**2.1 Cross-breeding**

Hybridization is a fundamental breeding technique used in the production of colored cotton. It involves controlled cross-breeding between two cotton plants with specific color traits to create offspring with desired color characteristics. By crossbreeding different naturally colored cotton varieties with distinct color traits, breeders can generate hybrids that exhibit a wider range of colors and color intensities in their fibers. Since ancient times, these methods have been employed to create cotton types with certain colors, including brown, green or tints in between [4–6].

The breeding process for naturally colored cotton starts by identifying cotton plants with the desired color traits. This can be done by observing cotton plants in the field and selecting those that naturally exhibit the desired colouration. Now cross-pollinate the selected parent plants with each other, introducing genetic diversity. For example, if you want to develop brown cotton, you would cross-pollinate two brown cotton plants.

**Figure 1.** *Pictures of naturally colored cotton.*

*Naturally Colored Cotton: A Sustainable Innovation DOI: http://dx.doi.org/10.5772/intechopen.113290*

**Figure 2.** *Techniques for producing naturally colored cotton.*

The resulting offspring, known as the first generation or F1 generation, will have a mix of genetic traits from the parent plants. Some may display the desired color, while others may not. Select the plants from the F1 generation that match the desired color trait closely. In the case of brown cotton, select plants with brownish fibers. Now, cross the selected F1 plants with one of the original parent plants that possessed the desired color trait. This is known as backcrossing.

Backcrossing aims to reinforce the genetic traits responsible for the desired color while reducing the influence of other, unwanted genetic traits. This helps in stabilizing the color trait. This backcrossing is repeated for multiple generations until a cotton variety with stable and consistent color traits is achieved. This can take several generations of breeding. Once a stable, naturally colored cotton variety is achieved, scale up production by cultivating these plants on a larger scale for commercial purposes.

Pima Sutra Cotton is a naturally colored cotton variety developed through conventional breeding. It has a range of colors including shades of brown, green and red. Breeders selectively crossed different naturally colored cotton varieties to enhance color expression and fiber quality. These techniques require patience, careful observation and a deep understanding of genetics and plant biology. Over time, these techniques have been successful in producing naturally colored cotton varieties that are both commercially viable and environmentally friendly [6].

## **2.2 Genetic engineering**

Genetic engineering involves introducing specific genes into cotton plants to achieve desired traits, such as natural colouration [3, 5]. This method allows for more precise control over the color traits. Blue Cotton Researchers from CSIRO, an Australian research organization, used genetic engineering to produce blue cotton fibers. They introduced two genes from the pansy flower into cotton plants. These genes encode enzymes responsible for producing blue pigments called anthocyanins. The resulting cotton plants produced fibers with a light blue color [6–8].

Genetic engineering, also known as genetic modification (GM), offers a more targeted and rapid approach to producing naturally colored cotton by introducing specific genes responsible for pigment production into cotton plants. This method involves manipulating the genetic material of the plants to achieve the desired color traits.

In this technique firstly, the genes responsible for pigment production in other naturally colored plants are identified and selected. These genes encode enzymes or proteins involved in the synthesis of pigments like anthocyanins, carotenoids or chlorophyll. Now, isolate the selected pigment-producing genes from their natural sources and insert the isolated genes into a suitable vector, which is typically a small piece of DNA, such as a plasmid or a viral vector [2, 6, 7]. For example, if you want to create green cotton, you might isolate genes responsible for chlorophyll production from green plants like spinach. These vectors are used to carry the pigment-producing genes into the cotton plant cells which can be done through various methods, including Agrobacterium-mediated transformation or biolistics (gene gun).

Once inside the cotton cells, the vector carries the pigment-producing genes into the plant's genome. These genes are integrated into the cotton plant's DNA. Now the transformed cotton cells that have successfully integrated the pigment-producing genes are cultivated to regenerate whole cotton plants with the desired genetic modification.

Genetic engineering techniques offer a more precise and controlled method for producing naturally colored cotton compared to conventional breeding. However, it also involves regulatory and public acceptance considerations due to concerns about GMOs (genetically modified organisms). Careful testing and monitoring are essential to ensure the safety and stability of genetically modified cotton varieties [7, 9].

