Arab J. Biotech., Vol. 16, No. (2) July (2013):
Transformation of Egyptian wheat (Triticum aestivum L.) with
rice chitinase and bar genes for disease and herbicide
resistance
(Received: 02. 04. 2013; Accepted: 10. 06 .2013)
A. H. Fahmy*; R. A. Hassanein**; H. A. Hashem**; A. S. Ibrahim***; O. M. El Shihy***;
E. A. Qaid**** *Agricultural Genetic Engineering Research Institute, 9 Gamaa St. Giza, Egypt.
** Department of Botany, Faculty of Science, Ain Shams University, Abbassia, Cairo, Egypt.
***Department of Plant Physiology, Faculty of Agriculture, Cairo University, Egypt.
**** Department of Biology, Faculty of Science, Taiz University, Yemen.
ABSTRACT
Wheat (Triticum aestivum L.) is one of the most important food crops in the world. In the
present study, immature embryo derived calli of wheat cultivar Giza 164 was transformed by the
rice chitinase (RC7) and bar genes using particle bombardment to enhance disease and herbicide
resistance. Immature embryo-derived calli were co-bombarded with the plasmids pAHRC-7
harboring the rice chitinase (RC7) gene and pAB6 containing the gus reporter gene and the bar
selectable marker gene. Transient gus expression in calli and stable gus expression in transformed
nodes were observed. Transgenic calli were selected on phosphinothricin containing regeneration
medium and putative transformants were grown to maturity. Forty herbicide-resistant putative
transformants were selected after leaf painting with 0.2% Liberty. Presence and integration of
transgenes were assessed by subjecting the DNA of the transgenic plants to PCR analysis using
specific primers for gus, bar and RC7 genes. Transformation frequencies for gus, bar and RC7
were 5.5%, 6% and 4.21%; respectively. The incorporation of the rice chitinase gene in the genome
of the transformants was confirmed by the dot-blot analyses.
Keywords: Wheat, Triticum aestivum L., immature embryo, transformation, gus, bar, rc7.
INTRODUCTION
read wheat (Triticum aestivum L.) is an
important cereal crop and an essential
ingredient of the human diet
indisputably worldwide. Plants are exposed to
a wide variety of pathogens, i.e., fungal,
bacterial and viral. In nature, spikes, leaves
and roots of wheat plants can be infected and
that leads to substantial yield loss. Chemical
application of pesticides is not economical and
is also deleterious to the environment. The
most economical and efficient way to protect
wheat from rust is to develop genetically
resistant varieties. Wheat breeding program is
the primary and partial form of resistance
which depends on resistance to initial infection
and spread of disease (Rudd et al., 2001and
Kolb et al., 2001). While, the level of genetic
resistance provided is generally insufficient to
tolerate for epidemic diseases. Therefore,
genetic engineering is the best approach to
increase not only disease resistance but also
agronomic performance, resistance to biotic
B
Fahmy et al.
Arab J. Biotech., Vol. 16, No. (2) July (2013):
and abiotic stresses and enhancing yield and
grain quality (Bicar et al., 2008).
The fundamental problem is not the
delivery of genes but establishment of long-
term cultures from which transgenic plants
could be regenerated (Razzaq et al., 2011).
Use of enhanced and effective transformation
system to drive the genes of interest has drawn
attention of the researchers. Currently, the
successful method used for wheat
transformation is the direct delivery through
particle acceleration bombardment. Although
wheat is one of the most difficult crops to
transform, some reports have recorded
successful and practical transformation
systems for exogenous genes in wheat cells
and tissues (Patnaik and Khurana, 2003;
Delporte et al., 2005; Roy-Barman et al.,
2006; Mackintosh et al., 2007; Xing et al.,
2008; Lazzeri and Jones, 2009; Huang et al.,
2013 and Fahmy et al., 2013).
Vasil et al., (1992) produced the first
wheat transgenic plants by bombarding
embryogenic callus tissues with plasmid
pBARGUS harboring gus reporter gene and
the selectable bar gene which confers
resistance to the broad-spectrum herbicide
Basta. Immature embryo callus was the
suitable and the prosperous target tissue used
in transformation (Weeks et al., 1993; Wright
et al., 2001; Okubara et al., 2002; Gao et al.,
2005; Janni et al., 2008; Tamas et al., 2009;
Fahim et al., 2010; Huang et al., 2013 and
Fahmy et al., 2013).
In response to attacking pathogen, plants
synthesize and accumulate proteins which
inhibit the growth of invading pathogen
directly or degrade pathogen cell wall
components. Chitinases catalyze the hydrolytic
cleavage of the β-1,4-glycosidic bonds
between biopolymers N-acetyl-glucosamine
residues from the chitin molecule
(Schlumbaum et al., 1986; Leah et al., 1991;
Velazhahan et al., 2000; and Wani, 2010).
Several reports revealed that chitinase activity
in transgenic plants increased the inhibition of
fungal growth and improved resistance against
fungal attack (Ignatius et al., 1994; Chen et al.,
1998; and Datta et al., 2001). Some reports
indicated that the over-expression of the
chitinase gene in transgenic plants leads to
increased resistance to a wide range of disease
pathogens. Broglie et al. (1991) reported that
the expression of bean chitinase gene in
transgenic tobacco and canola enhanced
resistance to Rhizoctonia solani. Many reports
recorded the transformation of the rice
chitinase gene in different plants such as: in
wheat by Chen et al. (1998) and Huang et al.
(2013); in rice by Nishizawa et al. (1999);
Datta et al. (2001); Li et al. (2009); in barley
by Tobias et al. (2007); in Italian rye grass by
Takahashi et al. (2005) and in banana by
Kova´cs et al. (2013). Also, wheat plants were
transformed with the barley chitinase gene by
Oldach et al. (2001) and Shin et al. (2008).
