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

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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

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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).

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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.

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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