Wheat is the most widely grown and consumed food crop of the world. Conventional breeding coupled with improved farm management practices led to a significant increase in wheat production. But such gains/increase cannot continue to cope with increasing food demands of rapidly growing world population that will cause 40% greater demand for wheat than the current level (Rosegrant et al., 1997) in the year 2020. So the world wheat production will have to increase 1.6% to 2.6% annually. This does not seem feasible with conventional plant breeding strategies that are restricted due to gene pool availability, species barrier and other biological limitations in addition to being a slow process. Application of recombinant DNA technology and its allied disciplines certainly hold a great promise to augment wheat production.

DNA technology made it possible to genetically alter the crops through Agrobacterium. However genetic manipulation of monocotyledons especially cereals including wheat was found difficult through Agrobacterium and alternate transformation methods were applied to transform the cereals. Wheat was the last among cereals to be genetically modified due to its inherent difficulties associated with gene delivery into regenerable explants and recovery of plantlets with introduced transgenes in addition to its larger genome size.

Successful wheat crop transformation involves the transfer of a gene into a suitable explant, integration and expression of transgene into the host genome and regeneration of the fertile transgenic plants from the transformed tissues. The efficiency of stable transformation is strongly dependent on genotype, explant source, medium composition and transformation method. Initially wheat transformation was obtained by direct gene transfer methods into protoplast from cultured embryonic cells (Lröz et al., 1985) with low transformation efficiency. Difficulties involved in establishing and maintaining long-term culture conditions accompanied with a little plant regeneration limited transformation of wheat.

1 Transformation methods
A transformation method or delivery system involves the use of several technologies and requires a vector that has the capacity to shuttle isolated genetic fragments into a viable host cell. Several methods are employed to deliver foreign gene into wheat genome.

1.1 Agrobacterium mediated transformation
Agrobacterium mediated transformation is the most widely used indirect method for wheat transformation. Agrobacterium tumefaciens, a soil dwelling bacteria that naturally infects dicots and causes tumorous growth resulting in crown gall disease. Tumor formation results from incorporation of T-DNA, a part of small independent DNA molecule outside the bacterial genome called Ti (tumor inducing) plasmid. Small phenolic compounds exuded from plant wounds stimulate expression of vir genes, located on Ti plasmid and responsible for its excision, transfer and integration into plant genome. The natural capability of Agrobacterium was manipulated in plant transformation by replacing the genes causing tumorous growth by genes of interest. However, Agrobacterium has a natural tendency to infect dicot plants and monocots were considered recalcitrant to Agrobacterium transformation. Therefore, most of the Agrobacterium mediated transformation procedures were established for dicots and monocots including economically important cereals lagged behind for a considerable time. Low frequency of T-DNA transfer into the target genome was the major limitation. Nonetheless, improvements of co-cultivation conditions, selection and regeneration methods for transformed tissues, in addition to incorporation of super binary vectors helped in extending the host range of Agrobacterium to several recalcitrant cereals.

Transformation of wheat had been attempted since 1988 but stable transformation through Agrobacterium became possible through a reliable and relatively efficient transformation procedure and construction of a new plasmid vector (Cheng et al., 1997) with stable integration, expression and inheritance of transgene to next generation. Further improvement in the plasmid vector and wheat transformation procedure increased the transformation efficiency. Several investigators obtained subsequent successful transformation of wheat through Agrobacterium with more than 4% transformation efficiency. Multiple factors are involved in Agrobacterium mediated transformation that determine the success or failure of gene transfer, stable integration and expression into plants. Agrobacterium strain, plasmid vector, selected tissue, duration of pre-culture, extent of time and conditions of inoculation and co-cultivation, presence of acetosyringone in the medium, etc. are some of the factors that can affect the success of transformation. Following infection with Agrobacterium the calli remain for a long time on the media containing selective chemicals and antibiotics reducing the recovery of transformed plantlets. Supplementation of polyamine spermidine improves regeneration from the transformed calli (Khanna and Daggard, 2003). Increasing Agrobacterium cell density and duration of inoculation/co-cultivation also increases expression of foreign gene in wheat (Amoah et al., 2001).

Agrobacterium mediated gene transfer has remarkable advantage over direct methods. These include low copy number of transgene leading to fewer problems of co-suppression and instability, no requirement of special equipment and technique, defined and preferential integration of alien gene into transcriptionally active regions of chromosome (Hiei et al., 1997). Agrobacterium system is reproducible, has higher transformation efficiency as compared with particle bombardment for wheat (Hu et al., 2003). Agrobacterium also has a good promise for in planta transformation of wheat apical meristem. The newly growing seedling may be cut to expose the apical meristem followed by injury and infection with Agrobacterium solution to transfer the gene of interest into germ line precursor cells. The selection for the putative transformants can be done at the inflorescence stage or progeny level.

