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.