We continue to produce Agrobacterium-mediated mature transgenics for customers, test different approaches for increasing efficiency, & develop biolistics as a tool in citrus.� During this quarter, 11 mature transgenics were produced with Agrobacterium & have been micrografted, & a remaining 42 positive shoots were produced using Agrobacterium & must still be micrografted once/if they get larger. These two genetic constructs had GFP & were transformed into Hamlin (one of our better cultivars for transformation).� An inordinate number of transgenics were produced, which is very unusal in mature citrus & must have been the result of cultivar x genetic construct interaction. The GFP reporter makes identification of transgenics easy. A new initiative from the CREC Director is to produce mature citrus transgenics with stacked disease resistance genes using biolistics for faster deregulation.�We have been able to produce transgenics for Dr. Mou using a promising, new disease resistance gene & the genetic construct did not rearrange in mature citrus. (Unfortunately it rearranged for Dr. Orbovic in immature citrus).� We used a RecA- Agro strain because we suspected there might be a problem.� In a couple of transgenics, the gene of interest (GOI) was lost, but the majority of transgenics have the transgene. This is very similar to the deletion of the GOI from Dr. McNellis’ genetic construct using the mature citrus transformation protocol. During that instance, Dr. Orbovic was able to produce immature transgenics that had the GOI, while it rearranged in mature transformation, even using a RecA- Agro strain.� One advantage of biolistics over Agro transformation is that the GOI does not rearrange as easily as in Agro.��A manuscript was accepted & revised for In Vitro Cellular & Developmental Biology-Plant. This manuscript showed that improved selection with phosphomannose isomerase after biolistics in immature citrus drastically increased transformation efficiency after bombardment (a mean of ~2 transgenics per paired shot).� We are now testing phosphomannose isomerase (PMI) as a new selectable marker in mature citrus. Once the optimal mannose/sucrose concentrations have been determined in mature citrus, we will test PMI selection after Agrobacterium-mediated transformation.�We are testing a new protocol for precipitating DNA onto gold particles for biolistics that increased efficiency in other species.� This protocol will also be useful for gene editing using biolistic transformation.�We must buy a new soil sterilizer & a new ultra low -80C freezer.� Both are essential to this program.��An external review of the two transformation labs was conducted March 28 & 29 & presumably there will be a report issued at some time in the future.���