RESUMO
Efficient delivery of macromolecules into plant cells and tissues is important for both basic research and biotechnology product applications. In transgenic research, the goal is to deliver DNA molecules into regenerable cells and stably integrate them into the genome. Over the past 40 years, many macromolecule delivery methods have been studied. To generate transgenic plants, particle bombardment and Agrobacterium-mediated transformation are the methods of choice for DNA delivery. The rapid advance of genome editing technologies has generated new requirements on large biomolecule delivery and at the same time reinvigorated the development of new transformation technologies. Many of the gene delivery options that have been studied before are now being repurposed for delivering genome editing machinery for various applications. This article reviews the major progress in the development of tools for large biomolecule delivery into plant cells in the new era of precision genome engineering.
Assuntos
Edição de Genes/métodos , Técnicas de Transferência de Genes , Engenharia Genética/métodos , Genoma de Planta/genética , Agrobacterium/genética , Biotecnologia/instrumentação , Biotecnologia/métodos , Edição de Genes/instrumentação , Edição de Genes/tendências , Engenharia Genética/instrumentação , Engenharia Genética/tendências , Plantas Geneticamente Modificadas/genética , Transformação GenéticaRESUMO
Maize is an important food and feed crop in many countries. It is also one of the most important target crops for the application of biotechnology. Currently, there are more biotech traits available on the market in maize than in any other crop. Generation of transgenic events is a crucial step in the development of biotech traits. For commercial applications, a high throughput transformation system producing a large number of high quality events in an elite genetic background is highly desirable. There has been tremendous progress in Agrobacterium-mediated maize transformation since the publication of the Ishida et al. (1996) paper and the technology has been widely adopted for transgenic event production by many labs around the world. We will review general efforts in establishing efficient maize transformation technologies useful for transgenic event production in trait research and development. The review will also discuss transformation systems used for generating commercial maize trait events currently on the market. As the number of traits is increasing steadily and two or more modes of action are used to control key pests, new tools are needed to efficiently transform vectors containing multiple trait genes. We will review general guidelines for assembling binary vectors for commercial transformation. Approaches to increase transformation efficiency and gene expression of large gene stack vectors will be discussed. Finally, recent studies of targeted genome modification and transgene insertion using different site-directed nuclease technologies will be reviewed.
RESUMO
In recent years, there has been a rapid increase in the planting of transgenic crops with stacked traits. Most of these products have been formed by conventional breeding, i.e. the crossing of transgenic plant (event) containing individual transgenes with other event(s) containing single or double transgenic traits. Many biotech companies are developing stacked trait products with increasing numbers of insect and herbicide tolerance genes for controlling a broad range of insect pests and weeds. There has also been an increase in development of technologies for molecular stacking of multiple traits in a single transgene locus. In this review we look at the status of stacked trait products, crop trait stacking technologies and the technical challenges we are facing. We also review recent progress in developing technology for assembling large transgene arrays in vitro (molecular stacks), their delivery to crop plants and issues they pose for transgene expression.
Assuntos
Produtos Agrícolas/genética , Plantas Geneticamente Modificadas/genética , Transgenes/genética , Animais , Cruzamento , Produtos Agrícolas/parasitologia , Resistência à Doença/genética , Gossypium/genética , Gossypium/parasitologia , Insetos/crescimento & desenvolvimento , Resistência a Inseticidas/genética , Plantas Geneticamente Modificadas/parasitologia , Zea mays/genética , Zea mays/parasitologiaRESUMO
Agrobacterium tumefaciens T-DNA normally integrates into random sites in the plant genome. We have investigated targeting of T-DNA by nonhomologous end joining process to a specific double-stranded break created in the plant genome by I-CeuI endonuclease. Sequencing of genomic DNA/T-DNA junctions in targeted events revealed that genomic DNA at the cleavage sites was usually intact or nearly so, whereas donor T-DNA ends were often resected, sometimes extensively, as is found in random T-DNA inserts. Short filler DNAs were also present in several junctions. When an I-CeuI site was placed in the donor T-DNA, it was often cleaved by I-CeuI endonuclease, leading to precisely truncated targeted T-DNA inserts. Their structure requires that T-DNA cutting occurred before or during integration, indicating that T-DNA is at least partially double stranded before integration is complete. This method of targeting full-length T-DNA with considerable fidelity to a chosen break point in the plant genome may have experimental and practical applications. Our findings suggest that insertion at break points by nonhomologous end joining is one normal mode of entry for T-DNA into the plant genome.
