Researchers systematically assessed four key variables affecting transformation: culture medium, A. rhizogenes strain, seedling age and explant type. The selected protocol used strain K599 for infection and Hoagland-moistened vermiculite as the substrate. This combination produced successful hairy-root induction from all three explant types examined. The study did not describe the exact culture conditions, replicate numbers or statistical uncertainty in the supplied abstract, so the performance of the protocol across additional genotypes and experimental environments cannot be determined from this evidence.

Among nine developmental regulators tested, FsMP co-transformation increased leaf transformation efficiency to 95%. The abstract does not report efficiency values for the other eight regulators, meaning that 95% can be described as the reported performance of FsMP co-transformation but not as a demonstrated maximum across the full comparison. The broader objective was to reduce the low efficiency and long culture cycles associated with conventional A. tumefaciens-mediated transformation, which has hindered genomic and molecular-breeding studies in F. suspensa.

The authors characterized the resulting hairy-root system as a rapid, economical alternative to conventional tissue culture and proposed it as a platform for validating gene function and supporting molecular breeding in F. suspensa and other woody species. These are stated research conclusions rather than demonstrated field or breeding outcomes: the supplied evidence comes from an abstract and does not address genetic stability, regeneration of transformed shoots, field performance or long-term expression. The article was also identified as an early peer-reviewed version subject to further editorial changes.

Subject of Research: Optimization of hairy-root transformation and gene editing in Forsythia suspensa

Article Title: Establishment of a high-efficiency hairy root transformation system for Forsythia suspensa and enhancement of transformation efficiency by developmental regulators

Article References: Li, Q., Shao, Q., Wang, D., Zhang, N., Qiao, Y., & Song, Y. (2026). Establishment of a high-efficiency hairy root transformation system for Forsythia suspensa and enhancement of transformation efficiency by developmental regulators. BMC Plant Biology. https://doi.org/10.1186/s12870-026-09996-4

Image Credits: AI Generated

DOI: 10.1186/s12870-026-09996-4

Keywords: Forsythia suspensa, hairy-root transformation, Agrobacterium rhizogenes, FsMP, CRISPR/Cas9, RUBY reporter, leaf transformation, gene editing, woody plants, molecular breeding, Establishment, high-efficiency

RUBY was developed as a genetically encoded visual reporter that produces a vivid red betalain pigment, the same class of compounds that gives beets their characteristic color. Unlike green fluorescent protein or luciferase-based systems, RUBY requires no exogenous substrate, no specialized microscopy equipment, and no expensive imaging workflows. A researcher can simply look at a seedling, a leaf, or a callus and see whether a transgene is being expressed. That simplicity has made it attractive for tasks ranging from confirming transformation efficiency to screening genome-editing events, identifying haploid plants in maize and tomato breeding programs, monitoring transgene stability in lettuce, and even teaching genome editing in undergraduate classrooms.

The reporter works by reconstituting a three-enzyme betalain biosynthetic pathway borrowed from beet. The enzymes act sequentially on substrates drawn from the plant’s own tyrosine metabolism, ultimately producing the red pigment betanin. Because the pathway taps into central aromatic amino acid metabolism, the authors of the new study, led by Amandeep Cheema, Kasavajhala V. S. K. Prasad, Enamul Huq, and Anireddy S. N. Reddy at Colorado State University and the University of Texas at Austin, suspected that constitutive RUBY expression might not be metabolically neutral. Tyrosine sits at the head of the shikimate pathway, a hub that feeds the synthesis of a wide range of compounds, including the auxin precursor pool and other aromatic metabolites that influence growth.

To test that suspicion, the team generated independent transgenic Arabidopsis lines expressing RUBY and compared them with wild-type plants across the full life cycle. The differences were striking. RUBY-expressing plants were significantly smaller than controls and produced fewer leaves. They flowered later, a phenotype that in Arabidopsis often signals disruption of the hormonal and transcriptional networks that govern the transition from vegetative growth to reproduction. Perhaps most consequential for any breeding or seed-production application, the plants frequently developed malformed siliques, the seed-bearing pods of the flower, and produced a markedly reduced yield of seeds.

