Scalable production of soybean hairy roots: a reliable method for field trials

Scalable production of soybean hairy roots: a reliable method for field trials | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Scalable production of soybean hairy roots: a reliable method for field trials Luciano Nicolás Caraballo, Jesica Raineri, Raquel Lía Chan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8353559/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Mar, 2026 Read the published version in Plant Cell Reports → Version 1 posted 9 You are reading this latest preprint version Abstract Roots are the primary organs for sensing abiotic and biotic stress factors originating in the soil. A critical biological question is whether root plasticity in response to these stresses influences whole-plant development. The generation of soybean chimeric plants with an altered root transcriptome offers a powerful approach to address this question. The existing protocols for this strategy typically require sterile conditions and produce a limited number of chimeric plants. We optimized the technique by introducing several key modifications, significantly enhancing its efficiency. The new protocol eliminates the requirement for sterile conditions in most steps. Moreover, fresh bacterial transformation for each experiment was performed, overcoming the loss of infection ability associated with storing cultures at −80 °C, reaching 70-100 % efficiency. A previous transformation of cotyledons helped to select the colony with the highest infection ability. The optimal plant growth temperature was determined to be 22 °C. These combined changes and tips resulted in the consistent production of hundreds of chimeric plants, making them suitable for large-scale field trials. Results from subsequent field trials performed in three seasons confirmed that alteration of the root transcriptome significantly impacts whole-plant development. soybean hairy roots field trials GFP hairy roots Rhizobia infectivity loss Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Key message We optimized soybean hairy root production for field trials using newly transformed rhizobia, a 22°C growth temperature, and GFP flashlight visualization. Introduction The obtaining of composite plants with transgenic hairy roots is a powerful strategy for many research and biotechnological purposes. From a fundamental research point of view, such a strategy allows for revealing protein function by overexpressing or silencing a given gene, studying the effect of expression alteration on the whole plant performance or the transport of metabolites driven by specific proteins, among other objectives. Considering biotechnology, many metabolites used in medicine or industry can be obtained using hairy roots, which is a faster method compared with stable transformation. Moreover, composite plants with transgenic hairy roots are not considered GMO because seeds are not transgenic, allowing more freedom to do field trials. Hairy roots are obtained after plant infection by Rhizobium rhizogenes in a variety of species. A deep revision about the characteristics of Rhizobium rhizogenes and its differences with Agrobacterium tumefaciens has been published recently (Kiryushkin et al. 2021 ). Aside from transgene expression, they can be used as producers and secretors of a wide range of metabolites used in the cosmetic and pharmaceutical industries (Gutierrez-Valdes et al. 2020 ). Although the molecular mechanism involved in hairy root formation is not well understood, the whole process was divided into several steps, including the bacterial transformation and the transfer of T-DNA to the plant genome, ending in hairy root emergence (Georgiev et al. 2012 ). Many studies have focused on the use of hairy roots to produce proteins or metabolites (Peebles et al. 2009; Häkkinen et al. 2016 ; Robert et al. 2014 ). For these purposes, biomass production and culture stabilization have been the crucial improvement points, including testing different elicitors. Another common use of the hairy root technique is the study of nodulation capacity. For example, the expansin GmEXPA11 was highly induced by rhizobial infection and showed a positive role in nodule enlargement (Xing et al., 2025 ). Similarly, the gene encoding the phosphatase GmPP2C61A was upregulated by rhizobia inoculation, and its knockout reduced the number of nodules (Gao et al. 2024 ). On the contrary, TML encoding genes overexpressed in hairy roots resulted in a nodule number decrease, indicating a negative role in nodule organogenesis (Su et al. 2025 ). Moreover, symbiont perception and nodulation capacity under stress were studied through the characterization of plants with hairy roots transformed with Phosphatidylinositol 3-kinase (Robert et al. 2018 ). Several studies showed how the transformation of roots impacted morphological or physiological characters of the whole plant. For example, the ARF3 coding region was expressed in tobacco hairy roots, altering internode length, branch number, leaf blade, and flowering time, provoking dwarfism in plants grown in pots (Wang et al., 2016 ). The hairy roots technique was also combined with gene editing, potentiating the objectives that can be achieved through functional genomics (Kiryushkin et al. 2021 ). In that work, vectors, promoters, reporters, and other features of the editing system were compared and optimized (Kiryushkin et al. 2021 ). Soybean (Glycine max L. Merr), a dicot oleaginous species, is mostly used in animal and human feeding. Moreover, it is one of the most important crops worldwide, whose yield and seed quality are limited by abiotic and biotic stress factors (Hartman et al. 2012). Genetically modified soybeans were released to the market more than thirty years ago, exhibiting herbicide resistance and later insect tolerance. Despite its importance and negative impact, the only market-released event showing abiotic stress tolerance is soybean HB4 (Ribichich et al. 2020 ). There are others successfully tested in the field, albeit they have not been approved for commercialization, an expensive process that takes many years (Raineri et al. 2025 ). The reasons for this situation, not only with soybeans but with other important crops, are several. In general, there is a bad public perception of GMOs. However, this fact did not prevent 90% of the worldwide planted soybeans from being genetically modified ( www.isaaa.org ). A second problem is that abiotic stress-tolerant plants must be tested in multiple environments because they do not represent universal technologies. This means that the business for companies is not so lucrative as that of biotic-resistant crops (Chan et al. 2020 ). However, and no less important, one of the main issues to solve is that gene discovery experiments performed in model species are not always translatable to crops (Uauy et al. 2025 ). Field trials with crops can be carried out only once a year, after obtaining stable transgenics, which is costly and time-consuming. Given these reasons, the use of the hairy roots technique allows for testing different genes and their impact on yield and seed quality in controlled conditions throughout the year. Several protocols were already published to obtain soybean hairy roots (Araragi et al. 2025 ; Fan et al. 2020 ; Su et al. 2025 ) and used to study the crop tolerance of resistance to a wide range of abiotic and biotic stress factors and the involvement of varied genes in such responses (Jia et al. 2025 ; Li et al. 2025 ; Wang et al. 2025 ; Zhang et al. 2025 ). However, the protocols are time-consuming, tedious, require sterility, and usually allow for obtaining a very limited number of plants, considering that the reported efficiencies were mostly between 40 and 70%. That is also because the focus of such studies has not been the assessment in field trials, which demands a large number of transformed plants. Another significant limitation is the detection of transformed hairy roots, which is time consuming and may imply material destruction when using reporter genes detected by histochemistry, such as GUS. Reporter constructions take relevance because adventitious roots and hairy roots transformed only with the Ri helper plasmid also grow from the plants, even if the transformation with the gene of interest does not take place. When the efficiency is low, such events significantly reduce the transgene effects on the whole plant. We did not detect a protocol suitable for obtaining a good number of hairy root plants to perform a field trial. Moreover, the crucial bacterial infection capability during prolonged periods was not informed. Here we present an optimized protocol that allows us to obtain more than 200 soybean plants with transgenic hairy roots in three weeks and perform a field trial with them, using adequate controls. The main changes with other published methods are that the present transformation protocol eliminates the requirement for sterile conditions, the maintenance of bacterial infection capacity, the detection of the reporter GFP gene in situ and in vivo, and the growing conditions. This method is suitable both for overexpressing or silencing genes and would also be useful for performing gene editing. Material and methods Plant Material Soybean seeds from the genotype Williams 82 were used in this study. These were surface sterilised with 20% bleach in deionized water for 10 minutes, then rinsed five times with deionised water. Materials needed to carry out the hairy roots protocol Soybean seeds, 20% bleach, autoclaved deionised water, plastic trays, paper towels, autoclaved deionised water and foil film, autoclaved agar medium, 1% w/v in deionised water, sterile Petri dishes, autoclaved 15 mL Falcon tubes with 3 mL liquid LB medium each, and sterile syringes. Growth conditions and obtaining of hairy roots For chimeric whole plants transformation, the seeds were sowed on sterilised coarse vermiculite watered with deionised water. The seeds were then disposed in high density (up to 18 seeds per 13.5 cm x 10 cm plastic tray, extrapolated to up to 1300 seeds per m 2 ), and incubated in a growth chamber under long day cycle, 26 to 28°C temperature and using high intensity sodium lamps (16 h light/8 h dark cycle, 230–260 µmol. m − 2 .sec − 1 light intensity, 50–60% humidity). Transformed shoots were disposed on plastic recipients containing sterilised coarse vermiculite, previously irrigated until saturation with deionised water. Plant density was kept at approximately 40 plants in 46.5 x 34 cm containers (250 plants per m 2 ), and sealed with foil film to maintain high humidity at a height of at least 25 cm. These containers were cultured in a growth chamber under long day cycle, 22 to 24°C temperature and using low intensity led lamps (16 h light/8 h dark cycle, 60–70 µmol. m − 2 . sec − 1 light intensity, 50–60% humidity). Seeds germinated for cotyledon transformation were grown on plastic trays with paper towels, watered using deionised water and covered with a transparent foil film to maintain high humidity. Seeds were also disposed in high density, up to 30 seeds per 26 x 19 cm plastic tray (600 per m 2 ), and incubated in a growth chamber under long day cycle, 26 to 28°C temperature, and using high intensity sodium lamps (16 h light/8 h dark cycle, 230–260 µmol. m − 2 .sec − 1 light intensity, 50–60% humidity). Transformed cotyledons were disposed in plates containing 1% w/v agar in deionised water medium, up to 20 