The technologies for genetic transformation of cereals

In this review, the analysis of the most widely used technologies for genetic transformation of cereals is presented. The required conditions for transformation, regeneration, and testing of cereal crops are discussed, and a new Agrobacterium tumefaciens-mediated transformation approach is presented. This review can be useful for genetic engineers, biotechnologists, and other specialists involved in the production or studies of transgenic plants.     

Genetic transformation technology: Status and problems

Summary Transfer of genes from heterologous species provides the means of selectively introducing new traits into crop plants and expanding the gene pool beyond what has been available to traditional breeding systems. With the recent advances in genetic engineering of plants, it is now feasible to introduce into crop plants, genes that have previously been inaccessible to the conventional plant breeder, or which did not exist in the crop of interest. This holds a tremendous potential for the genetic enhancement of important food crops. However, the availability of efficient transformation methods to introduce foreign DNA can be a substantial barrier to the application of recombinant DNA methods in some crop plants. Despite significant advances over the past decades, development of efficient transformation methods can take many years of painstaking research. The major components for the development of transgenic plants include the development of reliable tissue culture regeneration systems, preparation of gene constructs and efficient transformation techniques for the introduction of genes into the crop plants, recovery and multiplication of transgenic plants, molecular and genetic characterization of transgenic plants for stable and efficient gene expression, transfer of genes to elite cultivars by conventional breeding methods if required, and the evaluation of transgenic plants for their effectiveness in alleviating the biotic and abiotic stresses without being an environmental biohazard. Amongst these, protocols for the introduction of genes, including the efficient regeneration of shoots in tissue cultures, and transformation methods can be major bottlenecks to the application of genetic transformation technology. Some of the key constraints in transformation procedures and possible solutions for safe development and deployment of transgenic plants for crop improvement are discussed.     

Agrobacterium-mediated transformation of cereals: a promising approach crossing barriers

Cereal crops have been the primary targets for improvement by genetic transformation because of their worldwide importance for human consumption. For a long time, many of these important cereals were difficult to genetically engineer, mainly as a result of their inherent limitations associated with the resistance to Agrobacterium infection and their recalcitrance to in vitro regeneration. The delivery of foreign genes to rice plants via Agrobacterium tumefaciens has now become a routine technique. However, there are still serious handicaps with Agrobacterium -mediated transformation of other major cereals. In this paper, we review the pioneering efforts, existing problems and future prospects of Agrobacterium -mediated genetic transformation of major cereal crops, such as rice, maize, wheat, barley, sorghum and sugarcane.     

Strategies for developing marker-free transgenic plants

The development of marker-free transgenic plants has responded to public concerns over the safety of biotechnology crops. It seems that continued work in this area will soon remove the question of unwanted marker genes from the debate concerning the public acceptability of transgenic crop plants. Selectable marker genes are co-introduced with genes of interest to identify those cells that have integrated the DNA into their genome. Despite the large number of different selection systems, marker genes that confer resistance to the antibiotics, hygromycin (hpt) and kanamycin (nptII) or herbicide phosphinothricin (bar), have been used in most transgenic research and crop development techniques. The techniques that remove marker gene are under development and will eventually facilitate more precise and subtle engineering of the plant genome, with widespread applications in both fundamental research and biotechnology. In addition to allaying public concerns, the absence of resistance genes in transgenic plants could reduce the costs of developing biotechnology crops and lessen the need for time-consuming safety evaluations, thereby speeding up the commercial production of biotechnology crops. Many research results and various techniques have been developed to produce marker-free transgenic plants. This review describes the strategies for eliminating selectable marker genes to generate marker-free transgenic plants, focusing on the three significant marker-free technologies, co-transformation, site-specific recombinase-mediated excision, and non-selected transformation.     

