TRANSGENIC EGYPTIAN WHEAT ( Triticum aestivum L . ) WITH Arabidopsis NPR 1 GENE via BIOLISTIC BOMBARDMENT

heat production needs to significantly increase to keep up with the growing demand (Bhalla, 2006). Unfortunately, this increase could not be achieved by plant breeding via conventional methods because of the limitation of available genetic pool. However, through genetic transformation technology this limitation can be overcome. Until date, the biolistic approach has been most successful in delivering foreign genes into wheat. Chawla et al. (1999) obtained transgenic wheat shoots following selection of cultures co-bombarded with a selectable herbicide resistance gene and gene of interest. Considerable progress has been made in the use of genetic engineering techniques for enhancing plant fungal disease resistance using this method. Chugh and Khurana (2003) bombarded two bread wheat (Triticum aestivum L.) varieties with pAHC25 (carries bar and gus genes). They recorded that transformation frequency of CPAN1676 variety was 4.4% while that for PBW343 was 3.6%. Fahmy et al. (2007) co-bombarded immature embryo-derived calli of wheat with two plasmids i.e. pK-Dy10 harboring Dy10 gene and pAHC25 plasmid containing gus and bar genes. They detected gus expression using a histochemical gus assay. Ding et al. (2009) pointed to that the gus gene could be used as a screen able marker gene, to assess the performance of DNA delivery in transformation of wheat and it was efficient and practical.

heat production needs to significantly increase to keep up with the growing demand (Bhalla, 2006).Unfortunately, this increase could not be achieved by plant breeding via conventional methods because of the limitation of available genetic pool.However, through genetic transformation technology this limitation can be overcome.Until date, the biolistic approach has been most successful in delivering foreign genes into wheat.Chawla et al. (1999) obtained transgenic wheat shoots following selection of cultures co-bombarded with a selectable herbicide resistance gene and gene of interest.Considerable progress has been made in the use of genetic engineering techniques for enhancing plant fungal disease resistance using this method.Chugh and Khurana (2003) bombarded two bread wheat (Triticum aestivum L.) varieties with pAHC25 (carries bar and gus genes).They recorded that transformation frequency of CPAN1676 variety was 4.4% while that for PBW343 was 3.6%.Fahmy et al. (2007) co-bombarded immature embryo-derived calli of wheat with two plasmids i.e. pK-Dy10 harboring Dy10 gene and pAHC25 plasmid containing gus and bar genes.They detected gus expression using a histochemical gus assay.Ding et al. (2009) pointed to that the gus gene could be used as a screen able marker gene, to assess the performance of DNA delivery in transformation of wheat and it was efficient and practical.
Plant pathogens are actual threats to worldwide agriculture.Significantly, fungal attacks cause unlimited yield losses for most of agricultural species.Fungi cause more than 70% of diseases in major crops (Agrios, 2005).Numerous of fungal pathogens that infect wheat plants are responsible for considerable yield loss.Chemical fungicide applications which are not economical and detrimental to the environment are generally used in management of fungal plant diseases.A method for protecting plants against diseases is constructing and employing pathogenresistant cultivars.Therefore, biotechnology integrated with traditional agricultural practices will be the backbone for W sustainable agriculture.Several classes of genes have been used in genetic engineering approaches to develop resistance in wheat to fungal pathogens utilized genes such as chitinase and Arabidopsis thaliana NPR1 genmimi.Exogenously applied jasmonic acid enhanced Arabidopsis thaliana resistance to many necrotrophic fungi (Thomma et al., 2000).Although JA and AtNPR1 cooperate, providing a clue to the activation of induced systemic resistance (ISR) (Koornneef and Pieterse, 2008).Arabidopsis engages SA signaling via AtNPR1 to govern diseases.Genetic engineering technology's future potential remains promising given the progress towards enhancing resistance for a wide range of pathogens in several plant species (Punja, 2006).Chern et al. (2001) transformed the Arabidopsis NPR1 gene in rice and this represents the first demonstration that Arabidopsis NPR1 gene can enhance disease resistance in a monocot plant.They stated that these results also suggest that monocot plants include a pathway controlling AtNPR1-mediated resistance.Chern et al. (2005) stated that Arabidopsis thaliana NPR1 gene is a key regulator of systemic acquired resistance (SAR), which confers lasting broad-spectrum resistance.Also, they confirmed that transformation of Arabidopsis NPR1 gene in rice resulted an enhanced resistance to Xanthomonas oryzae pv.Oryzae pathogen.Jiang et al. (2006) used optimized parameters for transforming into immature embryos of wheat cv.Yangmai 158 with genes for pathogenesis-related proteins and obtained forty-nine plants resistant to bialaphos.Two plants were identified to be positive by PCR.Makandar et al. (2006) showed that the Arabidopsis thaliana NPR1 gene (AtNPR1) regulates the activation of systemic acquired resistance (SAR), when expressed in the wheat cv.Bobwhite.Xing et al. (2008) transformed immature embryo-derived calli of common wheat via particle bombardment and after two rounds of bialaphos selection and regeneration; herbicide-resistant plants were obtained.Makandar et al. (2010) enhanced disease resistance in transgenic wheat and Arabidopsis plants that express the AtNPR1 gene.They declared that SA signaling through AtNPR1 is important for limiting the severity of diseases.On other hand, Fahmy et al. (2013) produced transgenic wheat developed using chi gene via particle bombardment of immature embryos and confirmed using polymerase chain reaction analysis that chi gene was transferred into 11 transformants with a transformation frequency of 1.8%.Recently, Silva et al. (2015) achieved genetic transformation in strawberry with the AtNPR1 gene and stated that the AtNPR1 gene confers broad-spectrum disease resistance when transformed.Introduction of disease resistance genes into wheat genome is considered necessary, especially when wheat production in Egypt is decreased severely due to infection with a wide range of fungal pathogens.This objective could be achieved via genetic transformation techniques.The goal of the current research is to improve Egyptian wheat (Triticum aestivum L.) cultivar cv.Giza 164 with improved disease resistance by introduction of AtNPR1 gene, which has a major role in plant defense using recent biotechnology tools.Here, we reported a reliable transformation of the Egyptian wheat cv.Giza 164 with AtNPR1 gene using genetic transformation via microprojectile bombardment.