1.2 Direct method
In the direct method there is no involvement of a biological vector or bacterial mediation for transfer of gene construct into explants. The gene construct is directly incorporated into plant cells/tissues. Variable efforts were made to introduce exogenous DNA into wheat in the last two decades. Initially attempts were made to deliver alien gene into wheat protoplast. Polyethylene glycol (PEG) mediated gene transfer was the first technique applied for genetic transformation of wheat. Transgenic fertile wheat plants were obtained using electroporation method with 0.40% efficiency (Sorokin et al., 2000). Use of silicon carbide fiber, microinjection and liposome fusion methods were also tried but no fertile transgenic plants were obtained. Production of transgenic wheat by pollen tube pathway method (Chong et al., 1998) seems very attractive but little success has been reported so far.

1.2.1 Bilolistic/microprojectile bombardment
Until 1988 there was no gene delivery procedure that could provide an alternative to the Agrobacterium transformation. Although the success of Agrobacterium vector was paramount, its inability to infect monocots and difficulties involved in protoplast culture lead to the invention of so called "biolistic" procedure, particle bombardment or gene gun. The main advantage of this method is to deliver DNA into intact regenerable plant cells eliminating the need for protoplast culture. This also minimized the abnormalities resulting from long-term protoplast cultures. Particle bombardment is a physical method of cell transformation in which high-density sub-cellular sized particles coated with DNA molecules are accelerated to high velocity in order to deliver DNA molecules to living cells. This method efficiently overcomes physical barriers and does not need long tissue culture and DNA can be introduced in all living tissues. Relatively higher number of transgenic plants can be obtained by this method. Despite its versatile nature, tissue damage and the relatively low yield of stable transformants (1%~5%), limited size of the DNA construct and inconsistency of bombardment replications are its limitations; consumable items are expensive and the method is sometimes laborious (http://ss.jircas.affrc.go.jp/engpage/jarq/32-4/hagio/hagio.htm)

The direct delivery of DNA by micro projectile is the most reliable and satisfactory method for the production of fertile transgenic wheat plants. There are two major requirements for efficient transformation; the efficient delivery of DNA-coated particles into larger number of target cells and high level of division and regeneration into seedlings. In most of the studies PDS-1000/He (BioRad) was used for delivery of transgene into primary explants or proliferating wheat callus. The cells are then induced to become embryogenic and regenerate. The influence of particle size and type, different procedures for coating DNA onto particles and distance of target tissue were studied in relation to the transformation efficiency. Vasil and others (Vasil et al., 1992) first time developed fertile transgenic wheat plants using particle bombardment. Efficiency of wheat transformation was commonly reported at around 1% (Alpeter et al., 1996). Instability of transgene after successful transformation was a major problem in addition to partial suppression of transgene. Optimum expression of genes in the target cells is important for achieving a high frequency of stable transformation. Considerable efforts were made to develop suitable gene expression vectors for transformation of wheat. The inclusion of intron between the promoters and coding region enhanced transient gene expression in wheat (Chibbar et al., 1991). Experiments were also conducted to optimize the culture conditions and DNA microprojectile delivery procedures for plant regeneration and gene expression in wheat calli. Osmotic treatment prior to bombardment, elimination of spermidine, replacement of CaCl₂ by Ca(NO₃)₂ and addition of thiosulfate enhanced the gus expression in the transformed calli. Development of improved protocol (Vasil et al., 1993) reduced the time to produce transformed plants. Need of establishing long term callus, cell suspension or protoplast culture was eliminated by introduction of a system for enhanced induction of somatic embryogenesis and regeneration of plants from isolated scutellum tissue of wheat (Becker et al., 1994). Modification in the particle bombardment and tissue culture procedures helped to increase the transformation efficiency up to 7% (Rasco-Gaunt et al., 2001; Zhang et al., 2000), facilitated quantitative production of multiple transgenic plants and significantly reduced the cost and labor. Low 2,4-D concentrations, increased sucrose content in callus induction and incorporation of silver nitrate in regeneration media were found to increase embryogenesis and shoot regeneration.

Efficient genetic transformation demands high regeneration capability of wheat tissues used for DNA delivery. Genotype is the primary factor for successful transformation. Several wheat genotypes were tested for callus induction and regeneration capability and a variety of explants were used in attempts to establish regenerable tissue cultures of wheat including caryopsis, mature/immature embryos, isolated scutellum, immature inflorescence, immature leaves, mesocotyl, apical meristem, coleoptile node and root. But culturing immature embryos proved more fruitful. Callus derived from mature embryos (http://www.biomedcentral.com) was also successfully transformed with a frequency of more than 8%. Fate of the transgene in wheat plants was also investigated (Mihaly et al., 2002). Transgenic wheat plants were crossed with non-transgenic ones and transgene inherited successfully to the next generation following Mendelian pattern.

