Aug 2026· Communications Biology· Vol 9· 0 citations· 92 references
Medicine
TL;DR
A new protoplast-based platform enables transient transformation and proof-of-concept CRISPR/Cas12a-mediated genome editing in European beech, a key but recalcitrant forest tree species.
Abstract
Fagus sylvatica L. (European beech) is a dominant hardwood forest tree species across Central Europe, supporting diverse ecosystem services and forming the basis of a significant market for high-value timber. However, climate change increasingly threatens beech vitality and productivity, making molecular insights into its stress resilience and functional validation of underlying genes urgently needed. Here, we report a protocol for protoplast isolation from seedling leaves and demonstrate transient genetic transformation and CRISPR/Cas-mediated genome editing in F. sylvatica. PEG-mediated transformation was sequentially optimized, achieving efficiencies of 59 ± 6.19% within distinct seasonal windows. Protoplast yield and transformation efficiency showed pronounced temporal variation throughout the year, indicating a strong seasonal influence on reproducibility of the workflow despite controlled growth conditions. A basic molecular toolkit for functional genomics and future biotechnological applications was established by testing a set of promoters and reporters. For proof-of-concept genome editing, we achieved 4.75 to 32.69% editing efficiencies in the PHYTOENE DESATURASE gene (FsPDS) using temperature-tolerant LbCas12a (ttLbCas12a). Although further optimization of transformation reproducibility and regeneration systems remains necessary, the presented protoplast platform provides a valuable foundation for transient functional assays and genome editing studies in this non-model tree species. A new protoplast-based platform enables transient transformation and proof-of-concept CRISPR/Cas12a-mediated genome editing in European beech, a key but recalcitrant forest tree species.
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.
Lucas Laflaquiere, H. Etienne, Thierry Joet et al.· Plant Cell Tissue and Organ...· 0 citations
The present study addresses optimization of in-vitro regeneration via direct organogenesis and Agrobacterium-mediated genetic transformation, enabling efficient multiplex CRISPR/Cas9-based genome editing of the phytoene desaturase (PsPDS) gene in pea. Pea (Pisum sativum L.) is an important legume crop valued for food, plant-based protein, vegetable, and green manure. Although genome editing offers a precise and rapid strategy for crop improvement, its application in pea remains challenging due to inherent recalcitrance to in-vitro regeneration and genotype-dependent transformation. The regeneration and Agrobacterium-mediated transformation systems were optimized, and the dicotyledonary node (DCN) was identified as the preferred explant for multiplex CRISPR/Cas9-based genome editing in pea. Among three explant types (embryonic axis, DCN and nodal segment), DCN showed the highest regeneration efficiency, producing 100% shoot bud induction and 39.70 shoots per explant on MS medium augmented with 6-benzylaminopurine (BAP; 6.00 mg/L) and kinetin (1.00 mg/L). Shoot elongation and rooting efficiencies were improved using GA3 (1.00 mg/L), BAP (1.00 mg/L), IAA (0.10 mg/L), and NAA (0.5 mg/L), respectively. Manipulating explant type, Agrobacterium optical density, vacuum infiltration, acetosyringone concentration, infection time, and co-cultivation duration improved the transient transformation efficiency. We noted efficiency from 23.33% to 90.00% in DCN and from 6.66% to 93.33% in embryonic axis explants across 10 pea cultivars. Stable transformed lines generated from the DCN of cultivar Kashi Samridhi were confirmed by GUS staining and PCR. The optimized regeneration and transformation system facilitated targeted editing of phytoene desaturase (PsPDS) in pea, achieving ICE-estimated mutation frequencies of upto 97% in independent lines. The study provides a robust platform for functional genomics and accelerates the deployment of genome-editing technologies for pea improvement.
The first successful application of CRISPR/Cas9 genome editing in G. boninense is reported, establishing a robust platform for functional genetic analysis and dissecting pathogenicity in G. boninense, ultimately advancing strategies to mitigate basal stem rot disease in oil palm.
Anis Farhan Fatimi Ab Wahab, Mohd Azinuddin Ahmad Mokhtar, S. Vetaryan et al.· Journal of Fungi· 0 citations
This review synthesises the fundamental principles and limitations of multiple gene-editing technologies, with a particular emphasis on CRISPR systems (Cas9, Cas12, and Cas13), in the specific context of woody perennial biology, and highlights how synergising CRISPR technologies with multi-omics, genomic selection, and high-throughput phenomics can accelerate the development and application of climate-resilient woody perennials.
Tabeer Gulfam, Wanxin Li, Zhi-Yong Han et al.· Plant, Cell and Environment· 0 citations
It is demonstrated that Avr4 does not explain the resistance of Calcutta 4, suggesting that resistance is instead triggered by the recognition of other hitherto unknown effectors.
Maikel B. F. Steentjes, Gregory Ashe, Patricia Schöppl et al.· bioRxiv· 0 citations
A complete CRISPR-Cas9-mediated knockout of the BEL5 gene, encoding a transcription factor, is reported, known as one of the key regulators driving tuber formation, and a regulatory role of BEL5 in the timing of tuber onset but, unexpectedly, its dispensability for tuber development in modern cultivated potato is proposed.
Andrea Zounková, Daniele Chirivì, A. Přibylová et al.· bioRxiv· 0 citations
