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Triticeae Genomics and Genetics
TGG 2010, Vol.1, No.2
http://tgg.sophiapublisher.com
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caryopsis, mature/immature embryos, isolated
scutellum, immature inflorescence, immature leaves,
mesocotyl, apical meristem, coleoptile node and root.
But culturing immature embryos proved more fruitful.
Callus derived from mature embryos (http://www.biome-
dcentral.com) was also successfully transformed with a
frequency of more than 8%. Fate of the transgene in
wheat plants was also investigated (Mihaly et al., 2002).
Transgenic wheat plants were crossed with non-transgenic
ones and transgene inherited successfully to the next
generation following Mendelian pattern.
Since the first successful wheat transformation several
attempts have been made to transform different
varieties of wheat using genes for various traits of
common interest for instance bread and pasta making
quality, starch characteristics, resistance against
viruses, fungi, insects, herbicides, male sterility, heat,
cold, aluminum tolerance, etc. Expression of low
molecular weight glutenin (LMWG) subunits in the
starchy endosperm (Tosi et al., 2004) and acceleration
of flowering time by more than a month by insertion
of RNAi (Yan et al., 2004) (RNA-interference) gene
are recent advances in genetic improvement of wheat.
Improvement of wheat abiotic stress resistance
through genetic transformation was also attempted.
The plasmid containing aldolase reductase (
ALR
) gene,
for osmotic stress resistance isolated from alfalfa was
bombarded into wheat suspension culture (Pauk et al.,
2002) and its protective function was verified under
stress conditions.
DREB1A
(dehydration resistance
element binding-protein 1A), a gene for drought
resistance, was incorporated into callus induced from
immature embryos of wheat (Pellegrineschi et al.,
2002). The preliminary experiments revealed that the
transformed plants survived a short and intensive
water stress at the plantlet stage while the control
plants were completely desiccated. Drought tolerant
transgenic wheat has been sown in the field for
screening (CBI, 2004, www.whybiotec.com)
2 Promoters
The promoter region preceding a marker gene is one
of the most important factors affecting transformation
frequency. The expression of inserted genes is limited
to the activity of promoters. To date, the most of the
promoters used in transgenic wheat, such as
Adh1
(alcohol dehydrogenaseI),
CaMV35S
(cauliflo-wer
mosaic virus 35S),
Act1
(rice actin1),
ubi1
, (maize
ubiquitine1), etc. are constitutive causing gene
expression in all tissues throughout the plant life cycle.
ScBV
(sugarcane bacilliform badna virus) is another
promising promoter for use in wheat transformation
process (Tzafrir et al., 1998). Tissue specific and
developmentally regulated promoters that allow
expression of transgene only in specific tissues or
under certain development conditions are also used in
wheat transformation. Light regulated promoters such
as
LHCP
(light harvesting chlorophyll- binding
protein) and pathogen or wound induced promoters
such as
Vst1
(Vitis stilbene synthase1) from grapevine
and
RC24
(rice chitinase) in addition to stress
inducible promoters like
rd29a
(dehydration-resistance
29a) are potential promoters to facilitate genetic
transformation of wheat.
3 Genetic markers
Genetic transformation is comprised of delivery of
gene cassette followed by analysis of gene expression
in the transformants. As the event of transformation is
very low so a suitable and efficient selection system is
required to select these few transgenic cells. Therefore
marker genes are included to identify the transformed
tissues or cells. Genetic markers employed in genetic
engineering are of two types, screenable/scorable
markers (reporter genes) and selectable markers.
3.1 Scorable/screenable markers
Scorable markers encode gene products whose
enzyme activity can be assayed easily allowing not
only the detection of transformants but also an
estimation of the level of foreign gene expression in
the transgenic tissue. The most useful reporter genes
encode an enzyme activity not found in the plant
being studied.
CAT
(chloramphenical acetyltransferase)
obtained from
E. coli
was initially used for study of
introduced alien gene into wheat genome (Chibbar et
al., 1991). However, difficulties associated with
enzymatic analysis of this gene limited its use in
wheat transformation.
GUS
(
β
-glucuronidase) a
hydrolase that catalyses the cleavage of a wide variety
of
β
-glucuronide compounds, also derived from
E.
coli
, is the most popular and effective reporter