Since the first successful wheat transformation several attempts have been made to transform different varieties of wheat using genes for various traits of common interest for instance bread and pasta making quality, starch characteristics, resistance against viruses, fungi, insects, herbicides, male sterility, heat, cold, aluminum tolerance, etc. Expression of low molecular weight glutenin (LMWG) subunits in the starchy endosperm (Tosi et al., 2004) and acceleration of flowering time by more than a month by insertion of RNAi (Yan et al., 2004) (RNA-interference) gene are recent advances in genetic improvement of wheat.

Improvement of wheat abiotic stress resistance through genetic transformation was also attempted. The plasmid containing aldolase reductase (ALR) gene, for osmotic stress resistance isolated from alfalfa was bombarded into wheat suspension culture (Pauk et al., 2002) and its protective function was verified under stress conditions. DREB1A (dehydration resistance element binding-protein 1A), a gene for drought resistance, was incorporated into callus induced from immature embryos of wheat (Pellegrineschi et al., 2002). The preliminary experiments revealed that the transformed plants survived a short and intensive water stress at the plantlet stage while the control plants were completely desiccated. Drought tolerant transgenic wheat has been sown in the field for screening (CBI, 2004, www.whybiotec.com)

2 Promoters
The promoter region preceding a marker gene is one of the most important factors affecting transformation frequency. The expression of inserted genes is limited to the activity of promoters. To date, the most of the promoters used in transgenic wheat, such as Adh1 (alcohol dehydrogenaseI), CaMV35S (cauliflo-wer mosaic virus 35S), Act1 (rice actin1), ubi1, (maize ubiquitine1), etc. are constitutive causing gene expression in all tissues throughout the plant life cycle. ScBV (sugarcane bacilliform badna virus) is another promising promoter for use in wheat transformation process (Tzafrir et al., 1998). Tissue specific and developmentally regulated promoters that allow expression of transgene only in specific tissues or under certain development conditions are also used in wheat transformation. Light regulated promoters such as LHCP (light harvesting chlorophyll- binding protein) and pathogen or wound induced promoters such as Vst1 (Vitis stilbene synthase1) from grapevine and RC24 (rice chitinase) in addition to stress inducible promoters like rd29a (dehydration-resistance 29a) are potential promoters to facilitate genetic transformation of wheat.

3 Genetic markers
Genetic transformation is comprised of delivery of gene cassette followed by analysis of gene expression in the transformants. As the event of transformation is very low so a suitable and efficient selection system is required to select these few transgenic cells. Therefore marker genes are included to identify the transformed tissues or cells. Genetic markers employed in genetic engineering are of two types, screenable/scorable markers (reporter genes) and selectable markers.

3.1 Scorable/screenable markers
Scorable markers encode gene products whose enzyme activity can be assayed easily allowing not only the detection of transformants but also an estimation of the level of foreign gene expression in the transgenic tissue. The most useful reporter genes encode an enzyme activity not found in the plant being studied. CAT (chloramphenical acetyltransferase) obtained from E. coli was initially used for study of introduced alien gene into wheat genome (Chibbar et al., 1991). However, difficulties associated with enzymatic analysis of this gene limited its use in wheat transformation. GUS (β-glucuronidase) a hydrolase that catalyses the cleavage of a wide variety of β-glucuronide compounds, also derived from E. coli, is the most popular and effective reporter extensively used as a scorable marker gene for transformation of wheat. Some visual marker genes such as GFP (green fluorescent protein), LUC (luciferase) and anthocyanin are also used in wheat transformation. GFP seems to have considerable promise for use in transformation of cereals. The LUC gene isolated from firefly requires ATP and luciferin substrate to produce light (Ow et al., 1986). The breakdown of luciferin results in the emission of light and does not harm the plant tissue. So the tissue incorporated with vector containing the gene of interest and LUC gene emit a yellow green glow indicating its transformed status. Another reporter gene commonly used in the transformation of wheat is anthocyanin regulatory gene e.g. R gene from Zea mays, that stimulates the endogenous anthocyanin biosynthesis in the transformed tissues/cells. Upon activation, this reporter system produces a reddish-purple pigment in transformed tissue.

