Rhizogenes-mediated Hairy Root Research Articles

Enhancing the aervine and methylaervine production in in vitro cultures of Aerva javanica (Burm. F.) Schult via elicitors and Agrobacterium rhizogenes-mediated hairy root cultures

Enhancing the aervine and methylaervine production in in vitro cultures of Aerva javanica (Burm. F.) Schult via elicitors and Agrobacterium rhizogenes-mediated hairy root cultures

Development of a single transcript CRISPR/Cas9 toolkit for efficient genome editing in autotetraploid alfalfa

Alfalfa (Medicago sativa. L.) is a globally significant autotetraploid legume forage crop. However, despite its importance, establishing efficient gene editing systems for cultivated alfalfa remains a formidable challenge. In this study, we pioneered the development of a highly effective ultrasonic-assisted leaf disc transformation system for Gongnong 1 alfalfa, a variety widely cultivated in Northeast China. Subsequently, we created a single transcript CRISPR/Cas9 (CRISPR_2.0) toolkit, incorporating multiplex gRNAs, designed for gene editing in Gongnong 1. Both Cas9 and gRNA scaffolds were under the control of the Arabidopsis ubiquitin-10 promoter, a widely employed polymerase II constitutive promoter known for strong transgene expression in dicots. To assess the toolkit’s efficiency, we targeted PALM1, a gene associated with a recognizable multifoliate phenotype. Utilizing the CRISPR_2.0 toolkit, we directed PALM1 editing at two sites in the wild-type Gongnong 1. Results indicated a 35.1% occurrence of editing events all in target 2 alleles, while no mutations were detected at target 1 in the transgenic-positive lines. To explore more efficient sgRNAs, we developed a rapid, reliable screening system based on Agrobacterium rhizogenes-mediated hairy root transformation, incorporating the visible reporter MtLAP1. This screening system demonstrated that most purple visible hairy roots underwent gene editing. Notably, sgRNA3, with an 83.0% editing efficiency, was selected using the visible hairy root system. As anticipated, tetra-allelic homozygous palm1 mutations exhibited a clear multifoliate phenotype. These palm1 lines demonstrated an average crude protein yield increase of 21.5% compared to trifoliolate alfalfa. Our findings highlight the modified CRISPR_2.0 system as a highly efficient and robust gene editing tool for autotetraploid alfalfa.

Streamlined Agrobacterium rhizogenes-mediated hairy root transformation for efficient CRISPR/Cas9-based gene editing evaluation in diverse Citrullus varieties

The CRISPR/Cas9 genome-editing system serves as a pivotal tool for enhancing crop genetics. Within this system, single guide RNAs (sgRNAs) are instrumental in the precise cleavage of DNA double strands. However, the efficiency of gene editing varies among sgRNAs, emphasizing the need to meticulously select target sites, especially in the context of Citrullus lanatus, a species notorious for its challenging and inefficient generation of transgenic plants through stable transformation. This study employed an Agrobacterium rhizogenes-mediated hairy root method to assess effective target sites for gene editing of ClCIPK17 across various Citrullus species. Hairy roots were successfully induced in different plant tissues at diverse growth stages of Citrullus lanatus, Citrullus mucosospermus, and Citrullus amarus. Employing a vector with two sgRNAs (sgRNA1 and sgRNA5) positioned within conserved regions of exon 1 and exon 5 of ClCIPK17 in the CRISPR/Cas9 system, targeted mutations were detected in 90.9% of accessions across the four Citrullus species. Notably, 73.94% of all examined hairy roots exhibited mutations at the sgRNA1 site, while the sgRNA5 site showed no mutations. Among the 31 different mutation types identified at the sgRNA1 site, base deletion was the most prevalent. Using the sgRNA1 site of ClCIPK17, stable transgenic watermelon buds were obtained from explants, and the targeted mutations of the sgRNA1 site were confirmed. These findings underscore the viability of the hairy root transformation system in assessing the editing efficiency of sgRNA targets in diverse Citrullus species.

Highly efficient Agrobacterium rhizogenes-mediated hairy root transformation in citrus seeds and its application in gene functional analysis.

