Efficient in-vitro regeneration and transformation for CRISPR/Cas9-mediated genome editing of phytoene desaturase (PDS) gene in pea (Pisum sativum L.)

Establishment of an efficient Agrobacterium-mediated transformation system and CRISPR/Cas9-mediated genome editing of the OvPDS1 gene in Oxalis vulcanicola ‘Sunset Velvet’

SAM-Targeted CRISPR-Cas9 RNP Delivery Combined with Leaf Regeneration Enables DNA-Free, Non-Chimeric Genome Editing in ‘Fuji’ Apple

iPB-REG is established as a practical strategy for producing uniform genome-edited fruit trees and provide a valuable platform for DNA-free genetic improvement and functional genomics in clonally propagated perennial crops.

Development of efficient methods for protoplast-to-plant regeneration in Coffea arabica and successful CRISPR-Cas9 editing in protoplasts

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.

CRISPR/Cas9-based gene-editing platform development by targeting genes encoding magnesium chelatase and phytoene desaturase in sugarbeet (Beta vulgaris L.)

Sugarbeet (Beta vulgaris ssp. vulgaris, L.), is a vital temperate crop, supplying nearly 40% of the world’s sugar. However, its high susceptibility to bacterial, fungal, and viral diseases creates an urgent need for improved, disease-resistant cultivars. The CRISPR/Cas9 system has rapidly advanced plant genetic engineering by enabling precise and targeted genome modifications. Our goal is to establish a gene-editing platform in sugarbeet to support future development of disease-resistant lines by targeting the candidate genes. In this study, we applied CRISPR/Cas9 to generate targeted mutations in two genes involved in chlorophyll biosynthesis and carotenoid-mediated leaf pigmentation: magnesium chelatase (Mg-chelatase) and phytoene desaturase (PDS). Two CRISPR/Cas9 constructs, each carrying an sgRNA targeting either Mg-chelatase or PDS, were developed and mobilized into Agrobacterium tumefaciens. A total of 233 and 200 hypocotyl explants were transformed with constructs targeting Mg-chelatase and PDS, resulting in regeneration efficiencies of 8% and 14% on kanamycin selection medium, respectively. Light green, yellow, variegated yellow-green, and albino phenotypes were observed among the putative transformants, whereas non-edited transformed lines resembled untransformed control plants. Targeted mutations, including insertions, deletions, and substitutions of nucleotides, were identified at both genomic loci, with editing efficiencies of 60.0% for Mg-chelatase and 68.75% for PDS underscoring the effectiveness of this approach in sugarbeet, a recalcitrant crop. Deletions ranged from 5 to 28 bp in Mg-chelatase and 2 to 21 bp in PDS, while insertion events consisted of single-base additions in Mg-chelatase edited lines and larger insertions of 7–16 bp in PDS mutants. The results demonstrate the successful deployment of CRISPR/Cas9 for targeted genome engineering in sugarbeet and establish a reliable platform for future gene-editing efforts aimed at enhancing resistance to a wide range of pathogens and diseases affecting the crop.

Development of plant regeneration and stable Agrobacterium tumefaciens–mediated transformation methods for Solanum retroflexum Dunal

Solanum retroflexum is cultivated for both its fruit and leaves in Africa, China, India, and Indonesia. However, it is still considered an underutilized crop due to limited genetic and genomic resources to support improvement. To enable gene function studies for future improvement by new breeding technologies, this study established efficient plant regeneration and Agrobacterium tumefaciens –mediated transformation approaches. Cotyledon and hypocotyl explants were used to test plant regeneration on a Murashige and Skoog salts-based medium supplemented with 0.5 mg L −1 zeatin. Both explant types showed 100% regeneration; however, cotyledons produced a greater number of whole plants than hypocotyls. For transformation experiments, cotyledon explants were infected with A. tumefaciens AGL1 carrying the pJL33 binary vector containing the Green Fluorescent Protein ( GFP ) reporter and the Neomycin Phosphotransferase-II ( nptII ) selectable marker genes. Following cocultivation, firstly, kanamycin at various concentrations (75.0 to 400.0 mg L −1 ) was evaluated to determine the optimal concentration for maximizing transgenic line recovery. However, a significant number of escapes even at 400.0 mg L −1 kanamycin were observed. Because the nptII gene also confers resistance to G418, in this study’s subsequent experiments the current authors tested the effectiveness of a range of G418 concentrations (25.0 to 150.0 mg L −1 ). Fluorescence microscopy and PCR analysis showed that 25.0 mg L −1 G418 resulted in the highest transformation efficiency at 88% and the fewest escapes. Ploidy levels of regenerated, transgenic, and wild-type plants were assessed using flow cytometry, confirming stable ploidy. The optimized selection medium facilitated an efficient and stable transformation system that will be utilized to support crop improvement efforts of S. retroflexum .

Establishment of a High-Efficiency In Vitro Regeneration and Agrobacterium-Mediated Genetic Transformation System for Ajania achilleoides (Turcz.) Poljakov

Ajania achilleoides, a wild species with strong environmental adaptability, is a valuable germplasm resource for genetic improvement and functional gene studies. However, efficient in vitro regeneration and genetic transformation systems for this species remain undeveloped, limiting functional gene characterization and genetic improvement of this valuable germplasm resource. In this study, leaf explants were used to establish an efficient regeneration system by evaluating different combinations of plant growth regulators and to develop an Agrobacterium tumefaciens-mediated genetic transformation system for A. achilleoides. MS medium supplemented with 2.0 mg/L 6-benzyladenine (6-BA) and 1.0 mg/L α-naphthaleneacetic acid (NAA) achieved the highest adventitious shoot regeneration rate (99%), while 1/2 MS medium containing 0.5 mg/L indole-3-butyric acid (IBA) was optimal for rooting, resulting in a 100% rooting rate. Critical hygromycin concentrations for shoot regeneration and rooting selection were determined to be 25 mg/L and 10 mg/L, respectively. For transformation, the best results were obtained with a 3-day preculture, bacterial suspension at OD600 = 0.2, 30 min infection, and 2-day co-cultivation. PCR and sequencing confirmed the successful integration of the target genes into the genome, yielding three positive transgenic lines with a molecular confirmation rate of 30%. This study establishes a stable and efficient regeneration and transformation system for A. achilleoides, providing a platform for functional gene analysis and molecular breeding of wild chrysanthemum species.