Cotton is one of the most important crops globally. The application of bioengineering technology has greatly improved the efficiency of molecular breeding. However, current cotton genetic transformation faces challenges such as genotype dependency, lengthy timelines, and limited transformation methods.

This study aims to establish an efficient Agrobacterium rhizogenes-mediated genetic transformation system for cotton to expand genetic breeding methodologies.

Using the common cotton receptor varieties WC and R18 as primary materials and mRUBY as a reporter gene, the root inducing process mediated by A. rhizogenes was optimized through screening hormone combinations (types and concentrations), analyzing differences in explant types and genotype-specific rooting systems. A stable genetic transformation system was subsequently developed and applied to gene editing.

The addition of naphthaleneacetic acid (NAA) and lovastatin to the root inducing medium (RIM) promoted more efficient root formation compared to NAA alone or combinations of NAA+indole-3-butyric acid (IBA) or NAA+Lovastatin+IBA. The optimal concentrations for inducing hairy roots were both 2 mg·L^-1 for NAA and lovastatin. Cotyledons were the most effective explants for root induction: WC cotyledons, cotyledon nodes, and hypocotyls exhibited rooting efficiencies of 398%, 72%, and 39%, respectively. Cotyledons required the shortest induction time (7 d), 3 d shorter than cotyledon nodes and 8 d shorter than hypocotyls. Cotyledons were also the optimal explants for R18, their rooting capacity differed. Genotype comparisons revealed that 20 days post-infection (dpi), the rooting efficiencies per cotyledon were 398% (WC), 116% (R18), 199% (NDM8), 103% (XLZ61), 57% (Gb-1), and 0 (Gb-2). Upland cotton varieties (WC, R18, NDM8, and XLZ61) exhibited rooting efficiencies above 100%, while sea island cotton varieties (Gb-1, Gb-2) were below 100%. Notably, Gb-2 began to root at 35 dpi. Receptor varieties of upland cotton generally showed slightly higher rooting efficiency than production varieties. There was a certain difference between the positive rate of genetic transformation and the rooting rate. The positive rates of NDM8, XLZ61, Gb-1 and Gb-2 at 20 dpi were 59.8%, 16.0%, 38.5% and 0, respectively. Using positive roots as explants, non-embryogenic and embryogenic callus induction yielded transgenic mRUBY-expressing plants, establishing a complete genetic transformation system. The intensity of plant coloration correlated positively with mRUBY expression levels. Additionally, cotton plants with edited GhGI genes were successfully obtained.

The study optimized the A. rhizogenes-mediated root induction process in cotton and established a robust genetic transformation system. This system was successfully applied to gene editing, generating transgenic cotton plants expressing mRUBY and edited GhGI genes.

The objective of this study is to confirm the target traits and genetic stability of transgenic glyphosate- resistant cotton GGK2 and provide technical support for its commercialization.

T₃, T₄, and T₅ transgenic cotton plants GGK2 were subjected to insertion site-specific PCR, Southern blot, ELISA, bioassays in the laboratory and field, analysis of target herbicide tolerance, and investigation of nutritional constituents.

The results indicated that the target genes, GR79 EPSPS and GAT, were integrated into the cotton genome as single copies and stably inherited in GGK2 plants. In GGK2 cotton, GR79 EPSPS, GAT, and NPTⅡ proteins were expressed at different stages and in different tissues, with relatively high expression levels in the leaves. At the four-leaf stage, bud stage and boll opening stag, the expression levels in leaves were 128.7-192.4 µg·g^-1, 24.4-35.0 µg·g^-1, and 17.0-23.9 µg·g^-1 fresh weight for GR79 EPSPS, GAT, and NPTⅡ, respectively. In the field, transgenic cotton GGK2 tolerated up to four times the recommended medium dose of glyphosate application. No significant differences were observed in agronomic traits and nutritional constituents compared to the control, Coker312.

These data demonstrate that transgenic cotton GGK2 is genetically stable and highly resistant to herbicides. Therefore, it can be utilized for breeding high-glyphosate- resistant commercial cotton varieties.

Upland cotton (Gossypium hirsutum) genetic transformation faces series of challenges such as a prolonged cycle and low efficiency, with the relatively slow proliferation rate of callus tissue being a critical factor contributing to the extended transformation period. This study aims to investigate the optimal light conditions for upland cotton callus growing. The establishment of this research endeavor is poised to accelerate callus proliferation and ultimately provide a technical foundation for shortening the period of cotton genetic transformation.

The hypocotyls of the upland cotton line WC were used as explants to induce callus tissues under four different lights: red, blue, red-blue (1:1), and white light (CK). The study aimed to investigate the varied effects of the different lights on callus induction and proliferation, determine the optimal light condition by comparing with the callus proliferation rate, morphological characteristics et al. under different light treatments.

Different light treatments had a significant impact on the callus induction and growth. The red-blue light treatment exhibited a most positive effect on both callus inducing and proliferation, which observed on the 7th and 15th day. The fresh weight of callus at 7 d under the red-blue light (0.39 g) was the heaviest, followed by the blue light (0.34 g), then the white light (0.24 g) and the red light (0.23 g). The same fresh weight order of the callus was observed at 15 d, with the treatment of the red-blue light (1.15 g) > blue light (0.98 g) > white light (0.69 g) > red light (0.51 g). The callus weight under red-blue light was 1.65 times heavier than the control, increased by 16.5% and 125.5% compared to the treatment of only blue or red light, respectively. In line with this, the callus proliferation rate in the second week under red-blue light was as high as 14.67%, which is twice to that of the red-light treatment (7.17%). The expression level of those genes promoted to cell proliferation and somatic embryo regeneration was consistent with the phenotype, the highest level was under the red-blue light treatment. Furthermore, the activity of the catalase (CAT) was significantly increased under the red-blue light treatment, while the content of reactive oxygen species (ROS) was lower than control.

Employing different light conditions could result in the varied proliferation rate of cotton callus. The optimal light treatment is by red-blue light in a 1∶1 ratio, followed by blue light and then white light. However, employing red light only does not favor the callus growth. The treatment of red-blue light (1∶1) induces the expression of phytochromes and cryptochromes in the callus, increases the expression level of those genes promoted to callus growth. It also enhances the activity of catalase, reduces the content of ROS, and finally promotes the callus proliferation.