Comparisons of methods measuring fiber maturity and fineness of Upland cotton fibers containing different degrees of fiber cell wall development

Abstract

Maturity and fineness are important physical properties of cotton fibers affecting qualities of fibers and yarns. A number of methods are used for measuring fiber maturity and fineness from developed fibers that are desiccated and harvested from open bolls. With the recent advent of molecular breeding and genomics, there is a growing need for measuring the physical properties of developing fibers that are living cells containing genetic materials. Unlike the developed fibers, the developing fibers are immature fibers composed of high levels of physiological sugars causing stickiness. Therefore, there is a challenge in measuring fiber properties from the developing fibers. To identify methods enabling the measurement fiber maturity and fineness from developing fibers, we compared various methods including the use of the USTER Advanced Fiber Information System (AFIS), high-volume instrument, Cottonscope, fiber cross-section image analysis microscopy, cellulose assay, and gravimetric fineness methods. Our results showed that maturity ratios (MR) measured from high maturity fibers correlated among all methods, whereas AFIS MR measured from low maturity fibers did not correlate with other methods. The fineness values measured by AFIS and Cottonscope were affected by the levels of physiological sugars in developing fibers. As a result, we conclude that pre-elimination of physiological sugars causing the stickiness was crucial to measure accurate fineness values from developing fibers. The results demonstrated strengths and weaknesses of various methods of measuring fiber maturity and fineness from immature and developing fibers. The information will help cotton scientists measuring and interpreting fiber properties from developing fibers.

Keywords

cotton fibers developing fibers fineness maturity physiological sugar stickiness

Fiber maturity and fineness of cotton fibers are important physical properties but they have not been well characterized as compared with other physical properties such as fiber length and strength.¹ Fiber maturity refers to the degree of fiber cell wall development in cotton fibers and plays a major role for dye uptake.^1,2 The term of fiber fineness has been used to define fiber perimeter, diameter, cross-sectional area, linear density (mass per unit length), and specific fiber surface. Among them, the linear density is most often used, since textile industries use the linear density data to determine the minimum numbers of fibers required to spin a certain size of yarn.¹ Both fiber maturity and fineness affect processing, yarn strength, and uniformity.³ Fiber maturity and/or fineness are measured with several methods including the USTER Advanced Fiber Information System (AFIS), Shirley Development Fineness Maturity Tester, polarized light microscopy, fiber cross-section image analysis microscopy (IAM), and near infrared spectroscopy.^1,2,4,5 The IAM and gravimetric fineness methods are considered as the reference methods that are the most accurate and direct methods of measuring fiber maturity and fineness, but they have not been frequently used due to a long and laborious process.^1,2 For a quick and automated assessment of fiber maturity and fineness, both textile industries and cotton breeders have mainly depended on the high-volume instrument (HVI) that is defined as a standardized instrument for testing cotton (SITC) by the International Cotton Advisory Committee (ICAC). HVI indirectly measures a combination of the fiber maturity and fineness of cotton fibers in the term of “micronaire” (MIC) that is determined by measuring air-flow resistance through a plug of cotton fibers of a given weight.^1,3 Cotton fibers with high MIC values are generally considered as highly mature fibers containing thick cell walls, but cotton fibers with low MIC values can have highly mature cotton fibers with very fine fibers.⁶ Thus, there is the limit in the MIC value for interpreting fiber maturity and fineness despite the fact that the United States Department of Agriculture (USDA) Agricultural Marketing Service (AMS) uses MIC as a key quality assessment parameter of cotton fiber.

To measure fiber maturity and fineness separately, the AFIS has been widely used by the textile industry and cotton breeders since it was developed to predict spinning performance and yarn quality in the 1980s.⁷ The AFIS uses an infrared beam and electro-optical technology with an aeromechanical separator for individualizing fibers within a sample. Fiber maturity is measured as the maturity ratio (MR) representing the relative amount of cellulose in the fiber cross-section in general. The MR in AFIS is calculated as the ratio between the high degree $(θ \geq 0.5)$ and the low degree $(θ \leq 0.25)$ of fiber cell wall thickening (θ) by measuring the shape of the fibers from two different angles. A higher MR indicates a more mature fiber. Fiber fineness is measured in millitex (mtex) that is milligrams per kilometer (mg km⁻¹) of fibers. Fiber MR and fineness measured with the AFIS were reported to have reasonably good correlation with the data obtained from the IAM that is used as a reference method for fiber maturity measurement.⁸ On the contrary, the MR and fineness measured with AFIS were also suggested to be biased, since they were poorly correlated with those measured using another method, the fineness maturity tester that was pre-calibrated with the results from the IAM.⁹

