Genetic Transformation and Gene Editing: Currently the MCF can produce mature citrus transgenics (gene comes from any organism) using a standard Agrobacterium-mediated transformation protocol or cisgenics (all citrus sequences) using a biolistic protocol. Biolistic transformation with citrus minimal expression cassettes is intended for the production of cisgenics without plant pest sequences, which avoids one step in the regulatory hurdle. We are also working on a DNA free gene editing protocol.
The mature scion genotypes currently amenable to Agrobacterium transformation include Valencia, Hamlin, Valencia-like B770 (EV1), Glenn Navel, and Pineapple. New, high yielding sweet orange cultivars from the UF Plant Improvement Team (OLL8, OLL4, Vernia, Valquarius) were introduced and are being tested with Agrobacterium and biolistic transformation. OLL8 and Vernia are relatively recalcitrant to Agrobacterium but can be transformed with the gene gun. OLL4 has a very high Agrobacterium transformation efficiency. We are currently testing Valquarius, and Ruby Red grapefruit with Agrobacterium and in biolistic transformation. We have also introduced Clementine mandarin for which an Agrobacterium protocol already exists. If seed is desired, mature rootstock cvs. Carrizo, Swingle, nematode resistant Kuharski are regularly transformed. The new UF rootstocks for which seed is scarce have also been introduced and will be tested soon. After transformation, transgenics will be reconfirmed by GUS assays and/or PCR for the gene of interest.
Procedures for Participation: To submit genetic constructs to the MCF, the following is required:
1. Evidence that the transgene(s) confers resistance/tolerance to a pest/pathogen (the priority is HLB) or confers another value-added trait of importance.
2. Source of the transgene and whether it is licensed to other parties.
3. The sequence of the transgene.
4. The identity of the binary or biolistic vector, a plasmid map delimiting the location and size of genetic elements, plasmid size, and location of restriction sites.
5. It is preferable to receive vector DNA rather than vectors mobilized in Agrobacterium. Gene fragments are also acceptable and the MCF will perform the sub-cloning (see Options for Transgenic Plants).
6. Additional information may be requested, depending on the submitted transgene or other special requests.
7. If you are a UF PI, please provide your UF EHS/IBC rDNA registration no.