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 in to plants was tested in tomato. Several variables affecting transformation efficiency were examined including insert size, Agrobacterium genetic backg round, and the presence of additional copies of the virG, virE1 and virE2 g enes. It was found that a helper plasmid containing extra copies of virG wa s an absolute requirement for obtaining tomato transformants with the BIBAC . MOG101 with the virG helper plasmid was found to be the most efficient st rain for transfer of high molecular weight DNA (150 kb). Selected high mole cular weight DNA transformants were advanced several generations (up to the R4) to assess T-DNA stability. This analysis showed that the T-DNA was sta bly maintained and inherited through several meioses regardless of whether it was in the hemizygous or homozygous state. Expression of a selectable ma rker gene within the T-DNA was also examined through several generations an d no gene silencing was observed. Thus, the BIBAC is a useful system for tr ansfer of large DNA fragments into the plant genome.