Protocol for generating and selecting transgenic tomato lines through Agrobacterium tumefaciens-mediated transformation

Here, we present a protocol for generating and selecting stable transgenic tomato lines through Agrobacterium tumefaciens -mediated transformation of cotyledon and hypocotyl explants. We describe steps for sterilizing and planting tomato seeds, cotyledon and hypocotyl excision and preculture, Agrobacterium tumefaciens preparation and co-cultivation with explants, and recovering explants and bacterial overgrowth restriction. We then detail procedures for selecting green calli; regenerating explant excision and growth in non-selective media; plant hardening, potting, and growing; and T-DNA insertion confirmation by PCR. For complete details on the use and execution of this protocol, please refer to Mountourakis et al. 1 , 2 , 3 • Steps for generating stable transgenic tomato lines in less than 6 months • Instructions for tomato explant regeneration using 1 hormone • Guidance on Agrobacterium elimination using Timentin • Procedures for T-DNA insertion confirmation by PCR and foliar spray Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Here, we present a protocol for generating and selecting stable transgenic tomato lines through Agrobacterium tumefaciens -mediated transformation of cotyledon and hypocotyl explants. We describe steps for sterilizing and planting tomato seeds, cotyledon and hypocotyl excision and preculture, Agrobacterium tumefaciens preparation and co-cultivation with explants, and recovering explants and bacterial overgrowth restriction. We then detail procedures for selecting green calli; regenerating explant excision and growth in non-selective media; plant hardening, potting, and growing; and T-DNA insertion confirmation by PCR.