Analysis of transgenic citrus (Citrus aurantium L.) plants expressing Citrus Tristeza Virus coat protein gene

Due to high quality of fruits, resistance to various pathogens and abiotic stress, Citrus aurantium is widely used and considered as the most favorable rootstock worldwide. Genetic engineering approaches such as pathogen-derived resistance (PDR), is a common practice in citrus breeding. Analysis of transgenic plants requires reliable and quick methods for early screening of T0 generation. In a PDR approach, a mosaic gene from Shiraz CTV strains was cloned and transferred into sour orange. Forty putative transformed shoots were isolated on selective medium, which were acclimatized and transferred to growth room. In the first screening method, leaf assay for Basta resistance was performed on liquid as well as solid selective medium. To further analyze the putative transgenic seedlings, PCR using CTV and BAR gene specific primers were performed and some of the PCR products were randomly chosen for sequencing. The transgene copy number(s) in individual genotypes was resolved using real-time PCR technique. Finally, the functionality of the transgene was provided using ELISA test, which confirmed that CTV amplification suppressed in five tested seedlings. In this experiment, the simple and economical screening methods were set up, which made unambiguous discrimination between transgenic and non-transgenic citruspossible.