1. Please state project objectives and what work was done this quarter to address them: Induce efficient flowering in citrus seedlings by overexpressing FT3 and knocking out negative regulators of flowering. Work done during this quarter:Due to failure of the first mixed infection of CTV-RNAi vector and CTV-FT3 vectors in the same C. macrophylla plants, we planned on setting up a second series of mixed infections of CTV RNAi vectors targeting negative regulators of flowering and CTV-FT3 vectors that positively regulate flowering. Before setting up new mixed infections, we ran RT-PCR stability assays onCTV-FT3 vector infected plants with primers within CTV upstream and downstream of the insertion sites. The assay revealed that despite inducing flowering, all CTV-FT3 constructs were primarily recombinants. This result pointed to a minimal amount of FT3 needed to induce early flowering in citrus and the lack of stability of FT3 expressed from the CTV vector. To manage the lack of stability of FT3 within the CTV vectors, we followed two strategies. The first strategy was dependent on replacing the p33 gene which is the furthest away from the 3′ end. This approach should decrease the expression of FT3 from the CTV vector; however, it should not affect the expression of the CTV gene especially the three silencing suppressors within the CTV genome, namely p20, p23 and CP, as the insertion of the FT3 gene is upstream of all endogenous CTV genes. The second strategy was to boost the expression of viral silencing suppressors, that play a role in minimizing recombination and keeping the integrity of viral genomes, by introducing a second gene cassette that either expressed a duplicated p20 silencing suppressor from CTV or a p24 silencing suppressor from the closely related Grapevine leaf roll associated virus-2 (GLRaV-2). We successfully generated the new plasmids and confirmed the right inserts by digestion and sanger sequencing.Transgenic Carrizo FT3 rooted were growing well at this time but had not flowered yet. 2. Please state what work is anticipated for next quarter: We will be working on Agroinfiltrating the new vectors into N. benthamiana and getting the newly generated CTV vectors into citrus to enable establishing mixed infection of CTV-FT3 vectors and CTV-RNAi vectors targeting negative regulators of flowering. 3. Please state budget status (underspend or overspend, and why): On budget