Biotechnological advancement in genetic improvement of broccoli (<Emphasis Type="Italic">Brassica oleracea</Emphasis> L. var. <Emphasis Type="Italic">italica</Emphasis>), an important vegetable crop

With the advent of molecular biotechnology, plant genetic engineering techniques have opened an avenue for the genetic improvement of important vegetable crops. Vegetable crop productivity and quality are seriously affected by various biotic and abiotic stresses which destabilize rural economies in many countries. Moreover, absence of proper post-harvest storage and processing facilities leads to qualitative and quantitative losses. In the past four decades, conventional breeding has significantly contributed to the improvement of vegetable yields, quality, post-harvest life, and resistance to biotic and abiotic stresses. However, there are many constraints in conventional breeding, which can only be overcome by advancements made in modern biology. Broccoli (Brassica oleracea L. var. italica) is an important vegetable crop, of the family Brassicaceae; however, various biotic and abiotic stresses cause enormous crop yield losses during the commercial cultivation of broccoli. Thus, genetic engineering can be used as a tool to add specific characteristics to existing cultivars. However, a pre-requisite for transferring genes into plants is the availability of efficient regeneration and transformation techniques. Recent advances in plant genetic engineering provide an opportunity to improve broccoli in many aspects. The goal of this review is to summarize genetic transformation studies on broccoli to draw the attention of researchers and scientists for its further genetic advancement.     

Citrus biotechnology: Achievements,limitations and future directions

Citrus is one of the most important commercial and nutritional fruit crops in the world, hence it needs to be improved to cater to the diverse needs of consumers and crop breeders. Genetic manipulation through conventional techniques in this genus is invariably a difficult task for plant breeders as it poses various biological limitations comprising long juvenile period, high heterozygosity, sexual incompatibility, nucellar polyembryony and large plant size that greatly hinder cultivar improvement. Hence, several attempts were made to improve Citrus sps. by using various in vitro techniques. Citrus sps are widely known for their recalcitrance to transformation and subsequent rooting, but constant research has led to the establishment of improved protocols to ensure the production of uniformly transformed plants, albeit with relatively low efficiency, depending upon the genotype. Genetic modification through Agrobacterium-mediated transformation has emerged as an important tool for introducing agronomically important genes into Citrus sps. Somatic hybridization has been applied to overcome self and cross-incompatibility barriers and generated inter-specific and inter-generic hybrids. Encouraging results have been achieved through transgenics for resistance against viruses and bacteria, thereby augmenting the yield and quality of the fruit. Now, when major transformation and regeneration protocols have sufficiently been standardized for important cultivars, ongoing citrus research focuses mainly on incorporating such genes in citrus genotypes that can combat different biotic and abiotic stresses. This review summarizes the advances made so far in Citrus biotechnology, and suggests some future directions of research in this fruit crop.Key words: Citrus sinensis, Citrus tristeza virus, Citrus regeneration, Citrus transformation     

转基因作物将为我国农业发展注入新动力

随着基因组学研究和生物技术的不断突破,应用生物技术改良农作物品种正成为引发新的农业技术革命的关键。由于转基因品种在生产上表现出的巨大效益,其研发和产业化在全球迅猛发展,并逐渐成为世界各国抢占的生物技术制高点。本文从转基因技术的内涵、国际转基因作物的研发态势、我国转基因作物的研发和产业化现状等方面进行阐述,探讨了大力发展转基因作物对于解决我国目前农业生产上面临的挑战、保障我国粮食安全和农业可持续发展的重要意义。     

Resistance that stacks up: engineering rust and mildew disease control in the cereal crops wheat and barley

Staying ahead of the arms race against rust and mildew diseases in cereal crops is essential to maintain and preserve food security. The methodological challenges associated with conventional resistance breeding are major bottlenecks for deploying resistance (R) genes in high-yielding crop varieties. Advancements in our knowledge of plant genomes, structural mechanisms, innovations in bioinformatics, and improved plant transformation techniques have alleviated this bottleneck by permitting rapid gene isolation, functional studies, directed engineering of synthetic resistance and precise genome manipulation in elite crop cultivars. Most cloned cereal R genes encode canonical immune receptors which, on their own, are prone to being overcome through selection for resistance-evading pathogenic strains. However, the increasingly large repertoire of cloned R genes permits multi-gene stacking that, in principle, should provide longer-lasting resistance. This review discusses how these genomics-enabled developments are leading to new breeding and biotechnological opportunities to achieve durable rust and powdery mildew control in cereals.     

Soybean transformation by electric discharge particle acceleration

By the use of a direct DNA-delivery system based on electric discharge particle acceleration we have been able to obtain transgenic soybean plants expressing the neomycin phosphotransferase II and the β-glucuronidase genes. Techniques for efficient introduction of foreign genes into agronomically important crop plants have been limited by the inability to regenerate fertile plants from single cells or by the limited hostrange of Agrobacterium vectors. In the case of soybean ( Glycine max ) the problem has been compounded due to the lack of a regeneration method compatible with existing transformation technology. In a commercial genetic engineering program the following points need to be considered: (a) not all crops/cultivars can be regenerated from transformed tissues, (b) long time frames are required for the regeneration of transgenic plants from callus, (c) Agrobacterium has a rather limited host specificity and (d) problems associated with somaclonal variation.     

