Efficiency and stability of high molecular weight DNA transformation: an analysis in tomato.
The efficiency of the binary bacterial artificial chromosome (BIBAC) vector for Agrobacterium-mediated stable transfer of high molecular weight DNA into plants was tested in tomato. Several variables affecting transformation efficiency were examined including insert size, Agrobacterium genetic background, and the presence of additional copies of the virG, virE1 and virE2 genes. It was found that a helper plasmid containing extra copies of virG was an absolute requirement for obtaining tomato transformants with the BIBAC. MOG101 with the virG helper plasmid was found to be the most efficient strain for transfer of high molecular weight DNA (150 kb). Selected high molecular weight DNA transformants were advanced several generations (up to the R4) to assess T-DNA stability. This analysis showed that the T-DNA was stably maintained and inherited through several meioses regardless of whether it was in the hemizygous or homozygous state. Expression of a selectable marker gene within the T-DNA was also examined through several generations and no gene silencing was observed. Thus, the BIBAC is a useful system for transfer of large DNA fragments into the plant genome.
Frary
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Plant Science Center, Center for Advanced Technology/Biotechnology, Cornell University, Ithaca, NY 14853, USA. af29@cornell.edu
Hamilton
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D001483:Base Sequence; D015139:Blotting, Southern; D022202:Chromosomes, Artificial, Bacterial; D017931:DNA Primers; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics*; D018551:Solanum lycopersicum / Q000235:genetics*; D008970:Molecular Weight; D010957:Plasmids; D012231:Rhizobium / Q000235:genetics
D017931:DNA Primers; D018744:DNA, Plant