Highly efficient genetic transformation technology is beneficial for plant gene functional research and molecular improvement breeding. However, the most commonly used Agrobacterium tumefaciens-mediated genetic transformation technology is time-consuming and recalcitrant for some woody plants such as citrus, hampering the high-throughput functional analysis of citrus genes. Thus, we dedicated to develop a rapid, simple, and highly efficient hairy root transformation system induced by Agrobacterium rhizogenes to analyze citrus gene function. In this report, a rapid, universal, and highly efficient hairy root transformation system in citrus seeds was described. Only 15 days were required for the entire workflow and the system was applicable for various citrus genotypes, with a maximum transformation frequency of 96.1%. After optimization, the transformation frequency of Citrus sinensis, which shows the lowest transformation frequency of 52.3% among four citrus genotypes initially, was increased to 71.4% successfully. To test the applicability of the hairy roots transformation system for gene functional analysis of citrus genes, we evaluated the subcellular localization, gene overexpression and gene editing in transformed hairy roots. Compared with the traditional transient transformation system performed in tobacco leaves, the transgenic citrus hairy roots displayed a more clear and specific subcellular fluorescence localization. Transcript levels of genes were significantly increased in overexpressing transgenic citrus hairy roots as compared with wild-type (WT). Additionally, hairy root transformation system in citrus seeds was successful in obtaining transformants with knocked out targets, indicating that the Agrobacterium rhizogenes-mediated transformation enables the CRISPR/Cas9-mediated gene editing. In summary, we established a highly efficient genetic transformation technology with non-tissue-culture in citrus that can be used for functional analysis such as protein subcellular localization, gene overexpression and gene editing. Since the material used for genetic transformation are roots protruding out of citrus seeds, the process of planting seedlings prior to transformation of conventional tissue culture or non-tissue-culture was eliminated, and the experimental time was greatly reduced. We anticipate that this genetic transformation technology will be a valuable tool for routine research of citrus genes in the future.

Highly efficient Agrobacterium rhizogenes-mediated transformation for functional analysis in woodland strawberry

BackgroundThe diploid woodland strawberry (Fragaria vesca) is an excellent model plant for investigating economically significant traits and several genetic resources within the Rosaceae family. Agrobacterium rhizogenes-mediated hairy root transformation is an alternative for exploring gene functions, especially the genes specifically expressed in roots. However, the hairy root transformation has not been established in strawberry.ResultsHere, we described an efficient and rapid hairy root transgenic system for strawberry using A. rhizogenes. Strain of A. rhizogenes MSU440 or C58C1 was the most suitable for hairy root transformation. The transformation efficiency was highest when tissues contained hypocotyls as explants. The optimal procedure involves A. rhizogenes at an optical density (OD600) of 0.7 for 10 min and co-cultivation duration for four days, achieving a transgenic efficiency of up to 71.43%. An auxin responsive promoter DR5ver2 carrying an enhanced green fluorescent protein (eGFP) marker was transformed by A. rhizogenes MSU440, thereby generating transgenic hairy roots capable of high eGFP expression in root tip and meristem of strawberry where auxin accumulated. Finally, this system was applied for functional analysis using jGCaMP7c, which could sense calcium signals. A significant upsurge in eGFP expression in the transgenic hairy roots was displayed after adding calcium chloride. The results suggested that this approach was feasible for studying specific promoters and could be a tool to analyze gene functions in the roots of strawberries.ConclusionWe established a rapid and efficient hairy root transformation in strawberry by optimizing parameters, which was adequate for promoter analysis and functional characterization of candidate genes in strawberry and other rosaceous plants.

Optimization of in vitro and ex vitro Agrobacterium rhizogenes-mediated hairy root transformation of soybean for visual screening of transformants using RUBY.