Moreover, transgenic wheat carrying the wheat
chitinase gene was produced by both Anand et
al. (2003) and Fahmy et al. (2013). In
addition, expressing the rice chitinase gene
(RC7), had improved disease resistance in rice
by Datta et al. (2002); and in sorghum by
Arulselvi et al. (2010).
The objective of this study was to
produce wheat (Triticum aestivum L.) cultivar
Giza 164 with improved disease resistance by
introducing the RC7 gene which confers a
wide range of disease resistance.
MATERIALS AND METHODS
Plant material and tissue culture
Immature embryos were the tissue
culture explants used in the present study.
Immature caryopsis of the cultivar Giza 164
was collected approximately 10-12 days post
anthesis. Seeds were surface sterilized with
20% commercial Clorox (5.25% Sodium
Transformation of Egyptian wheat with RC7 and bar genes
Arab J. Biotech., Vol. 16, No. (2) July (2013):
hypochlorite) supplemented with few drops of
Tween 20, and then washed five times with
d.d.H2O. Immature embryos, 1-1.25 mm in
diameter, were aseptically removed and
cultured scutellum side up on callus induction
medium containing MS salt (Murashige and
Skoog, 1962), supplemented with 2 mg/l 2,4-
dichlorophenoxyacetic acid as a source of
auxin, 150 mg/l of L-Asparagine, 100 mg/l of
myo-inositol, 20 g/l sucrose, adjusted to 5.8
pH with 1 M KOH solution and solidified by
2.5 g/l phytagel (Weeks et al., 1993).
Immature embryos were maintained in the
dark at 25°C for one week.
DNA constructs
Two different plasmids were used for the
co-bombardment experiments, i.e., pAHRC-7
(Fig. 1A) harboring the rice chitinase (RC7)
gene driven by the constitutive maize ubiquitin
promoter, and pAB6 (Fig. 1B) containing the
selectable bar gene under the control of
CaMV-35S promoter which confers resistance
to the herbicide BASTA and the screenable
gus gene which encodes the β-glucuronidase
(GUS) driven by the rice Act 1-intron
promoter (Christensen and Quail, 1996).
Bacterial strain
The highly efficient competent cells of E.
coli (DH10β) were used for the transformation
by the plasmids DNA and prepared according
to the method of Ausubel et al. (1987).
Plasmid transformation into E. coli
competent cells
Calcium chloride treatment of E. coli
(DH10β) was used to produce the competent
cells needed for transformation by the pAB6
or pAHRC-7 plasmid using heat shock step
according to Tu et al. (2005).
Plasmid purification
Bacteria harboring the pAB6 or pAHRC-
7 plasmid were grown in liquid LB-ampicillin
medium at 37°C in a shaker incubator. Mega
prep purification of DNA plasmid was
conducted using Wizard™ Megapreps DNA
Purification System (Promega, USA).
Preparation and coating of gold particles
with plasmid DNA
Microcarriers (o.6 µ gold particles) were
prepared and coated with plasmid DNA
according to the protocol of Sanford et al.
(1993).
Wheat transformation
The transformation procedure was
performed based on the bombardment method
described by Fahmy et al. (2006) immature
derived callus of cultivar Giza 164 used for all
transformation experiments. Embryo culture,
tissue culture selection and plant regeneration
were conducted according to Fahmy et al.
(2006). A 1:1 ratio of pAB6 and pAHRC-7
were co-transformed into Giza 164 calli.
Plant transformation was carried out by
particle bombardment using the Biolistic®
PDS-1000/He particle gun device (Bio-Rad,
USA). One week old immature-embryo
derived calli were transferred to a modified
callus induction medium (supplemented with
0.2 M mannitol and 0.2M sorbitol for four
hours before bombardment. Calli were
bombarded with 0.6 µ gold particles coated
with plasmid DNA. The distance between the
particle holder and target was 6 cm and helium
pressure was 1100 psi. Calli were kept for
additional 16 hrs on the same osmotic
treatment, and then transferred to recovery
medium for five days. Calli were then assayed
by the histochemical GUS activity assay
(Jefferson et al., 1987). The remaining calli
were transferred to selective medium
supplemented with 3 mg/l PPT. Calli showing
vigorous growth were sub-cultured twice
every three weeks onto selection medium, and
then transferred onto regeneration medium
supplemented with 1.5 mg/l TDZ until
Fahmy et al.
Arab J. Biotech., Vol. 16, No. (2) July (2013):
emerging of shoots. Vigorous shoots were
transferred to rooting medium with half
strength MS medium.
Acclimatization
After development of a root system,
regenerated putatively transgenic plantlets
were transferred to soil mixture of peat moss:
sand: clay with a ratio (1:1:1); respectively, in
small pots and covered with plastic bags and
placed in a controlled growth chamber at 25°C
for 3 weeks, then plants were transferred to
large pots and grown to maturity under green
house conditions. The seeds were then
collected from grown booted plants.
Assay of β-glucuronidase (gus) activity
GUS assay was carried out as described
by Jefferson et al. (1987). After recovery, calli
were incubated in X-Glue solution containing
1 mM (5-bromo-4-chloro-3-indolyl-β-D-
glucuronide), 0.1% (v/v) Triton X-100, 20%
methanol and 100 mM sodium phosphate
buffer (pH 7.0), 0.5 mM potassium
ferricyanide, 0.5 mM potassium ferrocyanide.
Transient GUS expression For observing transient GUS expression,
bombardment calli after 2 days on induction
medium were dipped in GUS staining solution
and incubated at 37°C for 2-3 days and then
photographed under microscope.