cotyledons per 14 cm-diameter Petri dish. These plates were then incubated in a growth chamber under long day cycle, 22 to 24°C temperature and using low intensity led lamps (16 h light/8 h dark cycle, 60–70 µmol. m − 2 . sec − 1 light intensity, 50–60% humidity). Genetic constructs and transformation To generate hairy roots, we cloned different coding sequences in the backbone of the plasmid binary vector pH7WG2D (Karimi et al. 2002 ) for the overexpression of homeodomain leucine zipper subfamily I (HD Zip I) genes. We also cloned fragments of these genes in the backbone of the plasmid binary vector pK7GWIWG2D (II) (Karimi et al. 2002 ) for the siRNA silencing of HD Zip I genes. Empty versions of both plasmids were generated by enzymatic digestion of the overexpressing cassette, containing the ccb death domain, in pH7WG2D; and by recombination of a dummy sequence in pK7GWIWG2D (II), for the correct assembly of the double stranded RNA and successive processing as dummy siRNA. All constructs had a GFP overexpressing cassette that allowed us to follow transformation efficiency in vivo and in situ , and were introduced in Rhizobium rhizogenes strain K599 using an electroporation transformation method. Transformed bacteria were stored at -80°C in 25% glycerol and then used to induce hairy roots formation on soybean explants. When selecting bacterial selection markers, if the plasmid bears a streptomycin/spectinomycin resistance, namely an aminoglycoside 3’ adenyltransferase (AadA) gene, normal growth inhibitory concentrations (100 µg spectinomycin/mL) are insufficient. In this case, we found that concentrations of 350 to 450 µg spectinomycin/mL generate a desired selection for the correct identification of positive colonies in LB agar plates. This scenario was not observed when using kanamycin as the selection agent for kanamycin resistance vectors. Whole plant hairy roots transformation protocol For whole plants transformation, soybean seeds were grown as previously described until their unifoliate leaves developed but remained unexpanded (Fig. 1 A), which usually takes between 5 to 7 days after sowing. Further development, as leaves expansion, may result in higher lethality of the transformation process, as expanded leaves grow bigger and then evaporate more water that the explants can withstand. Forty-eight hours before the transformation, selected high transformation efficiency bacterial colonies carrying the different vector constructs were cultured in sterile LB liquid medium overnight at 28°C and 200 rpm. The exponential grown cultures were then used to inoculate sterile LB agar plates, about 200 µL for a 9 cm-diameter plate. Bacteria were distributed evenly on the plate using a sterilised Drigalski spatula and left to dry for about 5 minutes. These plates were cultured for 24 hours at 28°C. Plates were grown until visible biomass appears on the surface. It is important to only use one selection antibiotic during these pre-transformation steps, as using more antibiotics, or higher concentrations, can lead to lower hairy roots transformation efficiency. In this case, using the plasmid selection marker antibiotic is recommended. In our hands, normal concentrations of 50 µg/mL kanamycin and 100 µg/mL spectinomycin do not seem to negatively affect transformation efficiency. From here onwards, sterile conditions were not needed. High humidity chambers were prepared the day before transformation, using approximately 5 cm of sterilised coarse vermiculite in plastic containers of at least 30 cm total height, to allow for unhindered shoot development during the transformation protocol. This vermiculite was then saturated with deionised water until about 1 cm of free water was obtained. On the transformation moment, an alcohol-flamed scalpel was used to generate an angled cut on the soybean plant’s hypocotyl, approximately 1 cm below the cotyledonary node (Fig. 1 B). The angled cut generates a wider section for the bacteria to transform, improving the transformation efficiency and hairy roots maximum population. A butter knife was alcohol flamed and air-cooled, then used to collect bacterial biomass from the surface of the plates (Fig. 1 C). This biomass was then immediately applied to the lesion (Fig. 1 D), and infected explants were introduced in the humid chambers previously prepared, planting them in vermiculite up to just below their cotyledons. These chambers were then covered with foil film (Fig. 1 E) and cultured as previously described, in a growth chamber under long day cycle, 22 to 24°C temperature and using low intensity led lamps. Cotyledons hairy roots transformation protocol For cotyledons explants transformation, soybean seeds were grown on germinators, as previously described, until greening, for about 4 to 5 days (Fig. 2 A). Twenty-four hours before transformation, K599 bacteria were inoculated on 15 mL Falcon tubes with 3 mL of sterile LB liquid medium and cultured overnight on a shaker at 28°C and 200 rpm. The media can be inoculated from fresh individual colonies, or directly from − 80°C glycerol stocks. The next day, bacterial biomass is collected by centrifugation at 2500 g for 5 minutes. Then, the residual LB of the supernatant was discarded, and the pellet is resuspended in 1 mL of autoclaved deionised water. These bacterial suspensions can be stored for up to an hour at 4°C without significantly affecting transformation efficiency. Agar plates for the transformed explants are prepared by pouring the autoclaved agar medium on sterile Petri dishes. From here onwards, there was no need for sterile working conditions. An alcohol-flamed scalpel was used to cut the cotyledons 1mm from their node, as can be seen on Fig. 2 B. Then, a syringe was loaded with bacterial suspension, one droplet was left in the cotyledon’s lesion, and it is then lightly stabbed repeatedly (about 10 times, Fig. 2 C) to facilitate infection. The infected explant was then placed on the agar plate, their abaxial face down, and the lesion facing outside. Subsequent explants were disposed contiguously, up to 10 cotyledons per 9 cm-diameter Petri dish (Fig. 2 D), or up to 20 cotyledons for 14 cm-diameter Petri dish. A negative control was done by stabbing cotyledons using a syringe loaded only with sterile deionised water. These plates are then cultured as previously described, in a growth chamber under long day cycle, 22 to 24°C temperature and using low intensity led lamps. During this period, microbial growth may occur and should be monitored. For instance, if fungal colonies develop on the cotyledons, the affected tissues should be removed to preserve the integrity of the remaining explants. Conversely, if the contamination affects the medium itself, it is advisable to transfer the transformed explants to a fresh agar plate. To avoid water condensation in the dishes, the plates were incubated on shelves without any heat source below, including another illuminated shelf. Between 7 and 10 days after transformation, infected explants should exhibit greenish callus growth at the lesion site, which may express reporter genes, if present (Figs. 2 E and F). At 12 to 14 days after transformation, cotyledons should have developed hairy roots from the infection site, which in turn may express the reporter gene (Fig. 2 G and H). The hairy roots formation must not occur in the negative control. Under these conditions, the plates may be incubated for up to an additional 7 days to promote further development of hairy roots, which can be advantageous for downstream applications such as nucleic acid extraction. Results Obtaining of soybean hairy roots plants and growing them until harvest Aiming at revealing the function of several genes expressed in soybean roots, we decided to obtain chimeric plants by the hairy root strategy (Figs. 1 A- 1 E). To do functional genomics of genes encoding transcription factors, the first challenge was to choose a suitable vector for overexpressing or silencing the same genes. For this objective, we selected the binary vectors pH7WG2D and pK7GWIWG2D for gene overexpression or silencing, respectively and the Rhizobium rhizogenes strain K599 strain for infection. Following the protocol described in the Methods section, we treated around 330 plants per experiment and obtained between 200 and 320 transformed plants per experiment that were visualized in vivo and in situ with a Fluorescence Flashlight System (Fig. 1 F). Transformed plants developed numerous new roots from the infection site and looked healthy (Fig. 1 G). A high transformation efficiency was corroborated visualizing GFP fluorescence in the same plants (Fig. 1 H). Then, several plants were transferred to 45 L pots and grown in greenhouse conditions until R2 stage, whereas other plants were transplanted to a microplot field trial, which carried out until reproductive maturity (R8). In both cases, transformed hairy roots GFP expression remained stable until harvest (Figs. 1 I- 1 K). A faster protocol can be used to test transformation ability of different constructs and bacteria Given that we detected that several transformed bacterial clones were less efficient to generate hairy roots, we applied different experimental strategies. The first one was to transform developed cotyledons and quickly evaluate the transformation ability. Such a protocol was less time consuming and effective than producing hairy roots chimeric plants. Cotyledons of 4 to 5-day-old seedlings were cut and inoculated with the bacteria suspension as described in the Methods section (Figs. 2 A- 2 C). Then, they were placed in a Petri dish (10–20 per dish) with agar medium and maintained in controlled conditions. Seven to 10 days post infection, a callous tissue started forming on the lesion site (Figs. 2 E and 2 F). At 12 to 14 days post infection, hairy roots emerged from the lesion site, and transformation efficiency was checked by GFP fluorescence using the flashlight system (Figs. 2 F and 2 H). This protocol allowed us to detect whether transformed bacteria were infective and if the introduced genetic construct was effective for transformation. Transformed bacteria lost their infective ability when stored at -80°C When doing several experiments to test the phenotype generated by different transcription factors in hairy roots and their impact on the whole plant performance, we detected that the transformation efficiency continuously decreased using the same constructs. Stating the hypothesis that bacteria losses their infection ability when stored, we tested such an efficiency in a parallel transformation experiment using bacteria stored at -80°C for one week and for eight months (Fig. 3 ). Independently on the construct, the original transformation efficiency of around 80% fells to 45–50% when bacteria were stored at -80°C for eight months (Fig. 3 A). Moreover, GFP positive hairy roots developed better (Figs. 3 B and 3 C), and the infection site showed more callous tissue generation (Figs. 3 E and 3 F) when using the recently transformed bacteria. Data collected over the course of several experiments was analysed performing a linear regression, which corroborated the observation (Fig. 3 D). Different − 80°C devices, all well-functioning were used for this assay, discarding a defect in temperature measurements. Similar results were obtained when bacteria was kept in Petri dishes with agar rich media and refreshed once a month. Finally, we decided to use newly transformed bacteria for each batch of experiments, which was the