Generation of lachrymatory factor synthase–suppressed onion (Allium cepa L.) by Agrobacterium-mediated gene transfer for CRISPR/Cas9 genome editing

Recovery of transgenic calli through Agrobacterium-mediated transformation in Quillaja lancifolia

Quillaja lancifolia D. Don (current correct taxonomic designation of the synonym Q. brasiliensis (A. St.-Hil. & Tul.) Mart.) is a promising source of triterpenoid saponins of high biotechnological value, including QS-21, a potent immunological adjuvant used in human vaccine formulations, producible in cell suspension cultures. Herein, an efficient Agrobacterium tumefaciens–mediated genetic transformation protocol for generating transgenic calli of Q. lancifolia was established. Transformation was performed using A. tumefaciens EHA105 harboring the binary vector pH7WG2D-GUS containing the β-glucuronidase (GUS), the green fluorescent protein (GFP), and hygromycin phosphotransferase (hpt) genes. Under the established conditions (OD₆₀₀ = 0.6, 30 min infection, 72 h co-cultivation), hygromycin-resistant callus sectors expressing reporter genes were consistently obtained on hygromycin-containing culture medium. Transgene presence was confirmed by PCR amplification of the hpt gene from genomic DNA. Histochemical GUS staining and GFP fluorescence were shown after confirmation of Agrobacterium elimination by successive subcultures on meropenem-containing medium, followed by cultivation on antibiotic-free conditions. The overall transformation efficiency reached 41.6 ± 4.6%, calculated as the percentage of original explants producing GFP-positive hygromycin-resistant callus. The protocol herein described constitutes a useful tool for the establishment of transgenic callus, enabling future metabolic engineering studies in Q. lancifolia cell cultures. An efficient Agrobacterium tumefaciens-mediated genetic transformation of Quillaja lancifolia was established, enabling transgenic callus generation.

CRISPR/Cas9-mediated transformation enables functional characterization of the effector Avr4 in the banana pathogen Pseudocercospora fijiensis

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.

Dual-gRNA CRISPR/Cas9 Deletion of CsDMR6 in Sweet Orange Supported by Improved In Vitro Regeneration

Huanglongbing (HLB), caused by Candidatus Liberibacter spp., remains the most destructive disease affecting citrus worldwide. To support host-directed genome-editing strategies aimed at reducing susceptibility, we optimized key regeneration steps in Citrus sinensis and validated a dual-gRNA CRISPR/Cas9 approach targeting the susceptibility gene CsDMR6. Juvenile explants of ‘Valencia’ and hybrid genotypes (CsH1–CsH3) were successfully established in vitro, and shoot elongation was markedly improved by supplementing Citrus Shoot Multiplication (CiSM) medium with 1 mg L−1 GA3. Callus induction was most efficient in Citrus Callus Induction (CiCM) medium under dark conditions, while a 48 h NAA pulse (100 µM) significantly enhanced rooting, increasing efficiencies to 37.1% in ‘Valencia’ and 52.9% in CsH1. Two guide RNAs targeting conserved regions of CsDMR6 were designed and shown to be identical across all evaluated genotypes. The dual-gRNA cassette was assembled into a CRISPR/Cas9 geminivirus-based vector and transiently delivered into sweet orange leaf tissue via Agrobacterium. GFP fluorescence verified construct expression, and PCR amplification across the target region produced a diagnostic ~447 bp fragment corresponding to the expected ~5.8 kb deletion. Sanger sequencing confirmed precise junction formation between the two cut sites. These results demonstrate efficient large-fragment deletion of CsDMR6 in sweet orange and establish an experimentally validated, genotype-compatible regeneration and editing platform. This study provides a transient validation of the dual-gRNA system and establishes the technical foundation required for future stable, non-transgenic edited lines. Together, these advances support the downstream functional evaluation of CsDMR6 loss-of-function alleles under HLB pressure.

Tiny but powerful: protoplast isolation in Solanum melongena L. and RNPs mediated genome editing

Transgene-free genome edited plants were regenerated from protoplasts, representing the first report of RNP mediated genome editing in eggplant protoplasts.

CRISPR/Cas9-mediated targeted mutagenesis of GmMYB100 to increase isoflavone content in soybean seeds

Homologous knock-in processes facilitated by CRISPR-Cas9 in Aurantiochytrium and disrupting the gene for DNA ligase IV prevents transformation events

The potential of these CRISPR-Cas9 systems to serve as a robust foundation for the functional genomics and metabolic engineering of A. limacinum is demonstrated.