Stable GUS expression For observing stable GUS expression,
regenerated shoot primordial were assayed by
dipping transformed shoot primordial into
GUS staining solution. The reaction mixture
was incubated at 37°C for 2-3 days.
Chlorophyll was extracted from the tissue by
incubation in 70% ethanol followed by100%
ethanol and regenerated shoot primordial
expressing gus were photographed under
microscope.
Assay of bar expression analysis
Leaf painting assay was used to study the
integration and expression of the bar gene in
T0 plants according to Cho et al. (1998).
Liberty solution (0.2%), containing 0.1% (v/v)
Tween-20, was applied to leaf sections using a
cotton swab. Resistance to the herbicide
solution was examined after seven days of
application by observing of leaf necrosis.
PCR analysis
Total genomic DNA was isolated from
wheat leaves using a cetyltriethyl-ammonium
bromide (CTAB) extraction method
(Sambrook et al., 1989). The PCR analysis
was used to confirm the presence or the
absence of the three transgenes in the
transformed plants. The specific primers used
to amplify the RC7 gene were 5'GCC GCG
GCC CCA TCC AAC TCT 3' and 5'CAT
CAC TGC TCC GCC AAC CCA ACC 3'. The
forward and reverse primers employed for
detection of bar gene were 5'CAG ATC TCG
GTG ACG GGC AGG C3`and 5` CCG TAC
CGA GCC GCA GGA AC -3`; and for the gus
gene were 5`AGT GTA CGT ATC ACC GTT
TGT GTG AAC 3`and 5`AGT GTA CGT
ATC ACC GT TTG TGT GAA C3`.The PCR
program profile for three genes was as
follows: initial denaturation at 94°C for 5 min,
followed by 30 cycles at 94°C for 30 sec,
annealing for 30 sec and 72°C for 1 min and
finally , an additional elongation step was
performed for 7 min at 72°C. the annealing
temperature for the amplification of RC7, bar
and gus genes were 66°C, 58°C and 62°C,
respectively. The PCR reaction mixture
contained 50 ng of template DNA, 0.5 µM of
each primer, 10 mM of dNTPs, 2.5mM of
MgCl2, PCR buffer and Taq polymerase in a
volume of 25 µl. The amplified products were
electrophoretically resolved on a 1% agarose
gel in TAE buffer.
Transformation of Egyptian wheat with RC7 and bar genes
Arab J. Biotech., Vol. 16, No. (2) July (2013):
Dot-Blot hybridization analysis
PCR products from transgenic plants,
non-transgenic plants (negative control) and
pAHRC-7 (positive control) were denatured
and neutralized with 0.4 M NaOH, 10 mM
EDTA, incubated at 96°C for 10 min, then
rapidly cooled in ice. Using a dot-blot
manifold, samples were spotted onto pre-
soaked nitrocellulose membrane. Membrane
was crosslinked under UV light. Hybridization
was performed overnight at 45°C in a buffer
containing 5X denhardt’s solution, 6X SSC,
0.5% SDS and 50% (v/v) deionized
formamide and followed by the addition of
pAHRC-7 as probe. Membrane was washed
twice at room temperature in 2X SSC/ 0.1%
SDS for 5 min followed by two washes in
0.1X SSC/ 0.1% SDS for 20 min at 70°C.
Direct detection system was carried out by the
Biotin Chromegenic Detection Kit (Thermo
Scientific). Blot was washed and product
detection was conducted by the addition of
BCIP/NBT solution, then the blot was exposed
to photography.
RESULTS AND DISCUSSION
In the present study, we used the particle
bombardment approach in wheat
transformation to produce transgenic plants,
harboring the RC7 and bar genes to enhance
resistance against a broad range of diseases
and herbicide. The first fertile transgenic
wheat was reported by Vasil, et al. (1992)
using immature embryos. There are numerous
reports employing immature and mature
embryo, embryo derived-calli and scutella
tissue as the explants for transformation by
biolistic (Weeks et al., 1993; Becker et al.,
1994; Nehra et al., 1994; Altpeter et al., 1996;
Zhang et al., 2000; Patnaik and Khurana,
2003; Fahmy et al., 2006 and 2013; Jia et al.,
2009 and He et al., 2010).
Callus was initiated after transferring
immature embryos on induction medium.
Stages of development of somatic embryos
were observed by examining embryogenic
cultures under a stereomicroscope (Fig. 2A).
The first step of somatic embryos is
longitudinal axis by cell division. Several
more rounds of cell division occured to
produce a compact globular somatic embryo
(Fig. 2B). Auxin (2,4-D) promotes cellular
division in plant tissue. The formation of
somatic embryos from immature embryos
differentiated into globular, scutella (Fig. 2C)
and coleoptilar shapes (Fig. 2D and E). The
different steps of the co-transformation
process are shown in Fig. (3). Eight hundred
immature embryos of wheat cultivar Giza 164
were used as explants (Table 1 and Fig. 3A).
After one week, 617 immature embryo-derived
calli were proliferated Fig. 3B), then were
transferred to the center of Petri-plate
containing osmotic medium for four hours
before co-transformation (Fig. 3C). The calli
were bombarded with gold particles coated
with plasmid pAHRC-7 harboring the RC7
gene and plasmid pAB6 containing the bar
(selectable gene) and gus (marker gene).
Bombarded calli were recovered following
bombardment on induction medium for five
days (Fig. 3D). The calli were transferred into
selection medium for three weeks containing 3
mg/L PPT for selection of resistant tissues
which showed active proliferation in the first
round of selection. In the second round of
selection of three weeks, some of the calli
grew healthy due to the expression of the bar
gene, while other calli turned dark brown (Fig.
