Cotton Genetics, Genomics, Breeding and Farming

**Chapter 2**

## Studies on Colored Cotton: Biochemical and Genetic Aspects

*Sathees Nagarajan, Yazhni Purushothaman, Monika Selvavinayagam, Pandidurai Govindharaj and Aasif Musthafa*

## **Abstract**

Cotton (*Gossypium hirsutum* L.) is a commercially important fiber crop used as the primary raw material in the textile industry and is cultivated throughout the world. Normally cotton fiber is white color and various dyes are used to color the fiber. In textile industry, the process of artificial dying is a major source of pollution to the environment and the cost of dying is also higher. Apart from the white fiber, several cotton species have colored fiber which can be used to reduce the dying process and its ill effects to the environment. The cotton fiber color inheritance pattern is an urgent problem. The physical and chemical properties of colored cotton are determined by its chemical composition. The naturally colored cotton contain some important properties such as, greater hygiene, hypoallergenic properties, lower flammability and higher ultraviolet protection value compared to traditional white cotton. The natural colored cotton loss their market value due to the poor fiber quality. Understanding of the colored cotton pigment composition, biochemical and genetic prospects of colored cotton will be useful for the development of high quality of colored cotton.

**Keywords:** colored cotton, fiber quality, colored pigment, biochemical and genetic property

## **1. Introduction**

In world, cotton is an important cash crop and it is a most traded commodity [1]. China, India, United States, Pakistan, and Brazil are the largest cotton producers [2]. With 312 lakh bales, India has the world's largest cotton area of around 12.7 million hectares and is now the world's second largest cotton producer (each of 170 kg) [3]. The cotton is a dicotyledon comes under the malvaceae family and *Gossypium* genus. Globally, *Gossypium* genus is spread in 5 continents. It contains 50 species in the world which are woody and herbaceous form [4]. The 50 species contain 45 diploid (2 n = 2x = 26) and five allotetraploid (2 n = 4x = 52) species [4]. Among these two diploid species (*G. arboreum* L. and *Gossypium herbaceum* L.) and two tetraploid species (*G. hirsutum* L. and *Gossypium barbadense* L.) comes under cultivated species. The 95 percentage of world cotton production was fulfilled by two tetraploid cotton cultivars such as *G. hirsutum* L. (upland cotton) and *G. barbadense* L. (Sea Island or

Egyptian cotton) because it contains good fiber yield and broad adaptation to several environments [5]. In global fiber market, the polyester and other synthetic fibers have the robust competition during 1990 s which increases the competition in beginning of 2000 s [1]. Most of textile products are manufactured by cotton fiber and lint [6]. In *Gossypium* genus, the formation of fibers is abnormal. The surface of the ovule has the outward elongated cells growing. Additionally, as the fiber matures, the protoplast dies, and the cell wall collapses inward to form a convoluted ribbon [5]. One of the most significant raw materials for the textile industry is cotton (*Gossypium hirsutum* L.). Nearly all cotton fiber used for textiles is white, dyes are necessary throughout the fiber processing process to color the cloth. The massive usage of dyes has resulted in pollution, which has had a significant impact on human health [7]. Cotton that is naturally colored is made up of pigmented fibers with color embedded in the lumen [8]. The colorful strain of *G. hirsutum* is crossed with a white linted strain to create hybrids that outperform the color parents in terms of fiber length, strength, and color fastness. Natural colored cotton will be the next big thing in the market as the world shifts toward pollution-free organic fabrics and products. This is due to the fact that the production of naturally colored cotton avoids the most polluting activity of textile product manufacturing (dyeing) [9]. In ecology textiles, naturally colored cotton is an important raw material which eliminates the dying during the processing. It would significantly decrease the processing cost, environmental pollution and chemical residue [8, 10]. Furthermore, when compared to standard white cotton, naturally colored cotton may have a reduced flammability and a greater UV protection rating [11]. One of the most efficient solutions is to breed cotton varieties that naturally contain colored cotton fibers (CCFs), which are environmentally safe. Cotton plants with colorful fibers have been cultivated for a long time [12]. However, for the following two reasons, their development has been slower than that of white fiber cotton. To begin with, colored fiber cotton yields far less than white fiber cotton [13]. Various dye products that have been employed in the textile industry since the industrial revolution are available, as well as the negative consequences of their use has long been forgotten [14]. Cotton fiber is the most important fabric material on the planet, with almost all the industrially used cotton coming from white cotton fiber (WCF). However, with rising environmental concerns and improved human life quality, interest in naturally colored cotton (NCC, *G. hirsutum*) fiber has steadily increased over the previous decade. Natural colors are used in NCC fibers. The use of NCC fiber in fabrics would cut textile processing and the generation of harmful chemical wastes significantly [15]. Nonetheless, poor fiber quality and drab colors have hampered NCC fiber adoption on a big scale [16]. The genetics and plant breeding of cotton goal is improving fiber quality while increasing the cotton yield [17]. Fiber quality is a complex trait which includes fiber length, strength, and fineness. The following traits determine the cotton yield such as number of bolls per plant, number of plants per unit area, lint percentage, and single boll weight [18]. The fiber quality and yield have the negative correlation, so the synchronous improvement of that traits through conventional breeding techniques is difficult. So, understanding of the colored cotton biochemical and molecular aspects will be used to improve that cotton.

