without selection. With certain traits, such as herbicide resistance, the gene of interest may
be directly selectable without the need for a separate selectable marker gene.
The easiest method for generating marker-free plants is by cotransformation of the
gene of interest with a marker gene followed by segregation of the unlinked genes into
separate lines [Fig. 9.13; reviewed by Ebinuma et al. (2001)]. Although effective, this
requires the production of many transgenic lines initially because so many are discarded.
Furthermore, the technology is restricted to transgenic plants that are propagated through
seeds. This would exclude vegetatively propagated species, such as trees.
Cotransformation can be achieved in many ways. For instance, the two genes can be intro-
duced on two separate plasmids. IfAgrobacterium-mediated transformation is used, this
can be achieved by infecting tissues with separate plasmids in separateAgrobacterium
strains or by separate plasmids in one strain. Cotransformation could also be achieved
using a single strain carrying a single plasmid with two separate T-DNA regions. The fre-
quencies of cotransformation may be very high (.50%, depending on the situation)
allowing the selection of transgenic material carrying both the selectable marker and
the gene of interest. The segregation of transgenic lines carrying the gene of interest
from lines carrying the selectable marker gene may occur at frequencies sufficiently
high to be practical in species that are efficiently transformed.
Figure 9.12.Novel fluorescent proteins whose genes were recently cloned from corals and expressed
in tobacco (a) andArabidopsis(b) plants. See color insert.
9.6. MARKER-FREE STRATEGIES 235