Paramjit Khurana*
Centre for Plant Molecular Biology
University of Delhi South Campus
New Delhi-110 021, India
Tel: 91-11-4675096
Fax: 91-11-6885270
E-mail: paramjitkhurana@hotmail.com

* Corresponding author

Financial support: Department of Biotechnology, Government of India.

Keywords: biotechnology, transgenic wheat, wheat improvement, wheat transformation.

Due to the inherent difficulties associated with gene delivery into regenerable explants and recovery of plantlets with the introduced transgene, wheat was the last among cereals to be genetically transformed. This review attempts to summarize different efforts in the direction of achieving genetic transformation of wheat by various methods. Particle bombardment is the most widely employed procedure for the introduction of marker genes and also for the generation of transformed wheat with introduction of agronomically important genes for quality improvement, engineering of nuclear male sterility, transposon tagging, resistance to drought stress, resistance against fungal pathogens and insect resistance. The other methods of choice of gene delivery into wheat tissues include electroporation and co-cultivation with Agrobacterium. Several alternative approaches including microinjection, direct imbibition, permeabilization, silicon carbide fiber-mediated and pollen tube pathway have also been attempted for introduction of foreign DNA with varied degrees of success. In future, use of marker assisted selection and genomics approaches will increase the effectiveness and efficiency of wheat breeding programs, and will also provide insights into genetic control of key traits to be used for genetic manipulation.

Wheat is a member of the family Poaceae which includes major cereal crops of the world such as maize, wheat, and rice. Among the food crops, wheat is one of the most abundant sources of energy and proteins for the world population. Ninety-five percent of wheat grown today is of the hexaploid type, used for the preparation of bread and other baked products. Nearly all of the remaining 5% is durum (tetraploid) wheat, which is mainly used for making pasta, macaroni and biscuits. Wheat is characterised by a large genome size (approximately 17000 Mb), thus making the improvement process by any method genetically challenging.

Modern bread wheat is a true breeding hybrid with its ancestry linked to three wild grass species still growing in the Middle East. Bread wheat first originated in the 'fertile crescent', an area in the middle East, which stretches from Israel and Lebanon into Syria, Turkey, Iraq and Iran. Diploid einkom types of wheat are the earliest and the most primitive, while the hexaploids including the bread wheat, Triticum aestivum, constitute the most recent and latest step in the evolution of the wheat complex. Millions of years ago, the first hybridisation event is thought to have occurred when the wild grass Aegilops speltoides crossed with the wild diploid wheat, Triticum monococcum. The resultant hybrid was the tetraploid emmer wheat, Triticum dicoccum. Domestication of emmer wheat lead to the evolution of the durum wheat. Hybridisation of tetraploid durum wheat, Triticum turgidum var. durum (2n=28, AABB) with the diploid wild goat grass, Aegilops tauschii, led to the origin and evolution of hexaploid wheat about 8000 years ago. Bread wheat is thus an allohexaploid, containing three distinct but genetically related (homoeologous) copies each of the three originally independent haploid genomes, the A, B and D (Gill and Gill, 1994). With the evolution of wheat, civilizations thrived and spread from the Middle East to new continents, resulting in massive wheat production feeding the growing world population. The tribe Triticeae comprising of over 300 species including wheat, rye and barley is considered as germplasm source for wheat improvement by classical breeding approaches.

Scientific approaches to crop improvement have their history in the rediscovery of Mendel's law at the beginning of this century. Consistently since then, breeders have been searching for technologies and converting this 'art' of breeding to a 'science'. Wheat breeders have able to introduce desirable traits that increased the grain yield and minimize the crop loss. However, conventional breeding techniques which are based on processes of crossing, back crossing and selection, proved to be time consuming and, therefore, could hardly keep pace with the rapid co-evolution of pathogenic micro-organisms and pests. The development of in vitro technologies have thus complemented the conventional methods of wheat breeding in generating genetic variability necessary for creating novel cultivars with desirable characters.

In recent years biotechnology is emerging as one of the latest tools of agricultural research. In concert with traditional plant breeding practices, biotechnology is contributing towards the development of novel methods to genetically alter and control plant development, plant performance and plant products. The term biotechnology is composed of two words bio (Greek bios, means life) and technology (Greek technologia, means systematic treatment). Biotechnology involves the systematic application of biological processes for the beneficial use. One of the areas of plant biotechnology involves the delivery, integration and expression of defined genes into plant cells, which can be grown in artificial culture media to regenerate plants. Thus biotechnological approaches have the potential to complement conventional methods of breeding by reducing the time taken to produce cultivars with improved characteristics. Conventional breeding utilizes domestic crop cultivars and related genera as a source of genes for improvement of existing cultivars, and this process involves the transfer of a set of genes from the donor to the recipient. In contrast, biotechnological approaches can transfer defined genes from any organism, thereby increase the gene pool available for improvement. The improvement of wheat by biotechnological approaches primarily involves introduction of exogenous genes in a heritable manner, and secondarily, the availability of genes that confer positive traits when genetically transferred into wheat.

The genetic improvement of wheat has received considerable attention over the years from plant breeders with the purpose of increasing the grain yield and to minimize crop loss due to unfavourable environmental conditions, and attack by various pests and pathogens. In the early 60's, conventional breeding coupled with improved farm management practices led to a significant increase in world wheat production thereby ushering in the green revolution. Subsequently, the targets of genetic improvement shifted to reducing yield variability caused by various biotic and abiotic stresses and increasing the input-use efficiency (Pingali and Rajaram, 1999). With this change in the global food policy in the last few decades, biotechnology offered a possible solution firstly, by lowering the farm level production costs by making plants resistant to various abiotic and biotic stresses, and secondly, by enhancing the product quality (i.e. by increasing the appearance of end product, nutritional content or processing or storage characteristics). The introduction of foreign genes encoding for useful agronomic traits into commercial cultivars has resulted in saving precious time required for introgression of the desired trait from the wild relatives by conventional practices and alleviating the degradation of the environment due to the use of hazardous biocides. In recent years, wheat improvement efforts have therefore focussed on raising the yield potential, quality characteristics, resistance to biotic stresses and tolerance to abiotic stresses depending on the regional requirement of the crop.

Approaches for wheat transformation

The last two decades have witnessed the widespread use of varied approaches for introduction of exogenous DNA into wheat. Wheat improvement by genetic engineering requires the delivery, integration and expression of defined foreign genes into suitable regenerable explants. Initial attempts at introducing transgenes into wheat employed protoplasts as explants due to the absence of cell walls. The introduction of marker gene constructs into protoplasts provided valuable information regarding the expression pattern and tissue specificity of various promoters and regulatory elements in the transformed tissue. However, the difficulties associated with plantlet regeneration from protoplasts have compelled researchers to look for alternate target cells/tissues with better regeneration capabilities. Therefore, attention shifted to embryogenic suspension cells and embryogenic callus cultures derived from scutellar tissue of mature and immature embryos. A detailed and in depth account of the in vitro culture response of wheat from different explants is available in numerous reviews and so has not been dealt with in detail here (Bajaj, 1990; Maheshwari et al. 1995; Vasil and Vasil, 1999). In recent years, with the development of suitable regeneration protocols, microspore embryos and immature inflorescences are emerging as suitable target tissues for genetic transformation experiments. Till date, the biolistics approach has been most successful in delivering foreign genes into wheat.

Scorable markers

Initial steps for genetic transformation involves delivery of a gene cassette into recipient cells followed by analysis of the expression of delivered gene. The results of the above events can be detected by assaying the expression of a reporter gene introduced into plant cell cultures or intact tissues. The reporter genes produce a visible effect, directly or indirectly, due to their activity in the transformed cells. Analysis of reporter gene expression does not require the integration of the transgene into the host genome and is commonly used to test promoter and gene functions. Initial studies on the introduction of foreign DNA to wheat have relied on the use of the E. coli gene for the enzyme chloramphenicol acetyl transferase (cat) (Hauptmann et al. 1988; Chibbar et al. 1991). However, the detection of transgene by enzymatic and immunochemical methods for measurement of CAT activity and amount of CAT protein, respectively, was tedious; and moreover, presence of inhibitors of CAT activity and endogenous CAT activity (Chibbar et al. 1991) hampered its use as a reporter gene in wheat. With the availability of a protocol for the rapid assay of b -glucuronidase (gus) gene from E. coli, this gene has emerged as the most widely used scorable marker in wheat transformation (Vasil et al. 1992; Vasil et al. 1993; Weeks et al. 1993; Becker et al. 1994). GUS enzyme hydrolyzes b glucuronide compounds and gives reaction products that can be quantified spectrophotometric or spectrofluorometrically (Jefferson et al. 1987). The gus reporter gene system is extremely useful for optimisation of parameters for genetic transformation, due to the availability of a simple histochemical detection procedure. One of the major limitations of gus reporter gene system, however, is the destructive nature of its assay. Thus, to study the fate of introduced transgenes in living cells, vital reporter genes encoding for anthocyanin biosynthesis, green fluorescent protein, and firefly luciferase have been used successfully. The R genes of Zea mays which stimulate endogenous anthocyanin accumulation in the vacuoles of plant tissues is useful as a scorable marker in mature and differentiated cells. Due to high sensitivity and ease of visualization, the use of R genes in wheat transformation have been reported by different groups (Kloti et al. 1993; Dhir et al. 1994; McCormac et al. 1998; Chawla et al. 1999a; Chawla et al. 1999b). A synthetic, spectrally modified, version of green fluorescent protein (gfp) from the jellyfish, Aqueorea victoria has also been used as a vital marker in wheat transformation (Pang et al. 1996; McCormac et al. 1998). Recently Jordan (2000) reported the use of a modified gfp as a visible marker for the detection of transgenic wheat plants on the basis of gfp expression alone. Amongst the other scorable markers, luciferase gene from the fire fly, Photinus pyranus, has been successfully used in stable transformation of wheat (Lonsdale et al. 1998; Harvey et al. 1999). Luciferase and modified versions of gfp permit non-destructive analysis of transgene activity, ease to follow the fate of introduced transgenes in individual tissue samples, facilitate rapid assessment and comparison of different transformation procedures, and provide valuable insight into the conditions influencing the efficiency of DNA integration and stable expression.

Selectable markers

The varied frequency of DNA delivery in cells of different explants has necessitated the development of methods for efficient selection of cells that carry and express the introduced gene sequences. The selection regimes for transformed cells are based on the expression of a gene termed as the selectable marker producing an enzyme that confers resistance to a cytotoxic substance often an antibiotic or a herbicide. The most commonly used selection marker in wheat transformation is the bar gene (bialaphos resistance gene) encoding for phosphinothricin acetyl transferase (pat). Both bar and pat genes isolated from different Streptomyces species, encode for phosphinothricin acetyl transferase. Amongst the antibiotic resistance markers, the bacterial neomycin phosphotransferase II (nptII) gene providing resistance to aminoglycoside antibiotics is commonly used in wheat transformation. Herbicide resistance genes offer an alternative to antibiotic-resistant markers. Genes coding for 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS), a critical enzyme for aromatic amino acid biosynthesis, and phosphinothricin acetyl transferase (PAT) provide tolerance to glyphosate and glufosinate ammonium herbicides, respectively. The enolpyruvylshikimate-phosphate synthase (CP4) gene isolated from Agrobacterium strain CP4 and the glyphosate oxidoreductase (GOX) also provide tolerance to glyphosate by degrading glyphosate into aminomethyl phosphoric acid, and have been added to the list of selectable markers available for wheat (Zhou et al. 1995). The hygromycin phosphotransferase (hpt) gene, widely used in rice transformation, has also been reported as an efficient selectable marker for achieving stable genetic transformation in wheat (Ortiz et al. 1996). The efficiency of hpt as a selectable marker is comparable to the other widely used selectable markers encoding resistance for antibiotics and herbicides (Table 1). Recently mannose-6-phosphate isomerase (MPI), encoded by manA from E. coli, has been developed as a selectable marker for wheat. This marker allows transformants to grow on mannose as a sole carbon source and has a positive mode of action which encourages the growth of the transformed tissues rather than just permitting it (Hansen and Wright, 1999). Recently, Weeks (2000) reported the use of cyanamide hydratase (Cah) gene as a selectable marker for wheat transformation. The Cah gene gives the transformed tissues the ability to grow on cyanamide-containing media by converting cyanamide into urea (which can be used as a fertilizer source).

