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

## TL;DR

This paper provides a step-by-step protocol for creating transgenic tomato plants using Agrobacterium tumefaciens in under six months.

## Contribution

A streamlined, efficient method for transgenic tomato line generation using a single hormone and Timentin for bacterial elimination.

## Key findings

- Transgenic tomato lines can be generated in less than six months using Agrobacterium-mediated transformation.
- Green calli selection and regeneration can be achieved using one hormone and non-selective media.
- PCR and foliar spray confirm T-DNA insertion in transgenic plants.

## Abstract

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

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.