Role of Biotechnology to Wheat Improvement

Wheat is characterized by a large genome size, thus making the improvement process by any method genetically challenging. 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. Scientists have placed a number of new tools into the hands of the plant breeder that enable him to: (a) Increase the amount of variability within the breeding population, (b) Increase the level of understanding of the factors involved with variability, (c) Introgress novel and valuable genes into the breeding population (d) And Increase the efficiency of the selection process. These diverse and often powerful tools can be placed on the shelf termed biotechnology. Biotechnology can be defined in many different ways, but for the purposes of wheat, all areas that use molecular and/or chemical approaches in order to manipulate or study the wheat genome will be considered biotechnology.

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. 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 biolistic approach has been most successful in delivering foreign genes into wheat. Different techniques used for transformation are:

    

Electroporation of protoplasts. Electroporation of organized tissues. Particle bombardment. Agrobacterium-mediated transformation. Alternative approaches (microinjection, direct imbibition, permeabilization, silicon carbide fibermediated and pollen tube pathway).

Introduction of useful traits by genetic transformation

1. 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 1. Increasing the protein content. 2. Increasing essential amino acids such as lysine. 3. Increasing the high molecular weight (HMW) glutenins to improve bread making properties of wheat flour. 4. Modifying starch composition. 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. 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. 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. 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. This work may thus open newer avenues for a wider applicability of wheat.

2. Engineering nuclear male sterility: The production of hybrids is an essential component of crop breeding programs but till date, hybrid wheat has remained elusive! The development of a suitable hybridization 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 ribonucleaseinhibitor 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. Expressing the barnase gene at specific stages of anther development destroys the tapetum, thereby preventing normal pollen development and causes pollen sterility.

3. Resistance to biotic stress: Most of the works on genetic engineering of wheat for resistance against biotic stress have focussed on developing protection against fungal pathogens . Introduction genes encoding for chitinases from barley resulted in increased resistance against Erysiphe graminis. 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. An increase in endogenous resistance against Tilletia tritici was achieved with the introduction of virally encoded antifungal protein. 4. 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. 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.

Gene mapping:
1. Wheat molecular linkage maps 2. Mapping of single or major genes 3. Quantitative trait mapping

Marker assisted selection in wheat breeding

MAS is one of the most widely used applications of molecular marker technologies. Biotechnology has provided additional tools that do not require the use of transgenic crops to revolutionize plant breeding progress. Molecular genetics has resulted in the development of DNA tags and marker assisted selection strategies for cultivar development. 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 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). Initial studies on the application of molecular markers in wheat relied on the hybridization 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. 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. RAPD markers provide a useful alternative to RFLP analysis for screening markers linked to a single trait within near isogenic lines and bulked segregants. 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. 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. 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. 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.

Using the Extended Gene Pool

Wide crosses, those involving a member of the primary and a member of the secondary or tertiary gene pools, are often marked by embryo abortion at certain stages of development following successful fertilization. This is due to improper development of endosperm in many cases. In order to avoid abortion and allow further growth and development of embryos a technique known as embryo rescue is used. Abortion of embryos at one or the other stage of development is a characteristic feature of crossing durum or bread wheat with species found in the tertiary and quaternary gene pools. Embryo rescue is the process where inherently weak, immature or hybrid embryos are rescued from degeneration. This technique also falls in the scope of biotechnology. Colchicine treatment to double the chromosome number is necessary to restore fertility of the hybrid plants because of an inability of the chromosome pairing between wheat and secondary/ tertiary species during meiosis. Colchicine treatment is used in the production of synthetic wheat varieties as well as in double-haploid production.

Synthetic Hexaploid Wheat

In recent times there has been a growing concern about the narrow genetic basis from which modern wheat cultivars are derived, as breeders generally use advanced breeding material in order to accelerate development of new cultivars. In order to alleviate this genetic bottleneck and to introgress diverse genes for resistance to biotic and abiotic stress; wheat breeders, including those at CIMMYT, have put forth an effort into utilizing a wider genetic base through interspecific hybridization. About 325 perennial and annual grasses are found within the Triticeae tribe. Although many of these grasses have been successfully hybridized with wheat, most success (in terms of easiness and speed for utilization in wheat breeding program) has been found when crossing Triticum turgidum (AABB) with Aegilops tauschii (DD) to form what is known as synthetic hexaploid wheat. To date, CIMMYT has produced around 1,100 synthetic wheat lines many of which are being used to introgress novel genes into advanced breeding lines. Of these lines 95 have been included in elite set one as these lines are considered to be agronomically good, and 34 have been included in elite set 2 as these lines are considered to hold a variety of stress resistances. Synthetic hexaploids are not produced for direct utilization as they often hold traits that are of a disadvantage to agricultural production such as tall height, lateness in maturity and difficult to thresh. Ideally synthetic hexaploid wheat should be used as bridges for easier introgression of traits from Ae. tauschii into bread wheat populations.

Doubled Haploid Production

There are a number of methods that the wheat breeder can use in order to produce doubled haploids. The production of double haploids with the help of modern tools reduces the time of breeding cycle.

Mutation Breeding
The FAO/IAEA database reports 200 Triticum aestivum and 30 Triticum duram mutant cultivars that have been released for use. Improved traits, due to induced mutations, held in these cultivars include: disease

resistance, drought tolerance, winter hardiness, earliness, lodging resistance, reduced height, increased seed size, grain color, and yield among others. It is estimated that three mutant derived bread wheat cultivars in Pakistan (Jaukar 78, Soghat 90, and Kiran 95) contributed to a combined increase of farmer income of $87.1 million U.S. over a period of nine years from 1991 to 1999. Screening for mutants has often in the past been carried out by visual means. With the advent of other biotechnology techniques, it is now possible to screen for changes in the genome of specific targets through the use of molecular techniques. The most successful uses of mutation breeding will complement conventional methods. Often a mutant genotype will not be directly usable as a cultivar but may hold a novel allele variant that is valuable and can be introgressed into the breeding population.