An effective transient expression system for gene function identification in Lotus japonicus

Transient transformation is a notably efficient and expeditious method for obtaining foreign gene expression products, making it a prominent avenue for investigating functional genes in pasture. The present study aimed to explore the non-in vitro transient transformation technique of Lotus japonicus , a leguminous herbage, to analyze functional genes in the future. This was achieved by evaluating the expression products of GUS reporter gene and the bioactivity of functional gene. The results indicated that the transient expression system was successfully optimized as follows: the utilization of parameters, including an OD 600 absorption of 0.8 for the bacterium, a vacuum pressure of 12 Kpa, and an infiltration time of 15 min, yielded a transient transformation rate of 57.78%. The GUS recombinant protein demonstrated optimal accumulation on the 8th day, while the functional evaluation and utilization of the exogenous gene were observed within a time frame of 2–12 days. The entire branch-leaf explant demonstrated an efficient expression of the GUS recombinant protein within a 12-day timeframe while maintaining viability. This finding suggests that this system not only facilitates the extended analysis of gene function but also accurately reflects the regulatory capabilities of the entire living plant. Furthermore, the results confirmed the viability of the transient transformation system by demonstrating improved tolerance to high temperature, drought, and salt stress in Lotus japonicus expressing foreign LjHsp70-1 gene. The successful establishment of this transient expression system offers a valuable way and framework for investigating the functional characteristics of vital genes in Lotus japonicus or other microphyllous plants.