In vitro and ex vitro Agrobacterium rhizogenes-mediated hairy root transformation (HRT) assays are key components of the plant biotechnology and functional genomics toolkit. In this report, both in vitro and ex vitro HRT were optimized in soybean using the RUBY reporter. Different parameters including A. rhizogenes strain, optical density of the bacterial cell culture (OD600), co-cultivation media, soybean genotype, explant age, and acetosyringone addition and concentration were evaluated. Overall, the in vitro assay was more efficient than the ex vitro assay in terms of the percentage of induction of hairy roots and transformed roots (expressing RUBY). Nonetheless, the ex vitro technique was deemed faster and a less complicated approach. The highest transformation of RUBY was observed on 7-d-old cotyledons of cv. Bert inoculated for 30 minutes with the R1000 resuspended in ¼ B5 medium to OD600 (0.3) and 150 µM of acetosyringone. The parameters of this assay also led to the highest percentage of RUBY through two-step ex vitro hairy root transformation. Finally, using machine learning-based modeling, optimal protocols for both assays were further defined. This study establishes efficient and reliable hairy root transformation protocols applicable for functional studies in soybean.

Rhizobium rhizogenes-mediated hairy root induction and plant regeneration for bioengineering citrus.

Rhizobium rhizogenes-mediated hairy root induction and plant regeneration for bioengineering citrus.

Establishment of an efficient transformation system and its application in regulatory mechanism analysis of biological macromolecules in tea plants

Tea (Camellia sinensis), one of the most important beverage crops originated from China and is now cultivated worldwide, provides numerous secondary metabolites that account for its health benefits and rich flavor. However, the lack of an efficient and reliable genetic transformation system has seriously hindered the gene function investigation and precise breeding of C. sinensis. In this study, we established a highly efficient, labor-saving, and cost-effective Agrobacterium rhizogenes-mediated hairy roots genetic transformation system for C. sinensis, which can be used for gene overexpression and genome editing. The established transformation system was simple to operate, bypassing tissue culture and antibiotic screening, and only took two months to complete. We used this system to conduct function analysis of transcription factor CsMYB73 and found that CsMYB73 negatively regulates L-theanine synthesis in tea plant. Additionally, callus formation was successfully induced using transgenic roots, and the transgenic callus exhibited normal chlorophyll production, enabling the study of the corresponding biological functions. Furthermore, this genetic transformation system was effective for multiple C. sinensis varieties and other woody plant species. By overcoming technical obstacles such as low efficiency, long experimental periods, and high costs, this genetic transformation will be a valuable tool for routine gene investigation and precise breeding in tea plants.

Metabolome and transcriptome analyses reveal the molecular mechanisms of LcMYB1 regulating anthocyanin accumulation in litchi hairy roots

Agrobacterium rhizogenes-mediated hairy root culture offer a promising approach for gene function analysis and production of plant secondary metabolites. Here, we obtained red litchi hairy roots using A. rhizogenes-mediated LcMYB1 transformation. Using high performance liquid chromatography, the main anthocyanins in the red hairy roots were determined to be cyanidin 3-rutinoside and cyanidin 3-glucoside. A total of 164 metabolites were significantly upregulated or downregulated in the red hairy roots, which were mostly involved in flavone and flavonol pathway, and flavonoid pathway. The transcriptome analysis revealed 472 differentially expressed genes (DEGs). Up-regulated genes were considerably enriched in anthocyanin, flavone and flavonol biosynthesis. Integrative metabolite profiling and transcriptome analyses showed that LcF3′H, LcUFGT1, and LcGST4 were key structural genes in anthocyanin biosynthesis. However, the expression of Cinnamyl-alcohol dehydrogenase (CAD) and Peroxidase (POD) leading to the production of lignin were significantly down-regulated, suggesting flavonoids and lignin compete with each other in the phenylpropanoid pathway. A total of 52 DEGs were identified as transcription factors. Correlation analysis showed that 8 transcription factors were positively correlated with LcUFGT1, and LcGST4, involving in anthocyanin biosynthesis. These findings clarify the molecular mechanisms of LcMYB1 regulating anthocyanin accumulation in litchi hairy roots.

Highly efficient Agrobacterium rhizogenes-mediated hairy root transformation for gene editing analysis in cotton.

