GENETIC TRANSFORMATION OF EGYPTIAN WHEAT (Triticum aestivum L.) WITH CHITINASE GENE VIA MICROPROJECTILE BOMBARDMENT

Plant diseases are caused by a variety of plant pathogens including fungi, and their management requires the use of techniques like transgenic technology and genetics. The chitinase gene, known to have a vital role in fungal disease defense, was introduced into the Egyptian wheat cv. Giza 164 via biolistic bombardment. Immature embryo derived calli were sub-cultured on CIMB medium containing bialaphos. After six weeks, the survived embryogenic calli were placed on MSRB regeneration selection medium. Finally, the green regenerated shoots produced were then transferred onto FMSB rooting selection medium. After the two weeks, healthy rooted plantlets were established under the rooting selection medium, and were then transferred into soil pots, then incubated in control growth chamber for acclimatization and subsequently trans-ferred to greenhouse until seed setting. In-tegration of the transgene with a transfor-mation frequency of 1.8% was confirmed by PCR and dot-blot analyses.

o introduce foreign genes, efficient protocols for embryogenic callus induction, transformation and selection and transgenic plants regeneration are considered necessary. In cereals, biolistic procedures have been used extensively in transformation (Cho et al., 2004). In wheat, poor tissue culture performance which limits the number of wheat genotypes that can be stably transformed has been the major hurdle (Varshney and Altpeter, 2002). Fahmy and El-Shihy (2006) developed an efficient protocol for regeneration of plants from long-term cultured immature embryo callus onto medium containing 2.0 mg/l 2, 4-D. Monostori et al. (2008) and Yu et al. (2008) suggested that regeneration system is an effective significant step in wheat genetic transformation. Several important reports have pointed to Murashige and Skoog's medium supplemented with 2,4-D as the ideal medium for embryogenic callus induction proliferation in wheat (Raja et al., 2008;Mahmood et al., 2009;Munazir et al., 2010) and that in comparing TDZ with other common growth regulators used in wheat regeneration. Moreover, it was suggested that TDZ was the best for in vitro regeneration (Fahmy et al., 2006;Chauhan et al., 2007;Biesaga et al., 2010). The last two decades witnessed the widespread use of biolistic bombardment for introduction of exogenous DNA into plant. The biolistic approach has been most successful in delivering foreign genes into wheat. Gene introduction into crop plants via genetic transformation is a better alternative to traditional breeding (Rashid et al., 2011). In the two past decades, the importance and steady use of gus gene as scrollable marker appeared as a necessary and many investigators accustomed to use in different co-bombardment procedures such as Yao et al. (2007), Fahmy et al. (2007, He et al. (2010) and Shi et al. (2011), since it produces a visible effect due to their activity in the transformed cells and the availability of a simple histochemical detection procedure. Also, the abundant utility of bar selectable gene in transformation experiments is not T less than gus scrollable gene. In the early trials, Chen et al. (1999) introduced chitinase gene (chi) into wheat cultivar Bobwhite with pAHG11 (harboring chi and bar) so as to use bar gene as a selectable marker, also, many researchers used the same approach such as Varshney and Altpeter (2002) and Wada et al. (2009). Transformation of a number of disease-resistance genes in transgenic plants has been reported. Chitin, an unbranched homopolymer of 1, 4-β-linked N-acetyl-D-glucosamine (GlcNAc), is widely distributed in nature. It is believed to be the second most abundant and renewable polymer on earth, next to cellulose. Chitinases are chitin degrading enzymes, and hydrolyze the β-(1, 4) linkages of chitin (Li 2006). The enzymes occur in a broad range of organisms including viruses, bacteria, fungi, insects, plant, and animals. Plant chitinases are participatory in defense and development (Graham and Sticklen, 1994). Moreover, chitinases have shown an immense potential application in agricultural, biological and environmental fields (Li, 2006). It has been proposed that chitinase transgene protein may function to provide fungal pathogen resistance at both direct and indirect levels. On the direct level it degrades chitin of growing hyphae, whereas, on the indirect level it results in the release of chitin oligomers which can act as elicitors of plant defense mechanisms (Collinge et al., 1993). Several authors indicated to the important role of chitinase gene in producing antifungal activity in plants, Furthermore, chitinase gene was introduced in numerous crops as a disease resistance gene as in rice by Nishizawa et al. (1999), Kim et al. (2003), Sridevi et al. (2003, Datta et al. (2004) and Nandakumar et al. (2007), and in sorghum by Krishnaveni et al. (2001), and also in barley by Tobias et al. (2007) and in sugarcane by Khamrit et al. (2012). Also, Shin et al. (2008) developed wheat cultivar cv. Bobwhite by biolistic bombardment of chitinase gene and produced seven transgenic plants. Recently, Huang et al. (2013) produced transgenic wheat developed by chitinase gene via particle bombardment of immature embryos and affirmed that polymerase chain reaction analysis indicated that chitinase gene was transferred into 17 transformants with transformation frequency of 1.8%. Introduction of disease resistance genes into wheat genome are considered necessary, since wheat production in Egypt is decreased drastically due to infection with wide range of fungal pathogens. This goal could be attained via gene transfer technology. The aim of the present investigation is to develop Egyptian wheat (Triticum aestivum L.) cultivar cv. Giza 164 with improved disease resistance by introduction of chitinase gene which has a role in plant defense via catalyzing the degradation of chitin, which is the major constituent of many fungal cell wall using modern biotechnology tools.

