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