This invention, using Agrobacterium gene clusters, enables passive regeneration of transgenic cells without the need for plant hormones
Background For plants which are recalcitrant (difficult) to transform, poor performance is usually associated with inadequate DNA delivery or difficult regeneration response. There is a need for improved methods of reducing transgenic plants. Production of transgenic plants requires the delivery of foreign DNA, and subsequent regeneration of cells with delivered DNA into intact plants. Typical regeneration methods utilize chemical hormone treatments in sterile culture to encourage plant cells to regenerate and vary widely in approach based on the species and genotype. Alternative methods have been developed which utilize genes co-delivered with the desired trait to accomplish plant regeneration. To date these mostly include plant-derived transcription factors which are tailored to induce shoot development or somatic embryogenesis. These can be co-delivered with the gene of interest and then removed by various approaches - or co-delivered on separate DNA molecules and rely on the cell-to-cell diffusion of the regeneration factors produced to promote other cells which contain only the trait of interest to regenerate.
Technology Description This invention uses Agrobacterium gene clusters to provide improved methods of shoot regeneration and plant transformation. The inventors have designed a system in which combinations of Agrobacterium-derived, shoot-inducing genes are delivered in an altruistic configuration with a trait gene of interest to enable passive regeneration of transgenic cells without the need for plant hormones, and thus can readily be used in non-sterile conditions. They isolated and amplified gene encoding DNA sequences from Agrobacterium isolates which passively induce shoots from infected gall tissue. The gene sets are cloned into DNA delivery vectors alongside fluorescent protein markers and antibiotic resistance genes. To accomplish transgenic shoot regeneration, separate Agrobacterium strains containing the regeneration or the trait vector cassettes are mixed and then transformed into plant tissues. For non-Agrobacterium-mediated delivery, such as through biolistics or other physical methods, the donor DNA can be mixed prior to delivery. Tissued are incubated without hormones and are selected using the trait vector resistance marker.
Figure 1: "Shooty" Agrobacterium oncogenes are valuable tools for transformation. **A)** Shoot-inducing and non-shoot inducing oncogene constructs for _in vitro_ altruistic transformation, testing the impact of oncogene donor source and addition of other T-DNA genes as regeneration tools. **B,C)** While shooty oncogenes were less effective to non-shoot inducing ones for transgenic callus, they were very effective at inducing altruistic shoot transformation. A subset of genes alone were insufficient for producing altruistic shoots. **D)** Altruistic transformation gave more efficient (2.3x) and faster (by 5 weeks) transformation over a hormone-based conventional protocol. **E,F)** High throughput hyperspectral imaging shows regenerating altruistic shoots transgenic for the trait vector but not the oncogene cluster. **G,H,I)** Fluorescent microscope images show altruistic shoots induced by non-cell autonomous signals from oncogene expressing calli.
Further Details: The bumpy road of DEV-geneassisted transformation of trees (presentation)
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Applications
Status Patent pending.