A citrus-derived peptide boosts transformation but suppresses shoot regeneration — here is how researchers fixed the trade-off

Citrus crops are among the world's most valuable fruits, yet their genetic improvement has long been hindered by notoriously low transformation and regeneration efficiencies. A new study reveals that citrus-derived REGENERATION FACTOR1 (REF1) peptides can boost transformation efficiency by more than fourfold — but they also suppress shoot regeneration. By applying the peptide only during the critical early stage and removing it later, researchers achieved a 4.1-fold increase in recovery of genetically modified shoots in sweet orange and a 3.3-fold increase in Carrizo citrange, paving the way for more efficient genome editing in recalcitrant perennial crops.

Traditional breeding in citrus faces steep obstacles: apomixis produces maternal clones, a juvenile phase lasting 5 to 10 years delays fruiting, and high heterozygosity complicates trait inheritance. Agrobacterium-mediated transformation offers a powerful alternative, but most citrus species remain highly recalcitrant, with transformation efficiencies often stuck around 5% or lower. Due to these challenges, there is an urgent need for deeper investigation into strategies that can break through the transformation bottleneck in woody perennial crops.

Now, a team from the University of Florida reports (DOI: 10.1093/hr/uhag134) in Horticulture Research (published April 7, 2026) that citrus-derived REF1 peptides — specifically CsREF1-2 — dramatically enhance early transformation events in Agrobacterium-mediated citrus transformation, while continuous exposure suppresses shoot regeneration. The researchers developed a staged application strategy that decouples these two processes, significantly improving the recovery of transgenic shoots.

The researchers identified two REF1 isoforms in sweet orange, CsREF1-1 and CsREF1-2, and tested them alongside tomato SIREF1. CsREF1-2 at 100 nM increased transformation efficiency by 4.3-fold, followed by CsREF1-1 at 4.1-fold. However, continuous REF1 exposure reduced shoot regeneration efficiency from 54.7% in the control to as low as 11.8–27.6%. To overcome this trade-off, the team applied 100 nM CsREF1-2 only during the first month of culture, then transferred explants to REF1-free regeneration medium. This staged approach yielded 4.1-fold more GFP-positive shoots in Hamlin sweet orange and 3.3-fold more in Carrizo citrange. Gene expression analysis revealed that CsREF1-2 strongly induced CsWIND1 and CsESR1 — key drivers of callus formation — but continuous exposure suppressed shoot-meristem regulators including WUS, STM, CSC1, and CSC2. Removing the peptide later restored the expression of these regeneration-essential genes. AlphaFold3 modeling further predicted that CsREF1-2 binds its receptor CsPORK1 with the highest affinity among the tested peptides, consistent with its superior activity.

“We found that CsREF1 peptides are excellent at pushing cells into a wound-reprogramming state that makes them more amenable to Agrobacterium infection and T-DNA integration,” the authors said. “But if you keep the signal on too long, the cells get stuck in a proliferative callus state and never move on to make shoots. The solution was surprisingly simple: use the peptide to drive transformation during the first month, then take it away so the tissue can complete the regeneration program.” The authors added that this staged approach offers a practical alternative to genetic co-expression strategies, which often require larger, more complex vectors that can reduce transformation efficiency.

The staged REF1 application strategy provides a broadly applicable tool for genetic improvement of citrus and potentially other transformation-recalcitrant woody crops. Unlike co-expression of regeneration-promoting genes — which increases plasmid size and T-DNA complexity — exogenous peptide treatment enhances transformation without permanently reprogramming gene expression. The approach also achieved 36% CRISPR/Cas9 editing efficiency in sweet orange, demonstrating its compatibility with modern genome-editing workflows. For an industry facing mounting threats from Huanglongbing (HLB), citrus canker, and climate-exacerbated abiotic stresses, this peptide-based strategy could accelerate the development of resilient citrus varieties.

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References

DOI

10.1093/hr/uhag134

Original Source URL

https://doi.org/10.1093/hr/uhag134

Funding ninformation

J. R. (Rip) Graves Endowment, Florida Citrus Initiative Program, Citrus Research and Development Foundation, USDA NIFA grants (2022-70029-38471, 2025-70029-44030, 2023-70029-41280), and Hatch project FLA-CRC-005979

About Horticulture Research

Horticulture Research is an open access journal of Nanjing Agricultural University and ranked number one in the Horticulture category of the Journal Citation Reports ™ from Clarivate, 2023. The journal is committed to publishing original research articles, reviews, perspectives, comments, correspondence articles and letters to the editor related to all major horticultural plants and disciplines, including biotechnology, breeding, cellular and molecular biology, evolution, genetics, inter-species interactions, physiology, and the origination and domestication of crops.