## **2. Genes regulating pigmentation and its inheritance pattern**

Knowledge on the genes responsible for pigmentation can help the breeders to develop colored cotton and to overcome the barriers in developing it. The natural colored

#### *Studies on Colored Cotton: Biochemical and Genetic Aspects DOI: http://dx.doi.org/10.5772/intechopen.104898*

lint of cotton shows great variability for lint color of which brown and green are the most stable and predominant types. The pigments are accumulated in the lumen of the lint [19] only when exposed to sunlight [20]. The developed color fades out when exposed to sunlight for a longer time and moisture content also affects the developed color [15]. The poor fiber quality *viz.,* fiber length, fiber percent and resistance is attributed to the pleiotropic effects of genes controlling fiber quality [21]. Presence of modifying genes also affects the color and quality of pigments. By expression analysis, *GhF3'5'H* and *GhCHS3* genes were found to be higher in colored cottons than in white cotton [22].

Green color results due to the deposition of caffeic acid in the suberin layer. Brown pigmentation results due to the pro anthocyanidin (PA) (condensed tannin) accumulation in cell vacuoles [23]. The prime sequences in PA biosynthesis were highly conserved in both white (*G. arboreum*) and brown (*G. raimondii* or *G. stocksii*) fibers [24]. Earlier it was reported that both the green and brown pigments are governed by single gene with incomplete dominance [25]. Later, in 1944, conventional approaches revealed the existence of six loci (Lc1- Lc6) for brown pigment, out of which Lc1 was located on chromosome 7, Lc2 on chromosome 6 and one locus (Lg) for green pigment. Further, colored cotton is dominant over white fiber cotton [26]. But in texas green and brown lint, they are controlled by single incompletely dominant gene [25]. Generally, these genes are pleiotropic in nature [10].

Genetically, green color genes are dominant over both brown and white colors in cotton. From the findings of several researchers, it is evident that, the brown color is controlled monogenically with incomplete dominance [27]. Also, the brown lint and brown fuzz color was found to be correlated and controlled by single gene with incomplete dominance [28, 29]. Further investigation advocated that the *GhTT2-AO7* gene of *LC 1* controls the brown color fiber trait [30]. Additionally, in brown color cottons, the flavanoid genes (*GhCH1, GhF3H, GhDFR, GhANS & GhANR*) were involved in proanthocyanidin flavanoid production which is responsible for the brown pigmentation [22]. The gene *Gh3GT* coding flavonoid 3- glucosyltransferase leads to green color even in brown fiber cotton [31]. Flavonoid biosynthesis is regulated by several transcription factors such as R2R3-MYB type factors, basic helixloop- helix and WD40 repeats [32]. The genes *GhMYB10* and *GhMYB36,* homologous to genes which encode the R2R3-MYB type transcription factors were noticed in cotton and they enhance the PA synthesis [33]. *GhTT2-A07* and *GhTT2-3A* also involve in the production of brown pigment. *GhTT2-3A* and *GhbHLH130D* drive the structural genes *GhANR* and *GhLAR* to accumulate PA in fiber [30]. Also, the genes *GhTTG1* and *GhTT3* genes play an inevitable role in PA synthesis and fiber development [34]. The genes which are responsible for anthocyanin pigment were also found to act in the regulation of PA synthesis [35].

