Barno Ruzimurodovna Rezaeva, Ingrid Otto, Götz Hensel, Pouneh Pouramini, Anton Peterson, Jochen Kumlehn
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引用次数: 0
Abstract
Background: Camelina is an oilseed crop with particularly useful fatty acid and amino acid profiles of its seeds, high resilience to abiotic and biotic stresses, and a short life cycle. Previous genetic engineering approaches in camelina have largely relied on the floral dip method which is, however, associated with genotype-dependent efficiency and incompatibility with methods of direct biomolecule delivery.
Results: Here, we established a novel method of transgenesis for camelina, taking advantage of the high regenerative capacity of immature embryos. Various culture conditions and treatments were experimentally validated, which included the duration of explant pre-cultivation, wounding of explants and its time of application, Agrobacterium strain and density of inoculum, acetosyringone concentration, duration of explant-Agrobacterium co-cultivation, as well as application time and concentration of the selective agent hygromycin. We provide convergent evidence of stable transgenicity and transgene inheritance by (1) selection for resistance to hygromycin, (2) PCR, (3) detection of the transgene product GFP, and (4) DNA gel blot analysis involving primary transgenic plants and segregating progeny. Primary transgenics examined in detail featured one to three T-DNA integration loci, with one to seven T-DNA copies being integrated in total per plant. The established method proved efficient across all three tested accessions including two current cultivars, whereby transformation efficiencies, determined as PCR-positive primary transgenic plants related to agro-inoculated explants, of between 13 and 17% were obtained.
Conclusion: With this method, we provide a viable platform for the functional validation of genes-of-interest and for biotechnological improvements of plant performance and quality features in camelina.
期刊介绍:
Plant Methods is an open access, peer-reviewed, online journal for the plant research community that encompasses all aspects of technological innovation in the plant sciences.
There is no doubt that we have entered an exciting new era in plant biology. The completion of the Arabidopsis genome sequence, and the rapid progress being made in other plant genomics projects are providing unparalleled opportunities for progress in all areas of plant science. Nevertheless, enormous challenges lie ahead if we are to understand the function of every gene in the genome, and how the individual parts work together to make the whole organism. Achieving these goals will require an unprecedented collaborative effort, combining high-throughput, system-wide technologies with more focused approaches that integrate traditional disciplines such as cell biology, biochemistry and molecular genetics.
Technological innovation is probably the most important catalyst for progress in any scientific discipline. Plant Methods’ goal is to stimulate the development and adoption of new and improved techniques and research tools and, where appropriate, to promote consistency of methodologies for better integration of data from different laboratories.