Bananas and plantains are an important source of food and income for millions of persons in the world. Their production is constrained by many biotic and abiotic stress factors but their improvement through traditional plant breeding methods is very difficult because they do not produce seeds, are polyploid and have a long generation time. Biotechnological approaches like genetic transformation...

Figure 1. Diagram of a split-marker gene replacement strategy using fusion-PCR (A) FPCR step 1 amplification of flanking regions and partial marker segments. Dotted tails indicate the overlap sequences on selected primers which allow fusion in step 2. (B) FPCR step 2 flanking regions are fused to the marker segments at the overlap sights, forming two constructs. The fusion products are then amp...

The ability to conveniently construct gene disruptions is an important methodology for genetic analysis of the fission yeast Schizosaccharomyces pombe. Because of the limited number of selectable markers available for generating gene disruptions in fission yeast, the construction of strains that contain multiple gene disruptions can be quite difficult. This becomes a particular problem when epi...

Phosphinothricin (PPT) is the active ingredient in bialaphos, which specifically inhibits glutamine synthetase in land plants. We isolated a novel PPT-resistant gene from a soil bacterium, Nocardia sp., and characterized it. The encoded protein, consisting of 177 amino acids, showed significant similarity to bacterial N-acetyltransferases, and we originally designated the gene MAT (methionine s...

A rapid method obviating the use of selectable markers to genetically manipulate large DNA inserts cloned into bacterial artificial chromosomes is described. Mutations such as single-base changes, deletions, and insertions can be recombined into a BAC by using synthetic single-stranded oligonucleotides as targeting vectors. The oligonucleotides include the mutated sequence flanked by short homo...