Abstract
BackgroundLong 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.ResultsWe 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.ConclusionsOur 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.
Highlights
Long distance signaling is a common phenomenon in animal and plant development
A split root technique demonstrates systemic AON Split-root experiments are valuable for the investigation of the autoregulation of nodulation, i.e. systemic suppression of subsequent root colonization by rhizobia through signaling from an already colonized part of the root system
This study presents two improved and reliable techniques of dissecting long distance autoregulatory mechanisms of nodule regulation in M. truncatula; (1) a split root experiment to assay AON inhibition that can be adapted to test long distance signaling in any combination of root environments and plants that can be grown on plates and moved to Perlite and (2) an inverted-Y graft experiment that in addition to the above benefits, can be used to differentiate signaling originating in the root from downstream roots responses to shoot signals and that can be used to generate reciprocal grafts
Summary
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. Plants and animals use long-distance signals to coordinate and adjust their growth in response to endogenous and environmental cues. These signals transmit messages throughout the whole organism to achieve biological homeostasis. Our lab uses Medicago truncatula to study long distance regulation; the rootto-shoot and shoot-to-root signals that control nodule number and to understand the regulatory network involved in this process. The bacterial and fungal symbionts of M. truncatula that lead to the fixation of nitrogen and the increased uptake of phosphorus are wellcharacterized [8,9] In both rhizobial and arbuscular mycorrhizal symbioses, the establishment and maintenance of symbiosis requires expensive plant resources, energy [10]. Very little grafting and split root work has been done in model legumes, with historical work in less genetically tractable plants such as bean, pea, soybean, clover and vetch, some of which have stems much larger than those in model systems
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.