Genetius

The study systematically examines sources of variability in Agrobacterium tumefaciens-mediated transient expression in Nicotiana benthamiana, analyzing a large dataset of 1,915 plants collected over three years. It demonstrates that normalization methods must be validated for each experimental context and provides a statistical model and power analysis framework to determine appropriate sample sizes for detecting specific effect sizes, offering practical guidelines to improve reproducibility in quantitative plant and synthetic biology studies.

The study engineered Tobacco rattle virus vectors incorporating distinct RNA secondary structures as mobility factors to improve guide RNA delivery to plant meristems. Using Nicotiana benthamiana plants expressing Cas9, optimal virus constructs were identified that generated both somatic and heritable edits, and these constructs were successfully applied to edit the emerging oilseed crop pennycress (Thlaspi arvense).

The authors introduce the ENABLE(R) Gene Editing in planta toolkit, a streamlined two‑step cloning system for creating CRISPR/Cas9 knockout vectors suitable for transient or stable transformation. Validation was performed in Oryza sativa protoplasts and Arabidopsis thaliana plants, and the toolkit includes low‑cost protocols aimed at facilitating adoption in the Global South.

The authors created a fast‑cycling, isogenic barley line (GP‑rapid) by introgressing the wild‑type Ppd‑H1 allele from Igri into the Golden Promise cultivar and performing two backcrosses to limit the donor genome, achieving a 25% reduction in generation time under speed‑breeding conditions while retaining high transformation efficiency. CRISPR/Cas9‑mediated editing of Ppd‑H1 showed regeneration and transformation rates comparable to the original Golden Promise, establishing GP‑rapid as a rapid platform for transgenic and gene‑edited barley research.

The study applied CRISPR/Cas9 gene editing to Physalis peruviana to modify plant‑architecture genes and create a compact growth ideotype. This compact phenotype is intended to increase per‑plot yield and support future breeding efforts for this nutritionally valuable minor crop.

The study systematically identified heterosis-associated genes and metabolites in rice, functionally validated three genes influencing seedling length, and integrated these molecules into network modules to explain heterosis variance. Predominant additive and partially dominant inheritance patterns were linked to parental genomic variants and were shown to affect 17 agronomic traits in rice, as well as yield heterosis in maize and biomass heterosis in Arabidopsis. The work highlights the quantitative contribution of transcriptomic and metabolomic variation, especially in phenylpropanoid biosynthesis, to hybrid vigor.

Using CRISPR‑Cas9, researchers knocked down CCD7 or CCD8 in Nicotiana benthamiana to suppress strigolactone synthesis, producing compact plants with a 45%–50% smaller spatial footprint while preserving recombinant protein yields (GFP and rituximab). The mutants showed altered leaf proteome, auxin/cytokinin balance, and metabolic fluxes without affecting overall growth rate, demonstrating suitability for indoor vertical farming biopharma production.

The authors created two Nicotiana benthamiana lines with CRISPR-mediated knockouts of two polyphenol oxidase genes, which exhibited slightly accelerated growth and retained normal transient GFP expression. These ppo-deficient plants produced leaf extracts that remained greener with markedly reduced enzymatic browning and protein crosslinking, leading to a nearly fourfold increase in yield and improved purity of a transiently expressed His‑tagged tomato protease. The study demonstrates that PPO depletion can enhance recombinant protein recovery from plant tissue.

The study used CRISPR/Cas9 to edit the downstream region of the Arabidopsis thaliana FLOWERING LOCUS T (FT) gene, identifying a 2.3‑kb segment containing the Block E enhancer as crucial for normal FT expression and flowering. Fine‑scale deletions pinpointed a 63‑bp core module with CCAAT‑ and G‑boxes, and revealed a cryptic CCAAT‑box that becomes active when repositioned, highlighting the importance of local chromatin context and motif arrangement for enhancer function.

The study optimized three wheat transformation methods—immature embryo, callus, and in planta injection—by systematically adjusting Agrobacterium strain, bacterial density, acetosyringone concentration, and incubation conditions, achieving transformation efficiencies up to 66.84%. Using these protocols, CRISPR/Cas9 knockout of the negative regulator TaARE1-D produced mutants with increased grain number, spike length, grain size, and a stay‑green phenotype, demonstrating the platform’s potential to accelerate yield and stress‑tolerance improvements in wheat.