Caffeic acid biosynthesis in green fiber takes place via phenyl propanoid pathway. The expression of the gene *GhPAL* was found higher at the initiation stage of secondary cell wall thickening. The genes *Gh4CL1- GhCL4* convert the caffeic acid to its ester form. Out of these four above mentioned genes, *Gh4CL2* showed higher expression in green fiber, which was confirmed by its expression level and enzymatic activity studies [36].

## **3. Biosynthetic pathway/molecular basis of pigment synthesis and deposition**

In brown fiber cotton, the amount of oxidized pro anthocyanidin increases with the maturation of bolls and its structure was observed to be modified by a galloyl

group [37]. MALDI- TOF MS proteomic analysis [38] in brown fiber revealed that out of 21 proteins responsible for pigmentation, 15 were the members of flavonoid biosynthesis process. PAs are polymers of polyhydroxy flavan-3-ol units and addition of leucoanthocyanidin (flavan-3, 4-diol) molecules. Digital gene expression (DGE) analysis showed that 34 PA synthase genes are involved out of which only 24 were upregulated [39]. These upregulated genes coding for the enzymes involved in the synthesis of PA including, 3- phenylalanine ammonia lyases (PAL), cinnamic acid-4-hydroxylase (C4H), 1, 4- coumarate CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavone 3- hydroxylase (F3H), flavonoid 3′ hydroxylase (F3'H) and flavonoid 3′ 5′- hydroxylase (F3'5'H), dihydroflavonol 4- reductase (DFR), anthocyanidin synthase (ANS) and anthocyanidin reductase (ANR) [39]. Out of these, CHI plays a major role in the coloration of brown fiber [31]. Individual biochemical pathways were identified in brown fiber cotton some involving the major role of ANR [23] and some with major role of leucoanthocyanin reductase (LAR) [39]. Also, the activity of PAL was found to be higher in brown fiber than white ones [40]. The accumulation of PA in the brown fiber was found to be in peak at 30 DAS and decreased due to their oxidation toward maturation [37]. The biosynthetic pathway involved in the synthesis of pro anthocyanins is illustrated in **Figure 1.**

Green color in fibers is the result of Caffeic acid (CA) accumulation. Nearly 70% ω- hydroxydocosanoic acid and 25% decosanedoic acid, which are the components of caffeic acid, were isolated from green cotton whereas only 0.5% was reported in white fiber. UV and Nuclear magnetic resonance (H-NMR) spectroscopic studies revealed that wax portion of green fiber is mostly composed of glycerol, CA and its esterified form. Isolated fatty acids from green fiber showed the presence of 22-O-caffeol-22 hydroxydocosanoic acid and 22-O-caffeol-22- hydroxydocosanonin. They were responsible for green and yellow pigmentation and increasing the concentration of

**Figure 1.**

*Biosynthesis of pro anthocyanins [41].*

the latter, leads to deep green color [23]. Another important point to be noted was the suberization of seed coat in the plant, producing green fiber, while it was absent in white and brown cotton [42].

