Peas in Rouge: Tyrosine Supplementation Enhances RUBY Reporter Visibility in

Genome modification of legumes, peas in particular, is accompanied by significant challenges. Establishing a reliable reporter system to identify tissue that expresses foreign DNA may help to optimize and develop transformation protocols for these species. The RUBY system, based on the synthesis of red betalain from tyrosine, offers a convenient solution for monitoring the efficiency of transgene introduction. To evaluate the effectiveness of RUBY application in pea tissue culture, we combined agrobacterial transformation with an in vitro cultivation system, inducing callus development. Transformed explants demonstrated RUBY pigmentation, but it disappeared during cultivation. We hypothesized that this issue is caused by tyrosine depletion. To check this suggestion, we tested whether tyrosine supplementation could maintain RUBY coloring. In the later stages, pigmentation still could not be preserved. However, our modified conditions increased the percent of colored shoot apex explants during the early cultivation stages. Thus, it is likely that some explants transformed with the RUBY cassette do not synthesize a sufficient amount of betalain due to the deficit of endogenous tyrosine. In this case, adding exogenous tyrosine would enhance betalain production and improve the detectability of tissues containing the RUBY cassette. These data can be used for the optimization of RUBY application conditions for peas and other species.