CRIPSR/Cas9 gene editing system is an effective tool for genome modification in plants. Multiple target sites are usually designed and the effective target sites are selected for editing. Upland cotton (Gossypium hirsutum L., hereafter cotton) is allotetraploid and is commonly considered as difficult and inefficient to transform, it is important to select the effective target sites that could result in the ideal transgenic plants with the CRISPR-induced mutations. In this study, Agrobacterium rhizogenes-mediated hairy root method was optimized to detect the feasibility of the target sites designed in cotton phytoene desaturase (GhPDS) gene. A. rhizogenes showed the highest hairy root induction (30%) when the bacteria were cultured until OD600 reached to 0.8. This procedure was successfully applied to induce hairy roots in the other three cultivars (TM-1, Lumian-21, Zhongmian-49) and the mutations were detected in GhPDS induced by CRISPR/Cas9 system. Different degrees of base deletions at two sgRNAs (sgRNA5 and sgRNA10) designed in GhPDS were detected in R15 hairy roots. Furthermore, we obtained an albino transgenic cotton seeding containing CRISPR/Cas9-induced gene editing mutations in sgRNA10. The hairy root transformation system established in this study is sufficient for selecting sgRNAs in cotton, providing a technical basis for functional genomics research of cotton.

Development of efficient Agrobacterium rhizogenes-mediated hairy root system in Curcuma longa L. and elicitation driven enhanced production of pharmaceutically important curcuminoids

Development of efficient Agrobacterium rhizogenes-mediated hairy root system in Curcuma longa L. and elicitation driven enhanced production of pharmaceutically important curcuminoids

A sequential transformation method for validating soybean genome editing by CRISPR/Cas9 system

This study was performed to evaluate the sequential transformation for soybean genome editing using the CRISPR/Cas9 system as well as to show a strategy for examining the activity of CRISPR/Cas9 constructs, especially the designed guide RNAs (gRNAs). The gRNAs for targeted mutations of an exogenous gene and multiple endogenous genes were constructed and transferred into a stably-overexpressed-Cas9 soybean line using Agrobacterium rhizogenes-mediated hairy root induction system. The targeted mutations were identified and characterized by the poly-acrylamide gel electrophoresis (PAGE) heteroduplex method and by sequencing. Induced mutations of the exogenous gene (gus) were observed in 57% of tested transgenic hairy roots, while 100% of the transgenic root lines showed targeted mutations of the endogenous (SACPD-C) gene. Multiple gRNAs targeting two endogenous genes (SACPD-C and SMT) induced mutation rates of 75% and 67%, respectively. Various indels including small and large deletions as well as insertions were found in target sites of the tested genes. This sequential transformation method could present the targeting efficacy of different gRNAs of each tested gene. Additionally, in this study differences in gRNA ratings were found between bioinformatics predictions and actual experimental results. This is the first successful application of the sequential transformation method for genome editing in soybean using the hairy root system. This method could be potentially useful for validating CRISPR/Cas9 constructs, evaluating gRNA targeting efficiencies, and could be applied for other research directions.

The First Report on Transgenic Hairy Root Induction from the Stem of Tung Tree (Vernicia fordii).

Tung tree (Vernicia fordii) is an industrially important oil-bearing woody plant of the Euphorbiaceae family. Functional studies of tung tree at the molecular level are limited by the lack of an efficient transgenic system. The Agrobacterium rhizogenes-mediated hairy root generation system is an alternative to typical plant transformation systems. However, its application in many plants has been blocked due to the inability of existing methods to induce hairy roots. Thus, it is critical to build a method suitable for the hairy induction of the specific plant of interest. In this study, a modified method for tung tree was developed, and it is the first report that hairy roots could be effectively induced in the stem of tung tree. With the method, an average of 10.7 hairy roots per seedling were generated in tung tree, approximately 67% of seedlings produced transgenic hairy roots and approximately 13.96% of the hairy roots of these seedlings were transgenic. This modified method is also suitable for the hairy root induction of two other oil-bearing plants of the Euphorbiaceae family, Ricinus communis and Vernicia montana. This modified method will accelerate functional studies of tung tree at the molecular level and also shed light on plants lacking a transgenic system.

