monitor the transmission of the linked transgenes among the progeny of the plant. One
hopes for a transgenic line or event with a simple 3 : 1 Mendelian segregation; however,
it is common for multiple insertion events to occur during a transformation experiment.
The genetic analysis of marker gene segregation is usually an important step in selecting
the homozygous transgenic lines with single insertions used for detailed studies of the
gene of interest. At this point the selectable marker gene is dispensable and can be elimi-
nated from the plant. A number of methods have been developed to remove them, and these
will be discussed later.
9.2.2 Reporter Genes: An Introduction
Whereas selectable marker genes help the researcher select transgenic tissue, reporter genes
usually report which cells are transgenic. Figure 9.4 illustrates the use of a typical con-
ditional, nonselectable (or reporter) gene. TheuidAgene (also calledgusA) codes for the
enzymeb-glucuronidase (GUS), which can react with a chemical substrate, 5-bromo-4-
chloro-3-indolyl glucuronide (X-gluc), to create a blue precipitate, thus providing a
histochemical stain that reflects the location of transgene expression (Fig. 9.4a)
(Jefferson et al. 1987). The staining patterns are used to understand the strength and
Figure 9.3.Selection of transgenic canola (Brassica napuscv Westar) on kanamycin-containing
tissue culture media (reproduced with permission from Pierre Charest, PhD thesis, Biology
Department, Carleton University, 1988). Stem explants were first infected with anAgrobacterium
tumefaciensstrain harboring a transformation vector with a chimericnptIIgene designed to confer
kanamycin resistance on transformed plant tissue. (a) After cocultivation of plant tissue and
Agrobacteriumto allow transformation to occur, the plant tissues were transferred to tissue culture
media containing kanamycin for growth of callus tissue and shoot differentiation. Much of the non-
transformed tissues turned white and stopped growing because they were sensitive to the antibiotic(s).
Transformed tissues remained green and continued to grow and differentiate because they were resistant
to kanamycin (r). (b) Transgenic shoots that differentiated in the presence of kanamycin were excised
from the callus and transferred to media for the regeneration of roots (r). Escapes that were not truly
kanamycin-resistant were unable to regenerate roots in the presence of the antibiotic(s). See color insert.
222 MARKER GENES AND PROMOTERS