Development of efficient methods for protoplast-to-plant regeneration in Coffea arabica and successful CRISPR-Cas9 editing in protoplasts
Abstract
Conventional coffee breeding is a long and complex process that limits genetic improvement, highlighting the need for advanced biotechnological tools. Protoplast-based CRISPR genome editing is a promising transgene-free approach, which enables precise genetic modifications and whole-plant regeneration while avoiding the stable integration of foreign DNA. However, reliable plant regeneration from protoplasts remains a significant challenge in most crops, especially in tropical tree species like coffee. Here, we present an efficient protocol for protoplast isolation from embryogenic calli and protoplast-to-plant regeneration in the allotetraploid species Coffea arabica. Culture conditions were optimised by comparing protoplast-derived cell growth in liquid medium versus alginate layers and by assessing medium supplementation with the peptide growth factor phytosulfokine-α (PSK) or polyamines (spermine and putrescine). The alginate-embedding system, combined with 0.1 µM or 1 µM of PSK, significantly enhanced early cell division frequencies (up to 50%) and facilitated the formation of up to 270 microcalli.cm−2, enabling subsequent whole-plant regeneration using somatic embryogenesis. Although spermine also favoured cell division, its effect was weaker than that of PSK, and no synergistic interaction was observed when combined with PSK. Furthermore, we demonstrated successful polyethylene glycol-mediated transfection of coffee protoplasts with a CRISPR–Cas9 plasmid targeting the coffee xanthosine methyltransferase (XMT) gene involved in caffeine biosynthesis. Subgenome-specific PCR and deep sequencing revealed insertion/deletion mutations at target sites in ca. 4% of protoplasts. Collectively, these findings pave the way for transgene-free genome editing and functional genomics studies based on cutting-edge technologies in coffee.
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BibTeX
@article{laflaquiere2026development,
title = {{Development of efficient methods for protoplast-to-plant regeneration in Coffea arabica and successful CRISPR-Cas9 editing in protoplasts}},
author = {Lucas Laflaquiere and Hervé Etienne and Thierry Joët and William Cresson and Stéphane Dussert and Estelle Remy and Lucie Kriegshauser and Frederic Georget},
journal = {Plant Cell, Tissue and Organ Culture},
year = {2026},
month = aug,
doi = {10.1007/s11240-026-03561-5},
url = {https://hal.science/hal-05759786v1},
note = {CC BY-NC-ND 4.0},
}
RIS
TY - JOUR
TI - Development of efficient methods for protoplast-to-plant regeneration in Coffea arabica and successful CRISPR-Cas9 editing in protoplasts
AU - Lucas Laflaquiere
AU - Hervé Etienne
AU - Thierry Joët
AU - William Cresson
AU - Stéphane Dussert
AU - Estelle Remy
AU - Lucie Kriegshauser
AU - Frederic Georget
PY - 2026
DA - 2026/08/11
JO - Plant Cell, Tissue and Organ Culture
DO - 10.1007/s11240-026-03561-5
UR - https://hal.science/hal-05759786v1
AB - Conventional coffee breeding is a long and complex process that limits genetic improvement, highlighting the need for advanced biotechnological tools. Protoplast-based CRISPR genome editing is a promising transgene-free approach, which enables precise genetic modifications and whole-plant regeneration while avoiding the stable integration of foreign DNA. However, reliable plant regeneration from protoplasts remains a significant challenge in most crops, especially in tropical tree species like coffee. Here, we present an efficient protocol for protoplast isolation from embryogenic calli and protoplast-to-plant regeneration in the allotetraploid species Coffea arabica. Culture conditions were optimised by comparing protoplast-derived cell growth in liquid medium versus alginate layers and by assessing medium supplementation with the peptide growth factor phytosulfokine-α (PSK) or polyamines (spermine and putrescine). The alginate-embedding system, combined with 0.1 µM or 1 µM of PSK, significantly enhanced early cell division frequencies (up to 50%) and facilitated the formation of up to 270 microcalli.cm−2, enabling subsequent whole-plant regeneration using somatic embryogenesis. Although spermine also favoured cell division, its effect was weaker than that of PSK, and no synergistic interaction was observed when combined with PSK. Furthermore, we demonstrated successful polyethylene glycol-mediated transfection of coffee protoplasts with a CRISPR–Cas9 plasmid targeting the coffee xanthosine methyltransferase (XMT) gene involved in caffeine biosynthesis. Subgenome-specific PCR and deep sequencing revealed insertion/deletion mutations at target sites in ca. 4% of protoplasts. Collectively, these findings pave the way for transgene-free genome editing and functional genomics studies based on cutting-edge technologies in coffee.
SN - 0167-6857
SN - 1573-5044
ER -