## **4. Characterization of colored cotton pigments**

Comparing to white cotton, naturally colored cotton has more flavonoids, which would reach 1 mg/g at maturity. This forms the major portion of pigments synthesized in colored cotton. pH values also tend to vary in the colored and white cotton, which was 5.60 in white fiber and 5.63 in colored cotton at 30 days post anthesis (DPA). But in brown cotton, it rose to 6.07 at 35 DPA and may reach 6.38. Generally, a drop in pH, favors cell elongation and secondary cell wall thickening during fiber cell development and this rise in pH may lead to poor development of fiber [13]. Cellulose content of colored cotton differs from that of white cotton after 20 DPA and this may be due to the fact that flavonoid synthesis in CCF may make use of the available simple carbohydrates. This in turn may affect the quality and quantity of fiber [43]. Pigment development in green cotton takes more time than that of brown cotton. The fibers of naturally colored cotton give low lint yield, produce short, weak and coarse fiber. Also, the distribution of pigment may not be uniform [10]. The brown pigmented cotton fiber was found to be superior to green. In addition, the green pigment deposits in fiber during 15 to 20 days post anthesis period. While testing the amount of Nitrogen in the colored cotton, it was higher in colored cotton. Potassium level was lower in the colored cotton particularly in green. Correlation studies indicated negative relationship between the pigment with fiber quality parameters. The pleiotropic nature of the color genes inhibits the fiber development and this becomes the reason in the difficulty of developing colored cotton with good fiber quality [13]. Thus, cotton with high cellulose level, low N and P and high K levels with acceptable level of pigment is desirable.

Suberin lamella was present in the cell wall of green cotton fiber cells [44]. Presence of glycerol also has been found in the green cotton fibers. The presence of some yellow green pigments due to the presence of caffeic acid derivatives [23].

## **5. Evaluation of quality of colored cotton**

Colored cotton fibers are currently available and can be combined with conventional white cottons. They are shorter, weaker, and finer than regular upland cotton fibers. Due to smaller bolls and low ginning outturn, color-linted cultivars are often low yielders with low productivity per unit area. Other issues with these cottons include high whiteness per cent, higher wax content, isolation distance requirements, the availability of only a few hues, and unpredictability and non-uniformity of fiber color across seasons and locales. To make these environmentally friendly color cottons commercially viable, researchers must focus their efforts on improving the genetics of agronomic features, fiber quality, and color uniformity [45]. The correct assessment of basic fiber properties and quality classification is a major issue for dealers, spinners, and farmers who are working to improve cotton production characteristics [46]. The degree of reflectance (Rd) and yellowness (+b) as specified by official criteria and measured by the high volume instrument determine the color grade (**Figure 2**). The equipment specification for Rd. and + b is specified in the **Table 1**. The brightness

#### **Figure 2.**

*The cotton color grading instrument [46].*


#### **Table 1.**

*Color cotton equipment specifications [47].*

or dullness of a sample is determined by its reflectance, while the degree of pigmentation is determined by its yellowness. Finding the intersection of the Rd. and + b values on the color chart for American Upland cotton yields a three-digit color code [47]. The color of cotton fibers can be affected by rainfall, freezes, insects, fungi, and staining through contact with soil, grass, or cotton-plant leaf. Color can also be affected by excessive moisture and temperature levels during storage, both before and after ginning. Color deterioration because of environmental conditions affects the fibers' ability to absorb and hold dyes and finishes and is likely to reduce processing efficiency [48].

Several tools and programs are in place to manage quality. These include laboratory conditioning, sample conditioning, equipment performance specifications, instrument calibration, in-house monitoring, and USDA's Quality Management Program.

#### **5.1 Laboratory conditioning**

The measurement of cotton fiber characteristics is influenced by atmospheric conditions. As a result, the classing laboratory's temperature and humidity must be strictly controlled. The temperature is kept at 70 degrees Fahrenheit plus or minus 1 degree Fahrenheit (about 21 degrees Celsius plus or minus 1/2 degree Celsius), and the relative humidity is kept at 65 percent plus or minus 2 percent.

