Agrobacterium Rhizogenes-mediated Transformation Research Articles

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.

Water-deficit stress induces prenylated stilbenoid production and affects biomass in peanut hairy roots: Exploring the role of stilbenoid prenyltransferase downregulation

The peanut plant is one of the most economically important crops around the world. Abiotic stress, such as drought, causes over five hundred million dollars in losses in peanut production per year. Peanuts are known to produce prenylated stilbenoids to counteract biotic stress. However, their role in abiotic stress tolerance has not been elucidated. To address this issue, hairy roots with the capacity to produce prenylated stilbenoids were established. An RNA-interference (RNAi) molecular construct targeting the stilbenoid-specific prenyltransferase AhR4DT-1 was designed and expressed via Agrobacterium rhizogenes-mediated transformation in hairy roots of peanut cultivar Georgia Green. Two transgenic hairy roots with the RNAi molecular construct were established, and the downregulation of AhR4DT-1 was validated using reverse transcriptase quantitative PCR. To determine the efficacy of the RNAi-approach in modifying the levels of prenylated stilbenoids, the hairy roots were co-treated with methyl jasmonate, hydrogen peroxide, cyclodextrin, and magnesium chloride to induce the production of stilbenoids and then the stilbenoids were analyzed in extracts of the culture medium. Highly reduced levels of prenylated stilbenoids were observed in the RNAi hairy roots. Furthermore, the hairy roots were evaluated in a polyethylene glycol (PEG) assay to assess the role of prenylated stilbenoids on water-deficit stress. Upon PEG treatment, stilbenoids were induced and secreted into the culture medium of RNAi and wild-type hairy roots. Additionally, the biomass of the RNAi hairy roots decreased by a higher amount as compared to the wild-type hairy roots suggesting that prenylated stilbenoids might play a role against water-deficit stress.

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.

Agrobacterium rhizogenes-mediated marker-free transformation and gene editing system revealed that AeCBL3 mediates the formation of calcium oxalate crystal in kiwifruit

The transformation and gene editing of the woody species kiwifruit are difficult and time-consuming. The fast and marker-free genetic modification system for kiwifruit has not been developed yet. Here, we establish a rapid and efficient marker-free transformation and gene editing system mediated by Agrobacterium rhizogenes for kiwifruit. Moreover, a removing-root-tip method was developed to significantly increase the regeneration efficiency of transgenic hairy roots. Through A. rhizogenes-mediated CRISPR/Cas9 gene editing, the editing efficiencies of CEN4 and AeCBL3 achieved 55 and 50%, respectively. And several homozygous knockout lines for both genes were obtained. Our method has been successfully applied in the transformation of two different species of kiwifruit (Actinidia chinensis ‘Hongyang’ and A.eriantha ‘White’). Next, we used the method to study the formation of calcium oxalate (CaOx) crystals in kiwifruit. To date, little is known about how CaOx crystal is formed in plants. Our results indicated that AeCBL3 overexpression enhanced CaOx crystal formation, but its knockout via CRISPR/Cas9 significantly impaired crystal formation in kiwifruit. Together, we developed a fast maker-free transformation and highly efficient CRISPR-Cas9 gene editing system for kiwifruit. Moreover, our work revealed a novel gene mediating CaOx crystal formation and provided a clue to elaborate the underlying mechanisms.Graphical abstract

An ex vitro hairy root system from petioles of detached soybeanleaves for in planta screening of target genes and CRISPR strategies associated with nematode bioassays.

The ex vitro hairy root system from petioles of detached soybeanleaves allows the functional validation of genes using classical transgenesis and CRISPR strategies (e.g., sgRNA validation, gene activation) associated with nematode bioassays. Agrobacterium rhizogenes-mediated root transformation has been widely used in soybean for the functional validation of target genes in classical transgenesis and single-guide RNA (sgRNA) in CRISPR-based technologies. Initial data showed that in vitro hairy root induction from soybean cotyledons and hypocotyls were not the most suitable strategies for simultaneous performing genetic studies and nematode bioassays. Therefore, an ex vitro hairy root system was developed for in planta screening of target molecules during soybean parasitism by root-knot nematodes (RKNs). Applying this method, hairy roots were successfully induced by A. rhizogenes from petioles of detached soybeanleaves. The soybean GmPR10 and GmGST genes were then constitutively overexpressed in both soybean hairy roots and tobacco plants, showing a reduction in the number of Meloidogyne incognita-induced galls of up to 41% and 39%, respectively. In addition, this system was evaluated for upregulation of the endogenous GmExpA and GmExpLB genes by CRISPR/dCas9, showing high levels of gene activation and reductions in gall number of up to 58.7% and 67.4%, respectively. Furthermore, morphological and histological analyses of the gallsweresuccessfully performed. These collective data validate the ex vitro hairy root system for screening target genes, using classical overexpression and CRISPR approaches, directly in soybean in a simple manner and associated with nematode bioassays. This system can also be used in other root pathosystems for analyses of gene function and studies of parasite interactions with plants, as well as for other purposes such as studies of root biology and promoter characterization.

