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

Abstract

A highly efficient genetic transformation system of <i>Liriodendron</i> hybrid embryogenic calli through <i>Agrobacterium</i>-mediated genetic transformation was established and optimized. The <i>Agrobacterium tumefaciens</i> strain EHA105, harboring the plasmid pBI121, which contained the <i>ß</i>-glucuronidase (<i>GUS</i>) gene and neomycin phosphotransferase II (<i>npt</i> 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 <i>Agrobacterium</i>-mediated genetic transformation of the <i>Liriodendron</i> hybrid, including the effects of various media, selection by different Geneticin (G418) concentrations, pre-culture period, <i>Agrobacterium</i> 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 <i>via</i> 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 <i>GUS</i> chemical tissue staining, polymerase chain reaction (PCR), and southern blot analysis demonstrated that the <i>GUS</i> gene was successfully expressed and integrated into the <i>Liriodendron</i> 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 <i>Liriodendron</i> hybrid reported here will facilitate the insertion of functional genes into the <i>Liriodendron</i> hybrid <i>via Agrobacterium</i>-mediated transformation.