Agriculture Reference
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versus only 17% of 77 transformants made by the
same laboratory using biolistics. One-half of the
Agrobacterium
transformants had 2 to 3 copies
(Cheng et al., 1997). The consensus of several
reports is that one-third to two-thirds of
Agrobac-
terium
transformants of wheat are single-copy
insertions (Cheng et al., 1997, 2003; Hu et al.,
2003; Khanna and Daggard 2003; Wu et al.,
2006).
For
Agrobacterium
-mediated transformation,
the DNA which resides between border sequences
usually integrates as a predictable structure
(Fig. 18.1b), but sometimes rearrangement or
truncation can occur. Moreover, one large study
found that the majority of
Agrobacterium
trans-
formants contained plasmid sequences from
beyond the left border (Fig. 18.4a) (Wu et al.,
2006). This would result whenever the transfer
DNA did not effi ciently and precisely end at the
left border sequence. Such DNA can include
antibiotic resistance genes from the plasmid
backbone.
Integration after biolistic transformation can
result in complex multi-transgene structures. The
most detailed studies of these integration sites
use fl uorescent
in situ
hydridization (FISH) (Color
Plate 34) and/or Southern blot hybridizations to
visualize the orientations and dispersal of trans-
genes (Abranches et al., 2000; Jackson et al., 2001;
Rooke et al., 2003). Genes that originally were on
separate plasmids usually become linked before or
during integration, forming tandem (Fig. 18.4b
top) or nontandem (Fig. 18.4b middle) arrays
in the chromosome. In some cases, the trans-
genes are interrupted by stretches of unknown
or presumably host genomic DNA (Fig. 18.4b
bottom). A study of eight independent wheat
lines transformed with a single plasmid showed
that in three lines, integration sites in metaphase
chromosomes were separated by more than
1 Mb of genomic DNA and in one line, the sites
were on opposite chromosome arms (Abranches
et al., 2000). The remainder of the lines had single
integration sites. In studies of 13 independent
a
b
c
Fig. 18.4
Complex integration structures that can result from
Agrobacterium
(a) or biolistic (b,c) transformations. Symbols
are as in Fig. 18.1, and drawings are not to scale. (a) In
Agrobacterium
-mediated transformations, plasmid DNA beyond the
T-DNA left border (bold dotted line) and/or multiple T-DNA copies can be integrated. (b) In biolistic transformations, multiple
copies can integrate in tandem (top drawing) or in inverse orientations (middle drawing). Gene copies can be separated by
genomic DNAs (slashed solid line), and/or genes can become disrupted or rearranged (bottom drawing). (c) Possible promoter
read-through from the integrated genes into adjacent wheat genomic DNA from the promoter of a truncated selection gene
lacking its transcription terminator and part of its coding region.
