Improved protoplast fusion technologies using metabolic inhibitors and microfluidics

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Plant breeding has dramatically increased agricultural productivity and will play an important role in adapting to a changing climate and feeding the growing population. In many cases the desired trait, be it disease/insect resistance, or adaptation to cold, heat, or drought stress, is not present in the crop but can be incorporated by hybridization with closely related wild relatives. However, this requires that the species are sexually compatible with each other, which is often not the case.***Protoplast fusion technologies, in which cells from two species can be fused together, can overcome sexual barriers and allow crops to be crossed with more distantly related species. This technology is particularly attractive for tree breeding, where traditional approaches can take decades and are not feasible. However, trees present a number of technical challenges that have not been fully addressed and limit the application of this technology. One example of this is American elm, where the native population was nearly eradicated by the invasive Dutch elm disease (DED). Early researchers found that the American elm population does not contain genes for DED resistance and despite over 50 years of traditional breeding there remains a need for true DED resistant trees. While some Asiatic elms are resistant, early attempts at hybridization failed due to sexual incompatibility and somatic hybridization was proposed as early as 1980 to overcome this. However, a number of problems related to isolation and regeneration of protoplasts have prevented success.***In recent work, we have identified the primary source of the problem and developed a novel approach that resolved some of these issues. This has led to the first protoplast to plant regeneration system for American elm and provides a framework for protoplast fusion. The key breakthrough that facilitated this was through manipulating one of the plant's metabolic pathways to alter the chemical composition of the cell wall, making it more amenable to the process. However, an in depth investigation of this system has not been conducted and a number of issues require further study to understand the underlying mechanism in American elm, evaluate if this approach will work in other tree species, and develop new approaches to improve fusion efficiency. This proposal will establish a research program to refine this technique and develop more controlled protoplast fusion technologies using a microfluidic approach for economically and ecological important plants, with a focus on woody species. These technologies could play an important role in the genetic improvement of plants to adapt to new climatic conditions and help feed the growing population. ********