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

Abstract

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.

Detailed description of four YAC contigs representing 17 Mb of chromosome 4 of Arabidopsis thaliana ecotype Columbia

“…These intervals vary in size along the chromosome, most of them are between 50 and 500 kb. Given the availability of cosmid libraries built in Agrobacterium binary vectors (Olszewski et al, 1988) and the relatively facile in p/anta transformation procedure for Arabidopsis (Bechtold et al, 1993), the isolation of genes from chromosome 4 using map-based cloning should now not be the limiting factor in the analysis of complex plant biological processes.…”

Signal transduction mutants of arabidopsis uncouple nuclear CAB and RBCS gene expression from chloroplast development

“…Construction of the pOCA107-2 Transgenic Line of Arabidopsis We created the pOCAl07 transgenic construct by modification of pOCA18 (Olszewski et al, 1988). Initially.…”

Gibberellins Are Required for Seed Development and Pollen Tube Growth in Arabidopsis

“…The pea 2ox2 cDNA encoding a full-length protein was cloned at the SalI and SacI sites of the pOCA28 vector (Olszewski et al, 1988) to generate the 35S:2ox2 construct. Standard molecular techniques were followed for DNA manipulations (Sambrook et al, 1989).…”

“…Agrobacterium tumefaciens GV3101 (a gift from J. Botella, Department of Botany, University of Queensland, Australia) was transformed with constructs in the pOCA vector (Olszewski et al, 1988;Robertson et al, 1998) by electroporation (Shen and Forde, 1989). The constructs were introduced into Arabidopsis using the Agrobacterium-mediated vacuum infiltration method (Ye et al, 1999).…”