Transformation of wheat protoplasts

Protoplasts are regarded as competent targets for gene transfer due to the absence of cell walls. During the early 80's protoplasts were the chosen explants for wheat transformation and their use as explants for direct gene transfer was facilitated by the development of suitable techniques for isolation of protoplasts from different tissues. However, due to limited success with culturing protoplasts from organized tissues (Mahalakshmi et al. 1993), cell suspension/calli derived protoplasts have been used in wheat. To overcome the hindrance exercised by the plasma membrane, protoplasts are subjected to either chemical treatment [polyethylene glycol (PEG)] or subjected to physical forces like electric pulses (electroporation), either alone or in combination with PEG, for the introduction of foreign DNA. For wheat, isolation of protoplasts from embryogenic callus cultures have been the most popular tissues of choice.

The first report of direct gene transfer in wheat protoplasts was by Lorz et al. (1985) from cultured cells Triticum monococcum by PEG-mediated uptake. The transformed cells were selected on a kanamycin-containing medium and identified by detection of nptII activity. Subsequently, Werr and Lorz (1986) reported the expression of the introduced genes but the majority of foreign DNA introduced was found to remain extra-chromosomal and transient gene activity was found to be dependent on the presence of the promoter fragment of the Shrunken gene of maize. This observation also indicated for the first time, the efficacy of a cereal promoter. Lee et al. (1989) introduced chimeric gene constructs having the cat reporter gene under the control of CaMV 35S promoter in aleurone protoplasts isolated from developing caryopses. The resultant gene activity detected in protoplasts from developing aleurone layers raised the possibility of investigating tissue- and development-specific control of genes for cereal seed proteins. By and large, the physiological age of the developing grain and the physiological state of the isolated protoplasts affected the level of transient gene expression. the significant factors affecting the transient activity of the reporter gene constructs were the presence of the divalent cation Mg²⁺ with PEG, time lapse after DNA uptake, pre-culture medium and the regulatory elements of the vector construct. Nonetheless, these reports yielded low transformation efficiencies in the order of 0.005% and the low regenerating ability of the explant hampered work in this area.

Introduction of the E. coli plasmid, pCGN1055, containing the hpt gene into wheat protoplasts, by cationic liposome-mediated transformation resulted in the production of transgenic albino plantlets. The presence and activity of the transgenes was confirmed by assay of enzymatic activity and southern hybridisation (Zhu et al. 1993) and the transformation efficiencies reported were in the order of 6%.

Electroporation of protoplasts

Electroporation is a technique that utilizes a high intensity electric pulse to create transient pores in the cell membrane thereby facilitating the uptake of macromolecules like DNA. Ou-Lee et al. (1986) reported the expression of the bacterial chloramphenicol acetyl transferase (cat) gene in three important graminaceous plants, i.e. rice, sorghum and wheat. The survival percentage of protoplasts after electroporation depended largely on the tissue of origin. The transient activity of cat gene was equal when it was fused to either CaMV 35S promoter or the copia long terminal repeat promoter of drosophila. This study also demonstrated, interestingly, that the Drosophila promoter (copia) was capable of directing the synthesis of functional CAT enzyme in plant cells as efficiently as one of the strongest constitutive promoters known to function in plant cells. Besides the promoter influencing the foreign gene expression levels, presence of an intron was also found to be helpful as shown by Oard et al. (1989) with a maize alcohol dehydrogenase (Adh) intron in chimeric constructs. This was evidenced by ribonuclease protection assays that demonstrated increased mRNA levels for the intron-containing constructs versus those without the intron. This report highlighted the use of regulatory elements for enhancing the transgene expression. Zaghmout and Trolinder (1993) optimised other conditions for a significant increase in gus activity which included the protoplast source, the promoter, the PEG concentration, as well as, electroporation parameters especially the electric field strength, preincubation with the plasmid, and recovery period on ice after the electric pulse.

Stable transformation by electroporation (Zhou et al. 1993) was reported by the use of the plasmid pBARGUS into protoplasts isolated from cell suspension initiated from an anther-derived callus. This was the first report of stable transformation of wheat protoplasts and also confirmed by Southern hybridization. Stable transformation of protoplasts was also reported by He et al. (1994) by electroporation employing the bar gene as the selectable marker, which also facilitated the selection of phosphinothricin-resistant colonies. Southern analysis and PAT assay revealed the presence of the bar gene and its product, respectively, in the regenerated colonies and plants. He et al. (1994) obtained transient expression of gus gene at a frequency of 1x 10^-5 which was more as compared to that observed by Lee et al. (1989). Electroporation of protoplasts thus considerably improved the gene transfer efficiency as compared to that achieved with PEG mediated approach.

Electroporation of organized tissues

The technique of electroporation, initially utilized for the introduction of foreign genes into protoplasts, was later extended for the transformation of organized tissues. In wheat, Kloti et al. (1993) reported the transfer of reporter genes by elecroporating zygotic wheat embryos. The embryos were arranged with the scutella side facing the negative electrode, so that the negatively charged plasmid DNA will contact the target cells while moving towards the positive electrode. Transient expression of the anthocyanin regulatory gene and the gus gene facilitated the identification of transformed scutellum cells. Introduction of gus gene was also reported by electroporating organized tissue from slow-growing, embryogenic calli (Zaghmout, 1994). A detailed analysis of the various parameters affecting DNA delivery was undertaken by He and Lazzeri, (1998). This study identified and optimised the conditions for tissue electroporation of scutellum cells. The factors which played a significant role in affecting the electroporation efficiency were the electroporation voltage, the pulse length, volume of the electroporation buffer, osmoticum of the electroporation buffer, and medium, the osmoticum of the pre-electroporation medium and the pre-electroporation incubation time and temperature. Recently, the production of fertile transgenic wheat plants by this method from intact immature embryos was reported by Sorokin et al. (2000) at a transformation frequency of 0.4%. Molecular evidence confirmed the stable integration and the inheritance of the gus and bar transgenes in the T₀ and T₁ plants. The electroporation efficiency was found to be dependent on a combination of factors including pretreatments of the recipient tissue and the culture conditions. It thus appears that tissue electroporation may emerge as a routine transformation method for wheat due to its technical simplicity and low cost associated with this procedure.

Particle bombardment

The development of methodologies for the delivery of genes into intact plant tissues by particle bombardment has, in fact, revolutionized the field of plant transformation. The concept of accelerating DNA-coated particles into cells and tissues has evolved from novelty to an established tool in plant molecular biology (Klein et al. 1987; Klein and Jones, 1999). This method of introducing DNA into cells by physical means was developed to overcome the biological limitations of Agrobacterium and the difficulties associated with plant regeneration from protoplasts. Initial studies on particle bombardment as a gene delivery method achieved transient expression of gus gene following bombardment of cell suspensions (Wang et al. 1988), leaf bases and apical tissues (Oard et al. 1990), immature embryos (Chibbar et al. 1991). Vasil et al. (1991) obtained stably transformed callus lines that expressed all the marker genes tested (gus, nptII and EPSPS). First successful generation of transgenic wheat plants was reported by Vasil et al. (1992) by particle bombardment of plasmid vector pBARGUS into cells of type-C, long-term regenerable embryogenic callus. Time required from isolation of immature embryos to the production of transgenic plants was 12-15 months. Subsequent improvement in procedures reduced the time required for production of transgenic wheat plants to 5-7 months by bombardment of immature embryos and embryogenic calli (Vasil et al. 1993), about 4 months by immature embryos (Weeks et al. 1993), 3 months by using an enhanced regeneration system using isolated scutella as the starting explant (Nehra et al. 1994), and more recently to 56-66 days by bombardment of cultured immature embryos (Altpeter et al. 1996a). Most of the subsequent gene delivery studies employed immature embryos, isolated scutellum, and calli initiated from immature embryos as the target tissue for bombardment (Table 2). The first successful transformation of durum wheat was reported by Bommineni et al. (1997) by particle bombardment of isolated scutella. Particle bombardment of explants like pollen embryos (Shimada et al. 1991), microspore-derived embryos (Loeb and Reynolds, 1994; Ingram et al. 1999) resulted in successful introduction of transgenes as evidenced by transient expression of gus gene, however regeneration of plantlets from these explants has not been reported. Iglesias et al. (1994) reported the direct delivery of foreign DNA to the meristem cells of immature embryos by microtargetting and transgenic sectors were obtained in both coleoptiles and leaf primordial. Notable here is the microtargetting of genes into meristematic cells which, nonetheless, were 10-fold lower in expression than the surrounding tissues. The effects of various bombardment parameters like amount of plasmid DNA, spermidine concentration, acceleration and vacuum pressure, osmotic pretreatment of target tissues on gene delivery into wheat tissues have been investigated in detail (Rasco-Gaunt et al. 1999) and most of the studies have found no clear correlation between transient expression and stable transformation. The transgenes introduced by biolistic approach display a considerable degree of stability in integration and expression in subsequent generations (Altpeter et al. 1996b; Srivastava et al. 1996).

Since multiple copy insertions of the transgene is common in transgenics obtained by particle bombardment, Srivastava et al. (1999) recently reported the use of a strategy based on site-specific recombination of Cre-lox system to achieve single copy insertion in transgenic wheat. Bombardment of immature embryos with a transformation vector that consists of a transgene flanked by recombination sites in inverted orientations, results in recombination between outermost sites thereby resolving the integrated molecules into a single copy in the transgenic plants.

In recent years, particle bombardment has emerged as a reproducible method for the introduction of various marker genes in wheat (Table 2) and this method is successfully being used for the generation of transformed wheat with introduction of agronomically important genes for quality improvement (Altpeter et al. 1996b; Blechl and Anderson, 1996), engineering of nuclear male sterility (De Block et al. 1997), transposon tagging (Takumi et al. 1999), resistance to drought stress (Sivamani et al. 2000), resistance against fungal pathogens (Leckband and Lorz, 1998; Bliffeld et al. 1999; Chen et al. 1999; Bieri et al. 2000) and insect resistance (Altpeter et al. 1999; Stoger et al. 1999).

Agrobacterium-mediated transformation

Agrobacterium-mediated transformation is a simple, low cost and highly efficient alternative to direct gene delivery methods. The advantages of the Agrobacterium system include the defined insertion of a discrete segment of DNA into the recipient genome. The initial studies on Agrobacterium and wheat were aided by the phenomenon of agroinfection, which for the first time demonstrated that the soil bacterium can interact in a limited manner with the cells of a monocot plant (Woolston et al. 1988; Dale et al. 1989) and transfer DNA. Agroinfection is the introduction of viral DNA residing between the T-DNA border sequences of Agrobacterium into plant cells and provide a suitable system to monitor DNA delivery into target explants by detection of disease symptoms. The wheat dwarf virus (WDV) acted as a sensitive marker for studying the wheat-Agrobacterium interaction due to amplification of viral DNA leading to the production of symptoms and detection by ELISA. The successful agroinfection of WDV paved way for a natural progression of techniques for the introduction of non-viral constructs with scorable and selectable markers, and also for a better understanding of parameters required for the introduction of foreign DNA into wheat.

