Embryo Specific Expression of a Visual Reporter Gene and the Regeneration of Reporter Gene Expression Free Transgenic Citrus

Development of marker free technologies is essential to producing plants that only express the gene of interest without an antibiotic resistance gene that is not acceptable in several parts of the world. As a functional proof of concept, a myb-related transcription factor cDNA, (VvmybA1) was cloned via RT-PCR from Vitis vinifera 'Ruby Seedless' berry RNA. The cDNA was introduced into a suspension of Citrus sinensis cv. ‘Hamlin’ cells and Citrus reticulata cv. ‘W Murcott’ cells under the control of a carrot embryo specific gene promoter (Dc3). A high level of anthocyanin production was observed in developing embryos. By switching gene expression off in germinating embryos, normal transgenic plants were obtained absent of any anthocyanin production. Because this promoter is potentially drought inducible, we conducted stress studies that demonstrated elevated levels of the myb transgene in leaves, but no significant amount of visual coloration. Following this successful demonstration, the myb-related transcription factor ruby cDNA (isolated from the Blood Orange cultivar ‘Moro’) was cloned. This cDNA was incorporated in between sequences obtained from the 5' and 3' regulatory regions of a sweet orange derived seed storage protein gene to create an all-citrus construct. This construct was incorporated into citrus cells via a protoplast transformation system developed previously by our program. Phenotypically normal intragenic plants were regenerated using this method. myb-related transcription factor genes provide great potential as a simple and non-destructive visual marker for citrus transformation when coupled with a tissue specific or inducible promoter.