Generation of transgenic Medicago truncatula roots
Agrobacterium rhizogenes is capable of inducing hairy roots on Medicago truncatula hypocotyls by transferring the TL-DNA of its Ri (root-inducing) plasmid. The TL-DNA encodes gene products involved either in plant hormone (auxin) biosynthesis or in the release of auxin from inactive precursors. As a result, the auxin/cytokinin ratio increases, that way inducing root development. Due to the production of endogenous hormones, hairy roots are able to grow independently, e.g. in root cultures. We are exploiting this natural gene transfer system to co-transfer reporter gene fusions into the hairy roots. After nodulation with Sinorhizobium meliloti or mycorrhization with Glomus fungi, the activity of reporter gene fusions in root endosymbioses can be studied histochemically.
Several protocols for the generation of hairy roots on Medicago truncatula exist in the literature. Since these usually require sterile conditions, we developed a protocol to generate hairy roots in open pots. The composite plants can directly be mycorrhized and nodulated in soil, vermiculite/sand or clay granules, allowing to study gene expression under more physiological conditions.
Similar to the expression of reporter gene fusions, RNAi fusions can be transferred to study the effect of downregulating or abolishing the expression of a particular gene on the formation or efficiency of root endosymbioses.
Geneneration of transgenic Medicago spec. plants
Several protocols for the generation of transgenic Medicago truncatula plants have been developed in different laboratories over the past years.
To generate transgenic plants expressing antisense or RNAi fusions, we have used the somatic embryogenesis protocol developed for Medicago truncatula cv. R108 at the ISV, CNRS Gif-sur-Yvette. Under our conditions, this protocol allowed the generation of transgenic plants of the T0 generation in appr. 6-8 months, followed by subsequent selfings to generate stable homozygous lines of the T1 and T2 generation.
A variant of this protocol can be used to transform the closely related forage legume Medicago sativa. Since Medicago sativa is allogamous, transgenic lines have to be maintained as cuttings.