Cottonscope was introduced in 2010 as a new fiber quality assessment instrument designed to measure fiber maturity and fineness using image analysis and polarized light microscopy technologies.¹⁰ In the Cottonscope measurement, a weighted sample (approximately 50 mg) is placed into a water bowl and agitated. As fiber segments pass under the camera/image analysis system, MR is measured by polarized light microscopy, and fineness is determined by image analysis of the total length of approximately 20,000 fiber segments of known total weight. Fiber MR and fineness values measured with Cottonscope were highly correlated with those measured with the IAM.^11–13

There has been a growing need for measuring physical properties of developing fibers for cotton breeding programs especially as major crops transition from conventional breeding to molecular breeding.^14,15 Identification of molecular markers, genes, and mRNAs responsible for important fiber properties is crucial to the process of molecular breeding. These genetic materials cannot be extracted from the developed fiber cells that are dead and dried. Therefore, the fiber property data of developing fibers that are living cells are often required for cotton scientists to identify cotton genes responsible for important fiber properties.^16–18 The cotton fibers are chronologically growing from the day of anthesis (DOA) to approximately 45 to 60 days post anthesis (DPA) until cotton bolls are opened.¹⁹ In this paper, the term “developing fibers” is used to define the growing fibers in the cotton bolls during fiber development. The developing fibers from most Upland cotton (Gossypium hirsutum L.) varieties grown in the U.S. typically reach the maximum length of 2.2–3.8 cm within 24–28 DPA, and secondary cell wall cellulose biosynthesis initiates approximately 14–16 DPA, overlapping with the final stage of fiber elongation.¹⁹ When cotton bolls are open in the 45–60 DPA, the fiber cells are desiccated. In this paper, the term “developed fibers” is used to define the dried fibers in the open bolls. Unlike the developed fibers that are mostly composed of highly mature fibers, developing fibers are thin and immature fibers containing high levels of physiological sugars causing stickiness among fiber individuals.²⁰ All cotton instruments are not suitable for measuring developing fibers with physiological sugars causing stickiness, since they were designed to measure fiber properties from the non-sticky developed fibers without the physiological sugars.²¹ Thus, it has been a challenge for cotton scientists to measure fiber properties from the developing fibers using the instruments invented to measure the developed fibers.

In this paper, we have compared the fiber maturity and fineness measured between the AFIS and Cottonscope in addition to the fiber property data measured by the HVI, IAM, cellulose assay, and gravimetric fineness methods to determine the strengths and weaknesses of each instrument for measuring both developed and developing fibers.

Experimental details

Cotton fiber materials and treatment

Five different Upland cotton varieties, SG 747 (PVP 9800118), MD 90ne (PI 634931), TM-1 (PI 607172), DP 90 (PI 564767), and Coker 312 (PI 529278) were grown in 5 gallon pots (three pots for each variety) with Metro-Mix 360 in the same environment conditions of a greenhouse. Two near-isogenic cotton lines (NILs), TM-1²² and immature fiber (im) mutant²³ were grown side by side in a row (50 plants) of the cotton field (2000 ft²) of the USDA-Agricultural Research Service (ARS) in New Orleans, LA, in 2007 and 2009. Ten bolls of developing TM-1 fibers were manually collected at each developmental stage (24, 28, 32, 36, and 40 DPA). Developing fibers were hand-ginned from ovules, and developed fibers were ginned using a laboratory roller gin. The soil type of the cotton field was Aquents dredged over alluvium in an elevated location to provide adequate drainage.

