An efficient Agrobacterium-mediated transient transformation system and its application in gene function elucidation in Paeonia lactiflora Pall

Abstract

<i>Paeonia lactiflora</i> Pall. is known as the king of herbaceous flowers with high ornamental and precious medicinal value. However, the lack of a stable genetic transformation system has greatly affected the research of gene function in <i>P. lactiflora.</i> The <i>Agrobacterium</i>-mediated transient gene expression is a powerful tool for the characterization of gene function in plants. In this study, the seedlings of <i>P. lactiflora</i> were used as the transformation receptor materials, and the efficient transient transformation system with a GUS reporter gene was successfully established by <i>Agrobacterium</i> harboring pCAMBIA1301. To optimize the system, we investigated the effects of germination time, <i>Agrobacterium</i> cell density, infection time, acetosyringone (AS) concentration, co-culture time, negative pressure intensity, Tween-20 concentration and different receptor materials on the transient transformation efficiency of <i>P. lactiflora.</i> The results showed that the highest transient transformation efficiency (93.3%) could be obtained when seedlings in 2-3 cm bud length were subjected to 12 h infection of resuspension solution comprising 1.2 OD₆₀₀ <i>Agrobacterium</i>, 200 μM AS and 0.01% Tween-20 under 10 of negative pressure intensity followed by 3 days of co-culture in darkness condition. This method is more suitable for the study of gene function in <i>P. lactiflora.</i> Subsequently, stress resistance genes <i>PlGPAT</i>, <i>PlDHN2</i> and <i>PlHD-Zip</i> were used to verify the effectiveness of this transformation system. These results can provide critical information for identification of key genes in non-model plants, such as <i>P. lactiflora</i>, and promote the development of molecular biology research for <i>P. lactiflora</i>.