The GmCYP2-GmHAL3 module regulates salt tolerance in soybean seedlings

Soybean (Glycine max L. Merr) production is severely affected by soil salinization as an important crop. The discovery of salt stress-responsive genes is essential for soybean breeding. To our knowledge, cyclophilins (CYPs) play important roles in salt stresses in crops such as rice and cotton, except in soybean. This study cloned GmCYP2 from soybean salt-tolerant cultivar Suxie No. 1 and obtained ten stable transgenic soybean lines overexpressing GmCYP2 by Agrobacterium rhizogenes-mediated transformation. Two of the higher-expressing transgenic lines (OE1 and OE2) were used to compare salt tolerance with the wild type (WT). Under the salt stress, the chlorophyll content, the maximum efficiency of PSII photochemistry (Fv/Fm) and actual photochemical quantum yield [Y(II)] of OE1 and OE2 lines were significantly higher than those of the WT. Meanwhile, the accumulation of malondialdehyde (MDA) in the OE1 and OE2 lines was significantly lower than that in the WT. In addition, the Na+/K+ ratio was also significantly lower than that of the WT in leaves and roots. The results also confirmed that the salt tolerance of GmCYP2-silencing soybean seedlings obtained by virus-induced gene silencing (VIGS) was reduced compared to the control. Subcellular localization showed that GmCYP2 was expressed in the plasma membrane and nucleus of Nicotiana benthamiana. RNA-seq results demonstrated that GmCYP2 is involved in key regulators of the ABA and Ca2+ signaling pathway. Yeast two-hybrid, bimolecular fluorescence complementation (BiFC) and split-luciferase complementation assay demonstrated that GmCYP2 protein interacts with salt-associated halotolerance 3 (HAL3) protein. These results suggest that the GmCYP2-GmHAL3 module enhances salt tolerance of soybean seedlings by maintaining the Na+/K+ ratio and good photosynthetic state and it may be involved in the ABA and Ca2+ signaling pathway.

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.

Fast, simple, efficient Agrobacterium rhizogenes-mediated transformation system to non-heading Chinese cabbage with transgenic roots

Non-heading Chinese cabbage, a variety of Brassica campestris, is an important vegetable crop in the Yangtze River Basin of China. However, the immaturity of its stable transformation system and its low transformation efficiency limit gene function research on non-heading Chinese cabbage. Agrobacterium rhizogenes-mediated (ARM) transgenic technology is a rapid and effective transformation method that has not yet been established for non-heading Chinese cabbage plants. Here, we optimized conventional ARM approaches (one-step and two-step transformation methods) suitable for living non-heading Chinese cabbage plants in nonsterile environments. Transgenic roots in composite non-heading Chinese cabbage plants were identified using phenotypic detection, fluorescence observation, and PCR analysis. The transformation efficiency of a two-step method on four five-day-old non-heading Chinese cabbage seedlings (Suzhouqing, Huangmeigui, Wuyueman, and Sijiu Caixin) was 43.33%–51.09%, whereas using the stout hypocotyl resulted in a transformation efficiency of 54.88% for the 30-day-old Sijiu Caixin. The one-step method outperformed the two-step method; the transformation efficiency of different varieties was above 60%, and both methods can be used to obtain transgenic roots for functional studies within one month. Finally, optimized ARM transformation methods can easily, quickly, and effectively produce composite non-heading Chinese cabbage plants with transgenic roots, providing a reliable foundation for gene function research and non-heading Chinese cabbage genetic improvement breeding.

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.

Adaptation of high-efficiency CRISPR/Cas9-based multiplex genome editing system in white lupin by using endogenous promoters.