Measurements of fiber properties

For measurements of fiber properties from the unwashed or washed cotton fibers, fibers were pre-equilibrated with 65 ± 2% humidity at 21 ± 1℃ for 48 h. Average MIC values were obtained from five replicates measured by HVI (USTER Technologies Inc., Knoxville, TN) and Fibronaire instrument (Motion Control Inc., Dallas, TX). All instruments for fiber property analyses were properly calibrated according to the manufacturers' instructions and standard cotton fibers obtained from USDA-AMS. Developed fibers were measured without pre-treatment for the MIC measurement, whereas developing fibers were teased to fluff fibers before the MIC measurement. AFIS maturity ratio and fineness were measured by Uster® AFIS-Pro (USTER Technologies Inc., Knoxville, TN). The average AFIS fiber data were obtained from five replicates with 5000 fibers per replicate. Cottonscope maturity ratio and fineness were measured by Cottonscope (Cottonscope Pty Ltd, Perth, Australia) according to the method described in Rodgers et al.¹¹ 20,000 fiber snippets (approximately 50 mg) were produced by cutting the fiber samples with a knife-blade cutter, and individualized in the Cottonscope water bowl by agitating the snippets. Four replicates were measured for each Cottonscope sample. For measuring gravimetric fineness (mtex, mg km⁻¹) of fibers, 300 fibers were combed, cut at the top and bottom to leave each 15 mm long, and measured by a microbalance.²⁴ Average gravimetric fineness was calculated from the three measurements. Statistical analyses and construction of graphs were performed using one- and two-way ANOVA and Prism version 5 software (Graph-Pad Software, Inc., San Diego, CA).

Fiber cross-section image analysis microscopy (IAM)

The fiber samples were embedded, thin-section cut, and photographed using a light microscope.²⁵ Average wall area (A) excluding lumen and perimeter (P) of the fiber cross sections were measured from three hundred cross-sections using the image analysis software according to Xu and Huang.⁸ Circularity (θ) representing the degree of fiber cell wall development was calculated using the equation, θ = 4 πA/P².² The obtained circularities from the cotton fibers were converted to maturity ratio (MR) using the equation, MR = θ / 0.577.²⁶

Measurement of cellulose content and water-insoluble fraction from developing fibers

Cellulose content from developing fibers at each developmental stage was measured by the method described by Updegraff with minor modifications.²⁷ Dried fiber samples were cut into small pieces. 10 mg of the fibers were placed to 5 mL of reacti-vials. Non-cellulosic materials in fibers were hydrolyzed with an acetic–nitric reagent. The remaining cellulose was hydrolyzed with sulfuric acid and measured by a colorimetric assay with anthrone. Avicel PH-101 (FMC, Rockland, ME) was used as a cellulose standard. The average cellulose content for fibers was obtained from two biological and three technical replications. The content of non-cellulosic component was calculated from the cellulose content and total fiber weight.

To measure water-insoluble fractions from developing fibers, the hand ginned fibers were dried in an incubator (37℃). Five hundred milligrams of the dried fibers were extensively washed in distilled water five times for 20–60 min per each washing. The washed fibers were dried and measured by a microbalance. The water-insoluble fractions of each of the developing fibers were calculated from two biological and three technical replications. The water-soluble fraction was calculated from the water-insoluble fraction and total fiber weight.

Results and discussion

Discrepancy of fiber maturity measured between AFIS and IAM

Upland cotton variety TM-1 and immature fiber (im) mutant are model cottons for studying cotton fiber maturity and fineness since they are genetically very similar as near-isogenic lines (NILs) showing different MIC values.^17,28–30 The developed fibers of the wild type TM-1 showed fluffy phenotype, whereas the im mutant showed non-fluffy phenotype (Figure 1(a)). IAM and HVI analysis showed that the TM-1 fibers consisted of thick and mature fiber cell walls with higher MIC value (4.35) whereas the im mutant fibers were composed of thin and immature fiber cell walls with lower MIC value (2.73) (Figures 1(b) and (c)).

Figure 1.

Comparisons of two Upland cottons, TM-1 and im mutant showing different fiber maturity. (a) Phenotypes of Upland cotton varieties, TM-1 showing a fluffy boll and im mutant showing a non-fluffy boll. (b) Microscopic images of cross-sections from TM-1 fibers with high maturity and im mutant fibers with low maturity. A scale bar represents 15 µm. (c) A comparison of average MIC values between TM-1 fibers (4.35) and im mutant fibers (2.73).

