Protocol for Agrobacterium-mediated transformation of tomato and flow cytometry-based ploidy confirmation

Here, we present an Agrobacterium-mediated transformation protocol for tomato, Solanum lycopersicum cv. Micro-Tom, which incorporates flow cytometric ploidy analysis, a critical yet often overlooked step. We detail steps for cotyledon explants, inoculating with Agrobacterium, and co-cultivating cotyledons. We then describe procedures for regenerating shoots/roots, identifying diploid transgenic plants, and screening transgenic plants. This protocol addresses the necessity of diploid selection for functional studies and breeding applications, ensuring reliable transgenic material generation. For complete details on the use and execution of this protocol, please refer to Feng et al.¹.