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During both 2010 and 2011 growing seasons, four genotypes cotton were planted in the research field by the Crop System Research Laboratory of USDA-ARS (USDAARSCSRL) at Lubbock, TX. Three irrigation levels (well-irrigated, semi-irrigated, dry-land) controlled by underground dripping irrigation systems were applied to study the effects of drought stress on fiber developmental, structural and cell wall compositional changes; three replications were randomly designed for each level. Ten cotton plants per replication per treatment (total of 30 plants per level) were randomly selected at the initiation of the first flower. On the day of flowering (0 dpa), individual flowers on these plants were tagged, and at least 3 developing bolls at each dpa per genotype, per irrigation level, and per replication were hand harvested at stages critical for secondary cell wall decomposition and fiber maturity: 10, 14, 16-21, 24, 27, 30, 35, 40, 47, and 56 dpa. Additionally bolls were collected from 10, 14, 16, 17, 18, and 19 dpas, due to the fact that only a smaller amount of fibers could be collected from each seed. Immediately after harvesting, the pericarp was removed; fiber tissues were transferred to cryogenic vials, frozen in liquid nitrogen, and then stored at -80 °C until analysis. Each replication was tested independently.
2.1.2 Sample preparation
Samples handling and preparation were critical for analytical testing. Firstly, the cotton ovules were thawed and the cotton fibers were separated from the seeds. The cotton fibers taken from several cotton bolls among replications were mixed manually. Afterwards, the fibers were rinsed with distilled water for several times. The procedure was performed to remove sugars and other water soluble substances that might interfere with the analytical testing procedures. Additional bolls were blended from 10, 14, 16, 17, 18, and 19 younger dpas, due to the fact that only a smaller amount of fibers could be obtained from each of these seed. The fibers were dried at room temperature (25°C) for 24 hours and conditioned in a laboratory maintained at 21°C± 1°C and 65% RH ± 2% at least for 2 days prior to any testing.
2.2 Analytical and Spectroscopic methods
2.2.1 Scanning Electron Microscopy (SEM)
A scanning electron microscope (SEM) images a sample by scanning its surface with a focused beam of electrons to generate a series of signals, which could reveal related information about the sample morphology and crystalline structure etc. TM-1000 SEM (Hitachi High Technologies America, Inc. Pleasanton, CA.), a type of environmental SEM, was performed to image the cotton fiber samples with a backscattered electrons detector at 15kV accelerating voltage. The SEM has a separation membrane between the specimen chamber and the electron gun, therefore, one chamber for the sample and one for the electron gun. The electron gun is at higher vacuum chamber, while the specimen is at lower vacuum chamber, which allows for the transfer of the electron beam to specimen chamber to image the samples at their natural status in a gaseous environment. The instrument is not only easy to use and maintain, but also has superior resolution and higher magnification. Samples from 30 dpa and 56 dpa were prepared and performed according to the protocol outlined in (Hequet, 2006). After the glass slides were well prepared, the SEM observations are performed under 7000 resolution, two replications were conducted for each sample.
2.2.2 Cross-section & Cotton-scope
Cotton fiber maturity was measured by cross-sections and cotton-scope.
Fiber cross-section is a direct and accurate measurement of fiber fineness and maturity, which is a process to extract useful information from images. A microscope and software were applied to analyze the cross-sections. Cotton cross-section followed by image analysis is used to increase the efficiency and accuracy of fiber separation and feature extraction. Fiber cross-sections were performed according to the protocol (Hequet, 2006): firstly, the fiber sample bundles were embedded in a methacrylate polymer, which holds the cotton fibers until they could be glued to a slide for observation. Then, the methacrylate polymer is dissolved in methyl ethyl ketone (MEK). After the 1Î¼m thick cross-sections slides are well prepared, the images were observed with a microscope and a Hitachi CCD Camera Model HVC-20 with a Coreco Oculus TCX Frame Grabber. Thereafter, the FIAS software developed by Xu & Huang (2004) was performed to analysis the image files. The limitation of this method is time consuming and tedious procedure for both preparing cotton samples and processing cross-sectional images, which need take five days to test one sample. Cotton fiber samples from 30 dpa and 56 dpa were prepared and performed, three replications for each sample. The samples from younger dpas (around 21 dpa or younger) were not possible to perform due to the collapse of the lumen and the lack of the secondary cell wall in their fibers.
Cotton-scope is another very useful instrument for cotton fiber development studies. It uses polarized light microscopy and image analysis to measure maturity. It can handle very small sample sizes, normally 50 mg per replication. Cotton fiber samples from 24 dpa to 56 dpa were carried out, three replications per sample. The same with the fiber cross-section methods, samples from younger dpas (around 21 dpa or younger) were not possible to perform due to the collapse of the lumen and the lack of the secondary cell wall in their fibers.