With the MR values obtained from IAM that is the reference method, we confirmed that the TM-1 fibers were mature (MR, 0.867) whereas the im mutant fibers were uncommonly immature (MR, 0.641) according to the rating system suggested by Cotton Incorporated (http://www.cottoninc.com/fiber/quality/US-Fiber-Chart/Ratings-Of-Fiber-Properties/) and USTER Technologies (Table 1 and Figure 2). On the contrary, the MR values measured by AFIS showed that both TM-1 (0.917) and im mutant fibers (0.873) were mature according to the both rating systems (Figure 2). The rating system suggested by Cotton Incorporated was established to classify cotton fibers according to the MR values obtained by the fineness maturity tester or IAM (Table 1). The other classification suggested by the USTER Technologies was developed to classify cotton fibers based on the AFIS MR values.³¹ AFIS analysis and its rating system classified the im mutant fibers as mature fibers although the im mutant fibers were uncommonly immature according to HVI and IAM measurements.

Figure 2.

Comparisons of fiber maturity between TM-1 and im mutant fibers. IAM maturity ratio was calculated from circularity values measured from fiber cross-section IAM, whereas AFIS maturity ratio was measured by AFIS-Pro.

Table 1.

Rating systems to classify cotton fibers based on fiber maturity ratio, fineness, and micronaire

Maturity ratio (MR)			Fineness (mtex)		Micronaire (MIC)
Class	Cotton Inc.	USTER	Class	Cotton Inc.	Class	USDA
Very mature	>1.0	>0.96	Very coarse	>230	Discount	>4.9
Mature	0.8–1.0	0.86–0.95	Coarse	200–230	Base	4.9–4.3
Immature	0.7–0.8	0.76–0.85	Average	175–200	Premium	3.7–4.2
Uncommon/very immature	<0.7	<0.75	Fine	135–175	Base	3.5–3.6
Very fine	<135	Discount	<3.5

For a direct fineness measurement, fineness was manually measured with the gravimetric fineness method, and compared with the fineness value obtained from AFIS (Figure 3). The gravimetric fineness of the im mutant fibers (136.3 mtex) was 22.4% lower than that of TM-1 fibers (175.6 mtex). The AFIS fineness of the im mutant fibers (156.3 mtex) was 5.9% lower than that of the TM-1 fibers (166.1 mtex). Since the latest AFIS application report from USTER Technologies does not provide the classification of raw cotton fibers on the basis of fineness, we used the current fineness rate suggested by Cotton Incorporated to classify cotton fibers (Table 1). Both gravimetric fineness method and AFIS classified the TM-1 fibers as average fineness and the im mutant fibers as fine fineness.

Figure 3.

Comparisons of fiber fineness measured between TM-1 and im mutant. Gravimetric fineness (GF) was measured from 300 fibers manually and calculated from three replicates. AFIS fineness was measured from 5000 fibers and calculated from five replicates.

As a result of the discrepancy of the im mutant fiber maturity measured by the different methods, we further compared fiber maturity and fineness covering a broad range of cotton fibers measured by various methods.

Comparisons of maturity and fineness from various developed Upland cotton fibers

To compare different methods measuring maturity ratio and fineness of developed fibers, we selected five Upland cottons (SG 747, MD 90ne, TM-1, DP 90, and Coker 312) among well-known cottons varieties. The MD 90ne was known as a mature and strong fiber with high MIC value.³² The Sure-Grow 747 (SG 747) is a commercial cultivar producing coarse and weak fibers.³³ The TM-1 is the Upland cotton genetic standard variety producing average fiber properties.²² Deltapine Acala 90 (DP 90) is an elite commercial cultivar with low MIC with high lint percentage.³² The Coker 312 is a cotton variety with high regeneration efficiency for transgenic cottons with low MIC value.³⁴ The fiber property data of the five varieties covering a broad range of MIC values (3.88–5.19) and their classified rates are summarized in Table 2. The comparisons of the MR values in Table 2 show that the pattern and order of the MR values among the tested varieties were identical between AFIS and Cottonscope measurements (Figure 4). The MR values of all cotton varieties measured with Cottonscope averaged 9.2% lower than those measured with AFIS. However, the Cottonscope MR range (0.179) was 33.6% greater than the AFIS MR range (0.134), indicating that the Cottonscope was more responsive to MR differences between samples compared to the AFIS. The MR differences between Cottonscope and AFIS could result from the different measurement methods. AFIS MR is indirectly calculated from a ratio of the fully mature fibers to immature fibers, whereas Cottonscope MR is directly measured from birefringence detected from the crystalline cellulose of the fibers using a polarized light microscopy. Therefore, Cottonscope is likely more directly measuring fiber maturity that is the relative amount of cellulose in the fibers. We classified the cotton fibers based on the AFIS MR values in Figure 4, since the USTER rating system was developed specifically for classifying AFIS MR values and the Cottonscope rating system has not been established yet.

