Optimization of Hairy Root Transformation and Application of RUBY as a Reporter in Lotus corniculatus

Lotus corniculatus is an important perennial legume forage species and has been widely used to study the relationships between plants and rhizobia or arbuscular mycorrhizal fungi. Composite plants (wild-type shoots with transgenic hairy roots) can be produced via genetic transformation mediated by Agrobacterium rhizogenes. The A. rhizogenes puncture method and a method of cutting embryonic roots and coating the hypocotyl incision with A. rhizogenes were used to induce hairy roots from seedlings. The identification of transgenic hairy roots from composite plants is also an important task. Here, young branches from adult plants were used as explants to produce composite plants instead of seedlings via the one-step cutting method in the natural environment. The results showed that the explants of young branches had higher transformation frequency than those of seedlings, and the composite plants obtained from the young branches had greater growth and were also more robust than the plants obtained from the seedlings. In this study, a system for the biosynthesis of betalains named RUBY was integrated into an expression vector as a reporter gene. It is very easy to distinguish transgenic roots from nontransgenic roots based on the red color of the hairy roots. Young branches were used as explants for hairy root transformation with higher transformation frequency than the seedlings. RUBY was used as a reporter gene to distinguish transgenic roots from nontransgenic roots based on the red color of the hairy roots.