Most of the experiments on Agrobacterium-mediated transformation employed incubation of suitable explants with the Agrobacterium inoculum (Table 3). Mooney et al. (1991) for the first time demonstrated that wounding is not necessary for adherence of bacteria to explants of wheat and reported an increase in adherence of bacteria at the wound site caused by mechanical and enzymatic treatments. Chen and Dale (1992) reported a higher frequency of infection in exposed apical meristems of dry wheat seeds as compared to intact seeds. Mahalakshmi and Khurana (1995) tested the suitability of various explants for Agrobacterium-mediated gene delivery and reported an increase in transient expression of GUS gene in mature seeds subjected to mechanical wounding by abrasion as compared to intact seeds. The induction of vir genes which is necessary for gene transfer has been achieved by wounding and also by using chemical inducers like acetosyringone. Other factors influencing this association are also discussed by Mahalakshmi and Khurana, (1997). Significant progress made in the area of Agrobacterium-mediated transformation of rice and maize (Chan et al. 1993; Hiei et al. 1994; Ishida et al. 1996) has contributed tremendously to an increasing understanding of various parameters necessary for the successful generation of transgenic cereals. Cheng et al. (1997) first reported the stable transformation of wheat by Agrobacterium-mediated co-cultivation and demonstrated the successful transmission of the transgene to the next generation. This study developed a system for the production of transgenic plants within a total time of 2.5 to 3 months by co-cultivating freshly isolated immaure embryos, precultured immature embryos and embryogenic calli. The factors which influenced the generation of transgenic plants included the explant tissue, inducers in the inoculation and co-cultivation media, and the surfactants present in the inoculation medium. Approximately 35% of the transgenic plants received a single copy of the transgene and one to five copies of the transgene were integrated into the wheat genome without rearrangement (Cheng et al. 1997). McCormac et al. (1998) used cell autonomous reporter genes (anthocyanin biosynthesis regulatory genes, gus, synthetic gfp for studying the localized transformation events. The localization of the transformed cells revealed a non-random distribution throughout each embryo and callus piece. Recently, Agrobacterium-mediated transformation of immature inflorescence tissue was reported by Amoah et al. (2001). This study achieved optimal T-DNA delivery (as measured by gus activity) in explants precultured for 21 days and sonicated (subjected to brief periods of ultrasound in presence of Agrobacterium). In near future, it can be expected that Agrobacterium will be employed as a reliable, efficient and economical vector for the introduction of exogenous genes into wheat by various laboratories throughout the world.

Alternative approaches

Most of the alternative approaches for wheat transformation have attempted to develop a genotype independent, cost effective procedure for the introduction of foreign DNA into wheat. Prominent among these methods are microinjection, direct imbibition, permeabilization, silicon carbide fiber-mediated and pollen tube pathway; which have been used for wheat transformation with varied levels of success.

The pollen-tube pathway method for transferring foreign DNA into recently pollinated floret was first reported for the successful transformation of rice (Duan and Chen, 1985; Luo and Wu, 1989). In this approach the stigma is cut off and a drop of DNA solution is applied to the cut end of the style. The DNA reaches the ovule by flowing down the pollen-tube. The pollen-tube pathway was used to introduce a verc203:gus fusion expression cassette into the winter wheat plants, and Chong and coworkers (Chong et al. 1998) investigated the function of a vernalization-related gene ver203, by expression of a complementary DNA as an antisense RNA, in transgenic plants. Molecular evidence indicated the successful integration of the transgenes and elucidated the role of the VER203 protein in controlling heading and flower development in winter wheat (Chong et al. 1998).

The uptake of plasmid DNA through membranes of dry plant tissue, the physiochemical characteristics of which change during natural desiccation, was thought to be an alternative way of gene transfer. Uptake of plasmid DNA, as well as, recombinant wheat dwarf virus genome by imbibition as reported in seed derived dry embryos of wheat (Töpfer et al. 1989).

Simmonds and coworkers reported the use of microcapillaries for the introduction of plasmid DNA into the germ line precursor cells of apical meristems by microinjection, (Simmonds et al. 1992). Despite a positive indication by PCR amplification of DNA isolated from injected apices, this approach has failed to yield any transgenic plants.

The successful use of silicon carbide fibers in stable transformation of maize (Frame et al. 1994) raised the possibility of its use for the introduction of foreign DNA into wheat. In wheat, the preferential expression of the gus gene in the scutellum and the epiblast of mature embryos was observed following vortexing of the explants in a solution containing silicon carbide fibers and plasmid DNA (Serik et al. 1996).

Recently, Mahalakshmi et al. (2000) exploited the altered membrane characteristics of dry tissues and coupled it with the use of membrane permeabilizing substances like saponin, toluene, triton, etc.; and reported gus gene expression in mechanically isolated seed-derived dry embryos of wheat by incubating in a buffer containing plasmid DNA along with various membrane permeabilizing agents. Although gus gene expression was reported as late as one month following permeabilization, recovery of plantlets was not reported. Nonetheless, all these methods promise the development of genotype independent methods of gene delivery and are thus of tremendous importance.

Introduction of useful traits by genetic transformation

Nutritional improvement

In addition to its basic calorific value, wheat with its high protein content is an important source of plant protein in the human diet. One of the prominent targets for the application of transgene technology for the nutritional improvement of wheat is targeted at enhancing the grain quality by, i) increasing the protein content, ii) increasing essential amino acids such as lysine, iii) increasing the high molecular weight (HMW) glutenins to improve breadmaking properties of wheat flour, iv) modifying starch composition and v) producing pharma- and neutraceuticals.

Amongst the cereals, the flour of bread wheat, Triticum aestivum, has a superior capability of forming leavened bread. This superiority stems from the structure and composition of its seed storage proteins, which upon hydration can interact to form gluten, an insoluble, but highly hydrated, visco-elastic aggregate that endows the wheat dough with its unique properties. Although the majority of wheat seed storage proteins participate in gluten formation, biochemical and genetic evidence has demonstrated that high molecular weight glutenin subunit (HMW-GS) plays a major role in determining the visco-elastic properties thereby determining bread making qualities. The HMW glutenins are necessary to create a strong dough, which is essential for making high quality, yeast-raised breads. Strong dough traps tiny bubbles of carbon dioxide gas formed naturally by yeast during mixing and subsequent raising thereby enabling the dough to rise forming leavened breads. Dough strength and the ability to contain gas bubbles is known as visco-elasticity and is an important characteristic of wheat with respect to its end product quality. Increasing understanding of the molecular basis of dough visco-elasticity would thus help in developing strategies for minimizing the effect of unfavourable environmental factors on wheat end-use quality.

Genetic transformation of wheat is a key component in a scheme proposing a complete set of approaches to apply biotechnology to improve wheat quality via direct manipulation of HMW-glutenin genes (Vasil and Anderson, 1997). To alter the amount and composition of these proteins by genetic engineering, a gene encoding a novel, hybrid-subunit of HMW-GS under the control of native HMW-GS regulatory sequences was inserted into wheat using the biolistic approach (Blechl and Anderson, 1996). The HMW-GS 1Ax1 gene which is known to be associated with superior bread making quality, was introduced into the cultivar (Bobwhite) lacking this gene (Altpeter et al. 1996b). The introduced 1Ax1 gene under the control of HMW-GS promoter was expressed at high levels and stability was maintained for several generations. The results demonstrate the feasibility of manipulating the composition of wheat kernels by genetic engineering and have successfully made changes in both the levels and the types of seed storage proteins. This work also demonstrated the usefulness of tissue-specific promoters for the expression of transgenic proteins in the endosperm tissue of wheat. The prospect of achieving the elusive goal of nutritional improvement of wheat brightened further with the improvement in functional properties of wheat dough due to transformation of wheat with high molecular weight subunit genes. Transformation with one or two subunit genes results in stepwise increase in dough elasticity (Barro et al. 1997). The expression of a recombinant protein with x- and y- type HMW-GS subunit in transgenic wheat has resulted in altered gluten polymer assembly and composition. This study thus made it feasible to change the glutenin composition by expressing modified HMW-GS in transgenic plants (Shimoni et al. 1997).

The quality improvement studies were extended to durum wheat by He et al. 1999 with the introduction of one of the two subunits of HMW-GS subunit genes (1Ax1 or 1Dx5). Analysis of the expression of the additional subunits in the T₂ generation using a mixograph, indicated an increase of dough strength and stability. This study demonstrated the feasibility of manipulating durum wheat for superior bread and pasta making qualities (He et al. 1999). Similarly, the overexpression of the HMW subunit 1Dx5, in transgenic wheat (T. aestivum) resulted in a four-fold increase in this proportion of component in the seed protein and also a corresponding increase in the proportions of the total HMW proteins and glutenins (Rooke et al. 1999). However, the overexpression of the 1Dx5 gene was found to be associated with a dramatic increase in dough strength by making it too strong, and, therefore, unsuitable for use in conventional breadmaking. Recently, Alvarez et al. (2000) introduced the HMW-GS genes 1Ax1 and 1Dx5 into a commercial cultivar of T. aestivum that already expresses five subunits. The overexpression of 1Dx5 gene increases the contribution of the HMW-GS to a level of 22% of the total protein content. In near future, molecular approaches including genetic transformation and marker-assisted selection will provide an opportunity for improving further the wheat processing qualities.

Modification of wheat starch is presently targeted in various laboratories to improve its potential utility. Wheat grain is predominantly composed of starch, which is a mixture of two polymers, the almost linear amylose molecules, and the heavily branched amylopectin molecules. The ratio of amylose to amylopectin in starch determines its physico-chemical characteristics and thereby its end-use. Wheat flour, low in amylose content is desirable for noodlemaking as it improves noodle texture. In wheat, scientists are thus working towards increasing the amylopectin content of starch thereby reducing amylose content leading to the formation of a value added, low-amylose flour. The starch branching enzymes catalyse formation of 1, 6-linkages in the glucan polymer and control the amount of amylopectin produced. In recent years, efforts are underway towards detailed characterization of the starch branching enzymes (Baga et al. 1999b; Baga et al. 2000) and starch synthases (Li et al. 1999; Gao and Chibbar, 2000; Li et al. 2000). This increasing information and characterization of the various components of starch biosynthesis will enable researchers to make rational design of novel starches and alteration in starch levels in other crop plants as well (Slattery et al. 2000).

Wheat is widely used as an animal feed also for non-ruminants in several developed countries of the world. The phytase of Aspergillus niger is used as a supplement in animal feeds to improve the digestibility and also to improve the bioavailabilty of phosphate and minerals. The phyA gene from Aspergillus niger, encoding for the phytase enzyme has been successfully expressed in transgenic wheat lines by the microprojectile bombardment of immature embryos. The constitutively overexpressed phytase was found to accumulate at high levels in the endosperm (Brinch-Pederson et al. 2000). This work may thus open newer avenues for a wider applicability of wheat.

Wheat is also an ideal system for the production of novel compounds due to its excellent storage properties and the existence of an efficient processing industry. The potential products include the high value, low-volume compounds such as biologically active proteins and peptides, as well as, high volume, low-cost raw materials for packaging and building (Shewry and Lazzeri, 1997). The production of recombinant antibodies in rice and wheat was recently reported with the expression of a medically important, single chain Fv recombinant antibody against the carcino-embryonic antigen. (Stoger et al. 2000). The recombinant antibody was targeted to the plant cell apoplast and endoplasmic reticulum and was detected in the leaves and seeds of wheat. More significantly, the recombinant antibodies remained active after prolonged seed storage at room temperature thus opening avenues for the further exploitation of wheat for producing high-value, novel compounds.

Engineering nuclear male sterility

The production of hybrids is an essential component of crop breeding programmes but till date, hybrid wheat has remained elusive! The development of a suitable hybridisation system for wheat requires a high degree of male sterility in all parts of the female parent to avoid self-fertilization. De Block and coworkers (De Block et al. 1997) have developed a nuclear male sterile system in wheat by introducing the barnase gene under the control of a tapetum specific promoter, the expression of which prevents normal pollen development at specific stages of anther development. This system employed the ribonuclease-inhibitor barstar gene to restore the fertility of male sterile plants. To avoid complicated gene integration patterns, the target tissues were incubated on niacinamide containing medium before bombardment. The authors suggest that the enzyme poly (ADP-robose) polymerase (PARP), which plays a key role in the processes of cell division and recombination, is inhibited by niacinamide, thereby resulting in simple integration pattern of the transgene (De Block et al. 1997). Expressing the barnase gene at specific stages of anther development destroys the tapetum, thereby preventing normal pollen development and causes pollen sterility.