Figure 4.

Comparisons of fiber maturity of developed fibers from five different Upland cotton varieties. Fiber maturity ratio values of developed fibers having a mature range of MIC values (3.88–5.19) were measured by AFIS and Cottonscope (CS).

Table 2.

Fiber maturity of developed fibers from of five Upland cotton varieties measured with HVI, AFIS and Cottonscope (CS)

Upland cotton varieties	MIC			AFIS MR			CS MR
Upland cotton varieties	Mean	SD	Range	Mean	SD	Rate	Mean	SD
MD 90ne	4.98	0.06	Discount	1.000	0.011	Very mature	0.967	0.010
SG 747	5.19	0.05	Discount	0.974	0.011	Very mature	0.875	0.006
TM-1	4.65	0.04	Base	0.952	0.021	Mature	0.860	0.012
DP 90	4.62	0.06	Base	0.940	0.059	Mature	0.801	0.020
Coker 312	3.88	0.08	Premium	0.866	0.036	Mature	0.796	0.022

In the same way, we classified the Upland varieties on the basis of the fineness values measured with AFIS (Table 3 and Figure 5). The order of cotton varieties sorted with the AFIS fineness values was identical to that sorted with the Cottonscope values. However, the fineness values between AFIS and Cottonscope measurements were significantly (p-value < 0.0001) different from each other. The average Cottonscope fineness values were 20.2% higher than the average AFIS fineness values. The Cottonscope fineness is calibrated to the fineness results from the Cottonscan instrument and normally has a higher fineness value than observed with cross-section image analysis (approximately 20 mtex higher).¹¹ Due to the significantly higher fineness values measured with Cottonscope over AFIS, the current fineness rates suggested by Cotton Incorporated were not used to classify the cotton fibers measured with Cottonscope (Figure 5).

Figure 5.

Comparisons of fineness values of developed fibers from five different Upland cotton varieties. Fiber fineness values of developed fibers having a mature range of MIC values (3.88–5.19) were measured by AFIS and Cottonscope (CS).

Table 3.

Fiber fineness of developed fibers measured between Cottonscope (CS) and AFIS

Upland cotton varieties	AFIS			CS
Upland cotton varieties	Mean (mtex)	SD	Rate	Mean (mtex)	SD
MD 90ne	201.0	7.9	Coarse	253.4	12.2
SG 747	192.2	6.8	Average	243.5	16.3
TM-1	181.8	10.7	Average	218.2	15.5
DP 90	179.8	8.3	Average	201.6	1.4
Coker 312	148.2	8.4	Fine	171.8	1.3

Due to the similar patterns of AFIS, Cottonscope, and IAM measured from the developed fibers (MIC values: 3.88–5.19), we concluded that AFIS MR measured from cotton fibers with high MIC values (≥3.88) were correlated with the MR values measured by Cottonscope and IAM. Thus, we further compared fiber properties measured by various methods from the developing fibers having low MIC values (<3.88).

Fiber properties of developing Upland cotton fibers

To measure fiber maturity and fineness of developing fibers with various methods, we selected TM-1, which is a genetic standard variety for cotton breeders and geneticists.²² For visualizing the fiber maturity referring the degree of cell wall development, we first cross-sectioned the developing fibers at the different developmental stages (24, 28, 32, 36, and 40 DPA). The images of thin-sectioned fiber bundles at six different developmental stages showed that the cell wall area of the cross-sectioned fibers was visually increasing from 24 to 44 DPA fibers during fiber development (Figure 6(a)). At 24 and 28 DPA, thin but noticeable secondary cell walls were detected from the developing fibers. At 32 DPA, secondary cell walls were well established. The fiber cell walls were thickening until 40 DPA when fiber maturation was completed. Cotton bolls were opened at 44 DPA and fibers began to be desiccated. Circularity (θ) referring to the degree of cell wall thickness was quantitatively calculated from average 300 cross-sections (Figure 6(b)). The average circularity increased from 24 (θ = 0.30) to 40 DPA (θ = 0.58) as fiber cell walls were thickening during fiber development.

Figure 6.

