The RUBY reporter for visual selection in soybean genome editing

Abstract Current systems to screen for transgenic soybeans ( Glycine max ) involve laborious molecular assays or the expression of fluorescent markers that are difficult to see in soybean plants. Therefore, a visual system for early screening of transgenic plants would increase the efficiency of crop improvement by genome editing. The RUBY reporter system, which consists of three genes encoding betalain biosynthetic enzymes, leading to the accumulation of purple pigment in transgenic tissue, has been employed in some plants and dikaryon fungi. Here, we assessed the RUBY reporter for visual verification during soybean transformation. We show that RUBY can be expressed in soybean, allowing for visual confirmation of transgenic events without the need for specialized equipment. Plants with visible accumulation of purple pigment in any tissue were successfully transformed, confirming the accuracy of the RUBY system as a visual indicator. We also assessed the genetic stability of the transgene across generations, which can be performed very early, using the cotyledons of the progeny. Transgene-free seedlings have a distinct green color, facilitating the selection of genome-edited but transgene-free soybean seedlings for harvest. Using the RUBY system, we quickly identified a transgene-free Gmwaxy mutant in the T1 generation. This system thus provides an efficient and convenient tool for soybean genome editing.