Below ground, the story was just as dramatic. Root architecture in the RUBY lines was severely impaired, with a pronounced reduction in the number of lateral roots. Lateral root formation is one of the most hormone-sensitive processes in plant development: auxin, the archetypal plant hormone, acts as a local morphogenetic trigger that specifies lateral root founder cells, and both auxin transport and gibberellin signaling feed into the regulatory network controlling lateral root organogenesis. A root system stripped of lateral roots is compromised in its ability to forage for water and nutrients, which likely compounds the above-ground growth defects.

One obvious hypothesis was that the plants were simply starved for auxin, and that supplementing the hormone would rescue the phenotype. The researchers tested this by applying indole-3-acetic acid, the principal natural auxin, to the RUBY-expressing plants. It did not help. The failure of IAA to restore normal development suggested that the disruption ran deeper than a straightforward hormone deficit, pointing instead to a broader perturbation of metabolic homeostasis that could not be corrected by adding back a single hormone.

Targeted metabolomic profiling of the RUBY lines confirmed that suspicion. The analysis revealed widespread alterations in amino acid and hormone pools. Levels of active indole-3-acetic acid were reduced, as were levels of gibberellins, the hormone family that promotes stem elongation, germination, and floral initiation. At the same time, the plants accumulated auxin precursors, indicating a bottleneck or diversion in the auxin biosynthetic pathway. Accumulation of indole-3-acetamide, an auxin precursor, has previously been shown to curtail growth in Arabidopsis by repressing ribosome-biogenesis and development-related transcriptional networks, and tryptophan deficiency is known to retard organ growth by slowing cell expansion. The metabolic signature of the RUBY lines is consistent with this kind of precursor-driven growth inhibition, layered on top of a gibberellin shortfall that would plausibly explain the delayed flowering and reduced stature.

The most convincing piece of evidence came from a genetic dissection of the reporter itself. The team disrupted RUBY expression by suppressing the third enzyme in the pathway, a glucosyltransferase that catalyzes the final step in betanin production. When that step was blocked, the RUBY-associated developmental phenotypes were restored to wild type. In other words, it is not the presence of the transgene or the act of transformation that damages the plants, but the flux of metabolites through the complete betalain pathway. Interrupting the pathway at its final step abolishes both the visible pigment and the collateral metabolic damage, cleanly linking the reporter’s biochemistry to its developmental side effects.

The study is not the first hint that heterologous betalain production can shake up plant metabolism. Recent work in tobacco showed that heterologous biosynthesis of betanin triggers broad metabolic reprogramming, and the relaxation of tyrosine pathway regulation is known to underlie the evolution of betalain pigmentation in the Caryophyllales, the plant order to which beets belong. What makes the new results consequential is the sheer breadth of RUBY’s adoption. The reporter has been built into T-DNA vectors for maize transformation, adapted into split-assay formats for detecting protein-protein interactions and DNA-protein interactions in planta, and used as a visual splicing reporter. If constitutive RUBY expression perturbs hormone and amino acid homeostasis in Arabidopsis, researchers using the system for developmental studies, particularly studies of growth, flowering time, or root architecture, may be measuring the reporter’s side effects alongside the biology they intended to study.

The authors argue that the way forward is not to abandon RUBY but to tame it. Because the damage is caused by pathway flux rather than by the reporter construct itself, regulated expression systems, such as inducible promoters or tissue-specific promoters that confine betalain production to a small subset of cells or a defined time window, should dramatically reduce the metabolic burden while preserving the reporter’s visual punch. Such strategies would let researchers keep the convenience that made RUBY famous, the naked-eye readout with no substrates and no instruments, while avoiding the hidden trade-offs now documented in Arabidopsis. For a tool that has been celebrated for making plant biology visible to the naked eye, the lesson of the new study is that some of what it reveals may be an artifact of its own chemistry, and that the next generation of RUBY experiments will need to be designed with that in mind.

