Specialized binary vector for plant transformation: expression of the Arabidopsis thaliana AHAS gene in Nicotiana tabacum

• Published in: Nucleic acids research Vol. 16; no. 22; pp. 10765 - 10782
• Main Authors: OLSZEWSKI, N. E, MARTIN, F. B, AUSUBEL, F. M
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 25.11.1988
• Subjects: Biological and medical sciences; Cloning, Molecular; Cosmids; DNA - genetics; DNA - isolation & purification; Drug Resistance - genetics; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; genes; Genetic Vectors; Genic rearrangement. Recombination. Transposable element; Herbicides - pharmacology; Molecular and cellular biology; Molecular genetics; Nicotiana - genetics; Nicotiana tabacum; Plants - drug effects; Plants - genetics; Plants, Toxic; Sulfonamides; Transformation, Genetic; Triazines - pharmacology; Escherichia coli; Agrobacterium tumefaciens; Gene library; Nicotiana tabacum; Arabidopsis thaliana; Proteins; Transfection; Cruciferae; Dicotyledones; Blotting assay; Angiospermae; Bacteria; Transgenic plant; Solanaceae; Molecular cloning; Escherichieae; Vector; Nucleic acid; Enterobacteriaceae; Enzyme; Gene expression; Resistance; Isolation; Spermatophyta; Rhizobiaceae; Experimental plant
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/16.22.10765

We constructed a cosmid vector, pOCA18, designed for transferring plant genomic libraries from Agrobacterium tumefaciens to plants. Clones from a genomic library of Arabidopsis thaliana DNA in pOCA 18 were propagated stably in both Escherichia coli and A. tumefaciens. Clones from the pOCA18 A. thaliana library were used to construct transgenic Nicotiana tabacum plants; the DNA inserts were transferred intact in 10 out of 16 transgenic N. tabacum plants examined but were partially deleted in six others. Transgenic N. tabacum plants constructed with a mutant A. thaliana acetohydroxy acid synthase gene (from the pOCA18 library) that encodes an enzyme resistant to the herbicide chlorsulfuron were resistant to chlorsulfuron. A statistical analysis indicated that if the A. thaliana library contains 10(7) members and if 10(7) A. tumefaciens transconjugants containing the library were used to transform plant cells, then 2 x 10(4) transformed plant cells must be generated to have a 95% probability of constructing a transgenic plant carrying a specific DNA sequence from the A. thaliana library.