best way to maintain good transformation efficiencies, suitable for the scale needed in field trial experiments. Before each field trials season, fresh bacteria cultures were transformed with the desired constructs. Their infection capabilities were checked using the faster cotyledon transformation protocol and selected bacterial colonies were stored in several small aliquots in glycerol 25% at -80°C. These bacteria were then used to obtain hairy roots chimeric plants. Temperature infection also strongly affects transformation efficiency Once seedlings were infected, different efficiencies were observed in the analysis. Usually, soybeans are grown at 28°C when experiments are carried out in culture chambers. Under these conditions, we observed overall healthy plants, but with fewer and smaller transformed roots. Hence, we decided to perform this post-infection incubation stage at 22°C, which in turn significantly increased the percentage of transformed hairy roots. To test this observation properly, we performed a parallel experiment at both temperatures (Fig. 4 ). Again, the results were independent on the constructs; plants grown at 28°C after transformation, generated half or less hairy roots than their counterparts at 22°C, indicating that a moderate temperature is needed in this step (Fig. 4 ). At 28°C, plants grew faster compared with the lower temperature, but the transformation efficiency significantly fell. Transformed hairy roots alter the whole plant performance depending on the silenced or overexpressed gene Our aim when starting this project was to reveal if and how the transcriptome alteration in roots could affect the whole performance of the plant. Roots are in direct contact with soil, which means that are the organs responsible for sensing drought, salinity, and other toxic elements. For this reason, we chose transcription factors from the homeodomain-leucine zipper family, strongly associated with development depending on stress conditions. For illustrative purposes, we show one example of the experiments performed with different transcription factors, leading to conclude that the technique was successful to study its effects in the whole plant when overexpressing an HD Zip I transcription factor, GmHDZ32 (Glyma.07g052100, Gm32OE). Firstly, we performed an experiment in pots in growth chamber, at 28°C, high intensity lamps, and long day cycle (Fig. 5 A and 5 B), and evaluated stem height, roots, and vegetative biomass accumulation. Gm32OE plants were smaller than those transformed with the Empty Vector (EV), used as controls (Fig. 5 C). Given the success in obtaining a large number of chimeric hairy roots plants, we did experiments transplanting the transformed plants in the field. Figure 5 shows three stages of such experiments, illustrating about 200 individuals at transplantation date, R1, and R6 developmental stages. Several traits were assessed, showing a significant difference in plant height at harvest for Gm32OE plants when compared with the EV controls (Fig. 5 E), but not on reproductive biomass production (Fig. 5 F). Moreover, aiming at comparing these chimeric plants aptitude with that of Williams82 non-transformed plants, we conducted several experiments over the course of a season, transplanting chimeric hairy roots plants on successive months, at the same time Williams82 seeds were sowed on the same plot. Figure 5 F shows that on all experiments William82 plants produced more reproductive biomass than the composite plants. Altogether these experiments illustrate that the technique was efficient enough to perform a microplot field trial and deeply study the effect of different transgenes expressed only in roots. Discussion Hairy roots methodology has been introduced in 1976 (De Cleene and De Ley 1976 ), improved since this date, and widely used with varied aims, including the production of metabolites and proteins for the pharmaceutical and other industries and fundamental research to functional characterize genes or test their biotechnological potential (Gutierrez-Valdés et al. 2020). Among the fundamental questions that can be addressed with this experimental strategy, we were particularly interested in revealing the impact of root transcriptome alteration in the whole plant development. Noticing that many results of gene characterization performed in controlled conditions and model plants were not translatable to crops in field conditions (Roeder et al. 2025 ; Uauy et al. 2025 ), we choose for our study soybean plants that can grow in chambers, greenhouses, and fields. We selected transcription factors from the HD-Zip I family previously associated with developmental regulation in response to abiotic stress (Perotti et al. 2021 ) to alter the root transcriptome as well as suitable overexpressing and silencing vectors (Karimi et al. 2002 ). Particularly, the selected vectors bear the reporter GFP gene that can be visualized under blue light without destructing plants (Karimi et al. 2002 ; Cheng et al. 2021 ). Regarding the already known methodologies to obtain hairy root plants, we selected the one described by Fan et al. ( 2020 ), which exhibits a significant innovation by carrying out all the process in one step. This change reduced contamination risk with good results. The efficiency obtained was about 50% counting the hereby transformed hairy roots, related to all the hairy roots (Fan et al. 2020 ). The visualization was done by GUS histology, which is a destructive procedure. Hence, we changed this by GFP detection (Fig. 1 ), which allowed us to conserve all the transformed plants, as it is used in several previous works (Cheng et al. 2021 ). Considering reporter genes that do not need plant destruction, RUBY was also used to obtain hairy roots in a two steps protocol (Niazian et al. 2023 ). Furthermore, we considered the technique described by Cheng et al. ( 2021 ), who transformed cotyledons instead of seedlings to select the more infective colonies (Fig. 2 ). Applying a particular combination of both methodologies and a few minor changes, we succeeded to have a large number of transformed soybean hairy root plants, enabling us to perform small field trials in microplots. The first transformation worked very well and we obtained a good number of plants with empty vectors and several constructs overexpressing or silencing endogenous genes encoding HD-Zip I transcription factors. However, when repeating the experiments to support conclusions about genes effects, transformation efficiency declined drastically in several months (Fig. 3 ), with losses approaching 20% across the points analyzed. Such reduction was independent on the genetic construct, including empty vectors used as controls, since we used four distinct ones with similar results (Fig. 3 ). Storing the transformed bacteria in Petri dishes at 4°C, and renewing the media each month, did not result in alleviating the efficiency loss. For field trials, the ability to generate a sufficient number of plants is critical to establishing plots of adequate size. This requirement, in turn, depends on maintaining a high transformation efficiency to ensure the production of chimeric plants bearing multiple hairy roots that carry the construct of interest. Addressing this limitation was therefore essential to secure a reliable and efficient source of composite plants. Even though previous works examined many characteristics strongly contributing to improve the methodology, such as nitrogen availability, sterilization technique, germination devices, explant age, and acetosyringone addition and concentration, etc. (Araragi et al. 2025 ; Niazian et al. 2023 ), we were not able to detect reports informing about the efficiency loss over the time we experienced. To overcome this situation, we tested the bacterial colonies one by one and detected a differential behavior. In the same dish, a few colonies were infective and succeeded in provoking hairy root emergence, whereas others were mostly unable (Fig. 3 ). The loss of efficiency prevented the obtaining of a significant number of transgenic plants. Hence, we tested another strategy that was to periodically retransform the bacteria before each batch of hairy root experiments, and it was a success. Even though we cannot explain the reason for this result, which is not a contamination of the mother Rhizobium culture, nor a bad temperature regulation of several ultrafreezers, it was a fact that many bacteria lost their infection ability after prolonged storage at -80°C or at 4°C (Fig. 3 ). Regarding recently infected plants growing temperature, we found that 22°C was rather better than the optimal temperature usually used to grow soybeans (28°C). Considering this condition, most reported protocols informed 28°C as cocultivation temperature (Araragi et al. 2025 ; Niazian et al. 2023 ; Kong et al. 2023 ) or a combination of 26°C day/22°C night (Song et al. 2021 ). Finally, we can state that transformed hairy roots expressing or silencing transcription factors from the HD-Zip I family impact on plant development, allowing to study many other genes and their effect on growing and stress tolerance. GmHDZ4, a soybean member of this transcription factor family, exhibiting sequence similarities with AtHB12 and Oshox12, was knocked out by edition in chimeric plants (Zhong et al. 2022 ). These chimeric plants were tested in pots and controlled conditions, showing significant differences in root architecture and enhanced tolerance to osmotic stress applied with polyethilenglycol in the vegetative stage (Zhong et al. 2021). It is tempting to speculate that this optimized protocol will allow to characterize the impact of many other genes when expressed in roots, at different developmental stages. We can state this after performing trials in microplots in open air during two seasons and several sowing dates, which added value to experiments performed only in pots and in controlled conditions. To the best of our knowledge, this is the first report describing field trials with composite soybean with hairy roots. CONCLUSIONS In this work, we describe a significant advancement over existing one-step Agrobacterium -mediated transformation protocols by enhancing transformation efficiency. Our improved method optimizes several key parameters: re-transforming bacteria before each experiment to maintain high infection viability, minimizing steps requiring strict sterility, enabling in vivo visualization of transformed roots via GFP detection with a flashlight, and standardizing growth at 22 ∘ C. These modifications enable reliable, high-throughput production of transgenic soybean roots, allowing us to perform crucial field trials in microplots. Assessing the effect of root transgenes under field conditions is vital for soybean research, as conclusions drawn solely from controlled environments may misrepresent a gene's true function in agriculturally relevant settings. Declarations CONFLICT OF INTEREST The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. FUNDING The work of the research group was supported by Agencia Nacional de Promoción Científica y Tecnológica (PICT 2020 0805, MINCYT (ex Ministerio de Ciencia y Tecnología) through the special grant “Redes de Alto Impacto”), CONICET, and Fundación Williams to RLC. LC is a CONICET Ph.D. Fellow. JR and RLC are CONICET Career members. Author Contribution Conceived by RLC. Made the experiments and illustrations: LC. Discussed the manuscript: LC, JR, and RLC. Wrote the paper: RLC. Revised and approved the manuscript: all authors. Acknowledgement We thank Dr. Germán Robert from Centro de Investigaciones Agropecuarias INTA-Córdoba for providing us the K599 bacterial strain and helpful advices and discussions. We also thank Dr. Ramiro Lascano for helpful advice. We thank Ana Belén Curi and Manuel Franco for technical assistance in field trials. References Araragi M, Songwattana P, Teaumroong N, Masuda S, Shibata A, Shirasu K, Kawaharada Y (2025) Improved rapid and efficient hairy root transformation using Rhizobium rhizogenes in legume crops. Plant Biotechnology (Tokyo) 42(3):279-288. doi: 10.5511/plantbiotechnology.25.0213a. Chan RL, Trucco F, Otegui ME (2020) Why are second-generation transgenic crops not yet available in the market? Journal of Experimental Botany 71(22):6876-6880. doi: 10.1093/jxb/eraa412. Cheng Y, Wang X, Cao L, Ji J, Liu T, Duan K (2021) Highly efficient Agrobacterium rhizogenes-mediated hairy root transformation for gene functional and gene editing analysis in soybean. 