GENETIC TRANSFORMATION OF EGYPTIAN WHEAT WITH 1DX5 HIGH-MOLECULAR-WEIGHT GLUTENIN SUBUNIT GENE

heat (Triticum aestivum L.) seedstorage proteins represent an important source of food and energy (Cooke and Law, 1998). The major endosperm storage proteins of wheat grains are prolamins. It consists of polymeric glutenins and monomeric gliadins. Under reducing conditions, polymeric glutenins are subdivided into high-molecular-weight glutenins (HMW-GS) and low-molecularweight glutenins (LMW-GS) according to their mobility by SDS-PAGE (Payne et al., 1987 & 1988). The composition of glutenin subunits in wheat is important for determining gluten and dough elasticity of wheat. There is a strong correlation between visco-elasticity and the relative amount of glutenin polymers with highest molecular masses (Payne et al., 1982; Thompson et al., 1983). HMW-GS coding genes in hexaploid bread wheat are located on three Glu-1 loci at the long arms of chromosomes 1A, 1B and 1D. Each locus is named as Glu-A1, Glu-B1 and GluD1, respectively, and contains two closely linked Glu-1-1 and Glu-1-2 genes encoding xand y-types, respectively. The gene products of Glu-1x and Glu-1y are distinguished from one another primarily on the basis of their size (Lawrence and Shepherd, 1981; Payne, 1987). A number of Glu-1 genes have been cloned, from bread wheat (Anderson and Greene, 1989; Anderson et al., 1989).


GENETIC TRANSFORMATION OF EGYPTIAN WHEAT WITH 1DX5 HIGH-MOLECULAR-WEIGHT GLUTENIN SUBUNIT GENE
A. H. FAHMY 1 , M. ABDALLAH 1 AND K. S. ABDALLA 2 1. Plant Genetic Transformation Dept., Agric. Genet. Eng. Res. Inst. (AGERI), ARC,Egypt 2. Plant Molecular Biology Dept.,Agric. Genet. Eng. Res. Inst. (AGERI), ARC, Egypt heat (Triticum aestivum L.) seedstorage proteins represent an important source of food and energy (Cooke and Law, 1998). The major endosperm storage proteins of wheat grains are prolamins. It consists of polymeric glutenins and monomeric gliadins. Under reducing conditions, polymeric glutenins are subdivided into high-molecular-weight glutenins (HMW-GS) and low-molecularweight glutenins (LMW-GS) according to their mobility by SDS-PAGE (Payne et al., 1987(Payne et al., & 1988. The composition of glutenin subunits in wheat is important for determining gluten and dough elasticity of wheat. There is a strong correlation between visco-elasticity and the relative amount of glutenin polymers with highest molecular masses (Payne et al., 1982;Thompson et al., 1983). HMW-GS coding genes in hexaploid bread wheat are located on three Glu-1 loci at the long arms of chromosomes 1A, 1B and 1D. Each locus is named as Glu-A1, Glu-B1 and GluD1, respectively, and contains two closely linked Glu-1-1 and Glu-1-2 genes encoding x-and y-types, respectively. The gene products of Glu-1x and Glu-1y are distinguished from one another primarily on the basis of their size (Lawrence and Shepherd, 1981;Payne, 1987). A number of Glu-1 genes have been cloned, from bread wheat (Anderson and Greene, 1989;Anderson et al., 1989).
Analyses of wheat cultivars have shown that HMW-GS differ in their impact on bread-making performance with subunits 1Ax1 and 1Ax2 and subunits 1Dx5 and 1Dy10 in particular being associated with high dough strength and good bread-making quality (Payne, 1987;Shewry et al., 2003). HMW-GS have been used to alter wheat grain quality by genetic transformation (Altpeter et al., 1996;Blechl and Anderson 1996;Barro et al., 1997;Fahmy et al., 2006). The HMW-GS 1Dx5 and 1Dy10 give better performances in dough strength and bread-making quality than the homeo-allelic subunits 1Dx2 and 1Dy12 (Payne et al., 1982;Popineau et al., 1994;Payne et al., 1987) which have relatively low-molecular-weights. Therefore, it is possible to improve the gluten quality of wheat by introducing novel copies of HMW-GS genes (Shewry, 1994). Many laboratories have reported the expression of HMW-GS transgenes (Altpeter et al., 1996;Blechl et al., 1997;Barro et al., 1997;Rooke et al., 1999). We have previously reported the transformation of maize, wheat and barley using W HMW-GS 1Dy10 (Abdallah et al., 2004;Fahmy et al., 2006;Abdalla et al., 2008).
Here we report our work on transgenic Egyptian wheat cultivar (Giza 164) expressing HMS-GS 1Dx5 gene using microprojectile bombarded with immature embryos.

Wheat transformation
Immature embryo-derived calli of the Egyptian wheat cultivar Giza 164 were co-transformed with plasmid pAHC25 (harboring gus and bar genes) and plasmid pK-Dx5 (BlueScript KS plasmid harboring HMW-GS 1DX5 gene driven by its own promoter). The transformation / regeneration procedure was done according to Fahmy et al. (2004 and2006).

DNA extraction
Genomic DNA was extracted from putative transgenic and control plants (non transgenic). DNA was isolated from leaf samples using DNeasy plant Mini Kit (QIAGEN, Germany).