Subject of Research: Metabolic and developmental side effects of the betalain-based RUBY visual reporter in Arabidopsis thaliana

Article Title: RUBY expression alters development and metabolism in Arabidopsis

Article References: Cheema, A., Prasad, K. V. S. K., Huq, E., & Reddy, A. S. N. (2026). RUBY expression alters development and metabolism in Arabidopsis. Plant Molecular Biology, 116(5), Article 102. https://doi.org/10.1007/s11103-026-01764-0

Image Credits: AI Generated

DOI: 10.1007/s11103-026-01764-0

Keywords: RUBY reporter, betalain, Arabidopsis thaliana, auxin, gibberellin, plant hormones, lateral roots, metabolomics, plant biotechnology, genetically encoded reporters, tyrosine metabolism, plant development

The research, led by Luying Chen and Shaojie Han of Zhejiang University together with colleagues at Northeast Agricultural University and Purdue University, centers on a mixture the team calls Agrobacterium Auxiliary Solution, or AAS. The solution combines B5 medium, sucrose, a MES buffering salt, the surfactant Silwet L-77, acetosyringone, and two plant hormones, the cytokinin 6-benzylaminopurine and gibberellin A3. Each ingredient has a rationale. Acetosyringone is a phenolic compound that induces the virulence genes Agrobacterium needs to transfer DNA into plant cells. The hormones encourage wounded tissues to regenerate roots. Silwet L-77, a detergent famously used in the floral-dip transformation of Arabidopsis, reduces surface tension so that the bacterial suspension can infiltrate plant surfaces and reach cells at wound sites.

To measure whether the solution actually worked, the researchers first had to solve a detection problem. Soybean roots naturally autofluoresce, which makes conventional green and red fluorescent protein markers difficult to distinguish from background signal. The team therefore built a panel of reporter vectors using Golden Gate assembly into the pAGM4673 binary backbone, all driven by a double cauliflower mosaic virus 35S promoter. Two of these carried the brighter fluorescent proteins ZsGreen and TdTomato, which proved clearly distinguishable from the roots’ own fluorescence under a stereomicroscope. The third was based on the Ruby reporter system, which packages the three betalain biosynthesis enzymes CYP76AD1, DODA, and glucosyltransferase into a single open reading frame. Because tyrosine, the substrate for betalain synthesis, is present in every plant cell, Ruby-positive roots turn visibly red without any microscope at all.

With reporters in hand, the team tested AAS on the cotyledon transformation method, in which excised soybean cotyledons are infected with Agrobacterium rhizogenes strain Ar.Qual and induced to sprout transgenic hairy roots. In the variety Wandou 28, adding only the hormone mixture to the solution produced a modest, statistically insignificant increase of roughly 1.9-fold in the proportion of positive roots among total roots. But when Silwet L-77 was included alongside the hormones, the transformation efficiency jumped to about 3.8 times the untreated control. The same pattern held when the researchers scored cotyledons rather than individual roots: the hormone mixture alone gave a 2.5-fold increase in the fraction of cotyledons producing positive roots, while hormones plus surfactant delivered a 4.0-fold enhancement. The combined treatment also raised the overall rate at which cotyledons produced any hairy roots at all, by about 1.4-fold, indicating that the solution stimulates both infection and regeneration.

Crucially, the benefit was not confined to a single variety. When the team applied AAS across Zhonghuang 39, Williams 82, Forrest, and Wandou 28, positive root conversion rates rose three to four times above baseline in every genotype tested. The effect on overall root induction varied more by variety; in Zhonghuang 39 and Williams 82 the proportion of cotyledons making roots was not significantly changed, whereas in Forrest and Wandou 28 it was. Even so, the consistent boost in positive transformation across genotypes suggests the solution addresses a bottleneck that is common to soybean rather than an idiosyncrasy of one cultivar. The full workflow, from seed sterilization to scored hairy roots, takes only about 22 days, and the cotyledon method routinely converted 90 to 99 percent of infected explants into hairy-root producers, of which 30 to 60 percent were genuinely transgenic.