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Zhong X, Hong W, Shu Y, Li J, Liu L, Chen X, Islam F, Zhou W, Tang G (2022) CRISPR/Cas9 mediated gene-editing of GmHdz4 transcription factor enhances drought tolerance in soybean (Glycine max [L.] Merr.). Frontiers in Plant Science 13:988505. doi: 10.3389/fpls.2022.988505. Additional Declarations No competing interests reported. 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08:42:46","extension":"html","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":121106,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8353559/v1/e3a7b034519cfe8939a2a30b.html"},{"id":99506695,"identity":"80d829d6-8ad1-44d6-ad36-d78941820deb","added_by":"auto","created_at":"2026-01-05 08:42:46","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4068668,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWorkflow to obtain soybean hairy roots\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Illustrative image of starting material: 7-day-old seedlings sowed at high-density. \u003cstrong\u003eB. \u003c/strong\u003eImage illustrating shoot cut made ~1 cm below the cotyledonary node. \u003cstrong\u003eC.\u003c/strong\u003e Petri dish containing \u003cem\u003eR. rhizogenes strain K599\u003c/em\u003e cultured until saturation in LB agar. Biomass was collected with a spatula or knife. \u003cstrong\u003eD.\u003c/strong\u003e Bacterial biomass applied on the incision, immediately after doing it. \u003cstrong\u003eE.\u003c/strong\u003e Infected explants were transferred to a recipient with at least 5 cm of wet vermiculite and an excess of distilled water. Then, the recipient was covered with a transparent film to maintain the humidity\u003cstrong\u003e F. \u003c/strong\u003e\u0026nbsp;Xite™ Fluorescence Flashlight System (\u003ca href=\"https://nightsea.com/products/xite-flashlights/\"\u003eXite\u003c/a\u003e\u003ca href=\"https://nightsea.com/products/xite-flashlights/\"\u003e Portable Fluorescence Flashlight System – NIGHTSEA\u003c/a\u003e), used to visualize GFP, consisting of a Royal Blue light lamp and both 500 nm long-pass (yellow) and 500-560 nm band-pass (green) filter glasses. \u003cstrong\u003eG. \u003c/strong\u003eHairy root composite plants 14 days after transformation. The picture was taken in bright field; \u003cstrong\u003eH. \u003c/strong\u003eThe same plants were photographed after illuminating with blue light to visualize GFP fluorescence; green arrows indicate GFP positive hairy roots, whereas white arrows indicate roots that don’t have GFP fluorescence. \u003cstrong\u003eI.\u003c/strong\u003e The same plants at R2 stage, grown in a pot in a greenhouse; \u003cstrong\u003eJ.\u003c/strong\u003eand \u003cstrong\u003eK.\u003c/strong\u003e Composite plants with hairy roots at R8 stage harvested from a field trial and photographed with bright light or blue light, respectively. Blue arrows indicate Williams 82 (WT) non-transformed control plants.\u003c/p\u003e","description":"","filename":"Fig1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8353559/v1/ef601bb8e0b6db38ecb8046a.jpg"},{"id":99506690,"identity":"161ee650-bd83-4788-b34f-0aa09d956d61","added_by":"auto","created_at":"2026-01-05 08:42:45","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1753255,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWorkflow to test bacterial infection viability in soybean cotyledons\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Illustrative image of starting material: soybean seeds germinated for about 4 days. \u003cstrong\u003eB.\u003c/strong\u003e Cotyledons were cut approximately 1mm from their node using a scalpel. \u003cstrong\u003eC. \u003c/strong\u003eSingle cotyledons were inoculated several times in the wounded zone with a bacterial suspension in distilled water, using a syringe. \u003cstrong\u003eD.\u003c/strong\u003e Cotyledons were arranged over distilled water-agar plates with their abaxial face down and the injury facing outside. Plates were cultured at 22 °C, in long-day cycle growth chamber for 10-14 days. \u003cstrong\u003eE. \u003c/strong\u003eand \u003cstrong\u003eF. \u003c/strong\u003eImages of calli grown in the infection site at 10 days after infection; photographs were taken in bright field and under blue light and yellow GFP filter, respectively. Green arrows indicate small GFP dots. \u003cstrong\u003eG.\u003c/strong\u003e and \u003cstrong\u003eH.\u003c/strong\u003e 14 days after infection, multiple transgenic hairy roots have already emerged from most transformed cotyledons. Photographs were taken in bright field and under blue light and yellow GFP filter.\u003c/p\u003e","description":"","filename":"Fig2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8353559/v1/2ee75a1ee6cfd69ee3b2bd3d.jpg"},{"id":99506692,"identity":"3337aed0-512c-46f9-a381-79844c7fb41d","added_by":"auto","created_at":"2026-01-05 08:42:46","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1487158,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTransformation efficiency is negatively affected by bacterial strain storage duration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Hairy roots transformed with GFP (HR GFP+) efficiency for K599 bacteria carrying four different constructs (EV-OE, Gm10OE, EV-RNAi, and Gm10RNAi) stored at -80 °C for 7 or 236 days. Bars indicate media and SEM of the group, and each dot represents technical replicates of transformation humid chambers containing at least 30 biological replicates, except for EV-RNAi at 236 days, which had only 11 biological replicates. HR GFP+ efficiency was calculated as the number of plants with at least one GFP-positive hairy root at 14 days after transformation over total transformed plants. \u003cstrong\u003eB\u003c/strong\u003e. Illustrative pictures of seedlings with hairy roots transformed with bacteria stored for 7 days (left) or 236 days (right) after 14 days of incubation at 22°C. \u003cstrong\u003eC.\u003c/strong\u003e The same plants visualizing GFP fluorescence. Green arrows indicate HR GFP+, whereas white arrows indicate non fluorescent roots.Photographs were taken with bright light (\u003cstrong\u003eB\u003c/strong\u003e) or blue lightwith yellow\u003cstrong\u003e \u003c/strong\u003efilter (\u003cstrong\u003eC\u003c/strong\u003e), respectively. \u003cstrong\u003eE\u003c/strong\u003e. and \u003cstrong\u003eF\u003c/strong\u003e. Zoom in of the images signaled in \u003cstrong\u003eB\u003c/strong\u003e.\u003cstrong\u003e D. \u003c/strong\u003eLinear regression of HR GFP+ efficiency as a function of storage time. Outliers were stained in red. Each dot represents technical 3 to 4 biological replicates obtained in a small humid chamber containing at least 4 and up to 40 biological replicates. All points were equally weighted. The solid black line is the estimated regression and the dashed lined marks the 95 % confidence bands for the curve fit.\u003c/p\u003e","description":"","filename":"Fig3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8353559/v1/6ff55c4285339ba51a91f171.jpg"},{"id":99791651,"identity":"08184913-28d8-4990-933f-823c92d6080f","added_by":"auto","created_at":"2026-01-08 13:06:55","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1461762,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTransformation efficiency depends on the growth temperature\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e HR GFP+ efficiency for K599 bacteria carrying four different constructs, as indicated in Figure 3, when seedlings were grown at 22 °C (left) or 28 °C (right). HR GFP+ efficiency was calculated as the number of plants with at least one GFP-positive hairy root at 14 days after transformation over total transformed plants. Each dot represents technical replicates of 3 to 4 biological replicates performed in small humid chambers. \u003cstrong\u003eB. \u003c/strong\u003eIllustrative picture of transformed plants incubated at 22 °C (left) or 28° C (right), 17 days after transformation, photographed with bright light. Root systems of representative composite plants from each group were displayed in bright field (\u003cstrong\u003eC\u003c/strong\u003e) and under blue light and with yellow GFP filter (\u003cstrong\u003eD\u003c/strong\u003e), in which green arrows indicate HR GFP+ and white arrows indicate non fluorescent roots.\u003c/p\u003e","description":"","filename":"Fig4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8353559/v1/7262507de33dd5604b62b706.jpg"},{"id":99506688,"identity":"85bb63cf-39ae-4b5f-b11a-f09eb1156410","added_by":"auto","created_at":"2026-01-05 08:42:45","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3024359,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenetic constructs used to obtain composite plants with transgenic hairy roots alter the whole plant performance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eA.\u003c/strong\u003e Illustrative picture of composite plants with hairy roots transformed with two different constructions (EV-OE and Gm32OE), incubated 15 days at 22°C, transplanted to a one-liter vermiculite pot and cultured at 28°C in a growth chamber for 23 days. \u003cstrong\u003eB.\u003c/strong\u003e Illustrative picture of one representative composite plant from each group. \u003cstrong\u003eC.\u003c/strong\u003e Dry biomass assessed 24 days after transplantation in the same growth chamber assay. Each group is comprised of four biological replicates. Asterisks indicate a significant difference in a Two-way ANOVA followed by Tukey’s post hoc test (**: p\u0026lt;0.01). \u003cstrong\u003eD.\u003c/strong\u003e Illustrative pictures of composite plants with hairy roots assessed in a field trial. Pictures were taken at the transplantation date (left), early flowering (R1, center) and grain filling stages (R6, right). \u003cstrong\u003eE. \u003c/strong\u003eStem height at harvest (R8 stage) in the field trial assay. \u003cstrong\u003eF\u003c/strong\u003e. Pods’ biomass per plant at harvest of plants transplanted on different, successive dates (1\u003csup\u003est\u003c/sup\u003e, 2\u003csup\u003end\u003c/sup\u003e and 3\u003csup\u003erd\u003c/sup\u003e), grown in well-watered conditions (CC) or subjected to water deficit (WD). Williams 82 non-transformed plants were used as controls. Each group is comprised of at least 18 biological replicates. Asterisks indicate significant differences in a Two-way ANOVA followed by Tukey’s post hoc test (****\u0026lt;0.0001).