the algorithm approach is the most attractive and interesting, because it can detect the slight difference in fibers with DPA larger than 25 days and also there is no need to perform PCA development that is regularly built from a spectral set containing at least five samples. From a limited 11 samples in two subsets in this study, along with previous results on diverse cotton fibers from simple algorithm approaches,13,14 it is expected that this ATR FT-IR protocol could be applicable to more fiber samples. Actually, similar studies on different cotton varieties are underway, and the promising results will be reported as available. On the basis of practical implementation of this ATR FT-IR procedure for rapid, nondestructive, routine, and direct investigation of fiber biosynthesis, this study reports simple procedures to acquire ATR FT-IR spectra and to analyze the spectra with simple algorithms. This procedure avoids the need to perform any pretreatments of cotton fibers, and this procedure has the advantages of using fewer fibers (as little as 0.5 mg) in analyzing the shorter DPA fibers with the majority of non-cellulosic components and of requiring only a short time (less than 2 min) for sample loading, spectral acquisition, and subsequent result reporting. ACKNOWLEDGMENTS The authors acknowledge Tracy Condon of USDA-ARS-SRRC for technical assistance in collecting the experimental samples and data. This research was partly supported by Cotton Incorporated-sponsored project 12-199. Mention of a product or specific equipment does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable. 1. Y.-L. Hsieh. ‘‘Chemical Structure and Properties of Cotton’’. In: S. Gordon, Y.-L. Hsieh, editors. Cotton: Science and Technology. Cambridge, UK: Woodhead Publishing Limited, 2007. Pp. 3-34. 2. J.M. Bradow, G.H. Davidonis. ‘‘Quantitation of Fiber Quality and the Cotton Production—Processing Interface: A Physiologist’s Perspective’’. J. Cotton Sci. 2000. 4(1): 34-64. 3. C.A. Beasley, I.P. Ting. ‘‘Effects of Plant Growth Substances on in Vitro Fiber Development from Unfertilized Cotton Ovules’’. Am. J. Bot. 1974. 61(2): 188-194. 4. H.J. Kim, B.A. Triplett. ‘‘Cotton Fiber Growth in Planta and in Vitro. Models for Plant Cell Elongation and Cell Wall Biogenesis’’. Plant Physiol. 2001. 127(4): 1361-1366. 5. S. Gordon. ‘‘Cotton Fiber Quality’’. In: S. Gordon, Y.-L. Hsieh, editors. Cotton: Science and Technology. Cambridge, UK: Woodhead Publishing Limited, 2007. Pp. 68-95. 6. I. Frydrych, D.P. Thibodeaux. ‘‘Fiber Quality Evaluation—Current and Future Trends/Intrinsic Value of Fiber Quality in Cotton’’. In: P.I. Wakelyn, M.R. Chaudhry, editors. Cotton: Technology for the 21st Century. Washington, DC: International Cotton Advisory Committee, 2010. Pp. 251-295. 7. D.P. Thibodeaux, J.P. Evans. ‘‘Cotton Fiber Maturity by Image Analysis’’. Text. Res. J. 1986. 56(2): 130-139. 8. N. Abidi, E. Hequet, L. Cabrales. ‘‘Changes in Sugar Composition and Cellulose Content During the Secondary Cell Wall Biogenesis in Cotton Fibers’’. Cellulose. 2010. 17(1): 153-160. 9. H.J. Kim, J. Rodgers, C. Delhom, X. Cui. ‘‘Comparisons of Methods Measuring Fiber Maturity and Fineness of Upland Cotton Fibers Containing Different Degrees of Fiber Cell Wall Development’’. Text. Res. J. 2014. 84(15): 1622-1633. 10. N. Abidi, L. Cabrales, E. Hequet. ‘‘Fourier Transform Infrared Spectroscopic Approach to the Study of the Secondary Cell Wall Development in Cotton Fiber’’. Cellulose. 2010. 17(2): 309-320.

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11. N. Abidi, L. Cabrales, C.H. Haigler. ‘‘Changes in the Cell Wall and Cellulose Content of Developing Cotton Fibers Investigated by FTIR Spectroscopy’’. Carbohydr. Polym. 2014. 100: 9-16. 12. Y.-L. Hsieh, X.-P. Hu, A. Nguyen. ‘‘Strength and Crystalline Structure of Developing Acala Cotton’’. Text. Res. J. 1997. 67(7): 529-536. 13. Y. Liu, D. Thibodeaux, G. Gamble. ‘‘Development of FTIR Spectroscopy in Direct, Non-Destructive, and Rapid Determination of Cotton Fiber Maturity’’. Text. Res. J. 2011. 81(15): 1559-1567. 14. Y. Liu, D. Thibodeaux, G. Gamble, P. Bauer, D. VanDerveer. ‘‘Comparative Investigation of Fourier Transform Infrared (FT-IR) Spectroscopy and X-Ray Diffraction (XRD) in the Determination of Cotton Fiber Crystallinity’’. Appl. Spectrosc. 2012. 66(8): 983-986.

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15. M.L. Nelson, R.T. O’Connor. ‘‘Relation of Certain Infrared Bands to Cellulose Crystallinity and Crystal Lattice Type. Part II. A New Infrared Ratio for Estimation of Crystallinity in Cellulose I and II’’. J. Appl. Polym. Sci. 1964. 8(3): 1325-1341. 16. H.A. Kra¨ssig. Cellulose Structure, Accessibility and Reactivity. Yverdon, Switzerland: Gordon and Breach Science Publishers, 1993. 17. P. Sanguansat. Principal Component Analysis—Engineering Applications. Rijeka, Croatia: Intech, 2012. 18. Y. Liu, D. Thibodeaux, G. Gamble. ‘‘Characterization of Attenuated Total Reflection Infrared Spectral Intensity Variations of Immature and Mature Cotton Fibers by Two-Dimensional Correlation Analysis’’. Appl. Spectrosc. 2012. 66(2): 198-207.