Challenges and suggestions for tissue culture-free transformation and genome editing in de novo grapevine meristems

Authors: KAYA, HILAL BETUL - Cornell University; Nasti, Ryan; WANG, LI - Cornell University; INOUE, AIDAN - Cornell University; ORAVEC, MADELINE - Cornell University; Cadle Davidson, Lance; BOGDANOVE, ADAM - Cornell University

Submitted to: PhytoFrontiers
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/6/2026
Publication Date: 2/9/2026
Citation: Kaya, H., Nasti, R.A., Wang, L., Inoue, A., Oravec, M., Cadle Davidson, L.E., Bogdanove, A. 2026. Challenges and suggestions for tissue culture-free transformation and genome editing in de novo grapevine meristems. PhytoFrontiers. https://doi.org/10.1094/PHYTOFR-01-26-0001-SC.
DOI: https://doi.org/10.1094/PHYTOFR-01-26-0001-SC

Interpretive Summary: Producing plants with transgenes or genome edits often requires tissue culture systems that take time, expertise, and resources. Some species and varieties work well in tissue culture, and others poorly or not at all. Tissue culture-free genome editing is an attractive potential alternative, particularly for difficult crops such as grapevine, where tissue culture remains a major bottleneck. Developmental regulators (DRs) have been shown to work in some species to enable genome editing without tissue culture. Here, we document attempts to use this DR system in soil-grown grapevine. Unfortunately, across more than 1,500 attempts involving multiple varieties and technical modifications, no genome-edited plants were recovered. Wound scarring and rapid activation of nearby shoots were identified as likely barriers to the DR system in grapevine. By documenting these negative but informative outcomes, we aim to help prevent fruitless repetition and to provide guidance for further research on this topic.

Technical Abstract: 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 axillary 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.