Abstract

BackgroundProtocols for Agrobacterium rhizogenes-mediated hairy root transformation of the model legume Lotus japonicus have been established previously. However, little efforts were made in the past to quantify and improve the transformation efficiency. Here, we asked whether effectors (nodulation outer proteins) of the nodule bacterium Sinorhizobium sp. NGR234 can promote hairy root transformation of L. japonicus. The co-expressed red fluorescent protein DsRed1 was used for visualization of transformed roots and for estimation of the transformation efficiency.ResultsStrong induction of hairy root formation was observed when A. rhizogenes strain LBA9402 was used for L. japonicus transformation. Expression of the effector gene nopP in L. japonicus roots resulted in a significantly increased transformation efficiency while nopL, nopM, and nopT did not show such an effect. In nopP expressing plants, more than 65% of the formed hairy roots were transgenic as analyzed by red fluorescence emitted by co-transformed DsRed1. A nodulation experiment indicated that nopP expression did not obviously affect the symbiosis between L. japonicus and Mesorhizobium loti.ConclusionWe have established a novel protocol for hairy root transformation of L. japonicus. The use of A. rhizogenes LBA9402 carrying a binary vector containing DsRed1 and nopP allowed efficient formation and identification of transgenic roots.

Highlights

  • Protocols for Agrobacterium rhizogenes-mediated hairy root transformation of the model legume Lotus japonicus have been established previously

  • Hairy root formation induced by A. rhizogenes LBA9402 carrying pISV‐DsRed1 We found in previous studies that pISV2678 is an effective binary vector for Agrobacterium transformation

  • The binary vector was mobilized into different A. rhizogenes strains (LBA1334, K599 and LBA9402) to test their capacity to transform L. japonicus roots

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Summary

Introduction

Protocols for Agrobacterium rhizogenes-mediated hairy root transformation of the model legume Lotus japonicus have been established previously. The co-expressed red fluorescent protein DsRed was used for visualization of transformed roots and for estimation of the transformation efficiency. Agrobacterium tumefaciens (Rhizobium radiobacter) causing formation of crown galls and Agrobacterium rhizogenes (Rhizobium rhizogenes) inducing hairy roots are powerful tools to express genes in plants. In these transformation systems, Agrobacterium harbors a plant expression vector (binary vector) with a given gene construct [1]. A. rhizogenes has been widely used to obtain composite plants that express genes in formed hairy roots. These roots can either be transgenic (i.e., express genes derived from the binary vector) or non-transgenic. Detection of transgenic hairy roots is facilitated by co-transformation of a marker gene that allows visualization of transgenic roots

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