\u003c/p\u003e","description":"","filename":"Fig5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8353559/v1/e51f60bdac97f11b66b35df0.jpg"},{"id":104252185,"identity":"9660f02d-e8c1-4bc5-8195-e8e6495b6c47","added_by":"auto","created_at":"2026-03-09 16:17:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12717357,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8353559/v1/6b614a74-7682-40d6-b7b0-4448a6fc5d16.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Scalable production of soybean hairy roots: a reliable method for field trials","fulltext":[{"header":"Key message","content":"\u003cp\u003eWe optimized soybean hairy root production for field trials using newly transformed rhizobia, a 22\u0026deg;C growth temperature, and GFP flashlight visualization.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eThe obtaining of composite plants with transgenic hairy roots is a powerful strategy for many research and biotechnological purposes. From a fundamental research point of view, such a strategy allows for revealing protein function by overexpressing or silencing a given gene, studying the effect of expression alteration on the whole plant performance or the transport of metabolites driven by specific proteins, among other objectives. Considering biotechnology, many metabolites used in medicine or industry can be obtained using hairy roots, which is a faster method compared with stable transformation. Moreover, composite plants with transgenic hairy roots are not considered GMO because seeds are not transgenic, allowing more freedom to do field trials.\u003c/p\u003e \u003cp\u003eHairy roots are obtained after plant infection by \u003cem\u003eRhizobium rhizogenes\u003c/em\u003e in a variety of species. A deep revision about the characteristics of \u003cem\u003eRhizobium rhizogenes\u003c/em\u003e and its differences with \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e has been published recently (Kiryushkin et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Aside from transgene expression, they can be used as producers and secretors of a wide range of metabolites used in the cosmetic and pharmaceutical industries (Gutierrez-Valdes et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough the molecular mechanism involved in hairy root formation is not well understood, the whole process was divided into several steps, including the bacterial transformation and the transfer of T-DNA to the plant genome, ending in hairy root emergence (Georgiev et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMany studies have focused on the use of hairy roots to produce proteins or metabolites (Peebles et al. 2009; H\u0026auml;kkinen et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Robert et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). For these purposes, biomass production and culture stabilization have been the crucial improvement points, including testing different elicitors.\u003c/p\u003e \u003cp\u003eAnother common use of the hairy root technique is the study of nodulation capacity. For example, the expansin \u003cem\u003eGmEXPA11\u003c/em\u003e was highly induced by rhizobial infection and showed a positive role in nodule enlargement (Xing et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Similarly, the gene encoding the phosphatase GmPP2C61A was upregulated by rhizobia inoculation, and its knockout reduced the number of nodules (Gao et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). On the contrary, TML encoding genes overexpressed in hairy roots resulted in a nodule number decrease, indicating a negative role in nodule organogenesis (Su et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Moreover, symbiont perception and nodulation capacity under stress were studied through the characterization of plants with hairy roots transformed with Phosphatidylinositol 3-kinase (Robert et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSeveral studies showed how the transformation of roots impacted morphological or physiological characters of the whole plant. For example, the ARF3 coding region was expressed in tobacco hairy roots, altering internode length, branch number, leaf blade, and flowering time, provoking dwarfism in plants grown in pots (Wang et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe hairy roots technique was also combined with gene editing, potentiating the objectives that can be achieved through functional genomics (Kiryushkin et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In that work, vectors, promoters, reporters, and other features of the editing system were compared and optimized (Kiryushkin et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSoybean (Glycine max L. Merr), a dicot oleaginous species, is mostly used in animal and human feeding. Moreover, it is one of the most important crops worldwide, whose yield and seed quality are limited by abiotic and biotic stress factors (Hartman et al. 2012). Genetically modified soybeans were released to the market more than thirty years ago, exhibiting herbicide resistance and later insect tolerance. Despite its importance and negative impact, the only market-released event showing abiotic stress tolerance is soybean HB4 (Ribichich et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). There are others successfully tested in the field, albeit they have not been approved for commercialization, an expensive process that takes many years (Raineri et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The reasons for this situation, not only with soybeans but with other important crops, are several. In general, there is a bad public perception of GMOs. However, this fact did not prevent 90% of the worldwide planted soybeans from being genetically modified (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.isaaa.org\" target=\"_blank\"\u003ewww.isaaa.org\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.isaaa.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). A second problem is that abiotic stress-tolerant plants must be tested in multiple environments because they do not represent universal technologies. This means that the business for companies is not so lucrative as that of biotic-resistant crops (Chan et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, and no less important, one of the main issues to solve is that gene discovery experiments performed in model species are not always translatable to crops (Uauy et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Field trials with crops can be carried out only once a year, after obtaining stable transgenics, which is costly and time-consuming.\u003c/p\u003e \u003cp\u003eGiven these reasons, the use of the hairy roots technique allows for testing different genes and their impact on yield and seed quality in controlled conditions throughout the year.\u003c/p\u003e \u003cp\u003eSeveral protocols were already published to obtain soybean hairy roots (Araragi et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Fan et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Su et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) and used to study the crop tolerance of resistance to a wide range of abiotic and biotic stress factors and the involvement of varied genes in such responses (Jia et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHowever, the protocols are time-consuming, tedious, require sterility, and usually allow for obtaining a very limited number of plants, considering that the reported efficiencies were mostly between 40 and 70%. That is also because the focus of such studies has not been the assessment in field trials, which demands a large number of transformed plants. Another significant limitation is the detection of transformed hairy roots, which is time consuming and may imply material destruction when using reporter genes detected by histochemistry, such as GUS. Reporter constructions take relevance because adventitious roots and hairy roots transformed only with the Ri helper plasmid also grow from the plants, even if the transformation with the gene of interest does not take place. When the efficiency is low, such events significantly reduce the transgene effects on the whole plant.\u003c/p\u003e \u003cp\u003eWe did not detect a protocol suitable for obtaining a good number of hairy root plants to perform a field trial. Moreover, the crucial bacterial infection capability during prolonged periods was not informed.\u003c/p\u003e \u003cp\u003eHere we present an optimized protocol that allows us to obtain more than 200 soybean plants with transgenic hairy roots in three weeks and perform a field trial with them, using adequate controls. The main changes with other published methods are that the present transformation protocol eliminates the requirement for sterile conditions, the maintenance of bacterial infection capacity, the detection of the reporter GFP gene in situ and in vivo, and the growing conditions. This method is suitable both for overexpressing or silencing genes and would also be useful for performing gene editing.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant Material\u003c/h2\u003e \u003cp\u003eSoybean seeds from the genotype Williams 82 were used in this study. These were surface sterilised with 20% bleach in deionized water for 10 minutes, then rinsed five times with deionised water.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMaterials needed to carry out the hairy roots protocol\u003c/h3\u003e\n\u003cp\u003eSoybean seeds, 20% bleach, autoclaved deionised water, plastic trays, paper towels, autoclaved deionised water and foil film, autoclaved agar medium, 1% w/v in deionised water, sterile Petri dishes, autoclaved 15 mL Falcon tubes with 3 mL liquid LB medium each, and sterile syringes.\u003c/p\u003e\n\u003ch3\u003eGrowth conditions and obtaining of hairy roots\u003c/h3\u003e\n\u003cp\u003eFor chimeric whole plants transformation, the seeds were sowed on sterilised coarse vermiculite watered with deionised water. The seeds were then disposed in high density (up to 18 seeds per 13.5 cm x 10 cm plastic tray, extrapolated to up to 1300 seeds per m\u003csup\u003e2\u003c/sup\u003e), and incubated in a growth chamber under long day cycle, 26 to 28\u0026deg;C temperature and using high intensity sodium lamps (16 h light/8 h dark cycle, 230\u0026ndash;260 \u0026micro;mol. m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e.sec\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e light intensity, 50\u0026ndash;60% humidity).\u003c/p\u003e \u003cp\u003eTransformed shoots were disposed on plastic recipients containing sterilised coarse vermiculite, previously irrigated until saturation with deionised water. Plant density was kept at approximately 40 plants in 46.5 x 34 cm containers (250 plants per m\u003csup\u003e2\u003c/sup\u003e), and sealed with foil film to maintain high humidity at a height of at least 25 cm. These containers were cultured in a growth chamber under long day cycle, 22 to 24\u0026deg;C temperature and using low intensity led lamps (16 h light/8 h dark cycle, 60\u0026ndash;70 \u0026micro;mol. m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e. sec\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e light intensity, 50\u0026ndash;60% humidity).