3E and F). After, the two rounds of selection,
391 resistant callus lines were regenerated on
regeneration medium containing 1.5 mg/L
TDZ (Fig. 3G).
Fahmy et al.
Arab J. Biotech., Vol. 16, No. (2) July (2013):
Table (1): Transformation characteristics of Egyptian wheat cv. Giza 164.
Regenerated shoots were then transferred
to culture tubes containing rooting medium
(half-strength MS) (Fig. 3H). A number of 264
plantlets were acclimated in growth chamber
(Fig. 3I and J). Transgenic plants were
generated to maturity stage in greenhouse (Fig.
3K). The transgenic plants had no phenotypic
abnormalities in comparison to the
untransformed control plants.
No. of
immature
embryos
No. of
induced
calli
No. of
bombarded
calli
No. of
surviving
calli on
selection
medium I
No. of
surviving
calli on
selection
medium
II
No. of
regenerated
shoots
No. of
acclamatized
plantlets
Gus
gene
+ve
PCR
plants
bar
gene
+ve
PCR
plants
RC7
gene
PCR
plants
RC7
gene
Dot
Blot
+ve
plants
50 30 30 24 22 14 3 2 3 3 3
50 28 28 22 16 12 3 2 3 1 1
50 33 33 27 24 16 2 2 2 2 2
50 29 29 24 18 13 4 3 2 1 1
50 32 32 25 21 15 3 2 3 3 3
50 35 35 27 22 18 2 2 2 2 2
50 30 30 25 20 11 3 3 3 1 1
50 33 33 28 25 20 4 3 3 3 3
50 45 45 36 29 17 1 1 1 1 1
50 44 44 33 25 13 2 2 2 1 1
50 46 46 38 32 21 3 2 3 1 1
50 45 45 33 24 11 1 1 1 0 0
50 48 48 39 32 26 3 3 3 3 3
50 45 45 35 26 21 3 3 3 2 2
50 46 46 32 25 12 1 1 1 0 0
50 48 48 38 30 24 2 2 2 2 2
800 617 617 486 391 264 40 34 37 26 26
- - - - - - - 5.51% 6.00% 4.21% 4.21%
Arab J. Biotech., Vol. 16, No. (2) July (2013):
Fig. (1): Schematic representation of plasmids pAHRC-7(A) and pAB6 (B) used for co-
bombardment.
Fig. (2): Different developmental stages of somatic embryos in Triticum aestivum L., cv. Giza
164: A) Initiation of embryogenic calli from cultured immature embryos, B) globular
stage, C) scutellar stage, D) early coleoptilar stage and E) late coleoptilar stage.
C B D E A
Fahmy et al.
Arab J. Biotech., Vol. 16, No. (2) July (2013):
Fig. (3): Co-transformation steps of Triticum aestivum L. cv. Giza 164 with pAHRC-7 and pAB6 by
particle bombardment. A) Immature embryos excised 10-12 d post anthesis on callus induction
medium. B) Proliferation of callus tissue from embryos after 7 d on induction medium. C) Calli
pooled to the centre of the Petri-plate to facilitate bombardment at a distance of 6 cm. D) Calli
after bombardment transferred to recovery medium for 5 days. E, F) Calli under first and second
stages of selection (3 mg/l phosphinothricin). G) Regenerated shoots from calli on TDZ medium.
H) The growth of shoots and roots from calli onto free half strength MS medium. I, J)
Acclimatized plantlets in growth chamber. K) A mature, putative transgenic plant at potting stage.
Forty (T0) transformed plants were
obtained from 617 immature embryo-derived
calli co-bombarded with the two plasmids.
Transformation efficiency of the three genes
was calculated based on the number of calli
bombarded and the number of plants showing
positive PCR for each gene. Among the three
genes studied, the bar gene had the highiest
efficiency of 6.0% followed by the gus gene
with 5.51% and then the RC7 with 4.21%.
(Table 1). The transformation efficiency of
wheat plants in the literature was low and
depended significantly on the genotype. Our
results were in agreement with previous
reports (2.4% by Bourdon et al., 2004; 0.6-
3.1% by Tosi et al., 2004; 1.4-3% by
Mackintosh et al., 2006 and 1.8%-2.7 by
Fahmy et al., 2013.) Plants that survived after
selection but revealed negative PCR for the
presence of transgene were considered
escapes. A number of 264 plantlets survived
after the two selection stages. Only forty plants
grew to maturity and thirty seven were
positive for bar gene. Our results are in
agreement with those of Becker et al. (1994);
Bieri et al. (2000); Roy-Barman et al. (2006);
Kasirajan et al. (2013) and Fahmy et al.
(2013).
Transformation of Egyptian wheat with RC7 and bar genes
Arab J. Biotech., Vol. 16, No. (2) July (2013):
Fig. (4): Histochemical assay showing gus gene expression: A) non-transformed callus, B) gus
expression in callus and shoot primordial after bombardment.
Fig. (5): leaves painted with A) Non-transgenic showing necrosis, B) transgenic plantlets
showing resistance to herbicide.
Histochemical assays of gus activity
were performed on bombarded calli and shoot
primordial as mention before. The presence of
blue color in bombarded calli and shoot
primordial compared to non-bombarded calli
indicated the successful delivery of DNA
using the particle bombardment (Fig. 4A and
B). Multi-cellular gus positive structures in
callus and shoot primordial seems to be due to
stable integration and expression in all cells
and this was in close agreement with Weeks et
al. (1993); Haliloglu and Baenziger (2002) and
Fahmy et al. (2006). The herbicide liberty was
applied on T0 plants carrying both genes and
untransformed plants as previously mention.