White lupin (Lupinus albus L.) is an important crop with high phosphorus (P) use efficiency; however, technologies for its functional genomic and molecular analyses are limited. Cluster regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) system has been applied to gene editing and function genomics in many crops, but its application in white lupin has not been well documented. Here, we adapted the CRISPR/Cas9-based multiplex genome editing system by using the native U3/U6 and ubiquitin (UBQ) promoters to drive sgRNAs and Cas9. Two target sites (T1 and T2) within the Lalb_Chr05g0223881 gene, encoding a putative trehalase, were selected to validate its efficacy in white lupin based on the Agrobacterium rhizogenes-mediated transformation. We found that the T0 hairy roots were efficiently mutated at T1 and T2 with a frequency of 6.25%-35% and 50%-92.31%, respectively. The mutation types include nucleotide insertion, deletion, substitution, and complicated variant. Simultaneous mutations of the two targets were also observed with a range of 6.25%-35%. The combination of LaU6.6 promoter for sgRNA and LaUBQ12 promoter for Cas9 generated the highest frequency of homozygous/biallelic mutations at 38.46%. In addition, the target-sgRNA sequence also contributes to the editing efficiency of the CRISPR/Cas9 system in white lupin. In conclusion, our results expand the toolbox of the CRISPR/Cas9 system and benefit the basic research in white lupin.

Agrobacterium rhizogenes-Mediated Transformation as a Means for Induction of Hairy Roots and Secondary Metabolites in Thymus daenensis

Agrobacterium rhizogenes-Mediated Transformation as a Means for Induction of Hairy Roots and Secondary Metabolites in Thymus daenensis

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.

A novel soybean hairy root system for gene functional validation.

Agrobacterium rhizogenes-mediated transformation has long been explored as a versatile and reliable method for gene function validation in many plant species, including soybean (Glycine max). Likewise, detached-leaf assays have been widely used for rapid and mass screening of soybean genotypes for disease resistance. The present study combines these two methods to establish an efficient and practical system to generate transgenic soybean hairy roots from detached leaves and their subsequent culture under ex vitro conditions. We demonstrated that hairy roots derived from leaves of two (tropical and temperate) soybean cultivars could be successfully infected by economically important species of root-knot nematodes (Meloidogyne incognita and M. javanica). The established detached-leaf method was further explored for functional validation of two candidate genes encoding for cell wall modifying proteins (CWMPs) to promote resistance against M. incognita through distinct biotechnological strategies: the overexpression of a wild Arachis α-expansin transgene (AdEXPA24) and the dsRNA-mediated silencing of an endogenous soybean polygalacturonase gene (GmPG). AdEXPA24 overexpression in hairy roots of RKN-susceptible soybean cultivar significantly reduced nematode infection by approximately 47%, whereas GmPG downregulation caused an average decrease of 37%. This novel system of hairy root induction from detached leaves showed to be an efficient, practical, fast, and low-cost method suitable for high throughput in root analysis of candidate genes in soybean.

Application of AtMYB75 as a reporter gene in the study of symbiosis between tomato and Funneliformis mosseae.

Composite plants containing transgenic hairy roots produced with Agrobacterium rhizogenes-mediated transformation have become an important method to study the interaction between plants and arbuscular mycorrhizal fungi (AMF). Not all hairy roots induced by A. rhizogenes are transgenic, however, which leads to requirement of a binary vector to carry a reporter gene to distinguish transgenic roots from non-transformed hairy roots. The beta-glucuronidase gene (GUS) and fluorescent protein gene often are used as reporter markers in the process of hairy root transformation, but they require expensive chemical reagents or imaging equipment. Alternatively, AtMYB75, an R2R3 MYB transcription factor from Arabidopsis thaliana, recently has been used as a reporter gene in hairy root transformation in some leguminous plants and can cause anthocyanin accumulation in transgenic hairy roots. Whether AtMYB75 can be used as a reporter gene in the hairy roots of tomato and if the anthocyanins accumulating in the roots will affect AMF colonization, however, are still unknown. In this study, the one-step cutting method was used for tomato hairy root transformation by A.rhizogenes. It is faster and has a higher transformation efficiency than the conventional method. AtMYB75 was used as a reporter gene in tomato hairy root transformation. The results showed that the overexpression of AtMYB75 caused anthocyanin accumulation in the transformed hairy roots. Anthocyanin accumulation in the transgenic hairy roots did not affect their colonization by the arbuscular mycorrhizal fungus, Funneliformis mosseae strain BGC NM04A, and there was no difference in the expression of the AMF colonization marker gene SlPT4 in AtMYB75 transgenic roots and wild-type roots. Hence, AtMYB75 can be used as a reporter gene in tomato hairy root transformation and in the study of symbiosis between tomato and AMF.

