Regulated expression of site-specific DNA recombination for precision genetic engineering of apple
Authors: Norelli John L. , Flaishman Moshe , Aldwinckle Herb , Gidoni David 
Published: December 2021

Objectives: The original objectives of this project were to: 1) evaluate inducible promoters for the expression of recombinase in apple (USDA-ARS); 2) develop alternative selectable markers for use in apple to facilitate the positive selection of gene excision by recombinase (Cornell University); 3) compare the activity of three different recombinase systems (Cre/lox, FLP/FRT, and R/RS)in apple using a rapid transient assay (ARO); and 4) evaluate the use of recombinase systems in apple using the best promoters, selectable markers and recombinase systems identified in 1, 2 and 3 above (Collaboratively). Objective 2 was revised from the development alternative selectable markers, to the development of a marker-free selection system for apple. This change in approach was taken due to the inefficiency of the alternative markers initially evaluated in apple, phosphomannose-isomerase and 2-deoxyglucose-6-phosphate phosphatase, and the regulatory advantages of a marker-free system. Objective 3 was revised to focus primarily on the FLP/FRT recombinase system, due to the initial success obtained with this recombinase system. Based upon cooperation between researchers (see Achievements below), research to evaluate the use of the FLP recombinase system under light-inducible expression in apple was then conducted at the ARO (Objective 4). Background: Genomic research and genetic engineering have tremendous potential to enhance crop performance, improve food quality and increase farm profits. However, implementing the knowledge of genomics through genetically engineered fruit crops has many hurdles to be overcome before it can become a reality in the orchard. Among the most important hurdles are consumer concerns regarding the safety of transgenics and the impact this may have on marketing. The goal of this project was to develop plant transformation technologies to mitigate these concerns. Major achievements: Our results indicate activity of the FLP\FRTsite-specific recombination system for the first time in apple, and additionally, we show light- inducible activation of the recombinase in trees. Initial selection of apple transformation events is conducted under dark conditions, and tissue cultures are then moved to light conditions to promote marker excision and plant development. As trees are perennial and - cross-fertilization is not practical, the light-induced FLP-mediated recombination approach shown here provides an alternative to previously reported chemically induced recombinase approaches. In addition, a method was developed to transform apple without the use of herbicide or antibiotic resistance marker genes (marker free). Both light and chemically inducible promoters were developed to allow controlled gene expression in fruit crops. Implications: The research supported by this grant has demonstrated the feasibility of "marker excision" and "marker free" transformation technologies in apple. The use of these safer technologies for the genetic enhancement of apple varieties and rootstocks for various traits will serve to mitigate many of the consumer and environmental concerns facing the commercialization of these improved varieties.