Resistance to biotic stress

The integration of transgenic approaches with classical breeding techniques offers a potential chemical-free and environment-friendly solution for controlling pests and pathogens. Wheat is attacked by a number of viral, bacterial and fungal pathogens and also by insect and nematode pests. Introgression of genes from the wild relatives exhibiting resistance to pests and pathogens has been successfully utilized over the years for generation of resistant varieties in wheat. With the development of plant transformation techniques, newer avenues for creating disease resistant and insect resistant crops have been created. Coupled with this is the availability of novel transgenes encoding highly potential anti-microbial peptides, defense-related proteins and enzymes for the production of anti-microbial compounds in crop plants, have greatly enhancing the possibility of engineering crop plants for resistance to pests and pathogens (Shah, 1997; Sharma et al. 2000).

Fungal pathogens of wheat cause severe crop damages by infecting the spikes, leaves and roots. Amongst the various strategies to introduce fungal resistance by transgenic approach, strengthening the host plant defense by genetic manipulation hold tremendous potential. Biochemical and structural responses against fungal attack include reinforcement of plant cell wall, accumulation of phytoalexins with microbial toxicity, ribosome-inactivating proteins (RIP) that inhibit protein synthesis, antimicrobial peptides and synthesis of other PR proteins, (Yang et al. 1997). Genetic engineering allows the expression of foreign genes from distant unrelated species as well as the modification of the usual pattern of expression of an already present gene.

Most of the works on genetic engineering of wheat for resistance against biotic stress have focussed on developing protection against fungal pathogens (Table 4). Introduction genes encoding for chitinases from barley resulted in increased resistance against Erysiphe graminis (Bliffeld et al. 1999). Bliffeld et al. (1999) reported the adverse effect of a ribosome inactivating protein on plant regeneration and development. Nonetheless, moderate protection against Erysiphe graminis was reported with the transformation of wheat with a gene encoding for a ribosome inactivating protein from barley (Bieri et al. 2000). The genes encoding for thaumatin like protein (TLP) and stilbene synthase in transgenic wheat have been shown to improve resistance of T₁, T₂ progeny plants against the fungal pathogens (Leckband and Lorz, 1998; Chen et al. 1999). An increase in endogenous resistance against Tilletia tritici was achieved with the introduction of virally encoded antifungal protein (Clausen et al. 2000).

For the engineering of resistance against different pests and pathogens in wheat, genes encoding for viral coat proteins, antifungal proteins, and proteinase inhibitors have been successfully introduced (Table 4). Most of the introduced genes confer increased resistance to the corresponding pests and pathogens in the transgenic plants. Introduction of canditate genes (lectin, proteinase inhibitor) for insect resistance into wheat have resulted in growth inhibition of insects on transgenic seeds, thereby decreasing the fecundity of insect population.

Resistance to abiotic stress

Traditional approaches at transferring resistance to crop plants are limited by the complexity of stress tolerance traits, as most of these are quantitatively linked traits (QTLs). Nonetheless, the direct introduction of a small number of genes by genetic engineering offers convenient alternative and a rapid approach for the improvement of stress tolerance. Although, present engineering strategies rely on the transfer of one or several genes that encode either biochemical pathways or endpoints of signalling pathways, these gene products provide some protection, either directly, or indirectly, against environmental stresses.

Drought is a major abiotic factor that limits crop productivity, thereby causing enormous loss. The genes encoding the late embryogenesis proteins (LEA) which accumulate during seed desiccation, and in vegetative tissues when plants experience water deficiencies have recently emerged as attractive candidates for engineering of drought tolerance. Transgenic approach has been used for successfully introducing and overexpressing the barley HVA1 gene encoding for a late embryogenesis abundant (LEA) protein by Sivamani et al. (2000) into wheat by particle bombardment. Most of the transgenic lines tested displayed improvement in important agronomic traits, including total dry mass and water use efficiency, shoot dry weight, root fresh and dry weights, when plants are grown under soil water deficit conditions. In general, this investigation showed that the transgenic lines expressing the HVA1 gene had improved growth characteristics including an enhanced biomass yield under water deficit conditions. The discovery of novel genes, determination of their expression pattern in response to abiotic stress and an improved understanding of their roles in stress adaptation (obtained by the use of functional genomics) will provide the basis of effective engineering strategies leading to greater stress tolerance (Cushman and Bohnert, 2000).

Transgene silencing in wheat

Success at developing improved wheat cultivars through genetic engineering depends on stable and predictable expression of the inserted gene. However, gene silencing is a common phenomenon in the production of transgenic plants and needs to be effectively controlled for the desired results. This is all the more important because gene silencing is an important phenomenon involved during natural plant defense. Gene silencing is a complicated phenomenon as it includes both transcriptional gene inactivation and post transcriptional gene inactivation (Khurana et al. 1998). The complex and the large genome size of wheat is expected to be prone to silencing by introduced transgenes. With the development of transformation methodologies for wheat, more information has started to accumulate regarding the inheritance and expression of the introduced transgenes. Information regarding the long term stability of transgenes is thus of immense significance for the use of genetic manipulation as a tool in wheat crop improvement and is necessary for the introduction and subsequent expression of desirable agronomic trait.

DNA methylation plays a significant role in establishing and maintaining an inactive state of the gene by rendering the chromatin structure inaccessible to the transcription machinery (Razin, 1988). Methylation of DNA is expected to result in reduced gene expression. Muller et al. (1996) studied the variability of transgene expression in clonal cell lines of wheat and found a negative correlation between the degree of methylation and marker gene expression. PEG-mediated approach was used to transform protoplasts isolated from suspension cultures of T. aestivum. The integration and expression of the selectable marker gene nptII fused with different promoters, namely, alcohol dehydrogenase, shrunken from maize, and actin1 from rice, was confirmed by Southern analysis and enzyme activity test. Transgenic cell lines under selection were 'protoplasted' and clonal callus lines were cultivated from genetically identical single cells without selection pressure. A reduction/loss of marker gene expression was observed due to a reduction in the nptII transcript level and was seen to be associated with hypermethylation of the integrated DNA. The silencing effect was reversed by a 4-week culture phase on media supplemented with demethylation agent, 5-azacytidine, thus confirming the role of methylation in transgene silencing

Demeke et al. (1999) studied the inheritance and stability of an Act1D-uidA: nptII expression cassette in T₄ and T₅ transgenic plants. based on the histochemical localization of GUS activity, this study demonstrated the lack of any cytoplasmic effect on the inheritance of the transgene. The transgenes maintained a multiple integration pattern similar to that observed in the T₁ generation. The transgenic plants which produced low gus and nptII activity in seeds had an intact expression cassette. Southern blot analysis of genomic DNA performed with the methylation sensitive enzyme, HpaII, showed the transgene in GUS negative plants to be highly methylated relative to the transgene in GUS positive plants. Some of the studies on gene silencing also indicate that multiple integration pattern and copy number is also associated with DNA methylation (Hobbs et al. 1993; Muller et al. 1996).

Cannell and coworkers studied the inheritance of gus and bar marker genes over three generations. The integration, inheritance and expression of these marker genes in the population studied were stable and predictable with a few exceptions (Cannell et al. 1999). The inheritance of integration patterns was stable, and transmission/ inheritance of the transgenes followed Mendelian ratios in a majority of lines. From this study, the authors claim that the transformation procedure, transgene integration, and marker gene expression had little effect on the transmission of transgenes to the subsequent progenies. However, over the three generations studied, a uniform, 'progressive' transgene silencing, specific to the gus gene, was observed. This gradual loss of the gus gene expression was not accompanied by a reduction in bar expression. Since in this case, the gus and the bar genes are located on the same plasmid, observations indicate a post-transcriptional gene silencing.

The silencing of HMW glutenins was also observed in transgenic wheat expressing extra HMW subunits (Alvarez et al. 2000). Characterization of six independent events involving the transformation of wheat HMW-GS genes, 1Ax1 and 1Dx5, in a cultivar expressing five subunits by particle bombardment resulted in partial or complete gene silencing. Silencing of all the HMW glutenin subunits was observed in two different events of transgenic wheat expressing the 1Ax1 subunit transgene and overexpressing the 1Dx5 subunit. Control of gene silencing thus remains a challenge for the immediate future.

Meyer, (1995) advocated that the problem of gene silencing in wheat can be minimized by optimising methods for simple integration patterns, use of promoters and gene sequences isolated from cereals, use of matrix associated regions (MARs) or scaffold attachment regions (SARs), which insulate transgenes from surrounding chromatin, might help in reducing the gene silencing problem (Baga et al. 1999a).

Transposon tagging in wheat

Transposon mutagenesis has been widely exploited in various organisms to isolate genes that encode unidentified products (Sundaresan, 1996). The maize activator (Ac) and Dissociation (Ds) elements are the best-studied transposable elements in heterologous host plants. Initial studies have reported the introduction and activation of the Ac/Ds elements into cultured wheat cells by using a wheat dwarf virus by particle bombardment (Laufs et al. 1990; Takumi, 1996). Takumi and coworkers (Takumi et al. 1999) has developed a transposon tagging system in wheat by introducing the Ac transposase gene under the CaMV 35S promoter into cultured wheat embryos by particle bombardment. For the development of a transposon tagging system, embryos isolated from a stable Ac line were bombarded with a plasmid containing the maize dissociator (Ds) element located between the rice Act1 and the gus genes. The transient expression of the gus gene was observed after the excision of Ds elements. Southern and northern analysis of the T₀ and T₁ plants has exhibited the stable expression and inheritance of the Ac transposase gene. These results have demonstrated the precise processing of the maize Ac transposase gene and the synthesis of the transposase protein in transgenic Ac lines. The Ac transposase gene causes the transactivation and excision of the Ds in the Ac transgenic lines transformed with the maize Ds elements. Thus in near future we can expect traits of commercial importance to be tagged for a wider utility in important crop plants.

Marker assisted selection in wheat breeding

Conventionally, plant breeding depends upon morphological/phenotypic markers for the identification of agronomic traits. With the development of methodologies for the analysis of plant gene structure and function, molecular markers have been utilized for identification of traits. Molecular markers act as DNA signposts to locate the gene(s) for a trait of interest on a plant chromosome, and are widely used to study the organization of plant genomes and for the construction of genetic linkage maps. Molecular markers are independent from environmental variables and can be scored at any stage in the life cycle of a plant. Over the last several years, there has thus been marked increase in the application of molecular markers in the breeding programmes of various crop plants. Molecular markers not only facilitate the development of new varieties by reducing the time required for the detection of specific traits in progeny plants, but also fasten the identification of resistance genes and their corresponding molecular markers, thus accelerating efficient breeding of resistance traits into wheat cultivars by marker assisted selection (MAS). The availability of back-cross derived near isogenic lines (NILs) have also facilitated the analysis of various lines by using different marker systems. The introduction of alien genetic variation into wheat is a valuable and proven technique for wheat improvement. Wild relatives of wheat provide an enormous resource of new genes for wheat improvement, particularly disease and stress tolerance. These genes can be of use if recombined into lines adapted to the conditions of a particular region.