Fiber cross-section image analysis microscopy of developing fibers. (a) Microscopic images of cross-sections from developing fibers (TM-1, 24-40 DPA) and developed fibers (44 DPA, Mature). A scale bar represents 15 µm. (b) Quantitative comparisons of circularity values (θ) from developing fibers.

Second, we measured the MIC values of developing fibers of TM-1 (Figure 7). Consistent with the circularity pattern during fiber development, the MIC values increased as fiber cell walls were thickening during fiber development. The MIC value of 24 DPA fibers was lower than the detection limit (<2.4) of HVI and Fibronaire. The MIC values of developing fibers increased while fibers were growing from 24 to 40 DPA (Figure 7 and Table 4).

Figure 7.

Micronaire measurement of developing fibers. Average MIC values of developing fibers (TM-1, 24-40 DPA) were obtained from five replicates by HVI. The 24 DPA developing fibers were not detected (N.D.) due to the detection limit of HVI.

Table 4.

Quantitative results from unwashed developing fibers measured by different methods

Developing fibers	MIC		Cellulose		IAM		CS		AFIS
Developing fibers	Mean	SD	Mean %	SD	Mean MR	SD	Mean MR	SD	Mean MR	SD
24 DPA	<2.4	–	58.2	1.5	0.52	0.06	0.353	0.004	0.860	0.000
28 DPA	3.12	0.01	77.3	3.1	0.57	0.15	0.509	0.004	0.875	0.007
32 DPA	3.58	0.01	79.6	1.0	0.82	0.13	0.654	0.004	0.930	0.028
36 DPA	4.3	0.01	91.2	1.5	0.83	0.18	0.791	0.005	0.960	0.017
40 DPA	4.75	0.01	95	2.6	1.00	0.14	0.826	0.005	0.980	0.010

Third, we measured non-cellulosic components and water-soluble fraction that are mainly composed of physiological sugars causing stickiness of the developing fibers.³⁵ The highest level of the non-cellulosic components (41.8%) and the water-soluble fraction (32.0%) were found at 24 DPA fibers (Figure 8(a)). The lowest levels of the non-cellulosic components (5.0%) and the water-soluble fraction (8.8%) were found in 40 DPA fibers (Figure 8(a)). The results showed that the developing fibers at younger stages contain more physiological sugars causing stickiness than the developing fibers at the older stages.

Figure 8.

Levels of non-cellulosic components and cellulose in developing fibers. (a) Contents of non-cellulosic components and water soluble fraction in developing fibers (TM-1, 24-40 DPA). (b) Contents of cellulose and water-insoluble fraction in developing fibers (TM-1, 24-40 DPA).

Fourth, we measured cellulose content and water-insoluble fraction that are mainly composed of the developed and mature fibers (Figure 8(b) and Table 4). The cellulose content of the developing fibers increased from 24 (58.2%) to 40 DPA (95.0%) as the fibers became mature. In the similar way, the water-insoluble fraction of the developing fibers increased from 24 (68.0%) to 40 DPA (91.2%).

From the results shown in Figures 6, 7, and 8, we concluded that 24 and 28 DPA developing fibers were very immature fibers with high levels of physiological sugars causing stickiness whereas 36 and 40 DPA developing fibers were mature fibers with high levels of cellulose.

Comparisons of maturity from unwashed developing fibers measured with AFIS and Cottonscope

Fiber MR values of developing fibers measured by AFIS and Cottonscope were compared with the reference IAM MR values that were converted from the circularity values from Figure 6(b). For the AFIS and Cottonscope measurements, the developing fibers containing physiological sugars were pre-treated with a teasing process for individualizing fibers. Despite the fact that the increasing order of AFIS MR values was the same as that of IAM reference values, the AFIS MR values at the early stages (24 and 28 DPA) of developing fibers were substantially higher than the reference IAM MR values (Figure 9 and Table 4). Based on the AFIS MR values, the 24 and 28 DPA developing fibers were classified as mature fibers despite the fact that they were uncommonly immature (Figure 9). In contrast, the MR values and rates of the developing fibers measured by the Cottonscope were similar to those by the IAM (Figure 9 and Table 4).

Figure 9.

Comparisons of maturity ratios measured with AFIS, Cottonscope (CS), and fiber cross-section image analysis microscopy (IAM) from unwashed developing fibers containing physiological sugars causing stickiness.

