appears to lead to transgene silencing, any method of controlling copy number would lead
to an improvement in consistency of transgene expression. To start, reducing the concen-
tration of DNA appears to reduce the copy number of the transgene in the target cell.
High concentrations of DNA are still used in many cases and are a remnant of early optim-
ization strategies. Use of high concentrations of DNA results in high levels oftransient
expression, which is used to optimize DNA delivery conditions and involves the rapid
expression of the some transgenes, often marker genes, which can be measured and quan-
tified within 24 h of DNA introduction (Fig. 10.9). As a result of these levels of high tran-
sient expression, DNA concentrations that are much higher than necessary are often used
for stable transformation studies. The beneficial effects of lower concentrations of DNA
on stability of transgene expression should be evaluated for each different target tissue.
Copy number of the introduced transgene can also be lowered by simplifying the form
of the introduced DNA. Simple integration patterns result if a fragment of DNA containing
only the gene of interest is used. When the backbone of the cloning plasmid is eliminated
from the bombardment precipitation mix, this results in low copy transgene integration
(Agrawal et al. 2005).
Transgene expression can also be stabilized using genes for certain viral proteins termed
“suppressors of silencing.” Suppressor proteins are produced by viruses to suppress the
defense system of plants against viruses. After a virus invades a plant cell, the plants try
to shut off or silence invading viral genes. The virus, in turn, evolved genes to turn off
or suppress the silencing mechanism. Viral silencing suppressors can potentially be used
to allow high levels of expression of transgenes through a similar mechanism. However,
the effect of these viral proteins on normal plant development is still unclear.
10.5 Other Methods
10.5.1 The Need for Additional Technologies
With the two main methods for DNA introduction, why are additional methods needed?
Isn’t this enough? In the scientific community (and for humanity in general), the theme
Figure 10.9.Particle bombardment-mediated transient GFP expression in lima bean cotyledonary
tissues. This target tissue is flat, nonpigmented, and ideally suited for tracking GFP expression in indi-
vidual transiently transformed cells. See color insert.
260 TRANSGENIC PLANT PRODUCTION