Contents - Faperta

212 Biotechnological Approaches for Pest Management and Ecological Sustainability
constraint in developing effective transgenic plants has been the lack of promoters that
offer a high level of site-specifi c gene expression in the crop species of interest. Generally,
transgene expression is driven by constitutive promoters such as caulifl ower mosaic virus
35S (CaMV35S) (Benfey and Chua, 1989, 1990) and Actin1 (McElory, Rothenberg, and Wu,
1990). Although the CaMV35S promotor has been widely used for transformation of
dicotyledons, it has low activity in monocotyledons. Moreover, the pattern of CaMV35S
promoter activity in different tissues of transgenic plants is diffi cult to predict (Benfey and
Chua, 1990). In general, it has been found that monocot promoters are more active in
monocot tissues than in dicot tissues (Wilmink, van de Ven, and Dons, 1995). However,
tissue-specifi c promoters have now been successfully employed for driving transgene
expression in pith tissue. Phosphoenolpyruvate carboxylase (PEPC) from maize can be
used for gene expression in green tissue (Hudspeth and Grula, 1989). From a crop yield
potential perspective, insect-resistant transgenes should be expressed only in those organs
likely to be attacked by the insects. Otherwise the plants may be highly resistant, but the
metabolic cost may substantially reduce the crop yield. Constitutive promoters such as
CaMV35S are effective in providing high levels of gene expression, but may have unanticipated
consequences towards nontarget organisms because of expression of the transgene
in all plant parts. Therefore, a more targeted expression of insecticidal genes by using
tissue- and organ-specifi c promoters can form an important component for developing
transgenic plants with resistance to insects (Wong, Hironaka, and Fischhoff, 1992; Svab
and Maliga, 1993; McBride et al., 1995).
Transposon-mediated repositioning of transgenes is another strategy to generate plants
that are free of selectable markers and T-DNA inserts (Cotsaftis et al., 2002). By using a
minimal number of transformation events, a large number of transgene insertions in the
genome can be obtained so as to benefi t from position effects in the genome that contribute
to higher levels of expression. The cry1B gene expressed under the control of maize ubiquitin
promoter between minimal terminal inverted repeats of the maize Ac-Ds transposon
system has been cloned, and the 5¢ untranslated sequence of a gfp gene used as an excision
marker. The results indicated that transposon-mediated relocation of the gene of interest is
a powerful method for generating transgenic plants, and exploiting favorable position
effects in the plant genome.
Genetic transformation typically involves a marker gene for resistance to antibiotics
(kanamycin – npt gene) or herbicides (phosphoinothricin – bar gene) (Table 7.1, Figure 7.1),
a replication site, and a multiple cloning site (MCS) with several restriction sites for DNA
insertion. Foreign DNA can be inserted into the vector using restriction enzymes that recognize
a specifi c DNA sequence. Insertion of foreign DNA interrupts gene expression of
TABLE 7.1
Selectable Markers Used in Genetic Transformation of Crops
Gene Markers
Antibiotic Dhfr: Dihydrofolate reductase—Methotrexate/trimethoprin
hpt: Hygromycin phosphotransferase—Hygromycin B
nptII: Neomycin phosphotransferase—Kanamycin, neomycin, G418
Herbicide als: acetolactate synthase—Chlorsulfuron, imidazolinones
aroA: 5-Enolpyruvylshikimate-3-phosphate synthase—Glyphosate
bar: Phosphoinothricin acetyltransferase—Phosphoinothricin
bar: Glufocinate—Basta