Initial studies on the application of molecular markers in wheat relied on the hybridisation based restriction fragment length polymorphism (RFLP) system. RFLP maps provided a more direct method for selecting desirable genes via their linkage to easily detectable markers thereby expediting the movement of desirable genes among varieties (Tanksley et al. 1989). The factors that had been instrumental for the use of RFLP in wheat was the limited number of polymorphisms observed among wheat lines and more significantly the availability of aneuploid stocks for the determination of chromosomal location of genes. In wheat, RFLP's have been used to map seed storage protein loci (Dubcovsky et al. 1997), loci associated with flour colour (Parker et al. 1998), cultivar identification (Vaccino et al. 1993), vernalization (Vrn1) and frost resistance gene on chromosome 5A (Galiba et al. 1995), intrachromosomal mapping of genes for dwarfing (Rht12) and vernalization (Vrn1) (Korzun et al. 1997), resistance to preharvest sprouting (Anderson et al. 1993), quantitative trait loci (QTL's) controlling tissue culture response (tcr) (Ben Amer et al. 1997), nematode resistance (Eastwood et al. 1994; Williams et al. 1996; Lagudah et al. 1997), milling yield (Parker et al. 1999), resistance to chlorosis induction by Pyrenophora tritici-repentis (Faris et al. 1997). RFLP markers are also useful in selection programs for resistance against pests and pathogens, which is otherwise labor and time consuming, and to detect homozygous individuals and have been used for resistance to barley yellow dwarf virus (Crasta et al. 2000), resistance to wheat spindle streak mosaic virus (Khan et al. 2000), resistance against powdery mildew (Hartl et al. 1993; Ma et al. 1994; Hartl et al. 1995), resistance against leaf rust (Dedryver et al. 1996; Autrique et al. 1995; Feuillet et al. 1995; Schachermayr et al. 1995; Schachermayr et al. 1994), resistance against cereal cyst nematode (Eastwood et al. 1994; Lagudah et al. 1997). The use of RFLP analysis in wheat has, however, been of limited use in the intervarietal analysis due to low level of polymorphism and the high cost for screening in breeding situations.

With the development of polymerase chain reaction (PCR) methodologies, Random Amplified Polymorphic DNA (RAPD) emerged as a convenient and effective technique for tracing alien chromosome segments in translocation lines (Williams et al. 1990; Devos and Gale, 1992). Rapd markers provide a useful alternative to RFLP analysis for screening markers linked to a single trait within near isogenic lines and bulked segregants. He et al. (1992) reported the development of a DNA polymorphism detection method by combining RAPD with DGGE (denaturing gradient gel elecrophoresis) for pedegree analysis and fingerprinting of wheat cultivars. RAPD markers can be converted to more user-friendly Sequence Characterized Amplified Region (SCAR) markers, that display a less complex banding pattern. SCAR markers linked to resistance genes against fungal pathogens have been characterized in combination with RAPD and RFLP (Procunier et al. 1997; Myburg et al. 1998; Liu et al. 1999). In recent years, RAPD and other PCR based markers like Sequence Characterized Amplified Regions (SCAR), Sequence Tagged Sites (STS) and Differential Display Reverse Transcriptase PCR (DDRT-PCR) are increasingly being used for identification of desirable traits in wheat and related genera. These markers have been used in particular for disease resistance against viral and fungal pathogens and also for insect and nematode pests and have the potential of pyramiding of resistance genes for effective breeding programs. PCR based markers have been extensively characterized for genes of resistance against common bunt, Tilletia tritici, (Demeke et al. 1996); powdery mildew, Erysiphe graminis (Hartl et al. 1995; Qi et al. 1996), leaf rust, Puccinia recondita (Dedryver et al. 1996; Feuillet et al. 1995; Seyfarth et al. 1999), resistance against Hessian fly, Mayetiola destructor (Dweikat et al. 1994) and Russian wheat aphid, Diuraphis noxia (Myburg et al. 1998; Venter and Botha, 2000).

Simple sequence repeats or microsatellites are more promising molecular markers for the identification and differentiation of genotypes within a species. The high level of polymorphism and easy handling has made microsatellites extremely useful for different applications in wheat breeding (Devos et al. 1995; Roder et al. 1995; Bryan et al. 1997; Korzun et al. 1997; Roy et al. 1999; Stachel et al. 2000). Microsatellites have also been used to identify resistance genes like Pm6 from Triticum timopheevii (Tao et al. 1999) and Yr15 from breadwheat (Chague et al. 1999). In near future, molecular markers can provide simultaneous and sequential selection of agronomically important genes in wheat breeding programs allowing screening for several agronomically important traits at early stages and effectively replace time consuming bioassays in early generation screens.

Genomics and future outlook

Genomics is the discovery and study of many genes simultaneously on a genome-wide scale. In the pregenomic era, gene discovery was the limiting factor for the improvement of crops by genetic engineering. For the upcoming postgenomic era, an exponential number of genes will be discovered through genome sequencing efforts on Arabidopsis and rice. Wheat genomes are large in comparision to the current plant models and are ideal targets for partial genome sequencing (Keller and Feuillet, 2000). However, due to a high level of genetic colinearity at a broader genetic level and also at the gene level, comparative genome analysis studies can serve as efficient tools for transferring information and resources from well studied genomes to the related genera (Devos and Gale, 1997; Schmidt, 2000). DNA mapping and sequence information generated in the rice genome project would be highly useful for the characterization of larger genomes like wheat. Molecular isolation of agronomically important genes and an understanding of their action will contribute immensely to breeding of cultivars. With the availability of detailed information regarding the location and function of gene(s) encoding for useful traits, scientists in future will be well equipped for efficiently creating varieties with exact combinations of desirable traits. However, genetic transformation will remain a significantly important tool for understanding gene functions and for testing the utility of new sequences. In near future, crop varieties could be tailor-made to meet both local consumer preferences and the demands of particular niche or environment. The new tools of biotechnology not only have the potential for increasing the effectiveness and efficiency of wheat breeding programs, and but will also provide insights into the genetic control of key traits to be used for genetic manipulation. Application of biotechnology will thus contribute greatly to improving yield stability by generating plants with improved resistance to biotic and abiotic stresses rather than raising the overall yield. The coming years will undoubtedly witness an increasing application of biotechnology for the genetic improvement of wheat.

D.P. wishes to thank the Council of Scientific and Industrial Research for the award of a Senior Research Fellowship (NET).

Altpeter, F.; Diaz, I.; McAuslane, H.; Gaddour, K.; Carbonero, P. and Vasil, I.K. (1999). Increased insect resistance in transgenic wheat stably expressing trypsin inhibitor CMe. Molecular Breeding 5:53-63.

Altpeter, F.; Vasil, V.; Srivastava, V.; Stöger, E. and Vasil, I.K. (1996a). Accelerated production of transgenic wheat (Triticum aestivum L.) plants. Plant Cell Reports 16:12-17.

Altpeter, F.; Vasil, V.; Srivastava, V. and Vasil, I.K. (1996b). Integration and expression of the high-molecular-weight glutenin subunit 1Ax1 gene into wheat. Nature Biotechnology 14:1155-1159.

Alvarez, M.L.; Guelman, S.; Halford, N.G.; Lustig, S.; Reggiardo, M.I.; Ryabushkina, N.; Schewry, P.; Stein, J. and Vallejos, R.H. (2000). Silencing of HMW glutenins in transgenic wheat expressing extra HMW subunits. Theoretical and Applied Genetics 100:319-327.

Amoah, B.K.; Wu, H.; Sparks, C. and Jones, H.D. (2001). Factors influencing Agrobacterium-mediated transient expression of uidA in wheat inflorescence tissue. Journal of Experimental Botany. 52: 1135-1142..

Anderson, J.A.; Sorrells, M.E. and Tanksley, S.D. (1993). RFLP analysis of genomic regions associated with resistance to preharvest sprouting in wheat. Crop Science 33:453-459.

Autrique, E.; Singh, R.P.; Tanksley, S.D. and Sorrells, M.E. (1995). Molecular markers for four leaf rust resistance genes introgressed into wheat from wild relatives. Genome 38:75-83.

Baga, M.; Nair, R.B.; Repellin, A.; Scoles, G.J. and Chibbar, R.N. (2000). Isolation of a cDNA encoding a granule-bound 152-kilodalton starch-branching enzyme in wheat. Plant Physiology 124:253-264.

Baga, M.; Chibbar, R.N. and Kartha, K.K. (1999a). Expression and regulation of transgenes for selection of transformants and modification of traits in cereals. In: Vasil I.K. ed. Advances in cellular and molecular biology of plants, Vol.5. Molecular Improvement of cereal crops. Kluwer Publ., Dordrecht, The Netherlands. pp. 83-132.

Baga, M.; Glaze, S.; Mallard, C.S. and Chibbar, R.N. (1999b). A starch-branching enzyme gene in wheat produce alternatively spliced transcripts. Plant Molecular Biology 40:1019-1030.

Bajaj, Y.P.S (1990). Biotechnology in wheat breeding. In: Bajaj YPS ed. Biotechnology in agriculture and forestry Wheat, Vol.3. springer-Verlag, Berlin, Heidelberg, New York, USA. pp 3-23.

Barro, F.; Cannell, M.E.; Lazzeri, P.A. and Barcelo, P. (1998). The influence of auxins on transformation of wheat and tritordeum and analysis of transgene integration patterns in transformants. Theoretical and Applied Genetics 97:684-695.

Barro, F.; Rooke, L.; Bekes, F.; Gras, P.; Tatham, A.S.; Fido, R.; Lazzeri, P.A.; Shewry, P.R. and Barcelo, P. (1997). Transformation of wheat with high molecular weight subunit genes results in improved functional properties. Nature Biotechnology 15:1295-1299.

Becker, D.; Brettschneider, R. and Lorz, H. (1994). Fertile transgenic wheat from microprojectile bombardment of scutellar tissue. Plant Journal 5:299-307.

Ben Amer, I.M.; Korzun, V.; Worland, A.J. and Börner, A. (1997). Genetic mapping of QTL controlling tissue-culture response on chromosome 2B of wheat (Triticum aestivum L.) in relation to major genes and RFLP markers. Theoretical and Applied Genetics 94:1047-1052.

Bieri, S.; Potrykus, I. and Futterer, J. (2000). Expression of active barley seed ribosome-inactivating protein in transgenic wheat. Theoretical and Applied Genetics 100:755-763.

Blechl, A.E. and Anderson, O.D. (1996). Expression of a novel high-molecular-weight glutenin subunit gene in transgenic wheat. Nature Biotechnology 14:875-879.

Bliffeld, M.; Mundy, J.; Potrykus, I. and Futterer, J. (1999). Genetic engineering of wheat for increased resistance to powdery mildew disease. Theoretical and Applied Genetics 98:1079-1086.

Brinch-Pederson, H.; Oleson, A.; Rasmussen, S.K. and Holm, P.B. (2000). Generation of transgenic wheat (Triticum aestivum L.) for constitutive accumulation of an Aspergillus phytase. Molecular Breeding 6:195-206.

Bryan, G.J.; Collins, A.J.; Stephenson, P.; Orry, A.; Smith, J.B. and Gale M. D. (1997). Isolation and characterisation of microsatellites from hexaploid bread wheat. Theoretical and Applied Genetics 94:557-563.

Bommineni, V.R.; Jauhar, P.P. and Peterson, T.S. (1997). Transgenic durum wheat by microprojectile bombardment of isolated scutella. Journal of Heredity 88:301-313.

Cannell, M.E.; Doherty, A.; Lazzeri, P.A. and Barcelo, P. (1999). A population of wheat and tritordeum transformants showing a high degree of marker gene stability and heritability. Theoretical and Applied Genetics 99:772-784.

Chan, M.; Cheng, H.; Ho, S.; Tong, W. and Xu, S. (1993). Agrobacterium-mediated production of transgenic rice plants expressing a chimeric a -amylase promoter/b -glucuronidase gene. Plant Molecular Biology 22:491-506.

Chawla, H.S.; Cass, L.A. and Simmonds, J.A. (1999a). Expression of anthocyanin pigmentation in wheat tissues transformed with anthocyanin regulatory genes. Current Science 76:1365-1370.

