Efficient production of transgenic citrus plants expressing the coat protein gene of citrus tristeza virus

The coat protein gene of citrus tristeza virus (CTV) has been introduced in to Mexican lime (Citrus aurantifolia Swing.) plants by using an improved Ag robacterium-mediated genetic transformation system. Internodal stem segment s from greenhouse-grown seedlings were co-cultivated with A. tumefaciens st rain EHA 105 carrying the binary plasmid pBI 121/CTV-CP in a medium rich in auxins that provided the explant cells with the proper treatment to shift them to a competent state for transformation. The transformation frequency was enhanced, and this allowed us to recover 42 transgenic plants from 1200 explants. Regenerated shoots were identified as transformants by performin g beta-glucuronidase (GUS) assays and subsequently by PCR amplifications of the CTV-CP transgene. Southern analyses revealed that at least one copy of the CTV-CP gene was integrated in all PCR positive plants. Interestingly, 70% of them had linked T-DNAs arranged at one locus. Copy number of the CTV -CP gene varied from one to six among the transgenic lines. Half of them sh owed truncated T-DNAs in which the left border was lost. Expression of the CTV-CP transgene was demonstrated in 38 out of 42 plants by western analysi s and DASI-ELISA. No correlation was found between coat protein expression and transgene copy number or integration pattern.