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.

Among the food crops, wheat is one of the most abundant source of energy for the world population. The genetic improvement of wheat has received considerable attention over the years from plant breeders with the purpose of increasing the grain yield, to minimize crop loss due to unfavourable environmental conditions, and attack by various pests and pathogens. The development of in vitro technologies have complemented the conventional methods of wheat breeding in generating genetic variability necessary for creating novel cultivars with desirable characters. Wheat is characterised by a large genome size thus making the improvement process by any method genetically challenging.

In recent years, biotechnology is emerging as one of the latest tools of agricultural research. Biotechnology involves the systematic application of biological processes for the beneficial use of mankind. 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. The introduction of foreign genes encoding for resistance against pests and pathogens can reduce the degradation of the environment due to the use of hazardous biocides.

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 steps for genetic transformation involves delivery of a gene cassette into recipient cells followed by analysis of expression of the 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. The gus gene of E. coli encoding for a ß-glucuronidase is most widely used as a reporter gene. To study the fate of introduced transgenes in living cells, vital reporter genes encoding for anthocyanin biosynthesis, green fluorescent protein and luciferase have also been used successfully.

The varied frequency of DNA delivery in cells of different explants requires the availabilty 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). The other selectable marker genes employed in wheat transformation confer resistance against aminoglycoside antibiotics [nptII (neomycin phosphotransferase), hpt (hygromycin phosphotransferase)] or herbicides [EPSPS (enolpyruvyl-shikimate-3-phosphate synthase) oxidoreductase, and glyphosate oxidoreductase]. Recently, the marker genes encoding for mannose-6-phosphate isomerase and cyanamide hydratase which confer ability to grow on mannose and cyanamide, respectively, have also been employed for achieving wheat transformation.

Initial attempts at introducing transgenes into wheat employed protoplasts as explants due to the absence of cell walls. To overcome the hindrance exercised by the plasma membrane, protoplasts are subjected to either chemical treatment [polyethylene glycol (PEG)] or 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 has been the most popular tissue of choice. 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, the technique of electroporation, initially utilized for the introduction of foreign genes into protoplasts, was recently extended for achieving stable transformation of organized tissues.

Subsequently, the development of methodology for the delivery of genes into intact plant tissues by bombardment of DNA-coated gold or tungsten particles revolutionized the field of plant transformation. This method of introducing DNA into cells by physical means was developed to overcome the biological limitations of Agrobacterium and the difficulties associated with plantlet regeneration from protoplasts. Particle bombardment-mediated transient gene expression of gus gene was achieved following bombardment of cell suspensions, leaf bases and apical tissues, immature embryos. First successful generation of transgenic wheat plants was achieved by particle bombardment of plasmid vector pBARGUS into cells long-term regenerable embryogenic callus. Improvements in procedures reduced the time required for production of transgenic wheat plants from an initial 12-15 months to 56-66 days. Later, most of the gene delivery studies employed immature embryos, isolated scutellum, and calli initiated from immature embryos as the target tissue for bombardment. Particle bombardment of explants like pollen embryos, microspore-derived embryos also resulted in successful introduction of transgenes as evidenced by transient expression of gus gene, however, regeneration of plantlets from these explants could not been reported. The transgenes introduced by biolistic approach display a considerable degree of stability in integration and expression in subsequent generations. Particle bombardment has thus emerged over the years as a reproducible method for the introduction of various marker genes in wheat and this method is successfully being used 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.

Agrobacterium-mediated transformation is a simple, low cost and highly efficient alternative to direct gene delivery methods. Significant progress made in the area of Agrobacterium-mediated transformation of rice and maize have contributed tremendously to an increasing understanding of various parameters necessary for the successful generation of transgenic cereals. The stable transformation of wheat by Agrobacterium-mediated approach has been possible by co-cultivating immaure embryos and embryogenic calli. In near future it can be expected that Agrobacterium will be employed as a more reliable, efficient and economical vector for the introduction of exogenous genes into wheat by various laboratories throughout the world.

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 with varied degrees of success for wheat transformation.

In addition to its basic calorific value, wheat with its high protein content is an important source of plant protein in the human diet. 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 mesh that endows the wheat dough with its unique properties. 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. 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. Modification of wheat starch is presently targeted in various laboratories to improve its potential utility. This increasing information and characterization of the various components of starch biosynthesis will enable researchers to create rational designs of novel starches and induce various alterations in starch levels in other crop plants as well.

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 the bioavailabilty of phosphate and minerals. The phyA gene from Aspergillus niger, encoding for the phytase enzyme has been successfully expressed in transgenic 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 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.

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. Engineering of nuclear male sterility in wheat has been undertaken 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.

Wheat is attacked by a number of viral, bacterial and fungal pathogens and also by insect and nematode pests. With the development of plant transformation techniques, newer avenues for creating disease resistant and insect resistant crops have been created. Most of the work on genetic engineering of wheat for resistance against biotic stress has focussed on developing protection against fungal pathogens. For the engineering of resistance against different pathogens in wheat, genes for viral coat proteins, antifungal proteins (ribosome inactivating protein, chitinases. thaumatin like protein, stilbene synthase, killer protein) have been successfully introduced. Introduction of canditate genes (lectin, proteinase inhibitor) for insect resistance into wheat has resulted in growth inhibition of insects on transgenic seeds, thereby decreasing the fecundity of insect population. Most of the introduced genes confer increased resistance to the corresponding pests and pathogens in the transgenic plants. Transgenic approach has been used for successfully introducing and overexpressing the barley HVA1 gene encoding for a late embryogenesis abundant protein into wheat by particle bombardment for drought tolerance.

Success at developing improved wheat cultivars through genetic engineering depends on stable and predictable expression of the inserted gene. The complex and the large genome size of wheat is expected to be prone to silencing of introduced transgenes. Studies have revealed that the transformation procedure, transgene integration, and marker gene expression has little effect on the transmission of transgenes to the subsequent progenies. The problem of gene silencing 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, etc.

Transposon mutagenesis has been widely exploited in various organisms to isolate genes that encode unidentified products. A transposon tagging system in wheat has been developed by introducing the Ac transposase gene under the CaMV 35S promoter into cultured wheat embryos by particle bombardment. Thus in near future we can expect traits of commercial importance to be tagged for a wider utility in important crop plants.

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. Initial studies on the application of molecular markers in wheat relied on the hybridisation based restriction fragment length polymorphism (RFLP) system. In wheat, RFLP's have been used to map seed storage protein loci, loci associated with flour colour, cultivar identification, vernalization and frost resistance gene, intrachromosomal mapping of genes for dwarfing and vernalization, resistance to preharvest sprouting, quantitative trait loci controlling tissue culture response, nematode resistance and milling yield. PCR based markers have been useful for characterization of genes for resistance against common bunt, powdery mildew, leaf rust, resistance against hessian fly, and Russian wheat aphid. 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 screening of subsequent generations.

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 environment or niche. The new tools of biotechnology thus not only have the potential for increasing the effectiveness and efficiency of wheat breeding programs, and but would also provide insights into the genetic control of key traits to be used for genetic manipulation. The coming years will undoubtedly witness an increasing application of biotechnology for the genetic improvement of wheat.