Chawla, H.S.; Cass, L.A. and Simmonds, J.A. (1999b). Developmental and environmental regulation of anthocyanin pigmentation in wheat tissues transformed with anthocyanin regulatory genes. In Vitro Cell and Developmental Biology 35:403-408.

Chague, V.; Fahima, T.; Dahan, A.; Sun, G.L.; Korol, A.B.; Ronin, Y.I.; Grama, A.; Roder, M.S. and Nevo, E. (1999). Isolation of microsatellite and RAPD markers flanking the Yr15 gene of wheat using NILs and bulked segregant analysis. Genome 42:1050-1056.

Chen, W.P.; Chen, P.D.; Liu, D.J.; Kynast, R.; Friebe, B.; Velazhahan, R.; Muthukrishnan, S. and Gill, B.S. (1999). Development of wheat scab symptoms is delayed in transgenic wheat plants that constitutively express a rice thaumatin-like protein gene. Theoretical and Applied Genetics 99:755-760.

Chen, W.P.; Gu, X.; Liang, G.H.; Muthukrishnan, S.; Chen, P.D.; Liu, D.J. and Gill, B.S. (1998). Introduction and constitutive expression of a rice chitinase gene in bread wheat using biolistic bombardment and the bar gene as a selectable marker. Theoretical and Applied Genetics 97:1296-1306.

Chen, D.F. and Dale, P.J. (1992). A comparison of methods for delivering DNA to wheat: the application of wheat dwarf virus DNA to seeds with exposed apical meristems. Transgenic Research 1:93-100.

Cheng, M.; Fry, J.E.; Pang, S.; Zhou, H.; Hironaka, C.M.; Duncan, D.R.; Conner, T.W. and Wan, Y. (1997). Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiology 115:971-980.

Chong, K.; Bao, S.; Xu, T.; Tan, K.; Liang, T.; Zeng, J.; Huang, H.; Xu, J. and Xu, J. (1998). Functional analysis of the ver gene using transgenic wheat. Physiologia Plantarum 102:87-92.

Chibbar, R.N.; Kartha, K.K.; Leung, N.; Qureshi, J. and Caswell, K. (1991). Transient expression of marker genes in immature zygotic embryos of spring wheat (Triticum aestivum L.) through microprojectile bombardment. Genome 34:453-460.

Clausen, M.; Krauter, R.; Schachermayr, G.; Potrykus, I. and Sautter, C. (2000). Antifungal activity of a virally encoded gene in transgenic wheat. Nature Biotechnology 18:446-449.

Crasta, O.R.; Francki, M.G.; Bucholtz, D.B.; Sharma, H.C.; Zhang, J.; Wang, R.C.; Ohm, H.W. and Anderson, J.M. (2000). Identification and characterization of wheat-wheatgrass translocation lines and localization of barley yellow dwarf virus resistance. Genome 43:698-706.

Cushman, J.C. and Bohnert, H.J. (2000). Genomic approaches to plant stress tolerance. Current Opinion in Plant Biology 3:117-124.

Dale, P.J.; Marks, M.S.; Brown, M.M.; Woolston, C.J.; Gunn, H.; Boulton, M.I.; Mullineaux, P.M.; Lewis, D.M.; Kemp, J.M.; Chen, D.F.; Gilmour, D.M. and Flavell, R.B. (1989). Agroinfection of wheat: Inoculation of in vitro grown seedlings and embryos. Plant Science 63:237-245.

Daniell, H.; Krishnan, M. and McFadden, B.F. (1991). Transient expression of b -glucuronidase in different cellular compartments following biolistics delivery of foreign DNA into wheat leaves and calli. Plant Cell Reports 9:615-619.

De Block, M.; Debrouwer, D. and Moens, T. (1997). The development of a nuclear male sterility system in wheat. Expression of the barnase gene under the control of tapetum specific promoters. Theoretical and Applied Genetics 95:125-131.

Dedryver, F.; Jubier, M.F.; Thouvenin, J. and Goyeau, H. (1996). Molecular markers linked to the leaf rust resistance gene Lr24 in different wheat cultivars. Genome 39:830-835.

Demeke, T.; Hucl, P.; Baga, M.; Caswell, K.; Leung, N. and Chibbar, R.N. (1999). Transgene inheritance and silencing in hexaploid spring wheat. Theoretical and Applied Genetics 99:947-953.

Demeke, T.; Laroche, A. and Gaudet, D.A. (1996). A DNA marker for the Bt-10 common bunt resistance gene in wheat. Genome 39:51-55.

Devos, K.M. and Gale, M.D. (1997). Comparative genetics in the grasses. Plant Molecular Biology 35:3-15.

Devos, K.M.; Bryan, G.J.; Collins, A.J.; Stephenson, P. and Gale, M.D. (1995). Application of two microsatellite sequences in wheat storage proteins as molecular markers. Theoretical and Applied Genetics 90:247-252.

Devos, K.M. and Gale, M.D. (1992). The use of random amplified polymorphic DNA markers in wheat. Theoretical and Applied Genetics 84:564-577.

Dhir S.K.; Pajeau M.E.; Fromm M.E. and Fry J.E. (1994). Anthocyanin genes as a visual marker for wheat transformation. In: Henry, R.J. and Ronalds, J.A., eds. Improvement of Cereal Quality by Genetic Engineering, Plenum Press, New York, USA. pp. 71-75.

Duan, X. and Chen, S. (1985). Variation of the characters in rice (Oryza sativa) induced by foreign DNA uptake. China Agricultural Science 3:6-9.

Dubcovsky, J.; Echaide, M.; Giancola, S.; Rousset, M.; Luo, M.C.; Joppa, L.R. and Dvorak, J. (1997). Seed-storage-protein loci in RFLP maps of diploid, tetraploid and hexaploid wheat. Theoretical and Applied Genetics 95:1169-1180.

Dweikat, I.; Ohm, H.; Mackenzie, S.; Patterson, F.; Cambron, S. and Ratcliffe, R. (1994). Association of a DNA marker with Hessian fly resistance gene H9 in wheat. Theoretical and Applied Genetics 89:964-968.

Eastwood, R.F.; Lagudah, E.S. and Appels, R. (1994). A directed search for DNA sequence tightly linked to cereal cyst nematode resistence genes in Triticum tauschii. Genome 37:311-319.

Faris, J.D.; Anderson, J.A.; Francl, L.J. and Jordahl, J.G. (1997). RFLP mapping of resistance to chlorosis induction by Pyrenophora tritici-repentis in wheat. Theoretical and Applied Genetics 94:98-103.

Feuillet, C.; Messmer, M.; Schachermayr, G. and Keller, B. (1995). Genetic and physical characterization of the LR1 leaf rust resistance locus in wheat (Triticum aestivum L.). Molecular and General Genetics 248: 553-562.

Frame, B.R.; Drayton, P.R.; Bagnall, S.V.; Lewnau, C.J.; Bullock, W.P.; Wilson, H.M.; Dunwell. J.M.; Thompson, J.A. and Wang, K. (1994). Production of fertile transgenic maize plants by silicon carbide whisker-mediated transformation. Plant Journals 6:941-948.

Galiba, G.; Quarrie, S.A.; Sutka, J.; Morgounov, A. and Snape, J.W. (1995). RFLP mapping of the vernalization (Vrn1) and frost resistance (Fr1) genes on chromosome 5A of wheat. Theoretical and Applied Genetics 90:1174-1179.

Gao, M. and Chibbar, R.N. (2000). Isolation. charcterization and expression analysis of starch synthase IIa cDNA from wheat. Genome 43:768-775.

Gill, K.S. and Gill, B.S. (1994) Mapping in the realm of polyploidy: The wheat model. BioEssays 16:841-846.

Guang-Min, X.I.A. and Zhong-Ti, L.I. (1999). Transgenic plant regeneration from wheat (Triticum aestivum L.) mediated by Agrobacterium tumefaciens. Acta Phytophysicologica Sinica 25:22-28. Available on the world wide web. (Digital periodical)

Hansen, G. and Wright, M.S. (1999). Recent advances in the transformation of plants. Trends in Plant Sciences 4:226-231.

Hartl, L.; Weiss, H.; Stephan, U.; Zeller, F.J. and Jahoor, A. (1995). Molecular identification of powdery mildew resistance genes in common wheat (Triticum aestivum L). Theoretical and Applied Genetics 90:601-606.

Hartl, L.; Weiss, H.; Zeller, F.J. and Jahoor, A. (1993). Use of RFLP markers for the identification of alleles of the Pm3 locus conferring powdery mildew resistance in wheat (Triticum aestivum L.). Theoretical and Applied Genetics 86:959-963.

Harvey, A.; Moisan, L.; Lindup, S. and Lonsdale, D. (1999). Wheat regenerated from scutellum callus as a source of material for transformation. Plant Cell Tissue and Organ Culture 57:153-156.

Hauptmann, R.M.; Vasil, V.; Ozia-Akins, P.; Tabaeizadeh, Z.; Rogers, S.G.; Fraley, R.T.; Horsch, R.B. and Vasil, I.K. (1988). Evaluation of selectable markers for obtaining stable transformants in the Gramineae. Plant Physiology 86:602-606.

He, G.Y.; Rooke, L.; Steele, S.; Bekes, F.; Gras, P.; Tatham, A.S.; Fido, R.; Barcelo, P.; Shewry, P.R. and Lazzeri, P.A. (1999). Transformation of pasta wheat (Triticum turgidum L. var durum) with high-molecular weight glutenin subunit genes and modification of dough functionality. Molecular Breeding 5:377-386.

He, G.Y. and Lazzeri, P.A. (1998). Analysis and optimization of DNA delivery into wheat scutellum and tritordeum inflorescence explants by tissue elctroporation. Plant Cell Reports 18:64-70.

He, D.G.; Mouradev, A.; Yang, Y.M.; Mouradeva, E. and Scott, K.J. (1994). Transformation of wheat (Triticum aestivum L.) through electroporation of protoplasts. Plant Cell Reports 14:192-196.

He, S.; Ohm, H. and Mackenzie, S. (1992). Detection of DNA sequence polymorphisms among wheat varieties. Theoretical and Applied Genetics 84:573-578.

Hess, D.; Dressler, K. and Nimmrichter, R. (1990). Transformation experiments by pipetting Agrobacterium into the spikelets of wheat (Triticum aestivum L.). Plant Science 72:233-244.

Hiei, Y.; Ohta, S.; Komari, T. and Kumashiro, T. (1994). Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant Journal 6:271-282.

Hobbs, S.L.A.; Warkentin, T.D. and DeLong, M.O. (1993). Transgene copy number can be positively or negatively associated with transgene expression. Plant Molecular Biology 21:17-26.

Iglesias, V.A.; Gisel, A.; Bilang, R.; Leduc, N.; Potrykus, I. and Sautter, C. (1994). Transient expression of visible marker genes in meristem cells of wheat after biollistic micro-targeting. Planta 192:84-91.

Ingram, H.M.; Power, J.B.; Lowe, K.C. and Davey, M.R. (1999). Optimisation of procedures for microprojectile bombardment of microspore-derived embryos in wheat. Plant Cell Tissue Organ Culture 57:207-210.

Ishida, Y.; Saito, H.; Ohta, S.; Hiei, Y.; Komari, T. and Kumashiro, T. (1996). High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nature Biotechnology 14:745-750.

Jefferson, R.A.; Kavanagh, T.A. and Bevan, M.W. (1987). GUS fusions: b -glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO Journal 6:3901-3907.

Jordan, M.C. (2000). Green fluorescent protein as a visual marker for wheat transformation. Plant Cell Reports 19:1069-1075.

Karunaratne, S.; Sohn, A.; Mouradev, A.; Janet, S.; Steinbib, H.H. and Scott, K.J. (1996). Transformation of wheat with the gene encoding the coat protein of barley yellow mosaic virus. Australian Journal of Plant Physiology 23:429-435.

Keller, B. and Feuillet, C. (2000). Colinearity and gene density in grass genome. Trends in Plant Science 5:246-251.

