Transgene organisation in potato after particle bombardment-mediated (co-)transformation using plasmids and gene cassettes.
Protocols for efficient co-transformation of potato internodes with genes contained in separate plasmids or gene cassettes (i.e., linear PCR fragments comprising a promoter-gene-terminator) using particle bombardment were established. Twenty-eight out of 62 (45%) and 11 out of 65 (17%) plants transformed with a plasmid containing the selectable marker contained one and two additional non-selected genes, respectively. When gene cassettes were used in transformation, six out of eight plants were co-transformed. Expression analysis showed that 75-80% of the plants transformed with two transgenes expressed both of them, irrespective of the use of plasmids or gene cassettes. Thirty-eight plants containing the gusA reporter-gene and the nptII selectable-marker have been characterised with respect to the molecular organisation of the donor DNAs. Seventeen out of 49 (35%) gusA sites of integration contained one copy of the gene. Only 11 gusA sites (22%) were linked to the site of integration of the selectable marker. When one site of integration contained several copies of the transgene, a predominance of 3'-3' inverted re-arrangement repeats was observed.
Romano
Andrea
A
Institute for Agrotechnological Research (ATO-BV), Wageningen University and Research Center, POB 17, 6700 AA, Wageningen, The Netherlands.
Raemakers
Krit
K
Bernardi
Jamila
J
Visser
Richard
R
Mooibroek
Hans
H
D001483:Base Sequence; D019470:Biolistics; D015139:Blotting, Southern; D017931:DNA Primers; D016254:Mutagenesis, Insertional; D030821:Plants, Genetically Modified / Q000235:genetics; D010957:Plasmids; D020133:Reverse Transcriptase Polymerase Chain Reaction; D011198:Solanum tuberosum / Q000235:genetics*; D014170:Transformation, Genetic; D019076:Transgenes
D017931:DNA Primers