Challenges and Suggestions for Tissue Culture-Free Transformation and Genome Editing in <i>De Novo</i> Grapevine Meristems

Producing plants with transgenes or genome edits often requires regeneration systems that take time, expertise, and resources. Some species, cultivars, and explants regenerate well and others poorly, inconsistently, or not at all. Moreover, tissue culture can introduce somaclonal variation, adding further complexity and unpredictability to regeneration outcomes. Tissue culture-free genome editing is an attractive potential alternative to conventional transformation methods, particularly for recalcitrant crops such as grapevine, where regeneration remains a major bottleneck. Developmental regulators (DRs) have been shown to enhance plant regeneration and transformation, and in some species, can induce de novo meristem formation to enable genome editing without tissue culture. Here, we present evaluation of DR-mediated, tissue culture-free transformation carried out independently by two research groups, using Agrobacterium-based delivery of ISOPENTENYL TRANSFERASE (IPT) in soil-grown grapevine. One group included gene editing reagents and the other a luciferase reporter. Across more than 1,500 total inoculations involving multiple cultivars, Agrobacterium strains, and tested protocol modifications, no stably transformed or genome-edited shoots were recovered. Wound scarring, lignification, and rapid activation of nearby prompt lateral meristems were identified as likely biological barriers to DR-mediated de novo meristem formation. By documenting these negative but informative outcomes, we aim to help prevent fruitless repetition and to provide guidance for further research toward robust in planta transformation and genome-editing in grapevine and other recalcitrant species.