\u003c/p\u003e \u003cp\u003eSeeds germinated for cotyledon transformation were grown on plastic trays with paper towels, watered using deionised water and covered with a transparent foil film to maintain high humidity. Seeds were also disposed in high density, up to 30 seeds per 26 x 19 cm plastic tray (600 per m\u003csup\u003e2\u003c/sup\u003e), and incubated in a growth chamber under long day cycle, 26 to 28\u0026deg;C temperature, and using high intensity sodium lamps (16 h light/8 h dark cycle, 230\u0026ndash;260 \u0026micro;mol. m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e.sec\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e light intensity, 50\u0026ndash;60% humidity).\u003c/p\u003e \u003cp\u003eTransformed cotyledons were disposed in plates containing 1% w/v agar in deionised water medium, up to 20 cotyledons per 14 cm-diameter Petri dish. These plates were then incubated in a growth chamber under long day cycle, 22 to 24\u0026deg;C temperature and using low intensity led lamps (16 h light/8 h dark cycle, 60\u0026ndash;70 \u0026micro;mol. m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e. sec\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e light intensity, 50\u0026ndash;60% humidity).\u003c/p\u003e\n\u003ch3\u003eGenetic constructs and transformation\u003c/h3\u003e\n\u003cp\u003eTo generate hairy roots, we cloned different coding sequences in the backbone of the plasmid binary vector pH7WG2D (Karimi et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) for the overexpression of homeodomain leucine zipper subfamily I (HD Zip I) genes. We also cloned fragments of these genes in the backbone of the plasmid binary vector pK7GWIWG2D (II) (Karimi et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) for the siRNA silencing of HD Zip I genes. Empty versions of both plasmids were generated by enzymatic digestion of the overexpressing cassette, containing the ccb death domain, in pH7WG2D; and by recombination of a dummy sequence in pK7GWIWG2D (II), for the correct assembly of the double stranded RNA and successive processing as dummy siRNA. All constructs had a GFP overexpressing cassette that allowed us to follow transformation efficiency \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein situ\u003c/em\u003e, and were introduced in \u003cem\u003eRhizobium rhizogenes strain K599\u003c/em\u003e using an electroporation transformation method. Transformed bacteria were stored at -80\u0026deg;C in 25% glycerol and then used to induce hairy roots formation on soybean explants.\u003c/p\u003e \u003cp\u003eWhen selecting bacterial selection markers, if the plasmid bears a streptomycin/spectinomycin resistance, namely an aminoglycoside 3\u0026rsquo; adenyltransferase (AadA) gene, normal growth inhibitory concentrations (100 \u0026micro;g spectinomycin/mL) are insufficient. In this case, we found that concentrations of 350 to 450 \u0026micro;g spectinomycin/mL generate a desired selection for the correct identification of positive colonies in LB agar plates. This scenario was not observed when using kanamycin as the selection agent for kanamycin resistance vectors.\u003c/p\u003e\n\u003ch3\u003eWhole plant hairy roots transformation protocol\u003c/h3\u003e\n\u003cp\u003eFor whole plants transformation, soybean seeds were grown as previously described until their unifoliate leaves developed but remained unexpanded (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), which usually takes between 5 to 7 days after sowing. Further development, as leaves expansion, may result in higher lethality of the transformation process, as expanded leaves grow bigger and then evaporate more water that the explants can withstand.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eForty-eight hours before the transformation, selected high transformation efficiency bacterial colonies carrying the different vector constructs were cultured in sterile LB liquid medium overnight at 28\u0026deg;C and 200 rpm.\u003c/p\u003e \u003cp\u003eThe exponential grown cultures were then used to inoculate sterile LB agar plates, about 200 \u0026micro;L for a 9 cm-diameter plate. Bacteria were distributed evenly on the plate using a sterilised Drigalski spatula and left to dry for about 5 minutes. These plates were cultured for 24 hours at 28\u0026deg;C. Plates were grown until visible biomass appears on the surface.\u003c/p\u003e \u003cp\u003eIt is important to only use one selection antibiotic during these pre-transformation steps, as using more antibiotics, or higher concentrations, can lead to lower hairy roots transformation efficiency. In this case, using the plasmid selection marker antibiotic is recommended. In our hands, normal concentrations of 50 \u0026micro;g/mL kanamycin and 100 \u0026micro;g/mL spectinomycin do not seem to negatively affect transformation efficiency.\u003c/p\u003e \u003cp\u003eFrom here onwards, sterile conditions were not needed. High humidity chambers were prepared the day before transformation, using approximately 5 cm of sterilised coarse vermiculite in plastic containers of at least 30 cm total height, to allow for unhindered shoot development during the transformation protocol. This vermiculite was then saturated with deionised water until about 1 cm of free water was obtained.\u003c/p\u003e \u003cp\u003eOn the transformation moment, an alcohol-flamed scalpel was used to generate an angled cut on the soybean plant\u0026rsquo;s hypocotyl, approximately 1 cm below the cotyledonary node (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The angled cut generates a wider section for the bacteria to transform, improving the transformation efficiency and hairy roots maximum population. A butter knife was alcohol flamed and air-cooled, then used to collect bacterial biomass from the surface of the plates (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). This biomass was then immediately applied to the lesion (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), and infected explants were introduced in the humid chambers previously prepared, planting them in vermiculite up to just below their cotyledons. These chambers were then covered with foil film (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE) and cultured as previously described, in a growth chamber under long day cycle, 22 to 24\u0026deg;C temperature and using low intensity led lamps.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCotyledons hairy roots transformation protocol\u003c/h2\u003e \u003cp\u003eFor cotyledons explants transformation, soybean seeds were grown on germinators, as previously described, until greening, for about 4 to 5 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Twenty-four hours before transformation, K599 bacteria were inoculated on 15 mL Falcon tubes with 3 mL of sterile LB liquid medium and cultured overnight on a shaker at 28\u0026deg;C and 200 rpm. The media can be inoculated from fresh individual colonies, or directly from \u0026minus;\u0026thinsp;80\u0026deg;C glycerol stocks.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe next day, bacterial biomass is collected by centrifugation at 2500 g for 5 minutes. Then, the residual LB of the supernatant was discarded, and the pellet is resuspended in 1 mL of autoclaved deionised water. These bacterial suspensions can be stored for up to an hour at 4\u0026deg;C without significantly affecting transformation efficiency. Agar plates for the transformed explants are prepared by pouring the autoclaved agar medium on sterile Petri dishes.\u003c/p\u003e \u003cp\u003eFrom here onwards, there was no need for sterile working conditions. An alcohol-flamed scalpel was used to cut the cotyledons 1mm from their node, as can be seen on Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB. Then, a syringe was loaded with bacterial suspension, one droplet was left in the cotyledon\u0026rsquo;s lesion, and it is then lightly stabbed repeatedly (about 10 times, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) to facilitate infection. The infected explant was then placed on the agar plate, their abaxial face down, and the lesion facing outside. Subsequent explants were disposed contiguously, up to 10 cotyledons per 9 cm-diameter Petri dish (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), or up to 20 cotyledons for 14 cm-diameter Petri dish. A negative control was done by stabbing cotyledons using a syringe loaded only with sterile deionised water. These plates are then cultured as previously described, in a growth chamber under long day cycle, 22 to 24\u0026deg;C temperature and using low intensity led lamps.\u003c/p\u003e \u003cp\u003eDuring this period, microbial growth may occur and should be monitored. For instance, if fungal colonies develop on the cotyledons, the affected tissues should be removed to preserve the integrity of the remaining explants. Conversely, if the contamination affects the medium itself, it is advisable to transfer the transformed explants to a fresh agar plate.\u003c/p\u003e \u003cp\u003eTo avoid water condensation in the dishes, the plates were incubated on shelves without any heat source below, including another illuminated shelf.\u003c/p\u003e \u003cp\u003eBetween 7 and 10 days after transformation, infected explants should exhibit greenish callus growth at the lesion site, which may express reporter genes, if present (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and F). At 12 to 14 days after transformation, cotyledons should have developed hairy roots from the infection site, which in turn may express the reporter gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG and H). The hairy roots formation must not occur in the negative control. Under these conditions, the plates may be incubated for up to an additional 7 days to promote further development of hairy roots, which can be advantageous for downstream applications such as nucleic acid extraction.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eObtaining of soybean hairy roots plants and growing them until harvest\u003c/h2\u003e \u003cp\u003eAiming at revealing the function of several genes expressed in soybean roots, we decided to obtain chimeric plants by the hairy root strategy (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). To do functional genomics of genes encoding transcription factors, the first challenge was to choose a suitable vector for overexpressing or silencing the same genes. For this objective, we selected the binary vectors pH7WG2D and pK7GWIWG2D for gene overexpression or silencing, respectively and the \u003cem\u003eRhizobium rhizogenes strain\u003c/em\u003e K599 strain for infection.