A robust in-vitro and ex-vitro Agrobacterium rhizogenes-mediated hairy root transformation system in mungbean for efficient visual screening of transformants using the RUBY reporter

Abstract

Background

Mungbean is one of the most economically important grain legume crops in Asia. Functional genomics studies in mungbean are necessary to understand the molecular mechanisms behind agronomic traits, to advance the crop improvement. However, this progress is significantly impeded by the absence of effective and extensive genetic analysis tools. Agrobacterium rhizogenes-mediated hairy root transformation has become a powerful tool for studying gene function and an efficient alternative for investigating root-specific interactions and processes in different species, due to its quick and simple methodology. Agrobacterium-mediated plant transformation, however, is known to be difficult in legumes, especially in mungbean.

Results

In this report, we developed an Agrobacterium rhizogenes-mediated mungbean transformation system using both in-vitro and ex-vitro approaches, with RUBY employed as a reporter gene. We optimized various parameters, including mungbean genotypes, explant age, optical density of the bacterial culture, co-cultivation medium, and acetosyringone concentration. Our findings indicated that in-vitro transformation was more efficient than ex-vitro in terms of hairy root induction percentage and the proportion of transformed hairy roots expressing the RUBY reporter gene. However, the ex-vitro transformation technique was faster and less complex than the in-vitro method. The highest transformation efficiency for RUBY expression was achieved using 5-day-old cotyledonary nodal explants of cv. K-851, inoculated for 30 min with A4 Agrobacterium cells resuspended in full-strength MS medium at an OD₆₀₀ of 0.5 and supplemented with 100 µM acetosyringone. A total of 60 composite plants were generated and evaluated through PCR, resulting in a transformation efficiency of 6.13%. These optimized parameters also led to the highest percentage of RUBY expression using the two-step ex-vitro hairy root transformation method.

Conclusion

We have developed a simple, rapid, low-cost, and labor-efficient Agrobacterium rhizogenes-mediated mungbean transformation protocol using both in-vitro and ex-vitro approaches, with RUBY as a reporter gene. This method enables the generation of composite mungbean plants that are easier to handle, exhibit higher transformation efficiency, and can be effectively used for root specific functional genomics studies. We expect this technology to be widely adopted for investigating root-related processes in mungbean and other plant species.