CRISPR/Cas9 in Planta Hairy Root Transformation: A Powerful Platform for Functional Analysis of Root Traits in Soybean.

Sequence and expression data obtained by next-generation sequencing (NGS)-based forward genetics methods often allow the identification of candidate causal genes. To provide true experimental evidence of a gene’s function, reverse genetics techniques are highly valuable. Site-directed mutagenesis through transfer DNA (T-DNA) delivery is an efficient reverse screen method in plant functional analysis. Precise modification of targeted crop genome sequences is possible through the stable and/or transient delivery of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (CRISPR/Cas) reagents. Currently, CRISPR/Cas9 is the most powerful reverse genetics approach for fast and precise functional analysis of candidate genes/mutations of interest. Rapid and large-scale analyses of CRISPR/Cas-induced mutagenesis is achievable through Agrobacterium rhizogenes-mediated hairy root transformation. The combination of A. rhizogenes hairy root-CRISPR/Cas provides an extraordinary platform for rapid, precise, easy, and cost-effective “in root” functional analysis of genes of interest in legume plants, including soybean. Both hairy root transformation and CRISPR/Cas9 techniques have their own complexities and considerations. Here, we discuss recent advancements in soybean hairy root transformation and CRISPR/Cas9 techniques. We highlight the critical factors required to enhance mutation induction and hairy root transformation, including the new generation of reporter genes, methods of Agrobacterium infection, accurate gRNA design strategies, Cas9 variants, gene regulatory elements of gRNAs and Cas9 nuclease cassettes and their configuration in the final binary vector to study genes involved in root-related traits in soybean.

Ribozyme-mediated CRISPR/Cas9 gene editing in pyrethrum (Tanacetum cinerariifolium) hairy roots using a RNA polymerase II-dependent promoter

BackgroundTraditional CRISPR/Cas9 systems that rely on U6 or U3 snRNA promoters (RNA polymerase III-dependent promoters) can only achieve constitutive gene editing in plants, hampering the functional analysis of specifically expressed genes. Ribozyme-mediated CRISPR/Cas9 systems increase the types of promoters which can be used to transcribe sgRNA. Therefore, such systems allow specific gene editing; for example, transcription of the artificial gene Ribozyme-sgRNA-Ribozyme (RGR) is initiated by an RNA polymerase II-dependent promoter. Genetic transformation is indispensable for editing plant genes. In certain plant species, including pyrethrum, genetic transformation remains challenging to do, limiting the functional verification of novel CRISPR/Cas9 systems. Thus, this study’s aim was to develop a simple Agrobacterium rhizogenes-mediated hairy root transformation system to analyze the function of a ribozyme-mediated CRISPR/Cas9 system in pyrethrum.ResultsA hairy root transformation system for pyrethrum is described, with a mean transformation frequency of 7%. Transgenic hairy roots transformed with the pBI121 vector exhibited significantly increased beta-glucuronidase staining as a visual marker of transgene expression. Further, a ribozyme-based CRISPR/Cas9 vector was constructed to edit the TcEbFS gene, which catalyzes synthesis of the defense-related compound (E)-β-farnesene in pyrethrum. The vector was transferred into the hairy roots of pyrethrum and two stably transformed hairy root transgenic lines obtained. Editing of the TcEbFS gene in the hairy roots was evaluated by gene sequencing, demonstrating that both hairy root transgenic lines had DNA base loss at the editing target site. Gas chromatography–mass spectrometry showed that the (E)-β-farnesene content was significantly decreased in both hairy root transgenic lines compared with the empty vector control group. Altogether, these results show that RGR can be driven by the CaMV35S promoter to realize TcEbFS gene editing in pyrethrum hairy roots.ConclusionAn A. rhizogenes-mediated hairy root transformation and ribozyme-mediated CRISPR/Cas9 gene editing system in pyrethrum was established, thereby facilitating gene editing in specific organs or at a particular developmental stage in future pyrethrum research.

Critical role for uricase and xanthine dehydrogenase in soybean nitrogen fixation and nodule development.