Red fluorescence protein (DsRed2) promotes the screening efficiency in peanut genetic transformation.

Peanut (Arachis hypogaea L.), one of the leading oilseed crops worldwide, is an important source of vegetable oil, protein, minerals and vitamins. Peanut is widely cultivated in Asia, Africa and America, and China is the largest producer and consumer of peanut. Genetic engineering has shown great potential to alter the DNA makeup of an organism which is largely hindered by the low transformation and screening efficiency including in peanut. DsRed2 is a reporter gene widely utilized in genetic transformation to facilitate the screening of transformants, but never used in peanut genetic transformation. In this study, we have demonstrated the potential of the red fluorescence protein DsRed2 as a visual reporter to improve screening efficiency in peanut. DsRed2 was firstly expressed in protoplasts isolated from peanut cultivar Zhonhua 12 by PEG, and red fluorescence was successfully detected. Then, DsRed2 was expressed in peanut plants Zhonghua 12 driven by 35S promoter via Agrobacterium tumefaciens-mediated transformation. Red fluorescence was visually observed in calli and regenerated shoots, as well as in roots, leaves, flowers, fresh pod shells and mature seeds, suggesting that transgenic screening could be initiated at the early stage of transformation, and continued to the progeny. Upon screening with DsRed2, the positive plant rate was increased from 56.9% to 100%. The transgenic line was then used as the male parent to be crossed with Zhonghua 24, and the hybrid seeds showed red fluorescence as well, indicating that DsRed2 could be applied to hybrid plant identification very efficiently. DsRed2 was also expressed in hairy roots of Huayu 23 via Agrobacterium rhizogenes-mediated transformation, and the transgenic roots were easily selected by red fluorescence. In summary, the DsRed2 is an ideal reporter to achieve maximum screening efficiency and accuracy in peanut genetic transformation.

MdERF114 enhances the resistance of apple roots to Fusarium solani by regulating the transcription of MdPRX63.

As the main fungal etiologic agent of apple (Malus domestica) replant disease (ARD), Fusarium solani seriously damages apple roots. Ethylene response factors (ERFs) play an important role in plant resistance to biotic stress. Here, we show that MdERF114 is expressed during F. solani infections and positively regulates the resistance of apple roots to F. solani. Yeast one-hybrid, dual-luciferase, electrophoretic mobility shift assays and determinations of lignin content indicated that MdERF114 directly binds the GCC-box of the MdPEROXIDASE63 (MdPRX63) promoter and activates its expression, resulting in lignin deposition in apple roots and increased resistance to F. solani. We identified a WRKY family transcription factor, MdWRKY75, that binds to the W-box of the MdERF114 promoter. Overexpression of MdWRKY75 enhanced resistance of apple roots to F. solani. MdMYB8 interacted with MdERF114 to enhance resistance to F. solani by promoting the binding of MdERF114 to the MdPRX63 promoter. In summary, our findings reveal that the MdWRKY75-MdERF114-MdMYB8-MdPRX63 module is required for apple resistance to F. solani and the application of this mechanism by Agrobacterium rhizogenes-mediated root transformation provides a promising strategy to prevent ARD.

Development of a set of novel binary expression vectors for plant gene function analysis and genetic transformation.