\u003c/p\u003e \u003cp\u003eFollowing the protocol described in the Methods section, we treated around 330 plants per experiment and obtained between 200 and 320 transformed plants per experiment that were visualized \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein situ\u003c/em\u003e with a Fluorescence Flashlight System (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Transformed plants developed numerous new roots from the infection site and looked healthy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). A high transformation efficiency was corroborated visualizing GFP fluorescence in the same plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). Then, several plants were transferred to 45 L pots and grown in greenhouse conditions until R2 stage, whereas other plants were transplanted to a microplot field trial, which carried out until reproductive maturity (R8). In both cases, transformed hairy roots GFP expression remained stable until harvest (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eA faster protocol can be used to test transformation ability of different constructs and bacteria\u003c/h2\u003e \u003cp\u003eGiven that we detected that several transformed bacterial clones were less efficient to generate hairy roots, we applied different experimental strategies. The first one was to transform developed cotyledons and quickly evaluate the transformation ability. Such a protocol was less time consuming and effective than producing hairy roots chimeric plants. Cotyledons of 4 to 5-day-old seedlings were cut and inoculated with the bacteria suspension as described in the Methods section (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Then, they were placed in a Petri dish (10\u0026ndash;20 per dish) with agar medium and maintained in controlled conditions. Seven to 10 days post infection, a callous tissue started forming on the lesion site (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). At 12 to 14 days post infection, hairy roots emerged from the lesion site, and transformation efficiency was checked by GFP fluorescence using the flashlight system (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). This protocol allowed us to detect whether transformed bacteria were infective and if the introduced genetic construct was effective for transformation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTransformed bacteria lost their infective ability when stored at -80\u0026deg;C\u003c/h2\u003e \u003cp\u003eWhen doing several experiments to test the phenotype generated by different transcription factors in hairy roots and their impact on the whole plant performance, we detected that the transformation efficiency continuously decreased using the same constructs. Stating the hypothesis that bacteria losses their infection ability when stored, we tested such an efficiency in a parallel transformation experiment using bacteria stored at -80\u0026deg;C for one week and for eight months (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Independently on the construct, the original transformation efficiency of around 80% fells to 45\u0026ndash;50% when bacteria were stored at -80\u0026deg;C for eight months (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Moreover, GFP positive hairy roots developed better (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), and the infection site showed more callous tissue generation (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF) when using the recently transformed bacteria. Data collected over the course of several experiments was analysed performing a linear regression, which corroborated the observation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Different \u0026minus;\u0026thinsp;80\u0026deg;C devices, all well-functioning were used for this assay, discarding a defect in temperature measurements. Similar results were obtained when bacteria was kept in Petri dishes with agar rich media and refreshed once a month. Finally, we decided to use newly transformed bacteria for each batch of experiments, which was the best way to maintain good transformation efficiencies, suitable for the scale needed in field trial experiments. Before each field trials season, fresh bacteria cultures were transformed with the desired constructs. Their infection capabilities were checked using the faster cotyledon transformation protocol and selected bacterial colonies were stored in several small aliquots in glycerol 25% at -80\u0026deg;C. These bacteria were then used to obtain hairy roots chimeric plants.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTemperature infection also strongly affects transformation efficiency\u003c/h2\u003e \u003cp\u003eOnce seedlings were infected, different efficiencies were observed in the analysis. Usually, soybeans are grown at 28\u0026deg;C when experiments are carried out in culture chambers. Under these conditions, we observed overall healthy plants, but with fewer and smaller transformed roots. Hence, we decided to perform this post-infection incubation stage at 22\u0026deg;C, which in turn significantly increased the percentage of transformed hairy roots. To test this observation properly, we performed a parallel experiment at both temperatures (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Again, the results were independent on the constructs; plants grown at 28\u0026deg;C after transformation, generated half or less hairy roots than their counterparts at 22\u0026deg;C, indicating that a moderate temperature is needed in this step (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). At 28\u0026deg;C, plants grew faster compared with the lower temperature, but the transformation efficiency significantly fell.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eTransformed hairy roots alter the whole plant performance depending on the silenced or overexpressed gene\u003c/h2\u003e \u003cp\u003eOur aim when starting this project was to reveal if and how the transcriptome alteration in roots could affect the whole performance of the plant. Roots are in direct contact with soil, which means that are the organs responsible for sensing drought, salinity, and other toxic elements. For this reason, we chose transcription factors from the homeodomain-leucine zipper family, strongly associated with development depending on stress conditions. For illustrative purposes, we show one example of the experiments performed with different transcription factors, leading to conclude that the technique was successful to study its effects in the whole plant when overexpressing an HD Zip I transcription factor, GmHDZ32 (Glyma.07g052100, Gm32OE). Firstly, we performed an experiment in pots in growth chamber, at 28\u0026deg;C, high intensity lamps, and long day cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), and evaluated stem height, roots, and vegetative biomass accumulation. Gm32OE plants were smaller than those transformed with the Empty Vector (EV), used as controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGiven the success in obtaining a large number of chimeric hairy roots plants, we did experiments transplanting the transformed plants in the field. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows three stages of such experiments, illustrating about 200 individuals at transplantation date, R1, and R6 developmental stages. Several traits were assessed, showing a significant difference in plant height at harvest for Gm32OE plants when compared with the EV controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), but not on reproductive biomass production (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Moreover, aiming at comparing these chimeric plants aptitude with that of Williams82 non-transformed plants, we conducted several experiments over the course of a season, transplanting chimeric hairy roots plants on successive months, at the same time Williams82 seeds were sowed on the same plot. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF shows that on all experiments William82 plants produced more reproductive biomass than the composite plants.\u003c/p\u003e \u003cp\u003eAltogether these experiments illustrate that the technique was efficient enough to perform a microplot field trial and deeply study the effect of different transgenes expressed only in roots.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eHairy roots methodology has been introduced in 1976 (De Cleene and De Ley \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1976\u003c/span\u003e), improved since this date, and widely used with varied aims, including the production of metabolites and proteins for the pharmaceutical and other industries and fundamental research to functional characterize genes or test their biotechnological potential (Gutierrez-Vald\u0026eacute;s et al. 2020). Among the fundamental questions that can be addressed with this experimental strategy, we were particularly interested in revealing the impact of root transcriptome alteration in the whole plant development.\u003c/p\u003e \u003cp\u003eNoticing that many results of gene characterization performed in controlled conditions and model plants were not translatable to crops in field conditions (Roeder et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Uauy et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), we choose for our study soybean plants that can grow in chambers, greenhouses, and fields. We selected transcription factors from the HD-Zip I family previously associated with developmental regulation in response to abiotic stress (Perotti et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) to alter the root transcriptome as well as suitable overexpressing and silencing vectors (Karimi et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Particularly, the selected vectors bear the reporter \u003cem\u003eGFP\u003c/em\u003e gene that can be visualized under blue light without destructing plants (Karimi et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Cheng et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRegarding the already known methodologies to obtain hairy root plants, we selected the one described by Fan et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), which exhibits a significant innovation by carrying out all the process in one step. This change reduced contamination risk with good results. The efficiency obtained was about 50% counting the hereby transformed hairy roots, related to all the hairy roots (Fan et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The visualization was done by GUS histology, which is a destructive procedure. Hence, we changed this by GFP detection (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), which allowed us to conserve all the transformed plants, as it is used in several previous works (Cheng et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Considering reporter genes that do not need plant destruction, \u003cem\u003eRUBY\u003c/em\u003e was also used to obtain hairy roots in a two steps protocol (Niazian et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurthermore, we considered the technique described by Cheng et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), who transformed cotyledons instead of seedlings to select the more infective colonies (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Applying a particular combination of both methodologies and a few minor changes, we succeeded to have a large number of transformed soybean hairy root plants, enabling us to perform small field trials in microplots.\u003c/p\u003e \u003cp\u003eThe first transformation worked very well and we obtained a good number of plants with empty vectors and several constructs overexpressing or silencing endogenous genes encoding HD-Zip I transcription factors. However, when repeating the experiments to support conclusions about genes effects, transformation efficiency declined drastically in several months (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), with losses approaching 20% across the points analyzed. Such reduction was independent on the genetic construct, including empty vectors used as controls, since we used four distinct ones with similar results (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Storing the transformed bacteria in Petri dishes at 4\u0026deg;C, and renewing the media each month, did not result in alleviating the efficiency loss. For field trials, the ability to generate a sufficient number of plants is critical to establishing plots of adequate size. This requirement, in turn, depends on maintaining a high transformation efficiency to ensure the production of chimeric plants bearing multiple hairy roots that carry the construct of interest. Addressing this limitation was therefore essential to secure a reliable and efficient source of composite plants. Even though previous works examined many characteristics strongly contributing to improve the methodology, such as nitrogen availability, sterilization technique, germination devices, explant age, and acetosyringone addition and concentration, etc. (Araragi et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Niazian et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), we were not able to detect reports informing about the efficiency loss over the time we experienced.