Crop improvement through tissue culture

Plant tissue culture comprises a set of in vitro techniques, methods and strategies that are part of the group of technologies called plant biotechnology. Tissue culture has been exploited to create genetic variability from which crop plants can be improved, to improve the state of health of the planted material and to increase the number of desirable germplasms available to the plant breeder. Tissue-culture protocols are available for most crop species, although continued optimization is still required for many crops, especially cereals and woody plants. Tissueculture techniques, in combination with molecular techniques, have been successfully used to incorporate specific traits through gene transfer. In vitro techniques for the culture of protoplasts, anthers, microspores, ovules and embryos have been used to create new genetic variation in the breeding lines, often via haploid production. Cell culture has also produced somaclonal and gametoclonal variants with crop-improvement potential. The culture of single cells and meristems can be effectively used to eradicate pathogens from planting material and thereby dramatically improve the yield of established cultivars. Large-scale micropropagation laboratories are providing millions of plants for the commercial ornamental market and the agricultural, clonally-propagated crop market. With selected laboratory material typically taking one or two decades to reach the commercial market through plant breeding, this technology can be expected to have an ever increasing impact on crop improvement as we approach the new millenium.D.C.W. Brown is with Agriculture and Agri-Food Canada, Central Experimental Farm, Plant Research Centre, Ottawa, Ontario, K1A 0C6, Canada. T.A. Thorpe is with the Plant Physiology Research Group, Department of Biological Sciences, University of Calgary, Calgary, Alberta, T2N 1N4, Canada     

Fast recovery of transgenic submergence tolerant rice cultivars of North-East India by early co-cultivation of <Emphasis Type="Italic">Agrobacterium</Emphasis> with pre-cultured callus

Agro-climatic conditions of North-East India are very complex and rice cultivars present in the region have been adapted to grow under harsh environmental conditions. Germplasm present in the region is considered to possess several important and unique traits that are of importance in rice improvement programs. Genetic engineering is a powerful tool to introduce new traits into crop plants. However, not much information is available on the methods to introduce foreign genes into North-East rice cultivars. Therefore, the main objective of this study is to develop transformation procedures for fast recovery of transgenic plants from North-East rice cultivars. To achieve this objective, a systematic study was carried out to identify media components and culture conditions for efficient embryogenic callus induction from the mature seeds and differentiation of callus into plantlets from two North-East deep water rice cultivars, Taothabi and Khongan. Also, role of preculture of callus on Agrobacterium-mediated transformation was studied. Co-cultivation of Agrobacterium with 1–5 days precultured callus was found to result in high frequency of transformation. Detailed characterization of transgenic lines confirmed stable integration of transgenes and expression of reporter gfp gene. The whole process starting from callus induction to regenerating of transgenic rice plants that can be established in the soil was achieved in about 35–45 days. The procedures developed were found to be applicable to a popular variety IR 64. Therefore, methods developed in this study should be useful not only to introduce new traits quickly but also to validate the function(s) of several candidate gene(s) identified under the functional genomics of rice.     

Nuclear and plastid genetic engineering of plants: Comparison of opportunities and challenges

Plant genetic engineering is one of the key technologies for crop improvement as well as an emerging approach for producing recombinant proteins in plants. Both plant nuclear and plastid genomes can be genetically modified, yet fundamental functional differences between the eukaryotic genome of the plant cell nucleus and the prokaryotic-like genome of the plastid will have an impact on key characteristics of the resulting transgenic organism. So, which genome, nuclear or plastid, to transform for the desired transgenic phenotype? In this review we compare the advantages and drawbacks of engineering plant nuclear and plastid genomes to generate transgenic plants with the traits of interest, and evaluate the pros and cons of their use for different biotechnology and basic research applications, ranging from generation of commercial crops with valuable new phenotypes to ‘bioreactor’ plants for large-scale production of recombinant proteins to research model plants expressing various reporter proteins.     

