Background

Genetic engineering has opened the doors to enhanced yield, disease resistance, and improved nutritional quality and quantity. Genetically modified (GM) crops ensure that the needs of the growing population are met worldwide. The altered attributes in GM crops are due to introduced transgenes, which is achieved by different tools as particle bombardment, chloroplast transformation, pollen tube pathway transformation, protoplast electroporation, Agrobacterium-mediated genetic transformation, and many more. Amongst these, Agrobacterium tumefaciens–mediated transformation is the preferred one due to its exceptional trait of transferring a DNA segment into the host cell via a specialized plasmid (Ziemienowicz, 2014). This technique has become favorable for scientists and industries because of its simple and easy approach, its ability to transfer a low copy number of the gene of interest, and its efficiency of transfer of a large DNA fragment into the host cell—all of this at a very low cost. Agrobacterium-mediated genetic transformation has been employed to transform many economically important crops using plant tissue culture, which helps to regenerate a large number of transgenic plants at a single time point (Somleva et al., 2002; Manickavasagam et al., 2004; Jones et al., 2005; Sun et al., 2006).

Transgenic cotton was one of the first genetically modified organisms to be globally accepted and commercially developed. Now and again, researchers have been trying to improve the quality of cotton crops, either by enhancing its fiber yield or by imparting pests resistance. Developing a transgenic cotton plant is tedious and time consuming because of its recalcitrant nature and high production of phenolics, which hinders the regeneration of the plant. Although Agrobacterium-mediated genetic transformation has enabled the development of stable transgenic cotton, various efforts have been made to improve the transformation process (Qandeel-E-Arsh et al., 2021). A successful transformation does not depend on a single parameter; instead, it heavily relies on multiple factors such as Agrobacterium strain, type and age of explants, duration of co-cultivation between Agrobacterium and the plant tissue, bacterial cell density during transformation, regeneration method for the plant, selection marker, and type of vector to use (Firoozabady et al., 1987; Satyavathi et al., 2002; Jadhav and Katageri, 2017). To develop transgenic cotton plants, scientists have used the hypocotyls, cotyledons, and shoot tips, as well as the embryogenic callus, pollen tube, embryo, and meristems as explants (Jin et al., 2005; Pathi and Tuteja, 2013; Wang et al., 2013). The use of meristems is very cumbersome and labor intensive and requires particle bombardment (with the drawback of high copy number). The use of the embryonic callus is a relatively good option, but these take several months to be obtained, and the subsequent process does not guarantee proper plantlet germination (McCabe and Martinell, 1993). Transformation of the shoot tip, the embryo, and the pollen tube is easier than others, but the potential for stable transgenic plants is low. Most of these techniques fall into the category of transient transformation, skipping the inserted gene in subsequent generations. Direct organogenesis has also been tried in order to facilitate regeneration, but when the transformation is involved, it becomes sluggish and dreary, and chimeric plants are often obtained. Many of these issues can be resolved by using hypocotyls or cotyledons as the explant, as the process involves somatic embryogenesis that produces somatic embryos, which are single cell–derived. Furthermore, this promotes high-end vegetative output, ensuring more transgenic plants.

Various regeneration protocols have been proposed by several researchers. They have fiddled with different concentrations and types of plant hormones to induce callus, manipulated growth media to induce embryogenesis in proliferating calli, and changed carbohydrate sources as well (Shoemaker et al., 1986; Finer, 1988; Hemphill et al., 1998; Aydin et al., 2004; Ikram-ul-Haq and Zafar, 2004; Kumar and Tuli, 2004). All these attempts have successfully regenerated cotton genotypes, but the development of stable transgenic lines requires an appropriate combination of cotton regeneration and transformation. Currently, the most commonly used approaches are the pollen tube pathway, embryo transformation, and meristem-mediated methods (Bajwa et al., 2015), but these do not guarantee stable gene transfer. Using these methods, elucidation of the applied hypothesis is possible, but only a few have been able to be applied in practical use for stable transgenic development.

Here, we propose a protocol for Agrobacterium-mediated genetic transformation and regeneration in cotton, resulting in transgenic production (Figure 1). This protocol is based on the approach described by Kumar and Tuli (2004), with various modifications that accelerate the achievement of the transgenic development. We have optimized the media for the Agrobacterium to be ready to infect the explants, the co-cultivation period, the appropriate quantity and type of phytohormones to be used, and the environment required for callus induction, its proliferation, and further embryogenesis, and we have reduced the abnormalities in the embryos produced. To summarize, this protocol is an approach towards obtaining a stable transgenic line, via transforming the explants with a gene of interest and regenerating them with early callus induction, improved embryogenesis, and culture requisites. We have used a 14 kb construct harboring a CamV35S promoter, the pectin methylesterase (PME) gene, the neomycin phosphotransferase II (nptII), and the nopaline synthase (Nos) terminator. The protocol has been used to develop several distinct transgenic lines and is currently being used to alter numerous unique genes in cotton for genome editing.

Figure 1. Schematic representation of Agrobacterium-mediated genetic transformation of cotton hypocotyl explants and regeneration (created with BioRender.com)