TL;DR
This work compares protoplast transfection with emerging alternatives, including tissue electroporation and nanomaterial-, viral-, and mRNA-based delivery, and argues that progress will depend less on editing chemistry than on integrating delivery, tissue culture, regeneration, and validation into genotype-responsive workflows.
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.
An efficient protocol for protoplast isolation from embryogenic calli and protoplast-to-plant regeneration in the allotetraploid species Coffea arabica is presented and polyethylene glycol-mediated transfection of coffee protoplasts with a CRISPR–Cas9 plasmid targeting the coffee xanthosine methyltransferase gene involved in caffeine biosynthesis is demonstrated.
Delivery, rather than nuclease chemistry, now sets the practical limit on plant genome editing. Conventional delivery depends on tissue culture and stable transformation, which remain slow, genotype-dependent and unavailable for most cultivated germplasm. Plant viruses offer an alternative because they replicate to high copy number, move systemically and can carry heterologous sequences into cells that no transformation protocol reaches. Virus-induced genome editing exploits this behaviour, and the field has moved within a decade from transient somatic mutagenesis in a model tobacco to heritable, transgene-free mutations in hexaploid bread wheat. This critical review evaluates the strength and the limits of that evidence. Vector chassis are compared on the properties that actually determine outcome, namely cargo capacity, insert stability, host range, movement behaviour and access to reproductive tissue, rather than on reported somatic editing percentages, which are shown to be poor predictors of germline transmission. The central unresolved problem is identified as the disjunction between efficient editing in infected somatic tissue and rare, stochastic entry of editing reagents into cells that give rise to gametes. Three engineering responses to that problem, namely fusion of guide RNAs to mobile RNA motifs, control of nuclease expression through meristem-competent promoters, and exploitation of axillary or adventitious growth points, are assessed against the evidence for each. Reagent miniaturisation, guide array design and virus-delivered precision editing are examined as partial solutions to the cargo constraint. Recurrent weaknesses in the literature are documented, including reliance on visible reporter loci, small progeny samples, inconsistent definitions of editing efficiency, near-absence of independent replication and the complete absence of field evaluation. The claim that virus-derived products are transgene-free is examined against the regulatory frameworks that will govern them, and the biosafety implications of releasing engineered, potentially insect-transmissible vectors are considered. Prioritised research directions are proposed, emphasising standardised reporting of progeny-level outcomes, biocontained vector design, and genotype-spanning validation in elite crop backgrounds.
A stepwise protocol for targeted gene editing in wheat via wide hybridization with transgenic maize expressing Cas9 and gRNA is described, which provides a one-step approach for generating DH lines with the target gene edited from any wheat genotypes of interest.
An RNA-guided bridge recombinase system is engineered through rational mutagenesis and AI-assisted directed evolution, enabling programmable chromosomal rearrangements in both plant and mammalian cells and achieving up to a 29.8-fold increase in activity.
A novel plant genome editing system is developed by delivering Cas9, Cas12f, and Cas12j nucleases along with their guide RNAs using a broad-host-range geminivirus, Wheat dwarf India virus (WDIV), in combination with Ageratum yellow leaf curl betasatellite (AYLCB).
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