High frequency transformation and regeneration of transgenic plants in the model legumeLotus japonicus

Abstract

The molecular analysis of plant genes involved in nodulation has been slowed by the inability to produce high numbers of transgenic legume lines. The high efficiency gene transfer and plant regeneration systems of the model legume Lotus japonicus is described. A collection of wild-type A. rhizogenes strains was tested for infectivity and the most virulent strains, 9402 and AR10, were selected for further use. Growth conditions for plantlets, induction of hairy roots and nodulation of composite plants were optimized for large-scale screening in Petri dishes. A cluster of 3–10 nodules was regularly formed on transgenic hairy roots 7–12 d after inoculation with the effective Rhizobium loti strain NZP2235. There were no apparent morphological differences between nodulation of hairy and wildtype roots. To test the applicability of the hairy root system for the trapping of symbiotic genes, transformation experiments with binary vectors possessing a β-glucuronidase (gus, uidA) or a luciferase (luc) reporter driven by a cauliflower mosaic virus (CaMV) 35S promoter were performed. The frequency of cotransfer of a binary T-DNA with a root-inducing (Ri) T-DNA was 70%. Positive expression suggests that gus and luc trap vectors can be used for gene tagging in L. japonicus. To open the possibility of searching for mutant phenotypes, a regeneration system has been developed enabling the regeneration of large numbers of transgenic plants from hairy root cultures in about 5–6 months. At the same time, the A. tumefaciens hypocotyl transformation regeneration in L. japonicus has been improved. This new version provides fertile transgenic plants in about 4 months.