2.2.3 High Volume Instrument (HVI) & Advanced Fiber Information System (AFIS)
High Volume Instrument (HVI) and Advanced Fiber Information System (AFIS) were applied to test fiber quality traits and determine cotton fibers micronaire, maturity ratio, and fineness.
HVI system is a widely used method based on fiber bundles testing to provide varied fiber qualities such as micronaire and so forth. Bundles of fiber samples with known weight are compressed and examined at one time in a chamber of given volume; a steam of air would be passing through the plug of fibers, the volume of air we got is the micronaire.
AFIS is an advanced and reliable measurement to predict detailed individual fiber qualities (length, neps, fineness, maturity, and so on). It works as the following: a sliver should be shaped by hand first and be put into a canister, fibers and trash would be separated into different popes (individualizer); when the fibers passing through the laser beam and detector, the size and length of the fiber quality would be acquired through the sensor and the fiber travel speed. The limitation of AFIS is time-consuming as the fibers need to be looked one by one. Meanwhile, the needles on the instrument gear, the friction between cotton, and the friction between cotton and instrument all might cause a biased result.
2.2.4 Thermogravimetric Analysis (TGA)
Thermogravimetric Analysis (TGA) provides quantitative results regarding the weight loss of a sample as a function of increasing temperatures. TGA of fiber samples was performed using the Pyris1-TGA equipped with a 20-sample autosampler (PerkinElmer Shelton, CT). TG temperature was calculated with the curie point of alumel and nickel alloys at 10 °C/min. TG curves were recorded between 37 and 600°C with a heating rate of 10°C/min in a flow of nitrogen at 20 mL/min. Cotton fiber samples were rolled into small bolls (between 1.5mg to 2.0 mg) by hand, and then placed on the sample pans. Samples from 14, 20, 24, 35, and 56dpa were performed, three replications for each sample. Pyris software was used to calculate the first derivatives of the thermo-grams and to calculate the percent weight loss of each sample. The first derivatives were adopted to compare thermo-grams; the inflection point at the first derivatives is the point where the degradation rate is the fastest. By using the inflection points, the thermo-grams of different cotton fiber samples were compared, which can be interpreted as the amount of water, the amount of non-cellulosic materials, the amount of cellulose and crystallinity. Cotton fiber samples were conditioned in the laboratory at 65±2 % relative humidity and 21±1°C temperature for at least 48 hours prior to the test. Data analyses were conducted using Statistica Software (Statsoft Inc., Tulsa). Factorial ANOVA (analysis of variance) was carried out to test any statistically significant effects.
2.2.5 Fourier Transform Infrared Spectroscopy (FTIR)
Fourier transform infrared spectroscopy (FTIR) can be used as a direct and nondestructive method suitable for the rapid investigation of isolated plant cell walls. FTIR spectra of the cotton fiber samples were recorded using the universal attenuated total reflectance Fourier transform infrared (UATR-FTIR) (Perkin Elmer, Waltham, MA). The UATR-FTIR was equipped with a Zn-Se Diamond crystal, allowing the spectra collection and analysis on the materials surface without any special preparations. A background scan of clean Zn-Se Diamond crystal was processed before the sample scanning procedures. Bunches of cotton fibers were twisted by hand, thereafter placed over the ATR crystal. Latex gloves should be worn to avoid the moisture transfer and contamination. There is a "pressure arm" attached to the UATR-FTIR; it is a necessary accessory to make sure a good contact pressure between the crystal and the samples positioned on its top. The pressure applied in the cotton fiber samples was 122N. All FTIR spectra were collected with spectrum resolution of 4 cm-1, with 32 co-added scans over wavenumber 4000-650 cm-1. The obtained spectra were baseline corrected, normalized and subjected to Principal Component Analysis (PCA). Before the testing, cotton fiber samples were all kept in the laboratory conditioning at 65±2 % relative humidity and 21±1°C temperature for at least two days.
Fifteen FTIR spectra per sample were obtained for every developmental stage to produce a sum of 180 spectra (15 spectra * 4 genotypes * 3 irrigation levels) for each dpa, each genotype and each irrigation level. Thus, there will be a total of 5400 spectra (15 developmental stages * 2 years * 180 spectra) acquired from fifteen developmental stages of two years samples. The Perkin-Elmer software Spectrum v 6.2 was used to perform spectra baseline correction, normalization, and integrated intensities calculation of selected peaks at 3300, 1735, 1627,1543, 1141, 897, 710, and 667. The FTIR spectra are exported to Excel and then subjected to the statistical analysis on Statistica software (StatSoft Inc., Tulsa). Factorial ANOVA (analysis of variance) was proceeded to test the statistically significant effects.