A. Frary et Cm. Hamilton, Efficiency and stability of high molecular weight DNA transformation: An analysis in tomato, TRANSGEN RE, 10(2), 2001, pp. 121-132
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.