Page 8 - Triticeae Genomics and Genetics

Triticeae Genomics and Genetics
TGG 2010, Vol.1, No.2 http://tgg.sophiapublisher.com
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extensively used as a scorable marker gene for
transformation of wheat. Some visual marker genes
such as GFP (green fluorescent protein), LUC
(luciferase) and anthocyanin are also used in wheat
transformation. GFP seems to have considerable
promise for use in transformation of cereals. The LUC
gene isolated from firefly requires ATP and luciferin
substrate to produce light (Ow et al., 1986). The
breakdown of luciferin results in the emission of light
and does not harm the plant tissue. So the tissue
incorporated with vector containing the gene of
interest and LUC gene emit a yellow green glow
indicating its transformed status. Another reporter
gene commonly used in the transformation of wheat is
anthocyanin regulatory gene e.g. R gene from Zea
mays, that stimulates the endogenous anthocyanin
biosynthesis in the transformed tissues/cells. Upon
activation, this reporter system produces a
reddish-purple pigment in transformed tissue.
3.2 Selectable Markers
Selectable markers are gene sequences that are
intentionally introduced into cells as developmental
tools to identify the cells those have successfully
incorporated gene for a desired trait. The selectable
markers are extremely important because they act as
genetic tags to easily identify successfully transformed
cells. However they have no actual function in the
normal organism. There are three categories of
selectable markers.
3.2.1 Antibiotic resistant markers
These are the selectable markers that confer resistance
to antibiotics so the cells/tissues incorporated with
these genes can be selected on the medium containing
the antibiotic whereas the non-transformed cell will
not survive thereby facilitating preferential selection
of the transformed cells. The nptII and hpt are the
commoly used markers for wheat. The nptII
(neomycin phosphotransferaseII) initially isolated
from Escherichia coli, is the most widely used
antibiotic resistance marker. The gene encodes for the
neomycin 3'-phosphotransferase that inactivates
aminoglycoside antibiotics such as kanamycin,
neomycin, geneticin (G418) and paromomycin by
phosphorylation. It binds 30s ribosomal subunit and
inhibits translation. The antibiotic paromomycin as a
selection agent in combination with npt II gene proved
very successful in recovering transgenic plants and
hence npt II gene became the most favored selection
marker for wheat transformation (Varshney and
Alpeter, 2002).
The choice of the selective agent is very crucial and is
based on the plant species to be transformed.
Kanamycin is the most widely used antibiotic for
plant transformation. It is very effective in inhibiting
the growth of untransformed cells. However
kanamycin is ineffective as a selection chemical for
several legumes and plants of gramineae. It interferes
with regeneration of transformed cells to green plants
and wheat tissues exhibit a high level of endogenous
tolerance to kenamycin. As an alternative, geneticin
(G418), another member of aminoglycosides, can be
used effectively for selecting npt II-transformed cells.
The hpt (hygromycin phosphotransferase) gene also
derived from Escherichia coli, encoding for hygromycin
phosphotransferase detoxifies the aminocyclitol antibiotic
hygromycin. It binds 30S ribosomal subunit and inhibits
translation. A large number of plants have been
transformed with the hpt gene and hygromycin as a
selective agent has been proved very effective in the
selection of transformed plants, including monocotyledons.
Most plants exhibit higher sensitivity to hygromycin than
to kanamycin, for instance cereals. So it is relatively
better and efficient selection system for wheat (Ortiz et
al., 1996).
3.2.2 Herbicide resistance markers
Genes conferring resistance to herbicides were used as
an alternate to antibiotic resistance genes for wheat
transformation. Herbicide resistance genes in fact
provide a more effective system for plant transformation.
The cell death in the presence of herbicide is generally
more rapid and complete thus providing more efficient
selection. It also provides a convenient and easily
assayable system whereby the transgenic material can
readily be identified/screened using simple techniques
such as leaf painting. Genes conferring resistance to a
number of herbicide groups including triazines,
sulfonylureas, bromoxynil, glyphosate and phosphinot-
hricin are readily available.