De novo purine biosynthesis is required for the incorporation of fixed nitrogen in ureide exporting nodules, as formed on soybean [Glycine max (L.) Merr.] roots. However, in many cases, the enzymes involved in this pathway have been deduced strictly from genome annotations with little direct genetic evidence, such as mutant studies, to confirm their biochemical function or importance to nodule development. While efforts to develop large mutant collections of soybean are underway, research on this plant is still hampered by the inability to obtain mutations in any specific gene of interest. Using a forward genetic approach, as well as CRISPR/Cas9 gene editing via Agrobacterium rhizogenes-mediated hairy root transformation, we identified and characterized the role of GmUOX (Uricase) and GmXDH (Xanthine Dehydrogenase) in nitrogen fixation and nodule development in soybean. The gmuox knockout soybean mutants displayed nitrogen deficiency chlorosis and early nodule senescence, as exemplified by the reduced nitrogenase (acetylene reduction) activity in nodules, the internal greenish-white internal appearance of nodules, and diminished leghemoglobin production. In addition, gmuox1 nodules showed collapsed infected cells with degraded cytoplasm, aggregated bacteroids with no discernable symbiosome membranes, and increased formation of poly-β-hydroxybutyrate granules. Similarly, knockout gmxdh mutant nodules, generated with the CRISPR/Cas9 system, also exhibited early nodule senescence. These genetic studies confirm the critical role of the de novo purine metabolisms pathway not only in the incorporation of fixed nitrogen but also in the successful development of a functional, nitrogen-fixing nodule. Furthermore, these studies demonstrate the great utility of the CRISPR/Cas9 system for studying root-associated gene traits when coupled with hairy root transformation.

The aquaporin gene PvXIP1;2 conferring drought resistance identified by GWAS at seedling stage in common bean.

A whole-genome resequencing-derived SNP dataset used for genome-wide association analysis revealed 12 loci significantly associated with drought stress based on survival rate after drought stress at seedling stage. We further confirmed the drought-related function of an aquaporin gene (PvXIP1;2) located at Locus_10. A variety of adverse conditions, including drought stress, severely affect common bean production. Molecular breeding for drought resistance has been proposed as an effective and practical way to improve the drought resistance of common bean. A genome-wide association analysis was conducted to identify drought-related loci based on survival rates at the seedling stage using a natural population consisting of 400 common bean accessions and 3,832,340 SNPs. The coefficient of variation ranged from 40.90 to 56.22% for survival rates in three independent experiments. A total of 12 associated loci containing 89 significant SNPs were identified for survival rates at the seedling stage. Four loci overlapped in the region of the QTLs reported to be associated with drought resistance. According to the expression profiles, gene annotations and references of the functions of homologous genes in Arabidopsis, 39 genes were considered potential candidate genes selected from 199 genes annotated within all associated loci. A stable locus (Locus_10) was identified on chromosome 11, which contained LEA, aquaporin, and proline-rich protein genes. We further confirmed the drought-related function of an aquaporin (PvXIP1;2) located at Locus_10 by expression pattern analysis, phenotypic analysis of PvXIP1;2-overexpressing Arabidopsis and Agrobacterium rhizogenes-mediated hairy root transformation systems, indicating that the association results can facilitate the efficient identification of genes related to drought resistance. These loci and their candidate genes provide a foundation for crop improvement via breeding for drought resistance in common bean.

Agrobacterium rhizogenes-mediated hairy root transformation as an efficient system for gene function analysis in Litchi chinensis