Sterilization and isolation of explants
Egyptian wheat cultivars Giza 164 seeds were obtained from the Department of Wheat, Field Crops Institute, Agricultural Research Center (ARC), Ministry of Agriculture and Land Reclamation, Egypt. Wheat spikes were collected from field grown plants approximately 10-12 days post anthesis. Immature caryopsis were removed from spikelets under aseptic condition in laminar flow hood (NUAIRE ™ , USA) where grains were surface sterilized with 20% (v/v) commercial Clorox® (5.25% Sodium hypochlorite) supplemented with few drops of Tween 20, followed by soaking for 2 min in sterile DD H 2 O, then rinsed five times in sterile DD H 2 O. Semi-transluscent immature embryos 1-1.25 mm in size were aseptically dissected under a stereo binocular microscope.

Culturing of explants
Immature embryos were cultured with scutellum side up onto callus induction medium (CIM) (Weeks et al., 1993) basically contains 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 incubated at dark for five days from culturing before bombardment in controlled growth chamber (Shel-Lab, USA) at 25C.

Biolistic gene transformation
After one week from culturing, twenty five immature embryo-derived calli were then transferred to CIMS osmotic medium (CIM medium supplemented with 0.4 M sorbitol) for four hours be-fore bombardment. Twenty five immature embryo-derived calli were placed in the center of a Petri dish (15x100 mm) and co-bombarded using the helium driven Biolistic Delivery system (Model PDS-1000/He, Bio-Rad) under a vacum of 25 in. Hg Vac with l.0 μ Golden microcarriers (BIO-RAD) coated with the two plasmids; the first plasmid is pAHC25 (10.0 kb), containing gus (uidA) (4.181 kb), (Jefferson et al., 1987) and bar (2.871 kb) cassettes (Thompson et al., 1987) both under control of Ubil promoter (Christensen et al., 1992) ( Fig. 1A) and the second plasmid is pAHC20Ubi383 containing chitinase gene, fragment containing the cauliflower mosaic virus transcription terminator (CaMV polyA) and the bar gene under the control of the maize ubiquitin promoter-intron (Fig. 1B). Target immature embryo-derived calli were bombarded at a distances of 6 cm from stopping screen with rupture disc strength of 1100 psi. After bombardment, calli were kept for additional 16 hr on the same osmotic medium (CIMS). Then, calli were transferred to CIM medium once more to recover for additional five days.

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

Selection and regeneration of transformed calli
After five days of recovery period, calli were then transferred to CIMB selection medium (CIM medium with 3 mg/l bialaphos) for two subcultures every three weeks. After the six weeks, the survived embryogenic calli were 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 growth conditions of 25C, 25-50 μl E/m 2 light intensity and 16 hr photoperiod. Finally, the green regenerated shoots produced were then transferred onto FMSB rooting selection medium (free Murashige and Skoog medium containing 1 mg/l bialaphos) for two weeks. Then, healthy rooted plantlets were established under the FMSB rooting selection medium, and were then transferred into soil pots, then incubated in controlled growth chamber (Conviron ® ) for acclimation and subsequently transferred to greenhouse until seed setting.

Leaf painting assay
In order to examine the expression of the bar gene in plants, 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 using a cotton plug swap according to Schroeder et al. (1993).

DNA isolation and analysis
Putative transgenic plants were subjected to molecular analysis to confirm the integration of the foreign genes (gus, bar and chi) into plant genome by PCR analysis. Total genomic DNA of putative transgenic plants and controls (nontransgenic) were isolated using DNeasy Plant Mini Kit (Qiagen, Germany).

Polymerase Chain Reaction (PCR)
DNA was subjected to PCR analysis for gus, bar and chi genes. Specific oligonucleotide primers for gus, bar and chi 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), 60C (for chi gene) and 2 min at 72C. One additional complete extension cycle was performed for 10 min at 72C. At the end of the cycles, 5 μ1 of the reaction products was mixed with 8 μ1 loading buffer and electrophoresed on 1% agarose gel at 80 Volt.

Dot blot hybridization analysis
Dot-Blot hybridization analysis was conducted to confirm the integration of chitinase gene in the transgenic plants. DNA from transgenic and non-transgenic plants (negative control) as well as the transformation vector pAHC20Ubi383 (positive control) were used in this experiment. DNAs were de-naturated by heating at 95C for 10 min., and then were directly spotted onto nitrocellulose membrane followed by a fixation step by exposure to UV waves for 1 min. The plasmid pAHC20Ubi383 was used as probe. Labeling and detection procedure was ac-complished using DNA Labeling and Biotin Chromogenic Detection Kit (Thermo Scientific) by random primed DNA labeling with digoxigenin-dUTP, alkali label and detection of hybrids by enzyme immunoassay according to the kit instruction manual.