#### **5.2 Sample conditioning**

The moisture content of the samples is conditioned to match the permitted atmospheric conditions. Moisture level in conditioned samples will range from 6.75 to 8.25 percent (on a dry-weight basis). The moisture content of the conditioned samples is examined at random to ensure that the correct moisture content has been achieved. Samples can be passively or actively conditioned. The samples are put in single layers in trays for passive conditioning.

## **6. Breeding methods for the development of Colored cotton**

Natural colored cotton cultivation dates back to 2300 BC [49]. Anciently, colored cottons were domesticated from *G. hirsutum* and *G. barbadense*. Subsequently, Due to the varied dyeing advantages in white cotton, the colored cotton was underrated and lost its preference among people and industrialist during the latter half of 19th century. But with the increasing environmental concern, demand for natural colored cotton gained its momentum during the past decade [16]. Generally the fiber color was negatively correlated with fiber yield, fiber quality [50] and limited color choice [51]. Correspondingly, the studies on genetics, inheritance and correlation of cotton fiber color and fiber yield, quality, pigmentation were stenuously carried out [29, 52]. These studies have led to the development of next generation colored cotton to overcome the short-comings encountered previously.

Primarily, colored cottons were identified as mutants of white cotton predominant from *G. hirsutum* and *G. barbadense* [53]. So far varieties of colored cotton were developed mostly by selection and recurrent crossing approaches from the germplasm [54]. Hybrids were also developed from crossing suitable germplasm with white cotton varieties to enhance the yield and fiber quality. Cocanada 1 & 2 and Red northers were the brown linted tree cotton varieties selected from *G. arboretum*. Vaidhehi 95 (MSH 53) is a introgressed cultivar developed from *G. hirsutum* which is also a brown linted variety produced by Central Intsitute for Cotton Research, Nagpur, India (CICR). CICR also produced some brown linted cotton varieties *viz.,* CNA 405, CNA 406 and CNA 407 but no green linted cotton varities were developed so far. In order to develop more vibrant, diverse fiber color, high yielding, quality colored cotton varieties needs the combined usage of conventional and biotechnological method of plant breeding. Also integrating the results of omics studies on pigmentation of colored cotton would help in a big leap in developing colored cotton varieties [16].

## **7. Conclusion**

To recapitulate, environmentalists are urging scientists and farmers to improve and grow NCC due to their concern about the effects of dyes and the advantages of naturally colored cotton over white cotton. Color development methods in green and brown cotton have been disclosed by the studies on the biochemical and molecular mechanisms of fiber color development in cotton and flavonoid biosynthesis genes.

*GhC4H*, *GhCHS*, *GhCHI*, *GhF3'H*, *GhDFR*, and *GhANR* are the genes responsible for the structural flavonoid biosynthesis pathway that play a major role in color formation in brown fiber cotton. Cloning and genome-wide association studies of the fiber color genes help us comprehend the complicated biological mechanism of color development in cotton fiber which would have been hampered if the cotton genome sequences were not developed. Changing the cellulose and flavonoid biosynthesis pathways to improve fiber quality will be a step toward manufacturing cotton fiber with a wide range of hues and high quality for the textile industry. The recent improvements in several NCC research areas offer opportunities to overcome barriers to commercial NCC breeding, while further studies including multi-omics techniques are needed. The ability to reduce the fiber-quality and yield gap between NCC and white-fiber cotton will determine its growth in the textile market.

## **Author details**

Sathees Nagarajan1 \*, Yazhni Purushothaman1 , Monika Selvavinayagam2 , Pandidurai Govindharaj3 and Aasif Musthafa4

1 Department of Plant Breeding and Genetics, Agricultural College and Research Institute, Tamil Nadu Agricultural University, Madurai, India

2 Centre for Plant Breeding and Genetics, Tamil Nadu Agricultural University, Coimbatore, India

3 Department of Food science and Nutrition, Community Science College and Research Institute, Tamil Nadu Agricultural University, Madurai, India

4 Department of Agronomy, Agricultural College and Research Institute, Tamil Nadu Agricultural University, Madurai, India

\*Address all correspondence to: sathees1063@gmail.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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## **Chapter 3**
