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

• Published in: Plant cell reports Vol. 19; no. 4; pp. 427 - 433
• Main Authors: Domínguez, A, Guerri, J, Cambra, M, Navarro, L, Moreno, P, Peña, L
• Format: Journal Article
• Language: English
• Published: Germany Springer Nature B.V 01.03.2000
• Subjects: ACP gene; Agrobacterium tumefaciens; Citrus aurantifolia; Citrus tristeza virus; CP gene; Genetic engineering; Transgenic plants; Agrobacterium tumefaciens; Citrus tristeza virus; Genetic transformation; Coat-protein-mediated resistance; Key words Lime
• ISSN: 0721-7714; 1432-203X
• DOI: 10.1007/s002990050751

The coat protein gene of citrus tristeza virus (CTV) has been introduced into Mexican lime (Citrus aurantifolia Swing.) plants by using an improved Agrobacterium-mediated genetic transformation system. Internodal stem segments from greenhouse-grown seedlings were co-cultivated with A. tumefaciens strain 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 performing β-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 showed 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 analysis and DASI-ELISA. No correlation was found between coat protein expression and transgene copy number or integration pattern.