Our progresses we have made for this project: 1) We have used the Kn1 gene to drastically improves shoot regeneration efficiently from transgenic cells of citrus. We have successfully used a maize knotted1 (KN1) gene to enhance genetic transformation efficiencies of juvenile tissues of six citrus varieties, Pineapple, Hamlin, Sucarri, Valencia, Carrizo and Eureka lemon via Agrobacterium-mediated infection. Our results demonstrate that expression of the KN1 gene improved transformation efficiencies from 3- to 15-fold compared to a control vector, 3- to 11-fold relative to the highest transformation efficiencies previously reported for the same citrus varieties. Stable incorporations of T-DNA into our transgenic plants have been confirmed with both histochemical staining of GUS activity and molecular analyses. The majority of KN1 over-expressing citrus plants grow and develop normally at young seedling stages, similar to those of the wild type plants. With all six genotypes of citrus tested including Eureka lemon, a cultivar difficult to transform, we have demonstrated that the kn1 gene can be an effective molecular tool for enhancing the genetic transformation of juvenile citrus tissues. Using mature shoot segments of Valencia and other cultivars as explants, we also found that the KN1 gene can improve transformation efficiencies compared to the control vector BUT an increase in efficiency is lower than what has been observed in juvenile citrus tissues. 2) We have demonstrated that manipulation of auxin transport can significantly enhances shoot regeneration of citrus. We have observed that the apical ends of epicotyl segments regenerated more shoots than the basal ends, and we therefore hypothesized that auxin transport and/or endogenous auxin concentration may play a key role in shoot regeneration of citrus explants. We tested some auxin transport modulators and identify one modulator that improved shoot regeneration. However, when the modulator was included in the transformation experiment, the transformation efficiency did not improve (i.e., number of transgenic shoots produced per explant). We hypothesize that the auxin modulator may inhibit Agrobacterium infection or T-DNA integration. 3) We have shown that an epigenetic modulator may be used to enhance shoot regeneration and transformation of mature citrus tissue. When we used an epigenetic modulator in transformation experiments with mature tissues, we observed increases in transformation efficiency of several citrus cultivars including Valencia and Washington Navel oranges. We have further demonstrated that the epigenetic modulator can lead to increases in shoot regeneration efficiency of mature citrus tissues when compared to the controls. 4) We have demonstrated that low Agrobacterium infection and T-DNA integration efficiencies are limiting factors for mature citrus transformation. As described above, we have developed some tools for enhancing shoot regeneration from mature citrus tissues. However, when these tools were used in mature citrus tissue transformation, the increase in transformation efficiency was lower than in juvenile tissues. We have further shown that the Agrobacterium infection and DNA integration are a major factor limiting transformation efficiency of mature citrus tissues, which provides a basis for our future experimentation to improve transformation efficiency of mature citrus tissues. We have published one manuscript reporting that Kn1 can drastically improve genetic transformation efficiencies of six citrus cultivars including a lemon cultivar: Hu et al (2016): Kn1 gene overexpression drastically improves genetic transformation efficiencies of citrus cultivars. Plant cell, Tissue and Organ Culture. 125: 81-91. The second manuscript reporting the effects of poplar transport of endogenous auxin and an auxin transport modulator on citrus regeneration and transformation will be submitted in 2-3 weeks. The third one is currently under preparation.