Establishment and Validation of an Efficient Agrobacterium Tumefaciens-Mediated Transient Transformation System for...

Salix psammophila, C. Wang & Chang Y. Yang, a desert-adapted shrub, is recognized for its exceptional drought tolerance and plays a vital role in ecosystem maintenance. However, research on S. psammophila has been limited due to the lack of an efficient and reliable genetic transformation method, including gene functional studies. The Agrobacterium-mediated transient overexpression assay is a rapid and powerful tool for analyzing gene function in plant vivo. In this study, tissue culture seedlings of S. psammophila were utilized as the recipient materials, and the plant expression vector pCAMBIA1301, containing the GUS reporter gene, was transferred into the seedlings via an Agrobacterium-mediated method. To enhance the efficiency of the system, the effects of secondary culture time, Agrobacterium concentration, infection time, and co-culture duration on the transient transformation efficiency of S. psammophila were explored. The optimal combination for the instantaneous transformation of S. psammophila tissue culture seedlings mediated by Agrobacterium was determined as follows: a secondary culture time of 30 d, a value of OD₆₀₀ of 0.8, an infection time of 3 h, and a co-culture duration of 48 h. Subsequently, the effectiveness of the transformation system was validated using the S. psammophila drought response gene SpPP2C80. To further confirm the accuracy of the system, SpPP2C80-overexpressing Arabidopsis was constructed and drought resistance analysis was performed. The results were consistent with the transient overexpression of SpPP2C80 in S. psammophila tissue culture seedlings, indicating that this system can be effectively employed for studying gene function in S. psammophila. These findings provide essential information for investigating gene function in non-model plants and pave the way for advancements in molecular biology research in S. psammophila.