Sterilization and isolation of explants
Grains of Egyptian wheat cultivar cv.Giza 164 were obtained from the Wheat Department, Field Crops Institute, Agricultural Research Center, Ministry of Agriculture and Land Reclamation, Egypt.Immature caryopses were removed from spikelets under aseptic conditions in a laminar flow hood.Grains were surface sterilized with 20% (v/v) commercial Clorox® (5.25% sodium hypochlorite) supplemented with few drops of Tween 20, and then rinsed five times with sterile double distilled H 2 O.After that, sterile immature embryos (1-1.25 mm) were aseptically dissected.

Culturing of explants
Immature embryos with scutellum side up were cultured on callus induction medium (CIM) (Weeks et al., 1993) containing Murashige and Skoog salts (Murashige and Skoog, 1962) supplemented with 0.15 g of L-Asparagine, 0.1 g of myo-inositol, 20 g sucrose, 2.5 g Phytagel as a solidifying agent and 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) as an auxin source.Then embryos were incubated in dark for five days before bombardment with gold particles coated with DNA.

Biolistic gene transformation
Twenty five immature embryoderived calli after one week from culturing were transferred to CIMS osmotic medium (CIM medium supplemented with 0.4 M sorbitol) for four hours before bombardment.Embryo-derived calli were placed in the center of a Petri dish (15x100 mm) and co-bombarded using the helium driven Biolistic Delivery system (Bio-Rad) with l.0 μ Golden microcarriers coated with the two plasmids; the first plasmid is pAHC25 containing the gus gene (uidA) and the bar gene that confers tolerance to phosphinothricin (ppt) both under control of the maize ubiquitin promoter (Fig. 1A), while the second plasmid pJS406 containing AtNPR1 gene (Fig. 1B).Target immature embryoderived calli were bombarded with rupture disc strength of 1100 psi.Calli were kept for additional 16 hr on the same osmotic medium (CIMS) after bombardment.Calli were then transferred to CIM medium for an additional five days of recovery.

Histochemical analysis
Histochemical staining analysis was conducted after seven days from bombardment to study gus gene expression according to Jefferson et al. (1987).Gus activity was assayed using callus tissues samples in gus buffer (Daniell et al., 1991).Then Petri dishes with calli were incubated at 37C overnight.Transient gus gene expression was photographed under the microscope.

Selection and regeneration of transformed calli
After the five days of recovery period calli were transferred to CIMB selection medium (CIM medium with 3 mg/l bialaphos) for two subcultures every three weeks.Survived embryogenic calli after the six weeks, were then placed on MSRB regeneration selection medium (Murashige and Skoog's medium supplemented with 3 mg/l bialaphos and 0.125 mg/1 TDZ) for two weeks at 25C and a 16 hr photoperiod.Green regenerated shoots were transferred for two more weeks onto FMSB rooting selection medium (Murashige and Skoog medium supplemented with 1 mg/l bialaphos).Finally, healthy rooted plantlets were transferred to soil pots in controlled growth chamber for acclimation and then transferred to greenhouse.

