Construction of a Genetic Transformation System for Populus wulianensis

Abstract

Transgenic technology is a potent tool for verifying gene functions, and poplar serves as a model system for genetically transforming perennial woody plants. However, the current poplar genetic transformation system is limited to a few genotypes. In this study, we developed an efficient transformation system based on the Agrobacterium-mediated transformation of Populus wulianensis, a rare and endangered tree species endemic to Shandong Province. Aseptic seedlings of P. wulianensis were used as experimental materials, and the optimal medium for inducing adventitious buds was explored as 1/2(NH4NO3) MS + 0.05 mg/L naphthalene acetic acid (NAA) + 0.5 mg/L 6-benzylaminopurine (6-BA), resulting in up to 35 adventitious buds. The selection resistance critical pressure of 300 mg/L for timentin can effectively inhibit the growth of Agrobacterium while promoting the induction of adventitious buds in leaves. The critical screening pressure for kanamycin for producing resistant adventitious buds and inducing resistant rooting seedlings was 100 mg/L. We optimized several independent factors, which significantly enhanced the efficiency of genetic transformation. The leaves were infected with Agrobacterium suspension diluted twice by adding 100 μmol/L acetylsyringone (β-AS) (OD600 = 0.6) for 15 min, followed by co-culture in the dark for 3 d. Using this improved transformation system, we obtained transgenic P. wulianensis clones overexpressing the enhanced green fluorescent protein (EGFP) gene through direct organogenesis. Among the 112 resistant buds obtained, 17 developed resistant rooting in seedlings. Eight positive plants were identified through DNA, RNA, and protein level analyses, with a positivity rate of 47.06%. This study provides a foundation for developing and utilizing P. wulianensis germplasm resources and lays the groundwork for resource improvement.