evaluated and then compared with an expected pattern for gene expression for the most
accurate results.
10.6.2.3. Transgene DNA.Ultimately, the transgenic nature of a plant relies on the
detection of the new transgene through DNA analysis. In some cases, DNA analysis has
become so sensitive that small amounts of contaminants in the laboratory can yield
false-positive results. Use of the polymerase chain reaction (PCR) must be cautiously
weighed as false positives are so common with this method. In addition, PCR does not
test for the integration of the transgenic DNA, only its presence in the sample. So, if
there is some DNA on the leaves from an adjacent plant or theAgrobacteriumremains
in/on the plant, there will be a positive signal. PCR is a great screening tool in the labora-
tory, but PCR results should never be presented as proof of transformation.
The best method for molecular analysis of integrated transgenic DNA is Southern
hybridization analysis (see Chapter 11 for details). Many publications present Southern
blots showing the same-sized band for all clones. If enzymes are used that cut a fragment
out of the transgene, a single band will be generated. A single band will also be generated if
the starting DNA is from a bacteria or DNA that is contaminating the sample. If a restriction
enzyme is used that cuts the foreign DNA at only one location, it will also cut somewhere in
the plant DNA, producing different sized fragments from each different transformation
event. More bands are typically generated from plants obtained using direct DNA introduc-
tion, whileAgrobacterium-mediated transformation yields fewer and less complex banding
patterns. Regardless of the method for DNA introduction, the presence of unique band sizes
and band numbers should be used to confirm transgene integration resulting from each
different transformation event. It is also important to analyze the progeny of putatively
transgenic plants. A transgenic plant should pass the transgene on to progeny with
Mendelian-expected frequencies. Non-Mendelian inheritance of transgenes suggests pro-
blems at some level.
10.7 A Look to the Future
In the early days of transgenic plant production, the major difficulty was the actual pro-
duction of transgenic plants. As transformation science progressed, the procedures for
gene delivery, gene selection, and transgenic plant production became more standardized
for most plants. Transformation systems for even the most difficult to transform plants
can now be termed, “consistent but inefficient.” This means that, if you know what you
are doing, you can count on the production of a few transgenic plants for each experiment.
Many plants that used to be difficult to transform are no longer even considered “difficult.”
So, for many plants, transformation is no longer limiting and the analysis of transgenics is
the new bottleneck. Can we even analyze fewer plants if we eliminate the variation in trans-
gene expression by developing more reliable methods to introduce the transgene into
exactly the same spot (in the genome) each time? Can we follow the lead of the automotive
industry by automating more of the process? Transformation science, as with science in
general, moves forward through the systematic optimization of known systems and the dis-
covery of new approaches. Hopefully, one of the young scientists reading this chapter will
take the lead to optimize or develop a new transformation technology that will eliminate one
or more of the remaining bottlenecks in transgenic plant production.
10.7. A LOOK TO THE FUTURE 269