\u003c/p\u003e \u003cp\u003eTo overcome this situation, we tested the bacterial colonies one by one and detected a differential behavior. In the same dish, a few colonies were infective and succeeded in provoking hairy root emergence, whereas others were mostly unable (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The loss of efficiency prevented the obtaining of a significant number of transgenic plants.\u003c/p\u003e \u003cp\u003eHence, we tested another strategy that was to periodically retransform the bacteria before each batch of hairy root experiments, and it was a success.\u003c/p\u003e \u003cp\u003eEven though we cannot explain the reason for this result, which is not a contamination of the mother \u003cem\u003eRhizobium\u003c/em\u003e culture, nor a bad temperature regulation of several ultrafreezers, it was a fact that many bacteria lost their infection ability after prolonged storage at -80\u0026deg;C or at 4\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRegarding recently infected plants growing temperature, we found that 22\u0026deg;C was rather better than the optimal temperature usually used to grow soybeans (28\u0026deg;C). Considering this condition, most reported protocols informed 28\u0026deg;C as cocultivation temperature (Araragi et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Niazian et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Kong et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) or a combination of 26\u0026deg;C day/22\u0026deg;C night (Song et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFinally, we can state that transformed hairy roots expressing or silencing transcription factors from the HD-Zip I family impact on plant development, allowing to study many other genes and their effect on growing and stress tolerance. GmHDZ4, a soybean member of this transcription factor family, exhibiting sequence similarities with AtHB12 and Oshox12, was knocked out by edition in chimeric plants (Zhong et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These chimeric plants were tested in pots and controlled conditions, showing significant differences in root architecture and enhanced tolerance to osmotic stress applied with polyethilenglycol in the vegetative stage (Zhong et al. 2021). It is tempting to speculate that this optimized protocol will allow to characterize the impact of many other genes when expressed in roots, at different developmental stages. We can state this after performing trials in microplots in open air during two seasons and several sowing dates, which added value to experiments performed only in pots and in controlled conditions. To the best of our knowledge, this is the first report describing field trials with composite soybean with hairy roots.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eIn this work, we describe a significant advancement over existing one-step \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation protocols by enhancing transformation efficiency. Our improved method optimizes several key parameters: re-transforming bacteria before each experiment to maintain high infection viability, minimizing steps requiring strict sterility, enabling \u003cem\u003ein vivo\u003c/em\u003e visualization of transformed roots via GFP detection with a flashlight, and standardizing growth at 22\u003csup\u003e∘\u003c/sup\u003eC. These modifications enable reliable, high-throughput production of transgenic soybean roots, allowing us to perform crucial field trials in microplots. Assessing the effect of root transgenes under field conditions is vital for soybean research, as conclusions drawn solely from controlled environments may misrepresent a gene's true function in agriculturally relevant settings.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCONFLICT OF INTEREST\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFUNDING\u003c/h2\u003e \u003cp\u003eThe work of the research group was supported by Agencia Nacional de Promoci\u0026oacute;n Cient\u0026iacute;fica y Tecnol\u0026oacute;gica (PICT 2020 0805, MINCYT (ex Ministerio de Ciencia y Tecnolog\u0026iacute;a) through the special grant \u0026ldquo;Redes de Alto Impacto\u0026rdquo;), CONICET, and Fundaci\u0026oacute;n Williams to RLC. LC is a CONICET Ph.D. Fellow. JR and RLC are CONICET Career members.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceived by RLC. Made the experiments and illustrations: LC. Discussed the manuscript: LC, JR, and RLC. Wrote the paper: RLC. Revised and approved the manuscript: all authors.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank Dr. Germ\u0026aacute;n Robert from Centro de Investigaciones Agropecuarias INTA-C\u0026oacute;rdoba for providing us the K599 bacterial strain and helpful advices and discussions. We also thank Dr. Ramiro Lascano for helpful advice. We thank Ana Bel\u0026eacute;n Curi and Manuel Franco for technical assistance in field trials.\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003eAraragi M, Songwattana P, Teaumroong N, Masuda S, Shibata A, Shirasu K, Kawaharada Y (2025) Improved rapid and efficient hairy root transformation using Rhizobium rhizogenes in legume crops. \u003cem\u003ePlant Biotechnology\u003c/em\u003e (Tokyo) 42(3):279-288. doi: 10.5511/plantbiotechnology.25.0213a. \u003c/p\u003e\n\u003cp\u003eChan RL, Trucco F, Otegui ME (2020) Why are second-generation transgenic crops not yet available in the market? \u003cem\u003eJournal of Experimental Botany\u003c/em\u003e 71(22):6876-6880. doi: 10.1093/jxb/eraa412. \u003c/p\u003e\n\u003cp\u003eCheng Y, Wang X, Cao L, Ji J, Liu T, Duan K (2021) Highly efficient Agrobacterium rhizogenes-mediated hairy root transformation for gene functional and gene editing analysis in soybean. \u003cem\u003ePlant Methods\u003c/em\u003e 17(1):73. doi: 10.1186/s13007-021-00778-7\u003c/p\u003e\n\u003cp\u003eDe Cleene M, De Ley J (1976) The host range of crown gall. \u003cem\u003eBotanical Review\u003c/em\u003e 42:389\u0026ndash;466 \u003c/p\u003e\n\u003cp\u003eFan YL, Zhang XH, Zhong LJ, Wang XY, Jin LS, Lyu SH (2020) One-step generation of composite soybean plants with transgenic roots by Agrobacterium rhizogenes-mediated transformation. \u003cem\u003eBMC Plant Biology\u003c/em\u003e 20(1):208. doi: 10.1186/s12870-020-02421-4. \u003c/p\u003e\n\u003cp\u003eGao Y, Qu D, Zhou M, Tang R, Ye J, Li X, Wang Y (2024) Rhizobial-induced phosphatase GmPP2C61A positively regulates soybean nodulation. \u003cem\u003ePhysiologia Plantarum\u003c/em\u003e 176(3):e14341. doi: 10.1111/ppl.14341. \u003c/p\u003e\n\u003cp\u003eGeorgiev MI, Agostini E, Ludwig-M\u0026uuml;ller J, Xu J (2012) Genetically transformed roots: from plant disease to biotechnological resource. \u003cem\u003eTrends in Biotechnology\u003c/em\u003e 30:528\u0026ndash;537. doi: 10.1016/j.tibtech.2012.07.001\u003c/p\u003e\n\u003cp\u003eGutierrez-Valdes N, H\u0026auml;kkinen ST, Lemasson C, Guillet M, Oksman-Caldentey KM, Ritala A, Cardon F (2020) Hairy root cultures-a versatile tool with multiple applications. \u003cem\u003eFrontiers in Plant Science\u003c/em\u003e 11:33. doi: 10.3389/fpls.2020.0003 3\u003c/p\u003e\n\u003cp\u003eRaineri J, Montero Bulacio E, Campi M, Portapila M, Otegui ME, Chan RL (2025) The sunflower transcription factor HaHB11 increases soybean grain number and heat tolerance across multi-season field trials. \u003cem\u003eJournal of Experimental Botany\u003c/em\u003e 76(17):5037-5055. doi: 10.1093/jxb/eraf088.\u003c/p\u003e\n\u003cp\u003eH\u0026auml;kkinen S, Moyano TE, Cusid\u0026oacute; RM, Oksman-Caldentey K-M (2016) Exploring the metabolic stability of engineered hairy roots after 16 years maintenance. \u003cem\u003eFrontiers in Plant Science\u003c/em\u003e 7:1486. doi: 10.3389/fpls.2016.01486\u003c/p\u003e\n\u003cp\u003eHartman GL, West ED, Herman TK (2011) Crops that feed the world 2. 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A critical biological question is whether root plasticity in response to these stresses influences whole-plant development. The generation of soybean chimeric plants with an altered root transcriptome offers a powerful approach to address this question. The existing protocols for this strategy typically require sterile conditions and produce a limited number of chimeric plants. We optimized the technique by introducing several key modifications, significantly enhancing its efficiency. The new protocol eliminates the requirement for sterile conditions in most steps. Moreover, fresh bacterial transformation for each experiment was performed, overcoming the loss of infection ability associated with storing cultures at −80 °C, reaching 70-100 % efficiency. A previous transformation of cotyledons helped to select the colony with the highest infection ability. The optimal plant growth temperature was determined to be 22 °C. These combined changes and tips resulted in the consistent production of hundreds of chimeric plants, making them suitable for large-scale field trials. Results from subsequent field trials performed in three seasons confirmed that alteration of the root transcriptome significantly impacts whole-plant development.\u003c/p\u003e","manuscriptTitle":"Scalable production of soybean hairy roots: a reliable method for field trials","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-05 08:42:40","doi":"10.21203/rs.3.rs-8353559/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-02T16:43:03+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-30T07:52:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"300754355416628872968345180664174504094","date":"2026-01-18T01:44:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-14T23:48:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"156404916491981587857229343117428323002","date":"2026-01-03T23:29:57+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-02T11:00:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-17T12:54:34+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-17T12:53:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell Reports","date":"2025-12-13T14:45:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a788b38e-f9e0-4ab3-b78c-f0694c88e1ee","owner":[],"postedDate":"January 5th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-09T16:14:26+00:00","versionOfRecord":{"articleIdentity":"rs-8353559","link":"https://doi.org/10.1007/s00299-026-03753-5","journal":{"identity":"plant-cell-reports","isVorOnly":false,"title":"Plant Cell Reports"},"publishedOn":"2026-03-02 15:58:44","publishedOnDateReadable":"March 2nd, 2026"},"versionCreatedAt":"2026-01-05 08:42:40","video":"","vorDoi":"10.1007/s00299-026-03753-5","vorDoiUrl":"https://doi.org/10.1007/s00299-026-03753-5","workflowStages":[]},"version":"v1","identity":"rs-8353559","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8353559","identity":"rs-8353559","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}