The study also uncovered a variable that transformation protocols rarely discuss explicitly: the size of the DNA payload. Markers such as GFP, at under one kilobase, and GUS, at roughly two kilobases, historically yielded high transformation rates. But when the researchers used the full three-gene Ruby cassette, totaling about four kilobases, efficiency dropped sharply. GFP and RFP constructs achieved root transformation efficiencies around 52 percent, while Ruby 1+2+3 managed only about 29 percent. At the cotyledon level the gap was similar, with fluorescent markers reaching 89 to 96 percent positive rates and the full Ruby cassette falling to 64 percent. A truncated two-gene version, Ruby 1+2, which still produces a red-root phenotype at 2.3 kilobases, performed better than the full cassette, confirming a size-dependent trend rather than a quirk of the betalain system itself.

The team pushed this observation further with a co-transformation experiment, splitting the Ruby system across two separate binary vectors, one carrying Ruby 1+2 and the other Ruby 3, and mixing two Agrobacterium strains to deliver both. A root only turned red if both vectors landed in the same cell, making co-transformation directly visible. The two-vector approach worked, producing red roots indistinguishable from those made by the single full-length cassette, but the efficiency was slightly yet significantly lower than the single-vector version, and fewer cotyledons produced roots at all. For researchers designing multi-gene constructs or genome-editing payloads, the message is clear: every additional kilobase and every extra vector carries a measurable cost, and compact designs will pay dividends in recovery of transgenic material.

AAS proved equally valuable in a second, even simpler system. In the hypocotyl method, used to generate so-called composite soybean plants with transgenic roots on wild-type shoots, six-day-old seedlings are soaked in an Agrobacterium suspension and then cut just below the cotyledons, allowing the solution to be drawn into the stem. This approach requires no sterile facilities at all. Testing Williams 82, Forrest, and Dongnong 50 with both Ruby vectors, the researchers found that AAS consistently raised both the positive root efficiency and the positive explant efficiency. Varietal differences were pronounced, with Forrest behaving distinctly from Williams 82 and Dongnong 50, a reminder that genotype remains a major determinant even with an optimized protocol. Interestingly, the strain Ar.Qual, which performed well on cotyledons, failed entirely in the hypocotyl system, underscoring that strain choice and tissue context matter as much as the additives themselves.

Perhaps most significantly for the future of stable soybean engineering, AAS also improved the classic Agrobacterium tumefaciens-mediated cotyledonary node transformation, the workhorse method for producing fully transgenic plants. Using the pCAMBIA3301-GUS vector with A. tumefaciens strain EHA105 on the variety Dongnong 50, the team found that adding AAS raised the GUS staining rate in cotyledons from 45.8 percent to 73.4 percent, and the staining rate in adventitious shoots from 43.2 percent to 67.5 percent. Because cotyledonary node transformation is the gateway to heritable, whole-plant lines, an improvement of this magnitude could shorten the timelines that currently make soybean functional genomics and genome editing so laborious.

The broader implications extend beyond soybean. Transgenic hairy roots generated by these systems are versatile tools for protein expression, subcellular localization, bimolecular fluorescence complementation, and the screening of guide RNAs before committing to full CRISPR/Cas9 plant production. The hairy root approach has already been adapted in tomato and other species, and the authors suggest the AAS-enhanced protocol could be tuned for root biology studies across a wide range of plant genotypes. For a crop in which narrow genetic diversity and long breeding cycles have slowed both basic research and applied improvement, a cheap, mix-and-soak solution that reliably multiplies transformation efficiency is the kind of unglamorous but consequential advance on which much of modern plant biotechnology depends.

Subject of Research: Improving Agrobacterium-mediated soybean transformation efficiency using an auxiliary solution with surfactant and plant hormones

Article Title: Enhancing Agrobacterium-mediated soybean transformation efficiency with an auxiliary solution

Article References: Chen, L., Wang, L., Zhang, L., Li, Y., & Han, S. (2024). Enhancing Agrobacterium-mediated soybean transformation efficiency with an auxiliary solution. Crop Health, 2(1), Article 17. https://doi.org/10.1007/s44297-024-00037-w

Image Credits: AI Generated

DOI: 10.1007/s44297-024-00037-w

Keywords: soybean, Agrobacterium rhizogenes, hairy root transformation, Silwet L-77, Ruby reporter, plant hormones, genetic transformation, cotyledon transformation, hypocotyl transformation, plant biotechnology, GUS assay, vector size