Simple and efficient methods to generate split roots and grafted plants useful for long-distance signaling studies in Medicago truncatula and other small plants.

Background: Long distance signaling is a common phenomenon in animal and plant development. In plants, lateral organs such as nodules and lateral roots are developmentally regulated by root-to-shoot and shoot-to-root long distance signaling. Grafting and split root experiments have been used in the past to study the systemic long distance effect of endogenous and environmental factors, however the potential of these techniques has not been fully realized because data replicates are often limited due to cumbersome and difficult approaches and many plant species with soft tissue are difficult to work with. Hence, developing simple and efficient methods for grafting and split root inoculation in these plants is of great importance.

Results: We report a split root inoculation system for the small legume M. truncatula as well as robust and reliable techniques of inverted-Y grafting and reciprocal grafting. Although the split root technique has been historically used for a variety of experimental purposes, we made it simple, efficient and reproducible for M. truncatula. Using our split root experiments, we showed the systemic long distance suppression of nodulation on a second wild type root inoculated after a delay, as well as the lack of this suppression in mutants defective in autoregulation. We demonstrated inverted-Y grafting as a method to generate plants having two different root genotypes. We confirmed that our grafting method does not affect the normal growth and development of the inserted root; the composite plants maintained normal root morphology and anatomy. Shoot-to-root reciprocal grafts were efficiently made with a modification of this technique and, like standard grafts, demonstrate that the regulatory signal defective in rdn1 mutants acts in the root.

Conclusions: Our split root inoculation protocol shows marked improvement over existing methods in the number and quality of the roots produced. The dual functions of the inverted-Y grafting approach are demonstrated: it is a useful system to produce a plant having roots of two different genotypes and is also more efficient than published shoot-to-root reciprocal grafting techniques. Both techniques together allow dissection of long distance plant developmental regulation with very simple, efficient and reproducible approaches.