Effect of physiological sugars on the fiber maturity measured with AFIS and Cottonscope

We tested how physiological sugars of developing fibers affected the MR values measured with AFIS and Cottonscope. Since most physiological sugars of the developing fibers were water-soluble, we eliminated the physiological sugars by washing the developing fibers extensively with distilled water, and measured the MR values with AFIS and Cottonscope (Figure 10 and Table 5). The AFIS and Cottonscope MR values from the washed developing fibers were similar to those from the unwashed developing fibers. The physiological sugars of the developing fibers very marginally affected the MR values measured by AFIS and Cottonscope. Despite the slight changes of the MR values of the washed developing fibers measured with AFIS and Cottonscope, the patterns of the MR values between unwashed (Figure 9 and Table 4) and washed developing fibers (Figure 10 and Table 5) were little changed.

Figure 10.

Comparisons of maturity ratios measured with AFIS, Cottonscope (CS), and fiber cross-section image analysis microscopy (IAM) from washed developing fibers with no physiological sugars.

Table 5.

Quantitative results from washed developing fibers measured by different methods

Developing fibers	CS				AFIS
	Maturity (MR)		Fineness (mtex)		Maturity (MR)		Fineness (mtex)
	Mean	SD	Mean	SD	Mean	SD	Mean	SD
24 DPA	0.390	0.003	107.6	2.8	0.800	0.000	115.0	2.8
28 DPA	0.520	0.006	114.6	5.1	0.850	0.007	133.0	1.4
32 DPA	0.770	0.008	156.0	3.0	0.900	0.007	156.0	1.4
36 DPA	0.940	0.005	176.0	3.4	0.937	0.006	163.3	4.0
40 DPA	0.900	0.008	195.6	1.1	0.973	0.023	183.3	10.7

From the results shown in Figures 9 and 10, we concluded that AFIS is not a suitable instrument of measuring the developing fibers (≤28 DPA) with low MIC values (≤3.12). We suspect that the shortcoming of AFIS to measure MR from the immature developing fibers may be caused by the way that AFIS MR is measured. The AFIS MR is calculated as the ratio between high circularity $(θ \geq 0.5)$ and the low circularity $(θ \leq 0.25)$ of fibers. In the immature fibers like 24 DPA developing fibers, there is no mature fiber having a θ value higher than 0.5 (Figure 6(b)). Thus, AFIS MR values cannot be accurately calculated with the very immature fibers. In addition, the aeromechanical action of AFIS may affect the AFIS MR values of the immature fibers by damaging the immature fibers more than the mature fibers.

Comparisons of fineness from unwashed developing fibers measured with AFIS and Cottonscope

The AFIS fineness values measured from the unwashed developing fibers containing physiological sugars showed that the fineness values increased during fiber development from 24 to 40 DPA (Figure 11). The AFIS fineness values of developing fibers at 24, 28, 32, 36, and 40 DPA were 143.5, 150.0, 170.5, 179.7, and 189.7 mtex, respectively. Thus, the 24–32 DPA developing fibers were rated as fine fibers, and the 36 and 40 DPA developing fibers were rated as average fibers according to the classification suggested by Cotton Incorporated. In contrast, the pattern and values of the Cottonscope fineness measured from developing fibers were very different from those of the AFIS fineness. Cottonscope fineness values measured from unwashed developing fibers containing physiological sugars showed that the finest 24 DPA fibers (263.2 mtex) among developing fibers were unrealistically coarser than the coarsest 40 DPA fibers (237.3 mtex) (Figure 11). We suspect that the unrealistic fineness values of the 24 DPA fibers may result from the high level of water-soluble physiological sugars (32.0%) causing stickiness among the 24 DPA fibers (Figure 8(a)). Thus, we re-measured fineness values from the washed developing fibers after eliminating the water-soluble physiological sugars from the developing fibers.

Figure 11.

Comparisons of fineness measured with AFIS and Cottonscope (CS) from unwashed developing fibers containing physiological sugars causing stickiness.

Effect of physiological sugars causing stickiness on the fiber fineness measured with AFIS and Cottonscope

After the elimination of the water-soluble physiological sugars causing stickiness from developing fibers, the unrealistic high fineness values of the 24 DPA fibers disappeared (Figure 12). Unlike the fineness values measured from the unwashed developing fibers containing physiological sugars causing stickiness (Figure 11), the washed developing fibers showed almost identical fineness values with AFIS and Cottonscope (Figure 12 and Table 5). The pre-washing step for eliminating physiological sugars causing stickiness marginally reduced AFIS fineness values and substantially reduced the Cottonscope fineness values of developing fibers (Figure 13). The fineness values of younger developing fibers containing higher levels of physiological sugars reduced more substantially than those of the older developing fibers containing lower levels of physiological sugars.