RESULTS AND DISCUSSION
In our present research, immature embryo derived calli were co-transformed with the two previously mentioned plasmids pAHC25 and pAHC20Ubi383. Embryo-derived calli (625 calli) were subjected to the co-transformation process via microprojectile bombardment at the condition previously mentioned before. Following bombardment, histochemical staining analysis for bombarded callus was performed in a separate experiment to study the gus gene expression. Consequently, gus gene expression was photographed under the microscope (Fig. 3a). After a recovery period of five days from bombardment, calli were subsequently transferred to selection medium (CIMB medium) for two subcultures every three weeks. Table (2) and Fig. (2) demonstrated that the total number of survived callus achieved after this selection phase onto CIMB medium was 175 calli. After six weeks, the 175 calli survived on CIMB selection medium were then placed onto regeneration selection medium (MSRB) for two more weeks, where, 71 calli out of the 175 survived calli on CIMB selection medium regenerated shoots. Subsequently, regenerated shoots were then transferred onto FMSB rooting selection medium for two more weeks. After the two weeks, a total number of 111 healthy rooted plantlets were successfully established under the FMSB rooting selection medium, and were then transferred into soil pots for acclimatization in the control growth chamber (Conviron ® ). Subsequently, a total number of 55 plants succeeded in acclimatization and reached seed setting in biocontainment greenhouse. Thereafter, leaf painting assay was conducted to examine the expression of the bar gene in plants as previously mentioned (Fig. 3b). Putative transgenic plants were subjected to molecular analysis to verify the integration of the foreign genes (gus, bar and chi) into plant genome by PCR analysis. Total genomic DNA of putative transgenic plants and control (non-transgenic) were isolated using DNeasy Plant Mini Kit (Qiagen, Germany). DNA was then subjected to PCR analysis for gus, bar and chi genes. The PCR products were separated on 1% agarose gel. PCR results revealed products of the expected sizes for all transgenes; 523 bp for chi gene, 1050 bp for gus gene and 443 bp for bar gene as shown in Fig.  (4 A, B and C). Also, PCR analysis affirmed that total number of 15, 17 and 11 plant's transgene insertion were positive for gus, bar and chi gene, respectively. In addition, Dot-Blot hybridization analysis was used to confirm the integration of the chi gene. Data confirmed the integration of chi gene in all eleven positive PCR plants (Fig. 5). Results of PCR and Dotblot analysis pointed to that the frequency of chitinase gene transformation process scored 1.8% (Table 1). All produced plants were fertile and set seeds (Fig. 2), which indicate that transgenes insertion did not affect the fertility of plants or seed setting. Also, Nandakumar et al. (2007) transformed four rice genotypes of with chitinase, and its integration was confirmed through polymerase chain reaction (PCR) with transformation efficiency ranged from 0.9 to 5.2%. Lately, Huang et al. (2013) produced transgenic wheat developed by chitinase gene via particle bombardment of immature embryos and affirmed that polymerase chain reaction analysis indicated that chitinase gene was transferred into 17 transformants with a transformation efficiency of 1.8%.
Wheat production in Egypt decreased drastically due to infection with wide range of pests and pathogens (e.g., rusts, sheath blight, scab and insects). Our gene of interest chitinase is an antifungal protein as classified by Wani (2010). Chitinase, limit fungal growth by degrading the major structural polysaccharide of fungal cell walls (Leah et al., 1991). Therefore, production of transgenic Egyptian wheat plants with improved disease resistance is a precious component for a disease management program, in this respect; it is desirable to transform wheat with genes that are capable for conferring broad and durable fungal resistance. Here, we transformed the Egyptian wheat cultivar cv. Giza 164 with chi gene, which plays an important role in improving wheat fungal resistance via catalyzing the degradation of cell wall chitin of many fungi. Thus, the objectives of the present investigation directly is the development of Egyptian wheat with improved disease resistance via chitinase gene introduction, and indirectly is increasing wheat production, and therefore, decreasing equivalently wheat importation, and accordingly saving a huge percentage from the Egyptian foreign currency.

SUMMARY
Plant diseases are caused by a variety of plant pathogens including fungi, and their management requires the use of techniques like transgenic technology and genetics. The chitinase gene, known to have a vital role in fungal disease defense, was introduced into the Egyptian wheat cv. Giza 164 via biolistic bombardment. Immature embryo derived calli were subcultured on CIMB medium containing bialaphos. After six weeks, the survived embryogenic calli were placed on MSRB regeneration selection medium. Finally, the green regenerated shoots produced were then transferred onto FMSB rooting selection medium. After the two weeks, healthy rooted plantlets were established under the rooting selection medium, and were then transferred into soil pots, then incubated in control growth chamber for acclimatization and subsequently transferred to greenhouse until seed setting. Integration of the transgene with a transformation frequency of 1.8% was confirmed by PCR and dot-blot analyses.

This research was supported by
The US -Egypt Science and Technology (S&T) Joint, Contract/Agreement No.: 301. We sincerely thank Biochemistry Department, Kansas State University, for giving us opportunity to work at their Labs providing us with the inputs, facilities and motivation for this study.