Agroinfiltration-mediated transient assay for rapid evaluation of constructs in pigeonpea

Abstract

The process of generating stable transformants is time-consuming, labor-intensive, and genotype-dependent. In contrast, transient gene expression techniques, such as agroinfiltration, offer a rapid assessment of gene function and expression. Agroinfiltration, widely employed for studying gene function, has been extensively applied in leaf tissues of Nicotiana benthamiana and various other plant species. Despite its broad utility in various plants, to our knowledge, no prior investigation has been reported in pigeonpea. In this study, we developed an agroinfiltration method for transiently expressing a green fluorescent protein (mGFP5) reporter gene in four pigeonpea genotypes using syringe infiltration at the seedling stage under greenhouse conditions. The expression of the reporter gene mGFP5 was assessed at 72-, 96-, and 120 h post-infiltration (hpi). Additionally, we assessed the effect of morphogenic genes, specifically growth-regulating factor 4 (GRF4) and GRF-interacting factor 1 (GIF1), from both rice and pigeonpea on the expression of mGFP5 in four pigeonpea genotypes. Our findings demonstrate that OsGRF4-GIF1 led to enhanced mGFP5 expression compared to CcGRF4-GIF1 in four diverse pigeonpea genotypes. Fluorescence could be detected till 120 hpi. Furthermore, PCR, RT-PCR, and fluorescence quantification confirmed the presence and expression of mGFP5 at 72 hpi. Our results highlight the efficacy of agroinfiltration in quickly evaluating candidate genes in four genetically diverse pigeonpea genotypes, thereby reducing the time required for the initial assessment of constructs suitable for diverse molecular biology analyses