Abstract
BackgroundFollowing genome sequencing of crop plants, one of the main challenges today is determining the function of all the predicted genes. When gene validation approaches are used for woody species, the main obstacle is the low recovery rate of transgenic plants from elite or commercial cultivars. Embryogenic calli have frequently been the target tissue for transformation, but the difficulty in producing or maintaining embryogenic tissues is one of the main problems encountered in genetic transformation of many woody plants, including Coffea arabica.ResultsWe identified the conditions required for successful long-term proliferation of embryogenic cultures in C. arabica and designed a highly efficient and reliable Agrobacterium tumefaciens-mediated transformation method based on these conditions. The transformation protocol with LBA1119 harboring pBin 35S GFP was established by evaluating the effect of different parameters on transformation efficiency by GFP detection. Using embryogenic callus cultures, co-cultivation with LBA1119 OD600 = 0.6 for five days at 20 °C enabled reproducible transformation. The maintenance conditions for the embryogenic callus cultures, particularly a high auxin to cytokinin ratio, the age of the culture (optimum for 7-10 months of proliferation) and the use of a yellow callus phenotype, were the most important factors for achieving highly efficient transformation (> 90%). At the histological level, successful transformation was related to the number of proembryogenic masses present. All the selected plants were proved to be transformed by PCR and Southern blot hybridization.ConclusionMost progress in increasing transformation efficiency in coffee has been achieved by optimizing the production conditions of embryogenic cultures used as target tissues for transformation. This is the first time that a strong positive effect of the age of the culture on transformation efficiency was demonstrated. Our results make Agrobacterium-mediated transformation of embryogenic cultures a viable and useful tool both for coffee breeding and for the functional analysis of agronomically important genes.
Highlights
Following genome sequencing of crop plants, one of the main challenges today is determining the function of all the predicted genes
We describe for the first time in the C. arabica species a highly efficient and reliable Agrobacterium-mediated transformation protocol that allows the generation of thousands transgenic coffee trees from multiple independent transformation events
Embryogenic callus cultures were revealed to be an excellent support for genetic transformation, since significantly higher transformation efficiency (17%) was obtained with four-month-old proliferating embryogenic calli in the same co-cultivation conditions
Summary
Following genome sequencing of crop plants, one of the main challenges today is determining the function of all the predicted genes. Agrobacterium-mediated transformation is a widely used and powerful tool for introducing foreign DNA into many plant species. It has several advantages over physical transformation methods including its tendency to generate single or a low copy number of transgenes with defined ends and preferential integration into transcriptionally active regions of the chromosomes [4]. For many important crop plants, including most woody species, a method for genetic transformation has either not yet been established or is still laborious and inefficient. Higher throughput transformation systems are needed to be able to fully benefit from the rapid development of plant genomics for basic research and for the design of new genetic improvement strategies including both conventional breeding and genetic transformation
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