High-efficiency Agrobacterium-mediated transformation of Lotus corniculatus using DsRed2 positive selection and vermiculite-based rooting method

Agrobacterium tumefaciens-mediated transformation is widely used for the genetic transformation of Lotus corniculatus. In this study, we optimized the genetic transformation system of L. corniculatus to improve the transformation efficiency. Firstly, besides the commonly used cotyledon, we successfully transformed and regenerated the hypocotyl as an additional explant. Secondly, we used DsRed2 instead of kanamycin as a screening marker to screen transgenic callus and shoots, and conducted PCR analysis to verify the selected plants based on the presence of red fluorescence conferred by DsRed2. The results confirmed the reliability and convenience of DsRed2 as a suitable reporter for the transgenic selection of L. corniculatus. Furthermore, we addressed the limitations of commonly used agar-based rooting media by introducing vermiculite as an alternative to agar. The results revealed that the vermiculite-based rooting medium effectively induced root initiation, and facilitated the growth of both roots and shoots in transformed seedlings. Thus, our vermiculite-based rooting medium could serve as an ideal substitute for traditional agar-based rooting medium. To sum up, we have developed a rapid, convenient, and efficient transformation system of L. corniculatus using both cotyledons and hypocotyls as explants, employing DsRed2 as a screening marker, and implementing a vermiculite-based MS medium as rooting medium.