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.)

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

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.