Putative transgenic wheat plants showed
resistance to herbicide application which was
strong evidence for the transmission of the
functional bar gene to T0 putative plants (Fig.
5A). Only control plants exhibited necrosis
when sprayed by herbicide liberty solution
after seven days (Fig. 5B).
Fahmy et al.
Arab J. Biotech., Vol. 16, No. (2) July (2013):
Fig. (6): PCR analysis of T0 plants. (A) Amplification product of RC7gene (421bp). (B)
Amplification product of bar gene (443 bp). (C) Amplification product of gus gene
(1050bp). Lane M is DNA marker (100 bp ladder). Lane 1: positive control (plasmid),
Lane 2 is non-transformed wheat cv. G164 (negative control). Other Lanes are the
transgenic wheat plants.
Fig. (7): Dot blot analysis of transgenic wheat cv. G164 plants; Dot 1 is pAHRC-7 plasmid
(positive control), Dot 2 is non-transformed wheat cv.G164 (negative control) and Dots
3-28 are the 26 wheat RC7 transgenic plants.
PCR analysis was performed to detect
the presence of the T-DNA in the genome of
the transgenic plants. DNA extracted from
leaves of the forty T0 plants was subjected to
PCR analysis with the primers specific for the
rc7, bar and gus genes. PCR results revealed
that there were twenty-six plants containing
the RC7 gene which scored an amplified
product of 421 bp (Fig. 6A). Thirty-seven
plants out of forty were found PCR positive
for the bar gene and amplified product was
443bp (Fig. 6B). Thirty-four transformants had
integrated the gus gene and amplified product
was 1050 bp (Fig. 6C). No amplified product
was detected in the samples containing
genomic DNA from untransformed plants.
PCR results revealed transformation efficiency
of 4.2 % for the RC7 gene, 6.0% for the bar
gene and 5.5 % for the gus gene. The stable
integration of the RC7 gene into plant genome
of 26 positive plants was confirmed by dot-
blot (Fig. 7).
In summary, we transferred the rice
chitinase RC7 gene and the bar gene into
immature embryo derived calli of Egyptian
wheat cultivar Giza 164 using biolistic device
Transformation of Egyptian wheat with RC7 and bar genes
Arab J. Biotech., Vol. 16, No. (2) July (2013):
to enhance resistance against pathogens and
herbicide.
ACKNOWLEDGMENTS
The authors thank appreciably Prof. Dr.
S. Muthukishnan (Department of
Biochemistry, Kansas State University,
Kansas, USA) for generously providing the
RC7 gene.
REFERENCES
Altpeter, F.; Vasil, V.; Srivastava, V.;
Stoger, E. and Vasil, I.K. (1996). Accelerated production of transgenic wheat
(Triticum aestivum L.) plants. Plant Cell
Rep., 16: 12–17.
Anand, A.; Zhou, T.; Trick, H.N.; Gill, B.S.;
Bockus, W.W. and Muthukrishnan, S.
(2003). Greenhouse and field testing of
transgenic wheat plants stably expressing
genes for thaumatin-like protein, chitinase
and glucanase against Fusarium
graminearum. J. Exp. Bot., 54, 1101–1111.
Arulselvi, I.; Micheal, P.; Umamaheswari,
S. and Krishnaveni, S. (2010). Agrobacterium mediated transformation of
Sorghum bicolor for disease resistance. Int.
J. Pharm Bio. Sci., 1(4): 272-281.
Ausubel, F.M.; Brent, R.; Kimgston, R.E.;
Moore, D.; Serdman, J.G.; J Smith, A.
and Struhle, K. (1987). Current protocols in
molecular biology. 1: 1-4, Green Publishing
Associates and Wiley Interscience, New
York, USA.
Becker, D.; Brettschneider, R. and Lorz, H.
(1994). Fertile transgenic wheat from
microprojectile bombardment of scutellar
tissue. Plant J., 5: 299–307.
Bicar, E.H.; Woodman-Clikeman, W.;
Sangtong, V.; Peterson, J.M.; Yang, S.S.;
Lee, M. and Scott, M.P. (2008). Transgenic
maize endosperm containing a milk protein
has improved amino acid balance.
Transgenic Res., 17: 59–71.
Bieri, S.; Potrykus, I. and Fuetterer, J.
(2000). Expression of active barley seed
ribosome-inactivating protein in transgenic
wheat. Theor. Appl. Genet., 100: 755-763.
Bourdon, V.; Wickhan, A.; Lonsdale, D.
and Harwood, W. (2004). Additional
introns inserted within the luciferase gene
stabilize transgene expression in wheat.
Plant Sci., 167: 1143-1149.
Broglie, K.; Chet, I.; Holliday, M.;
Cressman, R.; Biddle, P.; Knowlton, S.;
Mauvais, C.J. and Broglie, R. (1991).
Transgenic plants with enhanced resistance
to the fungal pathogen Rhizoctonia solani.
Sci., 254: 1194-1197.
Chen, W.P.; Gu, X.; Liang, G.H.;
Muthukrishnan, S.; Chen, P.D.; Liu, D.J.
and Gill, B. S. (1998). Introduction and
constitutive expression of a rice chitinase
gene in bread wheat using biolistic
bombardment and the bar gene as a
selectable marker. Theor. Appl. Genet., 97:
1296– 1306.
Cho, M.J.; Jiang, W. and Lemaux, P.O.
(1998). Transformation of recalcitrant barley
cultivars through improvement of
regenerability and decreased albinism. Plant
Sci., 138: 229-244.
Christensen, A.H. and Quail, P.H. (1996).
Ubiquitin promoter-based vectors for high-
level expression of selectable and/or
screenable marker genes in
monocotyledonous plants. Transg. Res., 5:
213–218.