#### **2.3 Marker-assisted selection (MAS)**

Marker-assisted selection (MAS) is a powerful breeding technique used to select plants with specific desirable traits, including natural colouration, based on genetic markers associated with those traits. When it comes to producing naturally colored cotton, MAS can be a valuable tool for breeders to efficiently and accurately identify cotton plants with the desired color characteristics [6, 10].

Initially, researchers identify genetic markers that are linked to the expression of natural color traits in cotton. These markers can be specific DNA sequences or variations (mutations) in certain genes that are consistently associated with the desired color trait. To effectively use MAS, a genetic map of the cotton genome is created. This map helps locate the positions of the genetic markers relative to one another and specific color traits. Then DNA samples are collected from cotton plants, including the ones under consideration for breeding. These samples can be from different cotton varieties or individual plants within the same variety The collected DNA samples are subjected to genetic analysis to identify which plants carry the specific markers associated with the desired color traits. Molecular techniques like PCR (Polymerase Chain Reaction) and DNA sequencing are often used for this purpose [10, 11].

Based on the genetic marker information and trait confirmation, breeders select cotton plants with the most promising genetic profiles for color traits. The selected parent plants are crossbred to create a new generation of cotton plants that carry the desired color traits. This process helps in combining the genetic markers associated with colouration. The offspring from the crossbreeding are then screened using genetic markers to identify individuals who have inherited the desired color traits. This process of selection and breeding is repeated over several generations to stabilize the color traits and improve other characteristics, such as fiber quality and yield [11].

MAS enables breeders to expedite the selection process by focusing on specific genes and markers associated with natural colouration, ultimately leading to the

development of naturally colored cotton varieties with enhanced traits. This technique enhances breeding efficiency, reduces the time required to develop new varieties and allows for precise selection based on genetic information [10, 12].

## **2.4 Mutation breeding**

Mutation breeding is a technique used to induce genetic mutations in plants to generate new traits, including natural color variations, that can be advantageous for agriculture or other purposes. In the context of producing naturally colored cotton, mutation breeding can be employed to generate cotton plants with unique color traits through the introduction of genetic mutations [9, 10, 12].

In mutation breeding, cotton seeds or young plants are exposed to mutagenic agents. These agents can include chemicals (e.g., ethyl methane sulfonate), radiation (e.g., gamma rays or X-rays) or even biological mutagens. The mutagenic treatment is carefully controlled to induce mutations without causing excessive damage to the plant's DNA. The goal is to create genetic variation in the cotton population.

After mutagen treatment, the treated seeds are germinated and grown into plants. Each plant represents a potential source of genetic variation. The plants that have undergone mutagenesis are carefully observed for any changes in their phenotypes (observable traits). In the context of natural colouration, breeders are looking for plants that display variations in color compared to the original cotton variety. Plants that exhibit the desired natural color traits are selected as mutants and the selected mutants are propagated through vegetative or generative methods to produce a stable population of cotton plants with the new color trait [4, 12, 13].

Mutation breeding is a powerful tool for introducing novel traits, such as natural colouration, into cotton varieties. It relies on the random creation of genetic variation and the subsequent selection of desirable mutants. While it can produce valuable results, it may also require extensive screening and breeding efforts to stabilize and commercialize the new trait. Additionally, regulatory considerations related to genetically modified organisms (GMOs) may apply in some cases, depending on the extent of the genetic changes induced by the mutagenic treatment [14].

## **2.5 Agronomic practices**

Agronomic practices also play a crucial role in producing naturally colored cotton by influencing the development and preservation of pigments responsible for the colouration of the cotton fibers. Every step, from careful variety selection to specific soil preparation, contributes to the deep and vivid colors that are unique to these cotton cultivars. Growers create a climate that promotes optimum pigment development by strategically fertilizing plants, providing sufficient watering, efficiently controlling pests and diseases and meticulously timing the harvest at peak maturity [2, 4–6].

These procedures protect the color integrity throughout the manufacturing process, together with careful post-harvest processing and quality control methods. These practices, coupled with attentive post-harvest processing and quality control measures, safeguard the integrity of the color throughout the production process. In concert with genetic advancements and environmental considerations, the application of agronomic expertise fosters the growth of naturally colored cotton, yielding fibers that embody the harmonious marriage of sustainable cultivation and captivating colouration [4, 6].