Leaf painting assay
A freshly prepared aqueous solution of 0.2% glufosinate-ammonium herbicide was applied on the mid-lamina portion (about 2.5 cm long) of the second/third youngest leaf according to Schroeder et al. (1993) for monitoring bar gene expression.

DNA isolation and Polymerase Chain Reaction (PCR)
Total genomic DNA of putative transgenic plants and controls (nontransgenic) were isolated using DNeasy Plant Mini Kit (Qiagen, Germany).Then, presence of transgenes (gus, bar and AtNPR1 genes) were detected, initially, by polymerase chain reaction (PCR).Specific oligonucleotide primers for gus, bar and AtNPR1 genes were used (Table 1).DNA amplifications were performed in a thermal cycler using initial denaturation at 94C for 5 min, followed by 30 cycles of 1 min at 94C, 1 min at 58C (for gus gene)/62C (for bar gene)/57C (for AtNPR1 gene) and 2 min at 72C.One additional complete extension cycle was performed for 10 min at 72C.

Dot blot hybridization analysis
To confirm AtNPR1 gene integration in the genomic DNA of transgenic plants, Dot-Blot hybridization analysis was conducted.DNA from transgenic and non-transgenic plants (negative control) as well as the transformation vector pJS406 (positive control) were used in this experiment.DNAs were denatured by heating at 95C for 10 min., and then directly spotted onto nitrocellulose membrane followed by a fixation step by exposure to UV waves for 1 min.The plasmid pJS406 was used as probe.Labeling and detection procedure was accomplished using DNA Labeling and Biotin Chromogenic Detection Kit (Thermo Scientific) by random primed DNA labeling with digoxigenin-dUTP and detection of hybrids by enzyme immunoassay according to the kit instructions manual.