Khan, A.A.; Bergstrom, G.C.; Nelson, J.C. and Sorrells, M.E. (2000). Identification of RFLP markers for resistance to wheat spindle streak mosaic bymo virus (WSSMV) disease. Genome 43:477-82.

Khurana, P.; Sehgal, A. and Chugh, A. (1998). Transgene interactions in transgenics. In: Srivastava, P.S., ed. Plant Tissue Culture and Molecular Biology-Applications and Prospects. Narosa Publishing House, New Delhi, India. pp. 728-750.

Klein, T.M. and Jones, T.J. (1999). Methods of genetic transformation: The gene gun. In: Vasil, I.K. ed. Molecular Improvement of Cereal Crops. Kluwer Academic Publishers, Dordrecht, The Netherlands. pp. 21-42.

Klein, T.M.; Wolf, E.D.; Wu, R. and Sanford, J.C. (1987). High-velocity microprojectiles for delivering nucleic acids into living cells. Nature 327:70-73.

Kloti, A.; Iglesias, V.A.; Wijnn, J.; Burkhardt, P.K.; Datta, S.K. and Potrykus, I. (1993). Gene transfer by electroporation into intact scutellum cells of wheat embryos. Plant Cell Reports 12:671-675.

Korzun, V.; Röder, M.; Worland, A.J. and Borner, A. (1997). Intrachromosomal mapping of genes for dwarfing (Rht12) and vernalization response (Vrn1) in wheat by using RFLP and microsatellite markers. Plant Breeding 116:227-232.

Lagudah, E.S.; Moullet, O. and Appels, R. (1997). Map-based cloning of a gene sequence encoding a nucleotide-binding domain and a leucine-rich region at the Cre3 nematode resistance locus of wheat. Genome 40:659-665.

Laufs, J.; Wirtz, U.; Kammann, M.; Matzeit, V.; Schafer, S.; Schell, J.; Czernilofsly, A.P.; Baker, B. and Gronenborn, B.H. (1990). Wheat dwarf virus Ac/Ds vectors: Expression and excision of transposable elements introduced into various cereals by a viral replicon. Proceedings of the National Academy of Sciences of the United States of America 87:7752-7756.

Leckband, G. and Lorz, H. (1998). Transformation and expression of a stilbene synthase gene of Vitis vinifera L. in barley and wheat for increased fungal resistance. Theoretical and Applied Genetics 96:1004-1012.

Lee, B.; Murdoch, K.; Topping, J.; Kreis, M. and Jones, M.G.K. (1989). Transient gene expression in aleurone protoplasts isolated from developing caryopses of barley and wheat. Plant Molecular Biology 13:21-29.

Li, Z.; Mouille, G.; Kosar-Hashemi, B.; Rahman, S.; Clarke, B.; Gale, K.R.; Appels, R. and Morell, M.K. (2000). The structure and expression of the wheat starch synthase III gene. Motifs in the expressed gene define the lineage of the starch synthase III gene family. Plant Physiology 123:613-624.

Li, Z.; Chu, X.; Mouille, G.; Yan, L.; Kosar-Hashemi, B.; Hey, S.; Napier, J.; Shewry, P.; Clarke, B.; Appels, R.; Morell, M.K. and Rahman, S. (1999). The localization and expression of the class II starch synthases of wheat. Plant Physiology 120:1147-1156.

Liu, Z.; Sun, Q.; Ni, Z. and Yang, T. (1999). Development of SCAR markers linked to the Pm21 gene conferring resistance to powdery mildew in common wheat. Plant Breeding 118:215-219.

Loeb, T.A. and Reynolds, T.L. (1994). Transient expression of the uidA gene in pollen embryoids of wheat following microprojectile bombardment. Plant Science 104:81-91.

Lonsdale, D.M.; Lindup, S.; Moisan, L.J. and Harvey A.J. (1998). Using firefly luciferase to identify the transition from transient to stable expression in bombarded wheat scutellar tissue. Physiologia Plantarum 102:447-453.

Lorz, H.; Baker, B. and Schell, J. (1985). Gene transfer to cereal cells mediated by protoplast transformation. Molecular and General Genetics 199:178-192.

Luo, Z. and Wu, R. (1989). A simple method for the transformation of rice via the pollen tube pathway. Plant Molecular Biology Reporter 7:69-77.

Ma, Z.Q.; Sorrells, M.E. and Tanksley, S.D. (1994). RFLP markers linked to powdery mildew resistance genes Pm1, Pm2, Pm3 and Pm4 in wheat. Genome 37:871-875.

Mahalakshmi, A.; Chugh, A. and Khurana, P. (2000). Exogenous DNA uptake via cellular perneabilization and expression of foreign gene in wheat zygotic embryos. Plant Biotechnogy 17:235-240.

Mahalakshmi, A. and Khurana, P. (1997). Agrobacterium-mediated cereal transformation: A critical appraisal. Indian Journal of Experimental Biology 35:416-426.

Mahalakshmi, A. and Khurana, P. (1995). Agrobacterium-mediated gene delivery in various tissues and genotypes of wheat (Triticum aestivum L.). Journal of Plant Biochemistry and Biotechnology 4:55-59.

Mahalakshmi, A., Maheshwari, S.C. and Khurana, P. (1993). High frequency divisions in leaf base protoplasts of wheat (Triticum aestivum L.). Journal of Plant Biochemistry and Biotechnology 2:61-65.

Maheshwari, N.; Rajyalakshmi, K.; Baweja, K.; Dhir, S.K.; Chowdhury, C.N. and Maheshwari S.C. (1995). In vitro culture of wheat and genetic transformation: Retrospect and Prospect. Critical Reviews in Plant Science 14:149-178.

Marks, M.S.; Kemp, J.M.; Woolston, C.J. and Dale, P.J. (1989). Agroinfection of wheat: A comparision of Agrobacterium strains. Plant Science 63:247-256.

McCormac, A.C.; Wu, H.; Bao, M.; Wang, Y.; Xu, R.; Elliot, M.C. and Chen. D.F. (1998). The use of visual marker genes as cell-specific reporters of Agrobacterium-mediated T-DNA delivery to wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.). Euphytica 99:17-25.

Meyer, P. (1995). Understanding and controlling transgene expression. Trends in Biotechnology 13:332-337.

Mi, Q.L.; Velazhahan, R.; Muthukrishnan, S.; Liang, G.H. (2000). Wheat transformation mediated by Agrobacterium tumefaciens. Annual Wheat Newsletter 45 [online]. Available from: http://wheat.pw.usda.gov/ggpages/awn/45/Textfiles/USKS.html#anchor367934

Mooney, P.A. and Goodwin, P.B. (1991). Adherence of Agrobacterium tumefaciens to the cells of immature wheat embryos. Plant Cell Tissue Organ Culture 25:199-208.

Mooney, P.A.; Goodwin, P.B.; Dennis, E.S. and Llewelleyn, D.J. (1991). Agrobacterium tumefaciens-gene transfer into wheat tissues. Plant Cell Tissue Organ Culture 25:209-218.

Mu, H.M.; Liu, S.J.; Zhou, W.J.; Wen, Y.X.; Zhang, W.J. and Wei, R.X. (1999). Transformation of wheat with insecticide gene of arrowhead proteinase inhibitors by pollen tube pathway and analysis of transgenic plants. I Chuan Hsueh Pao (Chinese) 26:634-642.

Muller, E.; Lorz, H. and Lutticke, S. (1996). Variabilty of transgene expression in clonal cell lines of wheat. Plant Science 114:71-82.

Myburg, A.A.; Cawood, M.; Wingfield, B.D. and Botha, A.M. (1998). Development of RAPD and SCAR markers linked to the Russian wheat aphid resistance gene Dn2 in wheat. Theoretical and Applied Genetics 96:1162-1169.

Nehra, N.S.; Chibbar, R.N.; Leung, N.; Caswell, K.; Mallard, C.; Steinhauer, L.; Baga, M. and Kartha, K.K. (1994). Self-fertile transgenic wheat plants regenerated from isolated scutellar tissues from microprojectile bombardment with two distinct gene constructs. Plant Journal 5:285-297.

Oard, J.H.; Paige, D.V.; Simmonds, J.A. and Gradziel, T.M. (1990). Transient gene expression in maize, rice and wheat cells using an air gun apparatus. Plant Physiology 92:334-339.

Oard, J.H.; Paige, D. and Dvorak, J. (1989). Chimeric gene expression using maize intron in cultured cells of breadwheat. Plant Cell Reports 8:156-160.

Ortiz, J.P.A.; Reggiardo, M.I.; Ravizzini, R.A.; Altabe, S.G.; Cervigni, G.D.L.; Spitteler, M.A.; Morata, M.M.; Elias, F.E. and Vallejos, R.H. (1996). Hygromycin resistance as an efficient selectable marker for wheat stable transformation. Plant Cell Reports 15:877-881.

Ou-Lee, T.M.; Turgeon, R. and Wu, R. (1986). Expression of a foreign gene linked to either a plant virus or a Drosophila promoter, after electroporation of protoplasts of rice, wheat and sorghum. Proceedings of the National Academy of Science of the United States of America 83:6815-6819.

Pang, S.; DeBoer, D.L.; Wan, Y.; Ye, G.; Layton, L.G.; Neher, M.K.; Armstrong, C.L.; Fry, J.E.; Hinchee, M.A.W. and Fromm, M.E. (1996). An improved green fluorescent protein as a vital marker in plants. Plant Physiology 112:893-900.

Parker, G.D.; Chalmers, K.J.; Rathjen, A.J. and Langridge, P. (1999). Mapping loci associated with milling yield in wheat (Triticum aestivum L.). Molecular Breeding 5:561-568.

Parker, G.D.; Chalmers, K.J.; Rathjen, A.J. and Langridge, P. (1998). Mapping loci associated with flour colour in wheat (Triticum aestivum L.). Theoretical and Applied Genetics 97:238-245.

Perl, A.; Kless, H.; Blumenthal, A.; Galili, G. and Galun, E. (1992). Improvement of plant regeneration and GUS expression in scutellar wheat calli by optimization of culture conditions and DNA-microprojectile delivery procedures. Molecular and General Genetics 235:279-284

Pingali, P.L. and Rajaram, S. (1999). Global wheat research in a changing world: Options for sustaining growth in wheat productivity. In: Cimmyt. World Wheat Facts and Trends. D.F., Mexico. pp. 1-18.

Procunier, J.D.; Knox, R.E.; Bernier, A.M.; Gray, M.A. and Howes, N.K. (1997). DNA markers linked to a T10 loose smut resistance gene in wheat (Triticum aestivum L.). Genome 40:176-179.

Qi, L.; Cao, M.; Chen, P.; Li, W. and Liu, D. (1996). Identification, mapping and application of polymorphic DNA associated with resistance gene Pm21 of wheat. Genome 39:191-197.

Rasco-Gaunt, S.; Riley, A.; Barcelo, P. and Lazzeri, P.A. (1999). Analysis of particle bombardment parameters to optimise DNA delivery into wheat tissues. Plant Cell Reports 19:118-127.

Razin, A. (1998). CpG methylation, chromatin structure and gene silencing- the three way interacion. EMBO Journal 7:4905-4908.

Roder, M.S.; Plaschke, J.; Konig, S.U.; Borner, A.; Sorrells, M.E.; Tanksley, S.D. and Ganal, M.W. (1995). Abundance, variability and chromosomal location of microsatellites in wheat. Molecular and General Genetics 246:327-333.

Rooke, L.; Bekes, F.; Fido, R.; Barro, F.; Gras, P.; Tatham, A.S.; Barcelo, P.; Lazzeri, P. and Shewry, (1999). Overexpression of a gluten protein in transgenic wheat results in greatly increased dough strength. Journal of Cereal Science 30:115-120.

