Agrobacterium-Mediated Genetic Transformation of Embryogenic Callus in a Liriodendron Hybrid (L. Chinense × L. Tulipifera)

Abstract

A highly efficient genetic transformation system of Liriodendron hybrid embryogenic calli through Agrobacterium-mediated genetic transformation was established and optimized. The Agrobacterium tumefaciens strain EHA105, harboring the plasmid pBI121, which contained the ß-glucuronidase (GUS) gene and neomycin phosphotransferase II (npt II) gene under the control of the CaMV35S promoter, was used for transformation. Embryogenic calli were used as the starting explant to study several factors affecting the Agrobacterium-mediated genetic transformation of the Liriodendron hybrid, including the effects of various media, selection by different Geneticin (G418) concentrations, pre-culture period, Agrobacterium optical density, infection duration, co-cultivation period, and delayed selection. Transformed embryogenic calli were obtained through selection on medium containing 90 mg L−1 G418. Plant regeneration was achieved and selected via somatic embryogenesis on medium containing 15 mg L−1 G418. The optimal conditions included a pre-culture time of 2 days, a co-culture time of 3 days, an optimal infection time of 10 min, and a delayed selection time of 7 days. These conditions, combined with an OD600 value of 0.6, remarkably enhanced the transformation rate. The results of GUS chemical tissue staining, polymerase chain reaction (PCR), and southern blot analysis demonstrated that the GUS gene was successfully expressed and integrated into the Liriodendron hybrid genome. A transformation efficiency of 60.7% was achieved for the regenerated callus clumps. Transgenic plantlets were obtained in 5 months, and the PCR analysis showed that 97.5% of plants from the tested G418-resistant lines were PCR positive. The study of the Liriodendron hybrid reported here will facilitate the insertion of functional genes into the Liriodendron hybrid via Agrobacterium-mediated transformation.