During this reporting period, we have constructed a gene vector that may reduce chimeras of mutant and wild-type plants of citrus using mature and juvenal tissues as explants. The vector contains a Cas9 gene and a sgRNA gene to target on a citrus gene that may lead to an endogenous chemical resistance. The gene may be useful to reduce wt and mutant chimaera when Agrobacterium-mediated transient expression of the Cas9 and sgRNA are used to produce transgene-free edited plants. We will start testing its efficiency in mature tissues of Valencia and juvenile tissues of Carrizo. We have been continuing to repeat some previously observed results using the proposed genes such as AGO and NPRD genes that showed some positive effects of transformation efficiencies. We have also repeated the experiments for the effects of PAT and SMZ we have previously observed some positive effects on transformation of both juvenile and mature tissues of Valencia and Washington navel. Our goals are to generate sufficient and repeatable for the genes and chemicals we have used results for scientific publications and patent applications. We have been working on production of fresh young shoots from mature citrus tissues and using them directly for regeneration and transformation studies. We have observed that reasonable amount of shoots can be produced from callus tissues derived from mature tissues of Valencia. We observed more shoots produced from these callus tissues if chemicals such as NPA or 5-Aza were used. When using the young shoot tissues derived from nature tissues of Valencia as explants for the transformation experiments, in addition to the genes originally described in the proposal, we will test the effects of the Wus and BBM genes. Over-expression of both genes has been shown very effective in enhancing transformation efficiencies of a large number of plants that are difficult to be transformed. We are designing and constructing Wus and BBM genes for citrus transformation and regeneration.