Genetic transformation of Rangpur lime (Citrus limonia Osbeck) with the b0 (bacterio-opsin) gene and its initial evaluation for Phytophthora nicotianae resistance

Transgenic plants expressing the bacterio-opsin (W) gene can spontaneously activate programmed cell death (pcd) and may enhance broad-spectrum pathogen resistance by activating an intrinsic defense pathway in plant species such as tobacco and potato. In this work, we produced transgenic Rangpur lime plants with the bO gene, via Agrobacterium tumefaciens-mediated transformation, and evaluated these plants for Phytophthora nicotianae resistance. Two transgenic lines were successfully regenerated and transformation was confirmed by GUS activity assay, PCR analysis, Southern, Northern and Western blot analyses, in addition to detecting the expressed bO protein by an immunological approach. Evaluation for Phytophthora nicotianae resistance was carried out by plant inoculations with the pathogen and quantification of the affected area. One of the two transgenic lines showed greater tolerance to the fungal pathogen as compared to the control, with significantly smaller stem lesions after pathogen challenge. This increase in pathogen tolerance is correlated with a significantly higher level of transgene expression in this line when compared with the other transgenic line. This is the first report of the introduction of a potentially important gene into Rangpur lime to provide novel pathogen tolerance.