Plant Biotechnology and Genetics: Principles, Techniques and Applications

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reporter gene linked together in the same DNA fragment that is transferred to the plant cell
(Fig. 9.1). As discussed above, the orientation and type of promoter fused to the reporter
gene must be carefully balanced, with the promoter fused to the selectable marker genes
(Fig. 9.6).
So far, we have been discussing insertions into the nuclear genome of plants and the
transcription of marker genes by RNA polymerase II in the nucleus. Technologies for
the insertion of genes into the chloroplast genome are also very advanced for some plant
species, such as tobacco (Svab and Maliga 1993). Some of the selectable marker genes
used in chloroplast transformation are functional in both nuclear and plastid transformation
(Miki and McHugh 2004); however, different promoters are needed for their expression in
the different genomes. For chloroplasts the ribosomal RNA operon promoter (Prrn) is par-
ticularly effective and allows transformation frequencies that are equivalent to those
achieved with nuclear transformation (Svab and Maliga 1993).


9.4 Selectable Marker Genes


Over 50 selectable marker gene systems have been described in the literature, primarily for
nuclear transformation (Miki and McHugh 2004), but only a small number have been
adopted for routine use. Having many different systems is important as they vary in effi-
ciency among plant species. Furthermore, experiments are often required in which different
transgene insertions are combined in single lines through genetic crosses using separate par-
ental transgenic lines or through consecutive transformation steps. Different selectable
marker genes allow the researcher to follow the segregation of each insertion event indepen-
dently. The underlying principles used to achieve selection differ widely among the select-
able marker genes, and the terminology for describing them in the literature has been
confusing. Table 9.1 provides a classification system, with selected examples, for the
various marker genes used in plants. A more comprehensive list can be found in Miki
and McHugh (2004).


9.4.1 Conditional Positive Selectable Marker Gene Systems


This category contains the largest number of and most widely used selectable marker gene
systems developed for plants. The genes code for an enzyme or product which provides
resistance to a substrate that selectively inhibits the growth and differentiation of the non-
transformed tissues (Fig. 9.2). The toxic substrate may be an antibiotic, herbicide, drug,
metabolic intermediate, or phytohormone precursor. The manner in which the substrate
is applied is very important because the ease with which transformed cells are allowed to
proliferate must be balanced with the stringency with which the nontransformed cells are
suppressed or killed. The accumulation of toxins from dead tissues can adversely affect
the ability of living tissues to survive, particularly if they are present in limited numbers
within a larger population of dying or dead material. The optimal selection conditions
tend to be specific for each plant species and tissue type. If not properly administered,
the proportion of transgenic material recovered may be disproportionately low relative to
the frequency of transformation events that actually occurred. Conversely, if the frequency
of “escapes” (i.e., nontransgenic tissues that the researcher believes to be transgenic since
they survived selection) is too high, then considerable effort and cost would be needed to
sort them out from the nontransformed material later.


9.4. SELECTABLE MARKER GENES 227
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