This category includes some recently developed systems that involve genes that provide
access to a nutrient source that can be utilized only by the transformed tissue with the con-
sequence that nontransformed tissues are eventually starved to death. An example is the
manAgene, which confers on plant cells the ability to use mannose as a carbon source
(Reed et al. 2001). The use of metabolic intermediates and drugs to achieve selection
has also been demonstrated. In some cases, the systems can distinguish transformed from
untransformed plants at the plantlet or whole-plant level but may not act efficiently
during the tissue culture steps needed for the selection of transformed tissue.
The scientific literature shows that only three of these selection systems have been
adopted routinely to generate transgenic plants for research or for commercialization.
They include thenptII(Fraley et al. 1983; Bevan et al. 1983; Herrera-Estrella et al.
1983) andhpt(Waldron et al. 1985) genes, which confer resistance to the antibiotics kana-
mycin and hygromycin, respectively, and thebarorpatgenes (De Block et al. 1989), which
confer resistance to the herbicide phosphinothricin (Table 9.1). In field trials, the most
frequently present selectable marker genes are thenptIIandbar/patgenes (Miki and
McHugh 2004).
9.4.1.1. Selection on Antibiotics.The aminoglycoside antibiotics include a number
of different antibiotics, includingkanamycin, neomycin, gentamycin, and paromomycin.
Kanamycin is produced in the soil actinomycete,Streptomyces kanamyceticus. These
molecules are very toxic to plant cells (Fig. 9.3) because they inhibit protein synthesis,
but a number of enzymes are found among microbes that will detoxify them. One
of these enzymes is a phosphotransferase that can confer resistance through the ATP-depen-
dent-O-phosphorylation of the kanamycin molecule.Neomycin phosphotransferase II
(NPTII) fromEscherichia coliis the bacterial aminoglycoside 3^0 -phosphotransferase II
[APH (3^0 ) II, EC 2.7.1.95] most often used with plant cells to generate kanamycin
resistance. It was originally selected for use in plants because prior work with
mammalian and yeast cells demonstrated its effectiveness as a selectable marker in
eukaryotic cells.
To function in plant cells the gene coding for NPTII (nptII, also designatedneooraphII)
was fused to regulatory elements from the nopaline synthase gene (nos) from the T-DNA of
theAgrobacterium tumefaciensTi plasmid. Thenosgene elements confer constitutive
expression of thenptIIgene and thus kanamycin resistance within all cells of the transgenic
plant. A stronger upstream promoter sequence from the cauliflower mosaic virus (CaMV),
which is responsible for transcription of the 35SRNA, generates a higher level ofnptIIgene
expression, which results in a higher level of kanamycin resistance. The nptII gene
can function as a selectable marker in both the nuclear and plastid genomes; however,
a member of another class of selectable marker gene, namely, aminoglycoside-
300 -adenyltransferase (aadA) is generally preferred for chloroplast transformation.
The popularity of kanamycin resistance, conferred by thenptIIgene, is because it is very
effective, functions in a wide range of plant species, and after extensive testing appears to be
very safe for use in food and feed crops. As it also functions effectively in a wide range of
microorganisms and eukaryotic cells, some initial concerns had been expressed about the
potential transfer of antibiotic resistance to other organisms; however, it has been used
since the mid-1980s in crops, and no adverse effects on humans, animals, or the environ-
ment have yet to appear (Flavell et al. 1992). It is also known that expression of thenptII
gene in plants does not alter the patterns of transcription in plants, so that transgenic plants
expressing it are essentially equivalent in composition to nontransgenic plants
228 MARKER GENES AND PROMOTERS