Transgenic Applications in Wheat Improvement - Wheat Science and Trade - page 387

Table 18.1 Selection systems used to identify wheat transformants.

Target in Plant
Cell

Selection
Gene

Selection Gene
Source

Reference for First Use
in Wheat Transformation Selection Agent(s) Encoded Enzyme

Geneticin,
paramomycin,
kanamycin

Inhibits protein
synthesis

nptII Neomycin

phosphotransferease
II

E. coli Nehra et al. (1994)

Phosphinothricin,
bialaphos,
glufosinate

Inhibits glutamine
synthetase

bar Phosphinothricin

acetyl transferase

Streptomyces
hygroscopicus

Vasil et al. (1992)

Hygromycin Inhibits protein

synthesis

hph Hygromycin

phosphotransferase

E. coli Ortiz et al. (1996)

Glyphosate Aromatic amino

acid synthesis,
specifi cally the
EPSPS a enzyme

aroA :CP4 Resistant EPSPS Agrobacterium Zhou et al. (1995)

GOX Glyphosate

oxidoreductase

Another
bacterium

Cyanamide Unknown cah Cyanamide hydratase Soil fungus

Myrothecium
verrucaria

Weeks et al. (2000)

Mannose None b pmi Phosphomannose

isomerase

E. coli Wright et al. (2001)

a 5-enopyruvylshikimate-3-phosphate synthase.
b Selection gene provides a metabolic advantage that allows nonphotosynthetic wheat cells in culture to utilize mannose as
a carbon source.

The common procedures for wheat transfor-
mation employ herbicides based on phosphi-
nothricin (Thompson et al., 1987), glyphosate
(Zhou et al., 1995; Hu et al., 2003), or the amino-
glycoside antibiotics G418, paramomycin, kana-
mycin, or hygromycin (Table 18.1). Partly driven
by a desire to avoid the use of antibiotic resistance
genes, nutrient conversion selection methods
have been developed. In these systems, cells that
acquire the selection gene have a metabolic advan-
tage and outgrow the cells that are not trans-
formed. An example of a metabolic selection
scheme that has been used for identifying wheat
transformants is the sugar mannose in conjunc-
tion with the phosphomannose isomerase ( pmi )
gene. Cells containing the pmi gene can use
mannose as a carbon source, while cells lacking it
cannot grow without some other sugar in the
medium (Wright et al., 2001; Gadaleta et al.,
2006). The cyanamide selection system combines
elements of both cytotoxicity and metabolic
advantage. Cyanamide is toxic to plant cells, but
the cyanamide hydratase ( cah ) gene enables them

to convert the herbicide to urea, simultaneously
detoxifying it and making it into a usable nitrogen
source (Weeks et al., 2000).
An alternative to using selection systems is to
employ a nondestructive screening method to
identify cells that have received new genes
and then to hand-pick those expressing cells.
This strategy often uses reporter genes that visu-
ally mark the presence and/or location of gene
activity, as pictured in Color Plate 32c and
described previously for gfp and the luciferase
gene (Table 18.2). Direct detection of the intro-
duced DNA by polymerase chain reaction
(PCR) can also be employed as a screening
method (Permingeat et al., 2003; Zhang et al.,
2006). For wheat transformation experiments, in
which only a few percent of cells are ultimately
stably transformed, such procedures are labor
intensive. In addition, DNA need not be inte-
grated to be detected by PCR or reporter gene
activity, so that transiently transformed tissues
are also identifi ed. Some researchers have
employed a combination of screening and