Plant Biotechnology and Genetics: Principles, Techniques and Applications

(Brent) #1

without the use of external substrates. Both allow the recovery of the transgenic cells that
may be present at a very low frequency in the cell population. The first selectable marker
gene used in plant biotechnology and still the one most often used in research is the neo-
mycin phosphotransferase II gene (nptII), which allows plant cells expressing it to survive
and grow on culture media containing the antibiotic kanamycin (Fig. 9.3).
As most transformation events occur at very low frequencies the selectable marker genes
are an essential component of most transformation protocols. This is particularly important
for plant species where the tissue culture methodologies are poorly developed or the effi-
ciency of transformation is low. The frequency of recovery of resistant tissues or plants pro-
vides a measure of transformation efficiency that can be used to optimize the components of
the transformation protocols in a stepwise manner. Chapters 5 and 11 describe this process
in greater detail. Once transgenic plants have been recovered, the selectable marker gene
can act as a genetic marker for subsequent genetic studies as it is linked to the gene of inter-
est (Fig. 9.1). For example, the selectable marker gene allows the researcher to predict the
number of segregating insertion events that have occurred in a transgenic line and also to


Figure 9.2.Comparison of a typical conditional positive selection system with a nonconditional
positive selection system. The conditional systems are the most abundant and extensively used,
whereas nonconditional systems are currently in early stages of research and development. The con-
ditional systems introduce a novel resistance trait often taken from bacterial sources. An external sub-
strate (indicated by shading of the medium) is added to the tissue culture media used to grow and
regenerate transformed material to suppress the growth of the nontransformed tissues. This differs
from the nonconditional systems, which introduce genes that alter the growth and differentiation of
the transgenic cells and tissues in a manner that allows them to be separated from untransformed
cells and tissues in the absence of external substrates. This latter approach fundamentally alters the
plant material by intervening in basic cellular processes. In this illustration the selectable marker
gene (sm) and gene of interest (goi) are linked in the transforming DNA. The process of plant regen-
eration depicted is organogenesis, which commonly involves the differentiation of the shoots followed
by the roots separately. It may also occur in a single step through the process of embryogenesis, which
may be induced in somatic cells or gametic cells.


9.2. DEFINITION OF MARKER GENES 221
Free download pdf