Datta, K.; Tu, J.; Oliva, N.; Ona, I.;
Velazhahan, R.; Mew, T.W.;
Muthukrishnan, S. and Datta, S.K. (2001). Enhanced resistance to sheath blight by
constitutive expression of infection-related
rice chitinase in transgenic elite indica rice
cultivars. Plant Sci., 160(3): 405-414.
Fahmy et al.
Arab J. Biotech., Vol. 16, No. (2) July (2013):
Datta, K.; Baisakh, N.; Thet, K.M.; Tu, J.
and Datta, K.S. (2002). Pyramiding
transgenes for multiple resistance in rice
against bacterial blight, yellow stem borer
and sheath blight. Theor. Appl. Genet., 106:
1-8.
Delporte, F.; Li. S. and Jacquemin, J.M.
(2005). Calluses initiated from thin mature
embryo fragments are suitable targets for
wheat transformation as assessed by long-
term GUS expression studies. Plant Cell,
Tissue and Organ Culture, 80 (2): 139-149.
Fahim, M.; Ayala-Navarrete, L.; Millar,
A.A. and Larkin, P.J. (2010). Hairpin RNA
derived from viral NIa gene confers
immunity to wheat streak mosaic virus
infection in transgenic wheat plants. Plant
Biotechnol J., 8(7): 821-834.
Fahmy, A.H.; El-Shihy, O.; El-Shafey, Y.H.
and Madkour, M.A. (2006). Genetic
transformation of Egyptian wheat cultivar
(Triticum aestivum L.) via biolistic
bombardment using different constructs.
American-Eurasian J. Agric. and Environ.
Sci., 1(2): 58-69.
Fahmy, A.H.; El Mangoury, K.; Abou El-
Wafa, W.; Barakat, H.M.S.; El-Khodary,
S. and Muthukrishnan, S. (2013). Genetic
transformation of Egyptian wheat (Triticum
aestivum L.) with chitinase gene via
microprojectile bombardment. Egypt. J.
Genet. Cytol., 42:233-245.
Gao, S.Q.; Xu, H.J.; Cheng, X.G.; Chen,
M.; Xu, Z.S.; Li, L.C.; Ye, X.G.; Du, L.P.;
Hao, X.Y. and Ma, Y. Z. (2005). Improvement of wheat drought and salt
tolerance by expression of a stress-inducible
transcription factor GmDREB of soybean
(Glycine max). China Sci. Bull., 50(23):
2714-2723.
Haliloglu, K. and Baenziger, P.S. (2002). Optimization of wheat (Triticum aestivum
L.) anther culture derived embryos
transformation by microprojectile
bombardment. Ziraat Fakultesi Dergisi,
Ataturk Universitesi, 33(4): 413-416.
He, Y.; Jones, H.D.; Chen, S.; Chen, X.M.;
Wang, D.W.; Li, K.X.; Wang, D.S. and
Xia, L.Q. (2010). Agrobacterium-mediated
transformation of durum wheat (Triticum
turgidum L. var. durum cv Stewart) with
improved efficiency. J. Exp. Bot., 61(6):
1567-1581.
Huang, X.; Wang, J.; Du, Z.; Zhang, C.; Li,
L. and Xu, Z. (2013). Enhanced resistance
to stripe rust disease in transgenic wheat
expressing the rice chitinase gene RC24.
Trans. Res., 22(5): 939-947.
Ignatius, S. M.J.; Huang, J.K.; Chopra,
R.K. and Muthukrishnan, S. (1994).
Isolation and characterization of a barley
chitinase genomic clone: expression in
powdery mildew infected barley. J. Plant
Biochem. Biotechnol., 3: 91–95.
Janni, M.; Sella, L.; Favaron, F.;
Blechl,A.E. ; De Lorenzo, G. and Ovidio,
R.D. (2008). The expression of a bean PGIP
in transgenic wheat confers increased
resistance to the fungal pathogen Bipolaris
sorokiniana. Mol. Plant Microbe Interact.,
21: 171–177.
Jefferson, R.A.; Kavanagh, T.A. and Bevan,
M.W. (1987). GUS fusions: β-glucuronidase
as a sensitive and versatile gene
fusionmarker in higher plants. EMBO J., 6:
3901–3907.
Jia, H.; Yu, J.; Yi, D.; Cheng, Y.; Xu, W.;
Zhang, L. and Ma, Z. (2009).
Chromosomal intervals responsible for tissue
culture response of wheat immature
embryos. Plant Cell, Tissue and Organ
Culture, 97(2): 159-165.
Kasirajan, L.; Boomiraj, K. and Bansal,
K.C. (2013). Optimization of genetic
transformation protocol mediated by biolistic
method in some elite genotypes of wheat
(Triticum aestivum L.). Afr. J. Biotech.,
12(6): 531-538.
Transformation of Egyptian wheat with RC7 and bar genes
Arab J. Biotech., Vol. 16, No. (2) July (2013):
Kolb, F.L.; Bai, G.H.; Muehlbauer, G.J.;
Anderson, J.A.; Smith, K.P. and Fedak,
G. (2001). Host plant resistance genes for
Fusarium head blight: Mapping and
manipulation with molecular markers. Crop
Sci., 41: 611-619.
Kova´cs, G.;Sa´gi, L.; Jacon, G.; Arinaitwe,
G.; Busogoro, J.P.; Thiry, E.; Strosse, H.;
Swennen, R. and Remy, S. (2013).
Expression of a rice chitinase gene in
transgenic banana (‘Gros Michel’, AAA
genome group) confers resistance to black
leaf streak disease. Transgenic Res., 22:
117–130.
