Stephanie Lütticke

Genetic improvement of wheat via alien gene transfer, an assessment

In recent years, with advent of the development of efficient plant regeneration systems in cereal crops, the field of recombinant DNA technology has opened up new avenues for genetic transformation of crop plants. Monocots particularly cereals were initially considered difficult to genetically engineer, primarily due to their recalcitrance to in vitro regeneration and their resistance to Agrobacterium. Continuous efforts and systematic screening of cultivars and tissues for regeneration potential, development of various DNA delivery methods and optimization of gene expression cassettes have led the development of reliable transformation protocols for the major cereals including wheat. Since the production of first fertile transgenic wheat plants in 1992s, microprojectile-mediated gene transfer has proved the most successful method for genetic transformation of wheat not only for the introduction of marker genes but also agronomically important genes for improving quality of wheat flour, transposon tagging, building resistance against fungal pathogen and insects, engineering male sterility, and resistance to drought stress. Despite tremendous successes in producing fertile transgenic wheat plants using various methods and approaches, elite cultivars of wheat still remain recalcitrant to transformation. Moreover, in comparison with other major cereals like rice and maize, the development of a high throughput wheat transformation systems has been slowed and severely affected by genotype effects on plant regeneration, low transformation efficiencies and problems with transgene inheritance and stability of expression. Majority of the researcher worldwide have used genetic engineering to tailor wheat for specific end-use by using immature embryos as the primary target tissue for the delivery of desired foreign genes. Hence, we have focused our attention to the work done on stable gene expression and transformation of wheat by employing microprojectile bombardment and Agrobacterium as a source of foreign DNA delivery into immature embryos. Recent advances in wheat transformation especially successes in genetic transformation of wheat with agronomically important genes and novel and innovative approaches for wheat transformation based on different selection schemes are also discussed.

Variability of transgene expression in clonal cell lines of wheat

A method for polyethyleneglycol (PEG) mediated direct DNA transfer into protoplasts was successfully established for transient and stable transformation of Triticum aestivum L. cell cultures. Transgenic cell lines, which had been transformed with the neomycin phosphotransferase II gene (nptII) fused to different promoters, were selected and integration and expression of the marker gene was shown by Southern analysis and enzyme activity test. For investigation of expression stability, five nptII positive cell lines maintained under selection were protoplasted and clonal callus lines were cultivated from the genetically identical single cells without selection pressure. Marker gene activity of 271 clonal callus lines was determined and compared with the corresponding parental line. A reduction or loss of marker gene expression in up to 50% of the clonal cell lines was observed. Detailed analysis of randomly selected clones showed that the observed variability in marker gene expression occurred due to a reduction in the nptII transcript level and was associated with hypermethylation of the integrated DNA. The silencing effect was reversible by a 4 week culture phase on media supplemented with the demethylation agent 5-azacytidin. These differences in marker gene expression could be observed regardless of copy number and position of the integrated nptII gene. The significance of such observations for a stable expression of foreign genes in plant cells is discussed.

An Ac/Ds -mediated gene trap system for functional genomics in barley

BackgroundGene trapping is a powerful tool for gene discovery and functional genomics in both animals and plants. Upon insertion of the gene trap construct into an expressed gene, splice donor and acceptor sites facilitate the generation of transcriptional fusions between the flanking sequence and the reporter. Consequently, detection of reporter gene expression allows the identification of genes based on their expression pattern. Up to now rice is the only cereal crop for which gene trap approaches exist. In this study we describe a gene trap system in barley (Hordeum vulgare L.) based on the maize transposable elements Ac/Ds.ResultsWe generated gene trap barley lines by crossing Ac transposase expressing plants with multiple independent transformants carrying the Ds based gene trap construct GTDs B. Upstream of the β-Glucuronidase start codon GTDs B carries splice donor and acceptor sites optimized for monocotyledonous plants. DNA blot analysis revealed GTDs B transposition frequencies of 11% and 26% in the F1 and F2 generation of gene trap lines and perpetuation of transposition activity in later generations. Furthermore, analysis of sequences flanking transposed GTDs B elements evidenced preferential insertion into expressed regions of the barley genome. We screened leaves, nodes, immature florets, pollinated florets, immature grains and seedlings of F2 plants and detected GUS expression in 51% (72/141) of the plants. Thus, reporter gene expression was found in 24 of the 28 F1 lines tested and in progeny of all GTDs B parental lines.ConclusionDue to the frequent transposition of GTDs B and the efficient expression of the GUS reporter gene, we conclude that this Ac/Ds-based gene trap system is an applicable approach for gene discovery in barley. The successful introduction of a gene trap construct optimized for monocots in barley contributes a novel functional genomics tool for this cereal crop.

