Highly efficient transient gene expression of three tissues in Osmanthus fragrans mediated by Agrobacterium tumefaciens

As one of the 10 most famous flowers, sweet osmanthus (Osmanthus fragrans) is widely utilized in landscaping and food processing in China. Owing to the high complexity and large size of its genome, it is still an enormous challenge to develop transgenic technology for O. fragrans. Therefore, it is necessary to develop an efficient transient expression protocol for the rapid identification of gene functions in O. fragrans. In this study, we selected three different tissues of O. fragrans, including calli, floral buds, and leaves, as explants for transient transformation mediated by Agrobacterium tumefaciens. However, we found that one protocol did not work for all tissues. Calli were induced and proliferated from the immature plumule of O. fragrans on one-half Murashige and Skoog media that contained 0.5 mg·L⁻¹ 6-benzylaminopurine and 1 mg·L⁻¹ naphthalene acetic acid. GUS (β-glucuronidase) was used as a reporter gene, where its level of transcription reflected the abundance of Agrobacterium-mediated T-DNA transferred into explants. To obtain the optimal transient expression protocol for each explant, we evaluated the effects of Agrobacterium load, acetosyringone concentration, co-culture duration, bud bract removal, and explant preculture on the transformation efficiency. The use of our transient expression protocols to overexpress a transcriptional repressor of OfXTH14, OfMYB4, in calli, floral buds, and leaves showed that the relative levels of expression of OfXTH14 decreased significantly in all three of our selected tissues. Since the transformed explants exhibited a transient high level of expression of the target gene, affecting the transcription of related genes as well as the synthesis of related metabolites, they can be widely applied to identify gene functions and study the molecular regulatory mechanisms of O. fragrans. Moreover, the critical steps to significantly increase the transformation efficiency varied from tissue to tissue, providing a reference for the development of transient expression protocols in other plant species.