2008 Annual Report 1a.Objectives (from AD-416) Analyze the two different 2-kb promoters derived from genomic clones of the apple alpha-farnesene synthase gene AFS1 to determine the mechanism of ethylene- and cold-induced expression in cold-stored apple fruit. Transform tissue from a scald-susceptible apple cultivar with an RNAi construct of AFS1, regenerate transformed plants, and test the transformants for supression of ethylene-and cold-induced alpha-farnesene synthesis. 1b.Approach (from AD-416) Analysis of the two different AFS1 promoter sequences cloned from leaf tissue genomic DNA of ‘Law Rome’ apple will entail testing the expression, in Arabidopsis, tomato, or petunia, of GUS fusion constructs including 0.5- to 2.0-kb fragments of the AFS1 5’ upstream sequence. Plants transformed with a sense construct of the AFS1 cDNA driven by the AFS1 promoter will be tested for ethylene-and cold-inducible alpha-farnesene synthesis. RNAi gene silencing constructs of AFS1 driven by the CaMV 35S, AFS1, or other suitable plant gene promoter will be used in Agrobacterium-mediated transformation of tissue from a scald-susceptible apple cultivar. Positive transformants screened by DNA gel blot analysis will be propagated and eventually compared with empty vector tranformed controls for ethylene- and cold-inducible alpha-farnesene production in leaf and fruit peel tissues. 3.Progress Report This collaborative project was initiated in March 2008 to address the end goals of objective 3 for the in-house project 1275-43000-009-00D. Specifically, the aim is to develop molecular genetic strategies for prevention of the costly storage disorder superficial scald in susceptible apple and pear varieties such as Granny Smith and d'Anjou. The disorder manifests as brown or black patches on the skin that render the fruit unmarketable. For many years the primary method of scald control used by the industry is a drench treatment including an antioxidant and a fungicide, which is expensive, restricts exports, creates chemical waste, and leaves chemical residues on the fruit. Oxidation products of alpha-farnesene, a natural volatile compound that accumulates in the fruit skin during cold storage, are implicated in the induction of superficial scald. Our prior work has shown that blocking the expression of AFS1, a key gene involved in a-farnesene synthesis, is likely to greatly diminish or eliminate the occurrence of scald in susceptible fruit. The long-range goal of the project is to test this hypothesis by employing molecular genetic methods to knock out (silence) AFS1 in fruit of a highly scald-susceptible apple variety. The first step in this process was to establish tissue of Granny Smith apple in sterile culture, which was accomplished several months ago. Next, an altered version of AFS1 (silencing construct), designed to turn off the gene in all tissues, was introduced into sterile leaflets, from which transgenic shoots were generated. Presently there are three putative transgenic cultures of Granny Smith, and the methods have been streamlined to produce additional transformed shoots more rapidly. Updates on research progress have been obtained regularly by e-mail, and were followed up by an on-site visit for discussion and observation of the experiments in early July.