Lazzeri, P.A. and Jones, H.D. (2009). Transgenic wheat, barley and oats:
production and characterization. Methods in
Mol. Biol., 478: 3–22.
Leah, R.; Tommerup, H.; Svendsen, I. and
Mundy, J. (1991). Biochemical and
molecular characterization of three barley
seed proteins with antifungal properties.J.
Biol. Chem., 266(3): 1564-1573.
Li, P.; Pei, Y.; Sang, X.; Ling, Y.; Yang, Z.
and He, G. (2009). Transgenic indica rice
expressing a bitter melon (Momordica
Charantia) Class I chitinase gene
(McCHIT1) confers enhanced resistance to
Magnaporthe grisea and Rhizoctonia solani.
Eur. J. Plant Pathol., 125: 533–543.
Mackintosh, C.A.; Garvin, D.F.; Radmer,
L.F.; Heinen, S.J.; and Muehlbauer, G.J.
(2006). A model wheat cultivar for
transformation to improve resistance to
Fusarium Head Blight. Plant Cell Rep., 25:
313- 319.
Mackintosh, C.A.; Lewis, J.; Radmer, L.E.;
Shin, S.; Heinen, S.J.; Smith, L.A.;
Wyckoff, M.N.; Dill-Macky, R.; Evans,
C.K.; Kravchenko, S.; Baldridge, G.D.;
Zeyen, R.J. and Muehlbauerm, G.J.
(2007). Over expression of defense response
genes in transgenic wheat enhances
resistance to Fusarium head blight. Plant
Cell Rep., 26(4): 479-488.
Murashige, T. and Skoog, F. (1962). A revised medium for rapid growth and Bio
assays with tobacco tissue cultures. Physiol.
Plant., 15: 473–497.
Nehra, N.S.; Chibbar, R.N.; Leung, N.;
Caswell, K.; Mallard, C.; Steinhauer, L.;
Baga, M. and Kartha, K.K. (1994). Self-
fertile transgenic wheat plants regenerated
from isolated scutellar tissues following
microprojectile bombardment with two
distinct gene constructs. Plant J., 5: 285–297.
Nishizawa, Y.Z.; Nishio, K.; Nakazono, M.;
Soma, E.; Nakajima, M.;Ugaki, T. and
Hibi, (1999). Enhanced resistance to blast
(Magnaporthe grisea) in transgenic Japonica
rice by constitutive expression of rice
chitinase. Theor. Appl. Genet., 99: 383–390.
Okubara, P.A.; Blechl, A.E.; McCormick,
S.P.; Alexander, N.J.; Dill-Macky, R. and
Hohn, T.M. (2002). Engineering
deoxynivalenol metabolism in wheat through
the expression of a fungal trichothecene
acetyl transferase gene. Theor. Appl. Genet.,
106: 74–83.
Oldach, K.H.; Becker, D. and Lorz, H.
(2001). Heterologous expression of genes
mediating enhanced fungal resistance in
transgenic wheat. Mol. Plant. Microbe.
Interact., 14: 832–838.
Patnaik, D. and Khurana, P. (2003). Genetic transformation of Indian bread
(Triticum aestivum) and pasta (Triticum
durum) wheat by particle bombardment of
mature embryo-derived calli. BMC plant
Biol., 3(5): 1-11.
Razzaq, A.; Hafiz, I.A.; Mahmood, I. and
Hussain, A. (2011). Development of in
planta transformation protocol for wheat.
Afr. J. Biotech., 10 (5): 740-750.
Roy-Barman, S.; Sautter, C. and Chattoo,
B.B. (2006). Expression of the lipid transfer
protein Ace-AMP1 in
Fahmy et al.
Arab J. Biotech., Vol. 16, No. (2) July (2013):
transgenic wheat enhances antifungal
activity and defense responses. Transgenic
Res., 5(4): 435-446.
Rudd, J.C.; Horsley, R.D.; McKendry, A.L.
and Elias, E.M. (2001). Host plant
resistance genes for Fusarium head blight:
Sources, mechanisms, and utility in
conventional breeding systems. Crop Sci.,
41: 620–627.
Sanford, J.C.; Smith, F.D. and Russell, J.A.
(1993). Optimizing the biolistic process for
different biological application. Methods
Enzmol., 217: 483-509.
Sambrook, J.; Fritsch, E. F. and Maniatis,
T. (1989). Molecular Cloning: a laboratory
manual, 2nd
edn. Cold Spring Harboor, NY:
Cold Spring Harbor Laboratory Press.
Schlumbaum, A.; Mauch, F.; Vogeli, U. and
Boller, T. (1986). Plant chitinases are potent
inhibitors of fungal growth. Nature, 324:
365–367.
Shin, S.; Mackintosh, C.A.; Lewis, J.;
Heinen, S.J.; Radmer, L.; Dill-Macky, R.;
Baldridge, G.D.; Zeyen, R.J. and
Muehlbauer, G.J. (2008). Transgenic wheat
expressing a barley class II chitinase gene
has enhanced resistance against Fusarium
graminearum. J. Exp. Bot., 59(9): 2371-
2378.
Takahashi, W.; Fujimori, M.; Miura, Y.;
Komatsu, T.; Nishizawa, Y.; Hibi, T. and
Takamizo, T. (2005). Increased resistance
to crown rust disease in transgenic Italian
ryegrass (Lolium multiflorum Lam.)
expressing the rice chitinase gene. Plant Cell
Rep., 23(12): 811-818.
Tamas, C.; Kisgyorgy, B.N.; Rakszegi, M.;
Wilkinson, M.D.; Yang , M.S.; Lang, L.;
Tamas, L. and Bedő, Z. (2009).
Transgenic approach to improve wheat
(Triticum aestivum L.) nutritional quality.