Figure 12.

Comparisons of fineness measured with AFIS and Cottonscope (CS) from washed developing fibers with no physiological sugars.

Figure 13.

Effect of physiological sugars on fiber fineness measured with AFIS and Cottonscope (CS). The ratio of reduced fiber fineness values after eliminating physiological sugars causing stickiness were presented as percent of the fineness values before eliminating water soluble components.

As a result, we concluded that the physiological sugars causing stickiness of developing fibers affected the fineness values measured with both Cottonscope and AFIS. The unwashed developing fibers containing high levels of physiological sugars causing stickiness were fiber “bundles,” not individual fibers, which gave the appearance of large, coarse fibers and resulted in higher fineness values. The stickiness has been known to cause difficulties of processing the developed fibers. Unlike the developing fibers whose stickiness caused by high levels (8.8–32%) of physiological sugars, the stickiness of developed fibers is mostly caused by honeydew and free sugars more than 0.3% of the fiber dry weight.²⁰ Due to the high level of physiological sugars causing stickiness in the developing fibers, the teasing treatment alone was not enough to individualize fibers from the fiber bundles formed by the physiological sugars for measuring the fineness from developing fibers. The complete elimination of the physiological sugars with extensive washing process was required to measure accurate fineness values from the developing fibers. The physiological sugar levels of the developing fibers affected AFIS fineness values marginally because an aggressive aeromechanical separation method of the AFIS might enable the partial separatation of individual fibers from fiber bundles. In contrast, a gentle motion in the water bowl for a few minutes during Cottonscope measurement was not enough to individualize the fibers from the fiber bundles caused by the high levels of physiological sugars in the developing fibers.

Conclusion

Measurements of fiber maturity and fineness from developing fibers have been challenging tasks among cotton scientists due to inconsistent values measured by either of the well-known and established methods such as AFIS, HVI, Cottonscope, and IAM, and the stickiness of developing fibers. Our results showed that fiber maturity ratios from immature and/or developing fibers were correlated well among the values that were measured by HVI, Cottonscope, and IAM, but not by AFIS. With cotton fibers with high MIC values (3.88–5.19), AFIS MR values were well correlated with those measured by HVI, AFIS, Cottonscope, and IAM. In contrast, the AFIS MR values measured from the immature fibers having MIC values below 3.58 were overly represented compared to the real maturity of the fibers. The results also showed that the physiological sugars causing stickiness were responsible for inaccurate fineness values measured from developing fibers by Cottonscope and AFIS. Therefore, the elimination of the physiological sugars causing stickiness from developing fibers was crucial to measure accurate fineness with both AFIS and Cottonscope. The removal of physiological sugars from developing fibers with water did not affect the MR value in this study and circularity values in the other study,³⁶ since both MR and circularity are measured based on the levels of secondary cell wall cellulose that is not water soluble. However, a question over how much the removal of physiological sugars would change fiber diameter, perimeter, and/or linear density values affecting fiber fineness remains to be answered in further studies. In addition to the IAM method providing the reference values in a long and laborious process, Cottonscope also provided quick, reliable, and sensitive maturity and fineness values from the immature and developing fibers when multiple replications (usually six replications) were measured from each fiber sample whose physiological sugars were pre-eliminated. The results presented here will help cotton scientists selecting appropriate instruments for measuring fiber maturity and fineness from the immature and developing fibers, preparing fiber samples for obtaining accurate fineness values from developing fibers containing high levels of physiological sugars causing stickiness, and interpreting fiber maturity and fineness data measured with various instruments that read different values from the same fiber samples.

Footnotes

Disclaimer

Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA, which is an equal opportunity employer.

Funding

This work was supported by the USDA-ARS CRIS (project number 6435-21000-016-00D) and Cotton Incorporated-sponsored (project numbers 12-199 and 12-216).

Acknowledgments

The authors acknowledge Jeannine Moraitis for measurements of Cottonscope and IAM, Holly King for microscopic images and AFIS measurements, and Tracy Condon for fiber property measurement and cellulose analyses from developing fibers.

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