With the advent of multiple omics and Genome-Wide Association Studies (GWAS) technology, genome-scale functional analysis of candidate genes is to be conducted in diverse plant species. Construction of plant binary expression vectors is the prerequisite for gene function analysis. Therefore, it is of significance to develop a set of plant binary expression vectors with highly efficient, inexpensive, and convenient cloning method, and easy-to-use in screening of positive recombinant in Escherichia coli. In this study, we developed a set of plant binary expression vectors, termed pBTR vectors, based on Golden Gate cloning using BsaI restriction site. Foreign DNA fragment of interest (FDI) can be cloned into the destination pBTR by one-step digestion-ligation reaction in a single tube, and even the FDI contains internal BsaI site(s). Markedly, in one digestion-ligation reaction, multiple FDIs (exemplified by cloning four soybean Glyma.02g025400, Glyma.05g201700, Glyma.06g165700, and Glyma.17g095000 genes) can be cloned into the pBTR vector to generate multiple corresponding expression constructs (each expression vector carrying an FDI). In addition, the pBTR vectors carry the visual marker, a brightness monomeric red fluorescent protein mScarlet-I, that can be observed with the unaided eye in screening of positive recombinants without the use of additional reagents/equipment. The reliability of the pBTR vectors was validated in plants by overexpression of AtMyb75/PAP1 in tomato and GUSPlus in soybean roots via Agrobacterium rhizogenes-mediated transformation, promoter activity analysis of AtGCSpro in Arabidopsis via A. tumefaciens-mediated transformation, and protein subcellular localization of the Vitis vinifera VvCEB1opt in tobacco, respectively. These results demonstrated that the pBTR vectors can be used in analysis of gene (over)expression, promoter activity, and protein subcellular localization. These vectors will contribute to speeding up gene function analysis and the process of plant molecular breeding.

Development of transgenic composite Stylosanthes plants to study root growth regulated by a β-expansin gene, SgEXPB1, under phosphorus deficiency.

A highly efficient transformation procedure to generate transgenic Stylosanthes roots was established. SgEXPB1 is involved in Stylosanthes root growth under phosphorus deficiency. Stylo (Stylosanthes spp.) is an important forage legume widely applied in agricultural systems in the tropics. Due to the recalcitrance of stylo genetic transformation, functional characterization of candidate genes involved in stylo root growth is limited. This study established an efficient procedure for Agrobacterium rhizogenes-mediated transformation for generating transgenic composite plants of S. guianensis cultivar 'Reyan No. 5'. Results showed that composite stylo plants with transgenic hairy roots were efficiently generated by A. rhizogenes strains K599 and Arqual, infecting the residual hypocotyl at 1.0cm of length below the cotyledon leaves of 9-d-old seedlings, leading to a high transformation efficiency of > 95% based on histochemical β-glucuronidase (GUS) staining. Notably, 100% of GUS staining-positive hairy roots can be achieved per composite stylo plant. Subsequently, SgEXPB1, a β-expansin gene up-regulated by phosphorus (P) deficiency in stylo roots, was successfully overexpressed in hairy roots. Analysis of hairy roots showed that root growth and P concentration in the transgenic composite plants were increased by SgEXPB1 overexpression under low-P treatment. Taken together, a highly efficient A. rhizogenes-mediated transformation procedure for generating composite stylo plants was established to study the function of SgEXPB1, revealing that this gene is involved in stylo root growth during P deficiency.

Zearalenone regulates microRNA156 to affect the root development of Tetrastigma hemsleyanum.

Zearalenone (ZEN) is a secondary metabolite from Fusarium species. It is also present in plants and regulates the photochemical reaction in Photosystem II, the stress response and root growth. To investigate the mechanism by which ZEN regulates Tetrastigma hemsleyanum root growth, differentially expressed microRNAs (miRNAs) were identified and verified by high-throughput sequencing and Agrobacterium rhizogenes-mediated transformation of the roots of T. hemsleyanum seedlings treated with and without ZEN. The predicted functions of microRNA156b (miR156b) and microRNA156f (miR156f) were confirmed in transgenic hairy roots. (i) A total of 70 miRNAs showed significantly different expression levels under ZEN treatment, including seven highly conserved miRNAs. (ii) The number of lateral roots and total root length of the transgenic hairy roots overexpressing miR156b and miR156f was significantly higher than the wild-type hairy roots, and thus the overexpression of miR156b and miR156f in T. hemsleyanum promoted lateral root development. (iii) Bioinformatics analysis predicted that the target genes of miR156b and miR156f were SPL9/10. As compared with the wild-type hairy roots, the expression of SPL9 was significantly lower in the hairy roots overexpressing miR156b, and the expression of SPL10 was significantly lower in the hairy roots overexpressing miR156f. Therefore, SPL9 could be the target gene of miR156b, and SPL10 could be the target gene of miR156f. This study shows that ZEN could increase the expression of miR156b and miR156f in T. hemsleyanum roots, which negatively regulated the expression of their putative target genes SPL9 and SPL10, consequently promoting the growth and development of the lateral 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.