<i>Agrobacterium rhizogenes</i>-Transformed Roots of <i>Medicago truncatula</i> for the Study of Nitrogen-Fixing and Endomycorrhizal Symbiotic Associations

TLDR

Medicago truncatula, a diploid autogamous legume, is being developed as a model plant for studying root endosymbiotic associations such as nodulation and mycorrhizal colonization. The study aims to enable rapid introduction and analysis of chimeric gene constructs in root tissues. The authors optimized an Agrobacterium rhizogenes‑mediated protocol that inoculates sectioned seedling radicles, yielding rapid hairy root organogenesis, selectable co‑transformation with kanamycin, and in vitro propagation of clonal lines capable of endomycorrhizal colonization. The composite hairy roots exhibit normal morphology, support successful nodulation by Sinorhizobium meliloti, display Nod‑factor‑responsive reporter expression indistinguishable from A.

Abstract

Medicago truncatula, a diploid autogamous legume, is currently being developed as a model plant for the study of root endosymbiotic associations, including nodulation and mycorrhizal colonization. An important requirement for such a plant is the possibility of rapidly introducing and analyzing chimeric gene constructs in root tissues. For this reason, we developed and optimized a convenient protocol for Agrobacterium rhizogenes-mediated transformation of M. truncatula. This unusual protocol, which involves the inoculation of sectioned seedling radicles, results in rapid and efficient hairy root organogenesis and the subsequent development of vigorous “composite plants.” In addition, we found that kanamycin can be used to select for the co-transformation of hairy roots directly with gene constructs of interest. M. truncatula composite plant hairy roots have a similar morphology to normal roots and can be nodulated successfully by their nitrogen-fixing symbiotic partner, Sinorhizobium meliloti. Furthermore, spatiotemporal expression of the Nod factor-responsive reporter p MtENOD11-gusA in hairy root epidermal tissues is indistinguishable from that observed in Agrobacterium tumefaciens-transformed lines. M. truncatula hairy root explants can be propagated in vitro, and we demonstrate that these clonal lines can be colonized by endomycorrhizal fungi such as Glomus intraradices with the formation of arbus-cules within cortical cells. Our results suggest that M. truncatula hairy roots represent a particularly attractive system with which to study endosymbiotic associations in transgenically modified roots.