Molecular wheat breeding by direct gene transfer

A method for efficient genetic transformation of wheat has been developed using immature embryos as targets for microprojectile-mediated gene transfer and a helium driven particle delivery system. Screening and selection of transgenic cells, somatic embryos and regenerated plants are performed with the gus-gene and the phosphinothricin acetyl transferase (bar) gene coding for Basta-resistance as the selectable marker. On average, one fertile transgenic plant can be obtained from about 100 microprojectile treated, immature embryos. The number of integrated copies of the transferred gene ranges from 1 up to about 10. Stable integrated genes are inherited in most of the transgenic lines in a normal mendelian fashion segregating 3:1 in the F2. Homozygous, as well as heterozygous, lines have been followed and analysed genetically at the molecular level and up to F5. Apart from normal stable gene expression, examples have also been found which showed a loss of gene activity or unexpected segregation pattern. For applied aspects, different genes are transferred aiming for improved disease resistance, modification of quality, or other characteristics. First results from these transgenic lines are reported, and problems still existing with the production of stable transgenic wheat lines are discussed.

A reporter gene under the control of tms or aux promoters is differentially expressed in tobacco and barley protoplasts.

SummaryAgrobacterium tumefaciens and some Agrobacterium rhizogenes strains possess auxin biosynthesis genes (tms and aux genes respectively), responsible for a de novo auxin biosynthetic pathway in transformed plant cells. A comparison is presented of the potential expression of these genes in a monocotyledonous (barley) and a dicotyledonous plant (tobacco). The promoters of the genes were translationally fused to the β-glucuronidase reporter gene and analysed in transient expression experiments. The tms and aux fusions were highly expressed in tobacco, but not in barley. However, the aux enhancer active in tobacco, conferred low β-glucuronidase expression in barley when fused to a truncated cauliflower mosaic virus 35S promoter. The results are discussed in relation to the differential responses to Agrobacterium infection in monocots and dicots.

Analysis of splice donor and acceptor function in a novel Ac -based gene trap construct

In this study, an Ac-based gene trap construct was engineered to increase gene trapping efficiency by an effective use of triple acceptor sites preceding a reporter gene. The target of the engineering process was a synthetic intron preceding the GUS reporter. Two different gene trap constructs were designed. In one construct, three of the sequence elements serving as signals for recognition of an intron 3′ boundary were systematically modified to allow for almost optimal acceptor site recognition, while these sequences remained unchanged in the other construct. To compare recognition of the engineered intron with that of the unmodified intron, tester constructs were transiently transformed into barley (Hordeum vulgare L.) tissue and the accuracy and efficiency of splicing was determined by mRNA mapping and reporter-gene expression frequency analysis. By employing this test system, we could show that systematic engineering of the intron sequence elements results in advanced intron recognition, compared to the unmodified intron, and that all three acceptor sites were activated, but with unequal frequency. The impact of our findings on reporter expression in a gene-trap approach is discussed.

Production of Transgenic Cereal Crops

Applied aspects set cereals as an attractive goal for transformation. Many efforts have been made during recent years towards the establishment of reliable transformation techniques. We report here the development of three transformation systems based on micro-projectile-mediated gene transfer and discuss alternative methods.