Protein extraction and separation
Protein was extracted from putative transgenic and control plants. Wheat grains were ground to a fine powder and extracted using 0.25M Tris-HCl buffer (pH 6.8) containing 5% (v/v) βmercaptoethanol, 2% (w/v) SDS, 10% (v/v) glycerol and 0.02 (w/v) bromophenol blue. The extracts were heated at 100C for 2 min and centrifuged for 2 minutes at 15000 rpm. Protein sam-ples were separated by 10% (w/v) polyacrylamide gel electrophoresis in the presence of sodium-dodecyl-sulfate (SDS-PAGE) according to Laemmli buffer system (Laemmli, 1970). The gel was fixed in 5% trichloroacetic acid for 30 min and then stained in Coomassie brilliant blue R250 for 0.5-2 h. Gel was destained with distilled water, until clear protein bands were detected and then dried between sheets of cellophane (Promega, USA).

Leaf painting assay
Leaf painting assay was performed to verify the expression of bar gene. The bar gene activity in putative transgenic plants was assayed according to Schroeder et al. (1993). The upper surface of a leaflet was thoroughly wet by painting with an aqueous solution of herbicide Basta (Aventis GmbH, Germany) with a final Phosphinothricin (PPT) concentration of 0.2 mg/L and 0.1% Tween 20. Leaves were scored for herbicide damage seven days after application.

PCR analysis
Genomic PCR was carried out using Taq  The cycling conditions were: one cycle at 95C for 3 min; followed by 35 cycles of 94C for 30 sec, 62C for gus and 57C for bar at 30 sec, extension at 72C for 1 min and final extension step at 72C for 7 min. The PCR products were separated in 1.2% agarose gels.

RESULTS AND DISCUSSION
The introduction of foreign genes into wheat is a powerful tool for research and to improve elite wheat cultivars. The successful application of genetic modification depends on an effective transformation system, the availability of genes for target traits and the use of regulatory sequences capable of driving appropriate levels of expression in the tissues and developmental stages required.
This work is a part of collective study on wheat HMW-GS and its effect on bread making quality in Egypt. According to a previous study (Abdalla et al., 2011), we have tested 17 Egyptian wheat cultivars for the distribution of HMW-GS. Accordingly, we selected cultivar Giza 164 to be transformed with 1DX5 gene because its genome does not contain such gene.

Transformation
Immature embryo-derived calli (1200 calli) were co-transformed with plasmid pAHC25 and plasmid pK-Dx5 using biolistic bombardment. After selection with bilaphose during regeneration process, seven putative transgenic plants were produced in vitro. Plantlets were transferred to pots for acclimatization. The obtained Plants were subjected to molecular and biochemical analysis to confirm the integration of the transgenes in their genome and to study the expression of the inserted genes.

Molecular analysis
PCR analysis revealed successful integration of the exogenous genes (bar, gus and 1DX5) in the genome of seven putative transgenic plants. PCR amplifications using primers specific to bar (Fig.  1A), gus (Fig. 1B) and 1DX5 (Fig. 1C) genes yielded the expected products of 443 bp, 1050 bp and 529 bp, respectively, in putative transgenic plants (Lanes 3-9). Plasmids pAHC25 (Fig. 1A, 1B, lane 1) and pK-Dx5 (Fig. 1C, lane 1) were used as a positive control templates and they revealed PCR products of the expected sizes. No amplified products were detected in non-transformed control plants (Fig.  1, lane 2).
Expressions of marker genes were examined histochemically for GUS activity and by leaf painting assay for bar gene. The GUS expression of the transgenic wheat plants transformed with pAHC25 was confirmed by GUS staining of wheat spikelet tissues. As shown in Fig. (2), transgenic tissues exhibit GUS expression (blue color) clearly distinguishable from those of the control, indicating stable gus gene integration and expression into the genome of the plants.
The expression of bar gene was confirmed by leaf-painting assay (Lonsdale et al., 1998). Leaf area, putative transformants and control plants were painted using a solution of phosphinothricin (150 mg/L) and 0.1% Tween-20 and examined after seven days.
Absence of necrotic damage in putative transgenic plants as compared to controls was taken as evidence for the expression of bar gene (Fig. 3). Several reports showed successful integration and expression of bar gene after transformation us-ing leaf painting assay (Wan and Lemaux, 1994;Cho et al., 1998;Harwood et al., 2000).
After screening with PCR, the plants were subjected to analysis for the expression of HMW-GS 1DX5 gene in grains of T 0 plants by SDS-PAGE. As indicated in Fig. (4), the total protein extracts from seven putative transgenic plants showed that five of the transgenic lines (samples 3, 4, 5, 6 and 8) contained additional HMW subunit band of the expected mobility for 1Dx5. However, samples number 2 and 7 did not exhibit such band, even though, these samples were confirmed by PCR for the integration of 1Dx5 gene. Several authors reported silencing expression of integrated genes (Blechl et al., 1997;Alvarez et al., 2000).
Also, DNA methylation may affect the level of transcription of the integrated genes (Muller et al., 1996;Razin, 1988).
Our results are in agreement with there of Barro et al. (1997) who showed the expression of the 1Dx5 transgene. They were also able to introduce the 1Ax1 transgene alone, or with 1Dx5 and analyzing the mixing properties of the resulting dough which showed that expression of the transgenes were correlated with increasing dough strength.
In summary, the importance of introducing HMW-GS such as 1Ax1, 1Dx5 and 1Dy10 genes in Egyptian wheat cultivars can improve bread-making quality (Altpeter et al., 1996;Blechl and Anderson, 1996;Barro et al., 1997;Shimoni et al., 1997). Therefore, in this study we produced transgenic Egyptian wheat cultivar (Giza 164) with Dx5 gene. This might contribute to produce elite wheat cultivars with enhanced bread making quality traits.