Plant Cell Rep., 28: 1085-1094.
Tobias, D.J.; Manoharan, M.; Pritsch, C.
and Dahleen, L.S. (2007). Co-
bombardment, integration and expression of
rice chitinase and thaumatin-like protein
genes in barley (Hordeum vulgare cv.
Conlon). Plant Cell Rep., 26(5): 631-639.
Tosi, P.; Ovidio, R.D; Napier, J.A.; Bekes,
F. and Shewry, P.R. (2004). Expression of
epitope-tagged LMW glutenin subunits in
the starchy endosperm of transgenic wheat
and their incorporation into glutenin
polymers. Theor. App. Genet., 108: 468-476.
Tu, Z.; He, G.; Li, K. X.; Chen, M. J.;
Chang, J.; Chen, L.; Yao, Q.; Li, D. P.;
Ye, H.; Shi, J. and Wu, X. (2005). An
improved system for competent cell
preparation and high efficiency plasmid
transformation using different Escherichia
coli strains. Elec. J. Biotech., 8(1): 113-120.
Vasil, V.; Castillo, A.M.; Fromm, M.E. and
Vasil, I.K. (1992). Herbicide resistant fertile
transgenic wheat plants obtained by
microprojectile bombardment of regenerable
embryogenic callus. Nat Biotechnol., 10:
667–674.
Velazhahan, R.; Samiyappan, R. and
Vidhyasekaran, P. (2000). Purification of
an elicitor-inducible antifungal chitinase
from suspension cultured rice cells.
Phytoparasitica, 28: 131–139.
Wani, S. H. (2010). Inducing fungus
resistance into plants through biotechnology.
Not Sci. Biol., 2(2): 14-21.
Weeks, J.T.; Anderson, O.D. and Blechl,
A.E. (1993). Rapid production of multiple
independent lines of fertile transgenic wheat
(Triticum aestivum L.). Plant Physiol., 102:
1077–1084.
Wright, M.; Dawson, J.; Dunder, E.; Suttie,
J.; Reed, J.; Kramer, C.; Chang, Y.;
Novitzky, R.; Wang, H. and Artim-
Moore, L. (2001). Efficient biolistic
transformation of maize (Zea mays L.) and
wheat (Triticum aestivum L.) using the
phosphomannose isomerase gene, pmi, as the
Transformation of Egyptian wheat with RC7 and bar genes
Arab J. Biotech., Vol. 16, No. (2) July (2013):
selectable marker. Plant Cell Rep., 20(5):
429–436.
Xing, L.P.; Wang, H.Z.; Jiang, Z.N.; Ni,
J.S.; Cao, A.Z.; Yu, L. and Chen, P. D.
(2008). Transformation of wheat thaumatin-
like protein gene and diseases resistance
analysis of the transgenic plants. Acta Agro.
Sinica., 34: 349–354.
Zhang, L.;Rybczynski, J.J.; Langenberg,
W.G.; Mitra, A. and French, R. (2000). An
efficient wheat transformation procedure:
transformed calli with long-term
morphogenic potential for plant
regeneration. Plant Cell Rep., 19: 241-250.
الملخص العربي
لمقاومة الامراض و مبيدات الحشائش barو جين rice chitinase قمح المصري بجينللالتحول الوراثي
اسامه محمد الشيحي ***, يفه احمد حسنين**, حنان احمد هاشم**, احمد شوقي ابراهيم***, ئاشرف حسين فهمي*, ر
*ابتسام احمد قائد*** لزراعية, شارع الجامعه, الجيزة , مصر.*معهد الهندسة الوراثية ا
**قسم النبات, كلية العلوم, جامعة عين شمس, العباسية, القاهرة, مصر.
*** قسم فسيولوجي النبات, كلية الزراعة, جامعة القاهرة, مصر.
, جامعة تعز, اليمن. قسم البيولوجي, كلية العلوم****
المى barو جمين rice chitinaseسة تمم ققمل جمين ية فى العالم. فى هذه الدراالقمح هو احد المحاصيل الغذائية الاكثر اهم
بواسمةة ررققمة المدفل الجينمي لاقتمات قباتمات معدلمة وراثيما 164جيمزة القممح س الناتج من الاجنة الغير قاضجة لصنفوالكال قسيج
لغيمر قاضمجة باسمتخدام المدفل المشمترز لبلا ميمد التحمول الجينمي لكمالس الاجنمة ا حيم تممع عمليمة مقاومة للامراض و المبيدات.
pAHRC-7 المذي قحممل جمينrice chitinase و بلا ميمدpAB6 المذي قحتموج جينماتbar وgus . التعبيمر و قمد لموح
الكمالس المحمور وراثيما تمم و قمد تمم اقتخما فمى الكمالس والعقمد فمى النباتمات المعدلمة وراثيما. gusالجيني و الثبمات الجينمي لجمين ال
نواربعم و قد تم اقتخما الى مرحلة النضج و تكوقن البذور. حي قمع النباتات المحورة وراثيا PPTتعرقضه لبيئة تحتوي على ب
وتمم التاكمد ممن الثبمات الجينمي .leaf painting % فى اختبمار0.2بتركيز Libertyقبات مقاوما للمبيد حي اظهرت مقاومة لمبيد
كفماةة التحمول تقنيمة التسلسمل البموليمري باسمتخدام با ئمات خاصمة لكمل جمين. و كاقمع عمن ررقم RC7و gus , barلجينمات
فمى النباتمات RC7 و تمم التاكمد ممن وجمو جمين % علمى التموالي. 4,1% و RC7 5,5 ,%6 وbar و gus الموراثي لجمين ال
.Dot-blotالمحورة وراثيا باستخدام تقنية