Roy, J.K.; Prasad, M.; Varshney, R.K.; Balyan, H.S.; Blake, T.K.; Dhaliwal, H.S.; Singh, H.; Edwards, K.J. and Gupta, P.K. (1999). Identification of a microsatellite on chromosomes 6B and a STS on 7D of bread wheat showing an association with preharvest sprouting tolerance. Theoretical and Applied Genetics 99:336-340.

Sautter, C.; Waldner, H.; Neuhaus-Url, G.; Galli, A.; Neuhaus, G. and Potrykus, I. (1991). Micro-targeting: high efficiency gene transfer using a novel approach for the acceleration of micro-projectiles. Biotechnology 9:1080-1085.

Schachermayr, G.M.; Messmer, M.M.; Feuillet, C.; Winzeler, H.; Winzeler, M. and Keller, B. (1995). Identification of molecular markers linked to the Agropyron elongatum-derived leaf rust resistance gene Lr24 in wheat. Theoretical and Applied Genetics 90:982-990.

Schachermayr, G.M.; Siedler, H.; Gale, M.D.; Winzeler, H.; Winzeler, M. and Keller, B. (1994). Identification and localization of molecular markers linked to the Lr9 leaf rust resistance gene of wheat. Theoretical and Applied Genetics 88:110-115.

Schmidt, R. (2000). Synteny: recent advances and future prospects. Current Opinion in Plant Biology 3:97-102.

Serik, O.; Ainur, I.; Murat, K.; Tetsuo, M. and Masaki, I. (1996). Silicon carbide fiber-mediated DNA delivery into cells of wheat (Triticum aestivum L.) mature embryos. Plant Cell Reports 16:133-136.

Seyfarth, R.; Feuillet, C.; Schachermayr, G.; Winzeler, M. and Keller, B. (1999). Development of a molecular marker for the adult plant leaf rust resistance gene Lr35 in wheat. Theoretical and Applied Genetics 99:554-560.

Shah, D.M. (1997). Genetic engineering for fungal and bacterial diseases. Current Opinion in Biotechnology 8:208-214.

Sharma, H.C.; Sharma, K.K.; Seetharama, N. and Ortiz, R. Prospects for using transgenic resistance to insects in crop improvement. EJB Electronic Journal of Biotechnology [online]. 15 August 2000, Available from: http://www.ejbiotechnology.info/content/vol3/issue2/full/3 ISSN 0717-3458.

Shewry, P.R. and Lazzeri, P. (1997). Molecular approaches to wheat quality improvement. Chemistry and Industry 21:559-562.

Shimada, T.; Seki, M. and Morikawa, H. (1991). Transient expression of glucuronidase (Gus) gene in wheat pollen embryos via microprojectile bombardment. Wheat Information Service 72:106-108.

Shimoni, Y.; Blechl, A.E.; Anderson, O.D. and Galili, G. (1997). A recombinant protein of two high molecular weight glutenins alters gluten polymer formation in transgenic wheat. Journal of Biological Chemistry 272:15488-15495.

Simmonds, J.; Stewart, P. and Simmonds, D. (1992). Regeneration of Triticum aestivum apical explants after microinjection of germ line progenitor cells with DNA. Physiologia Plantarum 85:197-206.

Sivamani, E.; Bahieldin, A.; Wraith, J.M.; Al-Niemi, T.; Dyer, W.E.; Ho, T.D.H. and Qu, R. (2000). Improved biomass productivity and water use efficiency under water deficit conditions in transgenic wheat constitutively expressing the barley HVA1 gene. Plant Science 155:1-9.

Slattery, C.J.; Kavakli, I.H.; Okita, T.W. (2000). Engineering starch for increased quantity and quality. Trends in Plant Science 5:291-298.

Sorokin, A.P.; Ke, X.Y.; Chen, D.F. and Elliott, M.C. (2000). Production of fertile transgenic wheat plants via tissue electroporation. Plant Science 156:227-233.

Srivastava, V.; Anderson, O.D. and Ow, D.W. (1999). Single-copy transgenic wheat generated through the resolution of complex integration patterns. Proceedings in National Academy of Science of the United States of America 96:11117-11121.

Srivastava, V.; Vasil, V. and Vasil, I.K. (1996). Molecular characterization of the fate of transgenes in transformed wheat (Triticum aestivum L.). Theoretical and Applied Genetics 92:1031-1037.

Stachel, M.; Lelley, T., Grausgruber H. and Vollman, J. (2000). Application of microsatellites in wheat (Triticum aestivum L.) for studying genetic differentiation caused by selection for adaptation and use. Theoretical and Applied Genetics 100:242-248.

Stoger, E.; Vaquero, C.; Torres, E.; Sack, M.; Nicholson, L.; Drossard, J.; Williams, S.; Keen, D.; Perrin, Y.; Christou, P. and Fischer, R. (2000). Cereal crops as viable production and storage systems for pharmaceutical scFv antibodies. Plant Molecular Biology 42:583-590.

Stoger, E.; Williams, S.; Christou, P.; Down, R.E. and Gatehouse, J.A. (1999). Expression of the insecticidal lectin from snowdrop (Galanthus nivalis agglutinin; GNA) in transgenic wheat plants: Effect on predation by the grain aphid Sitobion avenae. Molecular Breeding 5:65-73.

Sundaresan, V. (1996). Horizontal spread of transposon mutagensis: New uses of old elements. Trends in Plant Sciences 1:184-190.

Takumi, S.; Murai, K.; Mori, N. and Nakamura, C. (1999). Trans-activation of a maize Ds transposable element in transgenic wheat plants expressing the Ac transposase gene. Theoretical and Applied Genetics 98:947-953.

Takumi, S. (1996). Hygromycin-resistant calli generated by activation and excision of maize Ac/Ds transposable elements in diploid and hexaploid wheat cultured cell lines. Genome 39:1169-1175.

Takumi, S., and Shimada, T. (1996) Production of trangenic wheat through particle bombardment of scutellar tissue: frequency is influenced by culture duration. Journal of Plant Physiology 149:418-423.

Takumi, S.; Otani, M. and Shimada, T. (1994). Effect of six promoter-intron combinations on transient reporter gene expression in einkorn, emmer and common wheat cells by particle bombardment. Plant Science 103:161-166.

Tanksley, S.D.; Young, N.D.; Paterson, A.H. and Bonierbale, M.W. (1989). RFLP mapping in plant breeding: New tools for an old science. Biotechnology 7:257-264.

Tao, W.J.; Liu, J.Y.; Liu, D.J. and Chen, P.D. (1999). Identification of molecular markers linked to the Pm6 gene conferring powdery mildew resistance in wheat. I Chuan Hsueh Pao (Chinese) 26:649-656.

Töpfer, R.; Gronenborn, B.; Schell, J. and Steinbiss, H.H. (1989). Uptake and transient expression of chimeric genes in seed derived embryos. Plant Cell 1:133-139.

Uze, M.; Potrykus, I. and Sautter, C. (1999). Single-stranded DNA in the genetic transformation of wheat (Triticum aestivum L.): Transformation frequency and integration pattern. Theoretical and Applied Genetics 99:487-495.

Vaccino, P.; Accerbi, M. and Corbellini, M. (1993). Cultivar identification in T. aestivum using highly polymorphic RFLP probes. Theoretical and Applied Genetics 86:833-836.

Vasil, I.K. and Vasil, V. (1999). Transgenic cereals: Triticum aestivum (wheat). In: Vasil, I.K., ed. Molecular Improvement of Cereal Crops. Kluwer Academic Publishers Dordrecht, The Netherlands. pp. 137-147.

Vasil, I.K., and Anderson, O.D. (1997). Genetic engineering of wheat gluten. Trends in Plant Sciences 2:292-297.

Vasil, V.; Srivastava, V.; Castillo, A.M.; Fromm, M.E. and Vasil, I.K. (1993). Rapid production of transgenic wheat plants by direct bombardment of cultured immature embryos. Biotechnology 11:1553-1558.

Vasil, V.; Castillo, A.M.; Fromm, M.E. and Vasil, I.K. (1992). Herbicide resistant fertile transgenic wheat plants obtained by microprojectile bombardment of regenerable embryogenic callus. Biotechnology 10:667-674.

Vasil, V.; Brown, S.M.; Re, D.; Fromm, M.E. and Vasil, I.K. (1991). Stably transformed callus lines from microprojectile bombardment of cell suspension cultures of wheat. Biotechnology 9:743-747.

Venter, E. and Botha, A.M. (2000). Development of markers linked to Diuraphis noxia resistance in wheat using a novel PCR-RFLP approach. Theoretical and Applied Genetics 100:965-970.

Wang, Y.C.; Klein, T.M.; Fromm, M.; Cao, J.; Sanford, J.C. and Wu, R. (1988). Transient expression of foreign genes in rice, wheat and soybean cells following particle bambardment. Plant Molecular Biology 11:433-439.

Weeks, T. (2000). Transformation of wheat with the cyanamide hydratase gene (09/518,988). Patent. U.S. Department of Agriculture. Agricultural Research Service. Available on world wide web. http://ott.arsusda.gov/Inv/a518988.html

Weeks, J.T.; Anderson, O.D. and Blechl, A.E. (1993). Rapid production of multiple independent lines of fertile transgenic wheat (Triticum aestivum). Plant Physiology 102:1077-1084.

Werr, W. and Lorz, H. (1986). Transient gene expression in a Gramineae cell line. Molecular and General Genetics 202:471-475.

Williams, K.J.; Fisher, J.M. and Langridge, P. (1996). Development of a PCR-based allele-specific assay from an RFLP probe linked to resistance to cereal cyst nematode in wheat. Genome 39:798-801.

Williams, J.G.K.; Kubelik, A.R.; Livak, K.L.; Rafalsks, J.A. and Tinget, S.L. (1990). DNA polymorphism amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research 18:6531-6535.

Witrzens, B.; Brettell, R.I.S.; Murray, F.R.; McElroy, D.; Li, Z. and Dennis, E.S. (1998). Comparison of three selectable marker genes for transformation of wheat by microprojectile bombardment. Australian Journal of Plant Physiology 25:39-44.

Woolston, C.J.; Barker, R.; Gunn, H.; Boulton, M.I. and Mullineaux, P.M. (1988). Agroinfection and nucleotide sequence of cloned wheat dwarf virus DNA. Plant Molecular Biology 11:35-43.

Yang, Y.; Shah, J. and Klessig, D.F. (1997). Signal perception and transduction in plant defense responses. Genes and Development 11:1621-1639.

Zaghmout, O.M.F. (1994). Transformation of protoplasts and intact cells from slowly growing embryogenic callus of wheat (Triticum aestivum L.). Theoretical and Applied Genetics 89:577-582.

Zaghmout, O.M. and Trolinder, N.L. (1993). Simple and efficient method for directly electroporating Agrobacterium plasmid DNA into wheat callus cells. Nucleic Acids Research 21:1048.

Zhang, L.; Rybczynski, J.J.; Langenberg, W.G.; Mitra, A. and French, R. (2000). An efficient wheat transformation procedure: transformed calli with long-term morphogenic potential for plant regeneration. Plant Cell Reports 19:241-250.

Zhou, H.; Arrowsmith, J.W.; Fromm, M.E.; Hironaka, C.M.; Taylor, M.L.; Rodriguez, D.; Pajeay, M.E.; Brown, S.M.; Santino, C.G. and Fry, J.E. (1995). Glyphosate-tolerant CP4 and GOX genes as a selectable marker in wheat transformation. Plant Cell Reports 15:159-163.

Zhou, H.; Stiff, C.M. and Konzak, C.F. (1993). Stably transformed callus of wheat by electroporation-induced direct gene transfer. Plant Cell Reports 12:612-616.

Zhu, Z.; Sun, B.; Liu, C.; Xiao, G. and Li, X. (1993). Transformation of wheat protoplasts mediated by cationic liposome and regeneration of transgenic plantlets. Chinese Journal of Biotechnology 9:257-261.

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