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ISHS Acta Horticulturae 1135: III International Symposium on Citrus Biotechnology

Intragenic mediated genetic improvement of citrus: What have we learnt?

Authors:   M. Dutt, L. Soriano, J.W. Grosser
Keywords:   agrobacterium, genetic engineering, consumer friendly
DOI:   10.17660/ActaHortic.2016.1135.10
Abstract:
Most existing citrus cultivars lack the resistance to combat Huanglongbing (HLB), a serious bacterial disease that is usually fatal. However some wild relatives demonstrate resistance to this disease. Intragenic mediated genetic improvement of citrus obtained by improving the expression of genes that can potentially improve resistance in the cultivated varieties holds much promise. This can result in the development of a genetically modified sweet orange (Citrus sinensis (L.) Osbeck) acceptable to the consumer, juice processors and packing houses. This intragenic plant would have genetic material derived from the cultivated variety itself or from closely related species capable of sexual hybridization such as other wild citrus species, trifoliate oranges and kumquats. Use of this gene pool, which is similar to other genetic improvement methods such as conventional breeding and protoplast fusion, could lead to easier approval of a genetically modified product. We are exploring several strategies to create intragenic citrus. We have evaluated a single base pair mutant (A122V) of the Citrus sinensis acetolactate synthase gene as a herbicide resistance selectable marker gene. We have developed a transformation system to generate reporter gene expression free citrus by coupling a visual anthocyanin producing transcriptional factor gene with an embryo specific promoter to regenerate genetically modified plants that have the marker gene switched off. We are utilizing the cre-lox system to remove selectable marker genes from plants following transformation. In addition, we are also evaluating a large number of citrus derived promoters, genes and terminators to create an all citrus transformation vector, which is being incorporated into sweet oranges and mandarins by conventional Agrobacterium mediated transformation and our unique protoplast transformation methods.

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