BackgroundLitchi chinensis Sonn. is an economically important fruit tree in tropical and subtropical regions. However, litchi functional genomics is severely hindered due to its recalcitrance to regeneration and stable transformation. Agrobacterium rhizogenes-mediated hairy root transgenic system provide an alternative to study functional genomics in woody plants. However, the hairy root transgenic system has not been established in litchi.ResultsIn this study, we report a rapid and highly efficient A. rhizogenes-mediated co-transformation system in L. chinensis using Green Fluorescent Protein (GFP) gene as a marker. Both leaf discs and stem segments of L. chinensis cv. ‘Fenhongguiwei’ seedlings were able to induce transgenic hairy roots. The optimal procedure involved the use of stem segments as explants, infection by A. rhizogenes strain MSU440 at optical density (OD600) of 0.7 for 10 min and co-cultivation for 3 days, with a co-transformation efficiency of 9.33%. Furthermore, the hairy root transgenic system was successfully used to validate the function of the key anthocyanin regulatory gene LcMYB1 in litchi. Over-expression of LcMYB1 produced red hairy roots, which accumulated higher contents of anthocyanins, proanthocyanins, and flavonols. Additionally, the genes involving in the flavonoid pathway were strongly activated in the red hairy roots.ConclusionWe first established a rapid and efficient transformation system for the study of gene function in hairy roots of litchi using A. rhizogenes strain MSU440 by optimizing parameters. This hairy root transgenic system was effective for gene function analysis in litchi using the key anthocyanin regulator gene LcMYB1 as an example.

Highly efficient Agrobacterium rhizogenes-mediated hairy root transformation for gene functional and gene editing analysis in soybean

BackgroundAgrobacterium-mediated genetic transformation is a widely used and efficient technique for gene functional research in crop breeding and plant biology. While in some plant species, including soybean, genetic transformation is still recalcitrant and time-consuming, hampering the high-throughput functional analysis of soybean genes. Thus we pursue to develop a rapid, simple, and highly efficient hairy root system induced by Agrobacterium rhizogenes (A. rhizogenes) to analyze soybean gene function.ResultsIn this report, a rapid, simple, and highly efficient hairy root transformation system for soybean was described. Only sixteen days were required for the whole workflow and the system was suitable for various soybean genotypes, with an average transformation frequency of 58–64%. Higher transformation frequency was observed when wounded cotyledons from 1-day-germination seeds were inoculated and co-cultivated with A. rhizogenes in 1/2 B5 (Gamborg’ B-5) medium. The addition of herbicide selection to root production medium increased the transformation frequency to 69%. To test the applicability of the hairy root system for gene functional analysis, we evaluated the protein expression and subcellular localization in transformed hairy roots. Transgenic hairy roots exhibited significantly increased GFP fluorescence and appropriate protein subcellular localization. Protein–protein interactions by BiFC (Bimolecular Fluorescent Complimentary) were also explored using the hairy root system. Fluorescence observations showed that protein interactions could be observed in the root cells. Additionally, hairy root transformation allowed soybean target sgRNA screening for CRISPR/Cas9 gene editing. Therefore, the protocol here enables high-throughput functional characterization of candidate genes in soybean.ConclusionA rapid, simple, and highly efficient A. rhizogenes-mediated hairy root transformation system was established for soybean gene functional analysis, including protein expression, subcellular localization, protein–protein interactions and gene editing system evaluation.

Soybean Hairy Root Transformation: A Rapid and Highly Efficient Method.

New genetic engineering techniques have advanced the field of plant molecular biology, and Agrobacterium-mediated transformation has enabled the discovery of numerous molecular and genetic functions. It has been widely used in many plants, including the economically important crop, soybean. Large-scale genetic analyses are needed to comprehend the molecular mechanisms that underlie the agronomic traits of soybean, and the generation of stable transgenic plants involves a lengthy and laborious process. Agrobacterium rhizogenes-mediated hairy root transformation is a quick and efficient method for investigations of root-specific processes and interactions. Generation of composite plants with transgenic roots and wild-type shoots allows for the study of the genetic mechanisms involved in root biology, such as the Bradyrhizobium-soybean interaction. Here, we provide an updated protocol for generating hairy soybean roots in as little as 18 days in a cost- and space-effective manner and demonstrate possible uses of composite plants with soybean nodulation assays and gene expression analysis as examples. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Soybean hairy root transformation Basic Protocol 2: Soybean nodulation assay Alternate Protocol: Soybean nodulation assay in germination pouches Support Protocol: Bradyrhizobium japonicum culture preparation for inoculation Basic Protocol 3: Histochemical GUS staining for promoter analysis.