9.4.2 Nonconditional Positive Selection Systems
A very new area of research is the development of positive selection systems that do not
require any substrates for selection. The list was quite small as of 2002 [reviewed by
Zuo et al. (2002)]. They are based on the use of genes that confer a growth advantage, dis-
tinguishable morphology or that selectively induce the differentiation of transformed tissues
but do not necessarily kill nontransgenic tissues (Fig. 9.2). The use of shoot organogenesis
to select for transformed tissues is the most advanced example at this time (2007). Shoot
formation in culture depends on the presence of high cytokinin : auxin ratios. The T-
DNA of the Ti plasmids fromA. tumefacienscodes for the enzyme isopentenyl transferase
(IPT), which catalyses the synthesis of isopentenyl adenosine-5^0 -monophosphate, which is
the first step in cytokinin biosynthesis (Table 9.1). Expression of this gene alone in plant
cells results in a higher frequency of shoot regeneration and recovery of transformed
material. The difficulty is that the shoots have abnormal morphology due to the cytokinin
imbalance and cannot produce roots (Ebinuma et al. 2001). To overcome this obstacle an
inducible promoter, such as theb-estradiol-inducible promoter is needed to restrict the
timing of expression of theiptgene (Zuo et al. 2001). A number of alternatives have
been demonstrated to have potential; however, these need time to be fully evaluated and
developed. Again, this approach differs from most other systems in that it intervenes in
the basic processes of plant cell growth and differentiation.
9.4.3 Conditional Negative Selection Systems
Conditional negative selection systems can play an important role in experiments by elim-
inating unwanted transformation events or when selecting against expression in specific
tissues or under specific inducible conditions. Only a few systems have been described.
One example is the bacterialcodAgene (Stougaard 1993), which codes for cytosine dea-
minase (Table 9.1). It is interesting that it has been shown to be effective in nuclear and
plastid transformation. This class of selectable marker genes codes for enzymes that
convert a nontoxic substrate into a toxic substrate, thereby eliminating the transformed
cells that express it.
9.4.4 Nonconditional Negative Selection Systems
Nonconditional negative selection systems have not been used widely as a selectable
marker gene system, but they have been used effectively toablatespecific cell types in
transgenic plants. These systems may code directly for toxins or enzymes that disrupt
basic cellular processes causing cell death. Whereas most of the systems discussed so far
are used for the production of transgenic plants, a nonconditional negative selection
system kills cells in mature plants for biosafety or breeding purposes. An example is the
ribonuclease, barnase, fromBacillus amyloliquefaciens. As shown in Figure 9.7, when it
is expressed only in the tapetum, which gives rise to pollen cells, the tapetum is killed
and therefore pollen cells cannot differentiate and mature. The consequence to the plant
is the inability to produce pollen or male sterility (Mariani et al. 1990). The activity of
barnasecan be controlled by a specific protein inhibitor,barstar, which is also found in
the same bacteria. Although this technology is recognized in plant sciences mainly as a
molecular tool for generating male sterility and the commercial production of hybrid
seed, it can also be used in the functional analysis of specific cell types and the gene
230 MARKER GENES AND PROMOTERS