Plant Biotechnology and Genetics: Principles, Techniques and Applications

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regulatory elements that limit expression within them. As such, it may also be considered as
a complement to the reporter genes described below.


9.5 Nonselectable Marker Genes or Reporter Genes


As important as the selectable marker genes have been for the development of transformation
technologies, the nonselectable or reporter genes have played a different fundamental role in
the growth of our understanding of gene regulation mechanisms in plants. These kinds of
genes have formed an invaluable partnership with selectable marker genes in transgenic
research. For reasons that will soon be apparent, reporter genes are also sometimes called
“visible marker genes” since they change the appearance of plant tissues. Although
several reporter genes have been described, three have been particularly influential and
have dominated the scientific literature. These are the genes coding forb-glucuronidase
(GUS),luciferase(LUC), andgreen fluorescent protein(GFP). GUS and LUC are con-
ditional nonselectable marker genes as they require the use of an external substrate for detec-
tion of activity, whereas GFP is a nonconditional, nonselectable marker gene because the
protein encoded by the gene is directly detectable without the use of a substrate.


9.5.1 b-Glucuronidase

The bacterial enzymeb-glucuronidase (GUS), which is coded by theEscherichia coligene,
uidA(also known asgusA) has been the most widely used reporter system in plants
(Jefferson et al. 1987). It has a sensitive specific activity assay using 4-methylumbelliferyl
glucuronide (MUG) as substrate and permits histochemical localization using 5-bromo-4-
chloro-3-indolyl glucuronide (X-gluc) with specificity at the cellular level (Fig. 9.4). The
enzyme is stable in plant cells and can accumulate to high levels without toxicity to the
plant cell. It confers no apparent phenotype to plants in the absence of its substrates and
therefore can be used to study plant processes without concern of artifacts resulting from


Figure 9.7.Diagrammatic illustration on the use ofbarnaseas a negative selectable marker gene for
the ablation of the canola tapetal cells: (a) wild-type; (b) transgenic. Barnase codes for a ribonuclease
fromBacillus amyloliquefaciens.When it is expressed in plants under the control of the tapetum-
specific promoter (TA29), expression was restricted to the cells of the tapetum (t) in transgenic
plants. The ribonuclease activity in the tapetum resulted in failure of the tapetum to develop and col-
lapse of the pollen sac (ps). Because the tapetal cells are the precursors of the pollen cells, pollen (p)
cannot differentiate in the transgenic plants and the plants are therefore male sterile. Because the
pattern of ribonuclease expression was tapetum specific the rest of the plant was unaltered.
[Adapted from Mariani et al. (1990).]


9.5. NONSELECTABLE MARKER GENES OR REPORTER GENES 231
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