禾谷类作物抗倒性研究方法与谷子抗倒性评价

倒伏是植株与其生长的环境条件相互作用的结果,是禾谷类作物产量和品种的重要限制因素。诱发倒伏的自然条件不是年年发生,但发生的程度差异很大,难以在自然条件下重复评价作物的倒伏性,因而人工诱发倒伏对于作物抗倒性的评价十分重要。谷子是非常容易发生倒伏的禾谷类作物,抗倒性的强弱是决定谷子高产、稳产的关键因素之一。由于谷子抗倒性的研究相对较少,本文通过对禾谷类作物抗倒伏性研究方法和评价指标的述评,为谷子抗倒性的研究提供借鉴,并对谷子倒伏与植株和产量性状的关系、品种抗倒性评价的指标等相关研究的进展做一综述。     

Transformation of fruit trees. Useful breeding tool or continued future prospect?

Regeneration and transformation systems using mature plant material of woody fruit species have to be achieved as a necessary requirement for the introduction of useful genes into specific cultivars and the rapid evaluation of resulting horticultural traits. Although the commercial production of transgenic annual crops is a reality, commercial genetically-engineered fruit trees are still far from common. In most woody fruit species, transformation and regeneration of commercial cultivars are not routine, generally being limited to a few genotypes or to seedlings. The future of genetic transformation as a tool for the breeding of fruit trees requires the development of genotype-independent procedures, based on the transformation of meristematic cells with high regeneration potential and/or the use of regeneration-promoting genes. The public concern with the introduction of antibiotic resistance into food and the restrictions due to new European laws that do not allow deliberate release of plants transformed with antibiotic-resistance genes highlight the development of methods that avoid the use of antibiotic-dependent selection or allow elimination of marker genes from the transformed plant as a research priority in coming years     

Recent Progress in Model Grass Brachypodium distachyon (Poaceae)

Brachypodium distachyon, recently developed model system for temperate grasses, exhibited many traits with cereal crops and proposed to be an experimental system to access the biological approach. These traits have shown a surprised degree of phenotypic variation in many collected accessions. Like some important economical cereals, Bdistachyon also belongs to subfamily Pooideae, which make it become an unquestionable model system to research the economically important crops, such as wheat, barley and several potential biofuel plants. Recently, genome sequence and annotation of Bdistachyon has been finished. Associated with the development of the functional genomics and other experimental resources establishment, Bdistachyon will provide a key resource for improving cereal crops and facilitate the approach of sequence analyze gene expression and functional resources available for a variety of species. In this article we review and assess the current progress of Bdistachyon as a model system and then focus specifically on recent studies of comparative genomics, biological improvement, transformation and T DNA mutations.     

Production of insect-resistant transgenic rice plants for use in practical agriculture

Plant biotechnology provides a powerful solution to boost agricultural productivity and nutritional quality. The development process of a transgenic crop includes multiple steps that consist of gene isolation for a target trait, generation of T₀ transgenic crops, characterization of the transgene, evaluation of agronomic performance of transgenic crops, selection of elite transgenic lines and assessment of target trait efficacy. Here, we developed elite insect-resistant transgenic rice plants that may satisfy the standards of biosafety assessments. We made a construct with the insecticide cry1Ac gene for a target trait. A total of 310 T₀ transgenic lines were generated and underwent extensive analysis. We selected four T₃ lines that contain a single-copy transgene inserted into intergenic regions of the rice genome. During this process, we critically analyzed the transgenic lines with five checkpoints that include single copy of transgene, its integration into intergenic region, clean T-DNA arrangement, stability of transgene through generations and substantial equivalence of transgenic plants in agronomic traits other than insect resistance. Consequently, we obtained insect-resistant transgenic rice plants that can be used in practical agriculture.     

TILLING moves beyond functional genomics into crop improvement

Transgenic methods have been successfully applied to trait improvement in a number of crops. However, reverse genetics studies by transgenic means are not practical in many commercially important crops, hampering investigations into gene function and the development of novel and improved cultivars. A nontransgenic method for reverse genetics called Targeting Induced Local Lesions IN Genomes (TILLING) has been developed as a method for inducing and identifying novel genetic variation, and has been demonstrated in the model plant, Arabidopsis thaliana. Recently, TILLING has been extended to the improvement of crop plants and shows great promise as a general method for both functional genomics and modulation of key traits in diverse crops.