pattern conferred by gene regulatory elements fused to the chimeric reporter gene (Fig. 9.1)
and how the gene is controlled in the various cells of the plant. The specific activity of the
GUS enzyme can also be quantitatively measured, which reflects the relative level of gene
expression, which might in turn indicate the strength of the promoter fused to the marker
gene (Fig. 9.4b) (Jefferson et al. 1987). A generation of plant biotechnologists have
looked at blue “GUS” spots and staining patterns in plants to answer questions such as
Are my plants transgenic?
Is the gene expressed?
How is my promoter working?
These are also discussed in Chapter 6.Although any marker gene can be monitored using techniques for detecting and measur-
ing specific DNA, RNA, or protein sequences (Chapter 11), the reason for using a reporter
gene is the convenience of generating large volumes of data and working with large sample
sizes. This is important as transgenic plants vary considerably in the expression of trans-
genes because of position effects or the varying influences of the insertion sites on the
inserted genes (Fig. 9.4b). Large amounts of data are needed to overcome the variability
to extract meaningful information. It is also useful when looking for rare events. For
example, when plant transformation experiments are begun on a “new” plant species,
researchers need help in optimizing the system. Rare transgenic events can be found
using marker genes, and improvements can be carried out quickly. Because data generated
through conditional reporter genes are indirect, they can be subject to factors that can affect
interpretation. These include the stability of the product in various tissues, the presence of
inhibitors in certain tissues, or varying levels of background activity. Such factors can vary
greatly among different plant species. It is therefore common practice to confirm the find-
ings using techniques that independently detect the sequence of interest (Fig. 9.4c).
To ensure that the data accurately reflect the promoter activity, RNA blots or other
methods are often combined with marker gene activity to confirm the patterns of mRNA
accumulation (see Chapter 11). The advantages and disadvantages of the various reporter
gene systems will be discussed later.
9.3 Promoters
Chapter 6 illustrated the importance of promoter sequences in the spatio temporal regulation
of plants genes. Similarly, all transgene constructs, including genes of interest (GOIs) and
marker genes, require promoters to regulate their transcription reproducibly and predictably.
Transgenes can be cloned with a variety ofheterologous(from another source) promoters
during construction of the chimeric genes within the transformation vectors to provide bio-
technologists with a range of expression patterns to suit their experiments (see the previous
chapter). For optimal performance in regenerating transgenic plants, it is important that
selectable marker genes are expressed in all of the cells of the plant. Usually selection
pressure is applied shortly after the transformation phase and maintained throughout
growth and differentiation in tissue culture and during the stages of plantlet regeneration
(Figs. 9.2 and 9.3). For genetic analysis of the inserted DNA, selection may also be
applied during seed germination and seedling growth to measure the segregation of the
224 MARKER GENES AND PROMOTERS