During this reporting period, we have conducted the following studies. We have continued to work on method development to produce gene-edited and transgene-free plants derived from mature citrus tissues. For the approach to create a chemical resistance to reduce chimeras, we have not observed resistance. We are trying to determine what are possible reasons. We also started a different strategy to create chemical resistance. For that purpose, we have made some gene constructs and will test their efficiencies.
We have completed experiments to repeat some previously conducted experiments in which we observed inconsistent results using the proposed genes regarding their effects when used in mature citrus tissues. We have also completed to test the effects of several chemicals on transient and stable expression activities and also regeneration efficiencies in mature citrus tissues. Our results suggest that there are some improvement of shoot regeneration from calli derived from mature tissues of citrus. We will write a manuscript to report these results.
We have observed that fresh young shoot tissues propagated from mature citrus shoots through vegetative propagation can be regenerated but the efficiencies are somewhat low. The Agrobacterium-mediated transformation efficiency of young fresh tissues generated from mature tissues is very low. To circumvent these problems, we have constructed additional genes. We are starting to test their effectivities using both juvenile and mature citrus tissues.
To develop an in-planta transformation method for mature citrus tissues, we have done additional experiments from the last report. We observed low efficiencies for the Agrobacterium infection based on our transient or stable transformation assays. We have also observed low de novo regeneration efficiencies using mature citrus plants in our in-planta transformation experiments. Thus, we believe that new tools have to be incorporated into the in-planta transformation method in order to successfully use it in mature citrus plants. We are preparing tissues for test whether the new genes that can enhance shoot regeneration we have recently constructed can enhance regeneration efficiencies from mature plants in our in-planta transformation experiments.