RESULTS AND DISCUSSION
Fungal and bacterial diseases are serious and destructive diseases of wheat.
In severe epidemic years in Egypt, diseases may reach 50-100% incidence with yield reduction from 10-40%.Also, contamination of harvested grain by toxins, reduce the grain value for food or feed consumption.Genetic variation for fungal and bacterial resistance is low in wheat germplasm pools and inadequate to provide resistance through traditional breeding approaches.Therefore, genetic engineering will provide an additional approach to enhance disease resistance.The AtNPR1 gene plays a crucial role in systemic acquired resistance in plants.
Here, we report a successfully genetic transformation of the AtNPR1 gene into the Egyptian wheat cultivar cv.Giza 164.In this experiment, plasmids pAHC25 and pJS406 were co-transformed into immature embryo derived calli which was used as a target tissue.A total of 625 calli were subjected to microprojectile bombardment at the same conditions as described for the transformation process.
In a separate experiment, histochemical staining analysis of bombarded calli was achieved subsequent to the bombardment in order to study the gus gene expression which was monitored using a microscope and photographed (Fig. 2).
Following a recovery period of five days following bombardment, calli were sub-cultured twice at three weeks intervals onto selection medium (CIMB medium, Fig. 3).The total number of calli that survived after selection phase onto CIMB medium was 161 (Table 2 and Fig. 4).
The surviving calli (161) on CIMB selection medium were sub-cultured for more two weeks on regeneration selection medium (MSRB).Out of the 161 surviving calli, only 63 calli regenerated shoots on CIMB selection medium.Regenerated shoots were then transferred to FMSB rooting selection medium and maintained for two more weeks.Healthy rooted plantlets numbering 105 plantlets were established in the FMSB rooting selection medium, and were then transferred into soil pots and transferred to the controlled growth chamber (Conviron ® ) for acclimatization (Table 2 and Fig. 4 ).
During the acclimatization process, a total number of 48 plants survived and set seeds in a bio-containment greenhouse (Fig. 5B).All produced plants were fertile and set seeds which indicated that the insertion of the transgenes did not affect the fertility of plants or seed setting.Leaf painting assay was conducted to examine the expression of the bar gene in these plants as mentioned before (Table 2 and Fig. 6).
Putative transgenic plants were subjected to molecular analysis to verify the integration of foreign genes (gus, bar and AtNPR1) into plant genome by the PCR analysis and Dot-Blot hybridization analysis.Total genomic DNA of putative transgenic plants and control (nontransgenic) were isolated using DNeasy Plant Mini Kit (Qiagen, Germany).The DNA was then subjected to the PCR analysis for gus, bar and AtNPR1 genes using appropriate primer pairs for each gene.
PCR products were then separated on 1% agarose gel.PCR results revealed products of the expected sizes for all transgenes; 1050 bp for gus gene, 443 bp for bar gene and 439 bp for AtNPR1 gene, as shown in Fig. (7).PCR analysis confirmed that 12, 14 and 12 plant's transgene insertion were positive for gus, bar and AtNPR1 genes, respectively (Table 3).
To confirm the integration of the AtNPR1 gene, Dot-Blot hybridization analysis was used and confirmed the integration of the AtNPR1 gene in all the twelve positive PCR plants (Fig. 8).PCR and Dot-blot analysis results indicated that the frequency gene transformation process of AtNPR1 gene was 1.9%, while transformation frequency of both gus and bar genes were 1.9% and 2.2%, respectively (Table 2 and Fig. 8).Similar results were obtained by Chern et al. (2001) whom transformed rice plants with AtNPR1 gene and confirmed AtNPR1 gene integration using polymerase chain reaction (PCR) and Dot-Blot hybridization.Also, Makandar et al. (2006) produced AtNPR1 transgenic wheat plants by cotransforming embryogenic calli with plasmid pJS406 expressing AtNPR1 gene and plasmid pAHC20 that expresses the bar gene via particle bombardment and utilized PCR to monitor the presence of AtNPR1 and bar genes.
AtNPR1 gene is a key regulator of systemic acquired resistance (SAR) and can enhance disease resistance in plants such as Arabidopsis and monocot plants (Friedrich et al., 2001;Chern et al., 2005).
AtNPR1 gene confers lasting broadspectrum disease resistance to viral, bacterial and fungal pathogens (Chern et al., 2001).Also, AtNPR1 gene is as a positive regulator of the salicylic acid (SA).Salicylic acid signaling through AtNPR1 gene is important for limiting severity of fungal disease (Loake and Grant, 2007;Bari and Jones, 2009).In wheat, recently, it was proved that AtNPR1 is involved in SA signaling and that it plays a key role in both SAR signaling and disease resistance (Makandar et al., 2012).
AtNPR1 gene promotes the crosstalk between the SA signaling pathway and the jasmonic acid (JA) signaling pathway.Moreover, the antagonistic effect of SA on JA signaling requires AtNPR1 (Spoel et al., 2003 and2007;Liu et al., 2005).This is supported by the work of Makander et al. (2010) who reported that JA signaling promotes disease by attenuating the activation of SA signaling.Jasmonic acid (JA) also, contributes to defense presumably during the later stages of infection.Together SA and JAdependent defenses could provide maximal protection against fungal disease.
Here we report for the first time, transformation of AtNPR1 gene in our Egyptian cultivar cv.Giza 164.The objective of this study is the developing of wheat cultivar cv.Giza 164 in plant defenses using tools of modern biotechnology via transformation with the AtNPR1 gene, which will be a valuable alternative to the exogenous application of SARinducing chemicals, and furthermore will play a significant and a superior role in improving Egyptian wheat broadspectrum disease resistance, including fungal, bacterial and viral resistance.Since, transgenic improved disease resistance Egyptian wheat plants production is a valuable component in a disease management program; it is desirable to transform wheat with genes that are competent for conferring broad and durable fungal resistance even if this resistance is partial.
In conclusion, genetic transformation of Egyptian wheat cv.Giza 164 with AtNPR1 gene paves the way for further studies on gene expression and for achieving both biotic and abiotic stress tolerance.Moreover, leading to increased wheat production, and also filling the huge gap between consumption and production of wheat in Egypt.

SUMMARY
AtNPR1 gene plays a crucial role in biotic resistance in plants.Immature embryo derived calli of wheat cv.Giza 164 were co-transformed with the plasmid pAHC25 containing bar and gus genes and the plasmid pJS406 containing AtNPR1 gene via Biolistic bombardment.The produced calli were subcultured on bialaphos containing medium (CIMB).Surviving calli were then regenerated on selection medium (MSRB) to produce green regenerated shoots, which were then transferred onto rooting/selection medium (FMSB).Regenerated rooted plantlets were transferred to soil pots in the control growth chamber for acclimatization.Only 48 plants succeeded in acclimatization and reached seed setting in biocontainment greenhouse.Transgene integration was confirmed by PCR and dot-blot analyses.The overall transformation frequency was 1.9%.

Fig. ( 6
Fig. (6): Leaf painting assay examining the bar gene expression in transgenic AtNPR1 plants: the upper leaf from untransformed plant (control) showing leaf necrosis.The lower leaf from transgenic plant shows leaf resistance to 0.2% glufosinate-ammonium herbicide application.

Table ( 3
): Transformation characteristics of Egyptian wheat cultivar Giza 164 representing Gus, bar and AtNPR1 genes positive PCR plants and AtNPR1 gene Dot Blot positive plants.