Endogenous expression of the wound signal peptide ZmREF1 enhances Agrobacterium-mediated transformation of maize...

Ectopic ZmREF1 expression significantly enhances maize B104 transformation and regeneration using the QuickCorn Lite protocol and the ternary vector system, whereas exogenous peptide shows dose dependent, inconsistent effects. Genetic transformation is a vital tool for functional genomics and crop improvement, yet low regeneration efficiency remains a major obstacle. Recent studies have identified REGENERATION FACTOR 1 (REF1), a wound-induced signaling peptide, as a promising regulator of plant regeneration. In this work, we evaluated the role of maize REF1 (ZmREF1) in enhancing transformation and regeneration efficiency using a rapid Agrobacterium-mediated protocol in maize inbred B104. ZmREF1 was introduced either through endogenous expression from a T-DNA construct or by exogenous application to the tissue culture media. Endogenous expression via pCBL101-RUBY::ZmREF1 led to a modest increase in transient expression (71.8%) relative to the control pCBL101-RUBY (62.8%) but resulted in a significantly higher stable transformation frequency (33.6%). This represents an approximate fourfold increase compared with the control construct (8.7%), with no detectable effect on T-DNA copy number. Conversely, exogenous application of the synthetic ZmREF1 peptide to the culture media exhibited dose-dependent effects: low concentrations (0.1-1 nM) slightly improved transformation efficiency, whereas higher concentrations (10 nM) were detrimental, likely due to overactivation of stress-related pathways. These findings highlight the importance of maintaining a regulatory balance when leveraging stress-responsive mechanisms for regeneration. ZmREF1 represents a promising target for enhancing maize transformation and regeneration efficiency though further optimization of delivery strategies is required to maximize its potential.