Only a few studies have emerged in which transposons have been used to translocate
genes within the plant genome to break the linkage between selectable marker genes and
genes of interest (Fig. 9.13). An interesting strategy has been developed using the noncon-
ditional positive selectable marker gene (ipt) in combination with theAc transposase
element to remove theiptgene. In this multiautotransformation (MAT) process, theipt
gene first acts positively to generate a proliferation of morphologically abnormal shoots
with the “shooty” phenotype. They cannot regenerate because of the overproduction of
cytokinin; however, normal shoots emerge at low frequency several weeks later following
transposition of theiptgene to a distant locus that can segregate away in somatic cells or it
may be directly lost if not reinserted into the genome. [reviewed by Ebinuma et al. (2001)].
More recently, the use of site-specific recombinases has emerged as a versatile strategy
for the selective removal of marker genes from an insertion site. The recombinases
and their target sites include Cre/loxfrom bacteriophage P1, FLP/FRTfrom yeast
Figure 9.13.Processes for generating marker-free transgenic plants. Cotransformation is a practical
process for generating marker-free transgenic plants. It depends on the integration of the selectable
marker gene (sm) and gene of interest (goi) at separate chromosomal sites that can segregate away
from each other in the next sexual generation. This can also be achieved by the introduction of
both the selectable marker and gene of interest on the same vector and therefore insertion at the
same site followed by the subsequent transposition of the marker gene to a separate locus that can
segregate away from the gene of interest. A more recent advance is the use of excision recombinases
(R) under inducible promoters to autoexcise itself along with the selectable marker gene. This process
does not require a segregation step and has the potential for broader applications.
236 MARKER GENES AND PROMOTERS