3.2 Selectable Markers
Selectable markers are gene sequences that are intentionally introduced into cells as developmental tools to identify the cells those have successfully incorporated gene for a desired trait. The selectable markers are extremely important because they act as genetic tags to easily identify successfully transformed cells. However they have no actual function in the normal organism. There are three categories of selectable markers.

3.2.1 Antibiotic resistant markers
These are the selectable markers that confer resistance to antibiotics so the cells/tissues incorporated with these genes can be selected on the medium containing the antibiotic whereas the non-transformed cell will not survive thereby facilitating preferential selection of the transformed cells. The nptII and hpt are the commoly used markers for wheat. The nptII (neomycin phosphotransferaseII) initially isolated from Escherichia coli, is the most widely used antibiotic resistance marker. The gene encodes for the neomycin 3'-phosphotransferase that inactivates aminoglycoside antibiotics such as kanamycin, neomycin, geneticin (G418) and paromomycin by phosphorylation. It binds 30s ribosomal subunit and inhibits translation. The antibiotic paromomycin as a selection agent in combination with nptII gene proved very successful in recovering transgenic plants and hence nptII gene became the most favored selection marker for wheat transformation (Varshney and Alpeter, 2002).

The choice of the selective agent is very crucial and is based on the plant species to be transformed. Kanamycin is the most widely used antibiotic for plant transformation. It is very effective in inhibiting the growth of untransformed cells. However kanamycin is ineffective as a selection chemical for several legumes and plants of gramineae. It interferes with regeneration of transformed cells to green plants and wheat tissues exhibit a high level of endogenous tolerance to kenamycin. As an alternative, geneticin (G418), another member of aminoglycosides, can be used effectively for selecting nptII-transformed cells. 

The hpt (hygromycin phosphotransferase) gene also derived from Escherichia coli, encoding for hygromycin phosphotransferase detoxifies the aminocyclitol antibiotic hygromycin. It binds 30S ribosomal subunit and inhibits translation. A large number of plants have been transformed with the hpt gene and hygromycin as a selective agent has been proved very effective in the selection of transformed plants, including monocotyledons. Most plants exhibit higher sensitivity to hygromycin than to kanamycin, for instance cereals. So it is relatively better and efficient selection system for wheat (Ortiz et al., 1996).

3.2.2 Herbicide resistance markers
Genes conferring resistance to herbicides were used as an alternate to antibiotic resistance genes for wheat transformation. Herbicide resistance genes in fact provide a more effective system for plant transformation. The cell death in the presence of herbicide is generally more rapid and complete thus providing more efficient selection. It also provides a convenient and easily assayable system whereby the transgenic material can readily be identified/screened using simple techniques such as leaf painting. Genes conferring resistance to a number of herbicide groups including triazines, sulfonylureas, bromoxynil, glyphosate and phosphinot-
hricin are readily available.

There are three commonly used herbicide markers in plant transformation. The first one pat/ba, (phosphinothricin acetyltransferase) gene isolated from Streptomyces hygroscopicus has been widely used as an effective selectable marker in the presence of phosphinothricin (PPT) based formulations such as gluphosinate, bialaphos, basta, etc. by detoxifying these compounds for the selection of transformed tissues. It inhibits glutamine synthase. The enzyme inactivates phosphinothricin by the addition of an acetyl group from acetyl coenzyme A. This gene is freely available for research purposes and has proved particularly useful in cereals and grasses. PPT based selection has been the most common in cereals transformation (Kim et al., 1999). EPSPS (5-enolpyruvylshikimate, 3-phosphate synthase) oxidoreductase that inhibits aromatic acid biosynthesis is used as selectable marker with glyphosate as selection agent. GOX (glyphosate oxidoreductase) is another herbicide resistant marker that degrades glyphosate herbicides used in the transformation process of wheat (Zhou et al., 1995).

3.2.3 Metabolic/auxotrophic marker genes
This is a positive selection system that provides metabolic advantage to transformed cells and is environmentally safe. This kind of selectable markers enable transformed cells to synthesize an essential compound that the cells otherwise cannot synthesize. The medium is made intentionally to lack the essential component that is required for the cells to grow. So only successfully transformed cells with the selectable marker gene and the gene of interest will synthesize this compound and survive in the medium. The example of such markers is manA or pmi (mannose-6-phosphate isomerase). This is the most advanced positive selection system employed to date for wheat transformation (Negretto et al., 2000). Mannose is provided as a source of carbon and the medium lacks sucrose. So the transformed cells can use mannose for their carbon requirement whereas non-transformed cells will not survive. Cyanamide hydratase gene, also used for wheat transformation (Weeks, 2000), when inserted in the cells/tissues will make them able to grow on the medium containing cynamide whereas the non-transformed cells will die.

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