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Main conclusion: Long-term cultured calli may experience a biosynthetic shift due to the IAA-dependent expression of the rolA gene, which also affects ROS metabolism. The “hairy root” syndrome is caused by the root-inducing Ri-plasmid of Rhizobium rhizogenes, also known as Agrobacterium rhizogenes. The Ri-plasmid contains genes known as rol genes or root oncogenic loci, which promote root development. The important implications of the rolA gene from the T-DNA include reduced plant size, resistance to infections, and the activation of specialised metabolism. Nevertheless, rolA does not belong to the plast gene group because its function is still uncertain. Recent investigations have shown two important effects of the rolA gene. First, the production of secondary metabolites has changed in long-term cultivated rolA-transgenic calli of Rubia cordifolia L. Second, the expression of both the rolA and rolB genes has a strong auxin-dependent antagonistic effect on reactive oxygen species (ROS) homeostasis. In this work, we attempted to elucidate two rolA gene phenomena: what caused the secondary metabolism of long-term cultured calli to change? How does the individual expression of the rolA gene affect ROS homeostasis? We analysed SNPs in the 5′ untranslated region and coding region of the rolA gene. These mutations do not affect the known essential amino acids of the RolA proteins. Notably, in the promoter of the rolA gene, an ACTTTA motif for auxin-mediated transcription factors was identified. Using two separate cell cultures, we demonstrated the strong auxin dependence of rolA gene expression. The expression of genes involved in ROS metabolism decreased in response to an auxin-mediated increase in rolA gene expression. Two assumptions can be made. The long-term cultivation of calli may cause changes in the hormonal state of the culture over time, which may modulate the action of the RolA protein. Moreover, auxin-dependent expression of the rolA gene led to a decrease in ROS metabolism. It can be assumed that the antagonistic interaction between rolA and rolB prevents strong rolB-induced auxin sensitivity and oxidative bursts to balance the cell state. [ABSTRACT FROM AUTHOR]

SUMMARY Plant genetic transformation is essential for understanding gene functions and developing improved crop varieties. Traditional methods, often genotype‐dependent, are limited by plants' recalcitrance to gene delivery and low regeneration capacity. To overcome these limitations, new approaches have emerged that greatly improve efficiency and genotype flexibility. This review summarizes key strategies recently developed for plant transformation, focusing on groundbreaking technologies enhancing explant‐ and genotype flexibility. It covers the use of morphogenic regulators (MRs), stem cell‐based methods, and in planta transformation methods. MRs, such as maize Babyboom (BBM) with Wuschel2 (WUS2), and GROWTH‐REGULATING FACTORs (GRFs) with their cofactors GRF‐interacting factors (GIFs), offer great potential for transforming many monocot species, including major cereal crops. Optimizing BBM/WUS2 expression cassettes has further enabled successful transformation and gene editing using seedling leaves as starting material. This technology lowers the barriers for academic laboratories to adopt monocot transformation systems. For dicot plants, tissue culture‐free or in planta transformation methods, with or without the use of MRs, are emerging as more genotype‐flexible alternatives to traditional tissue culture‐based transformation systems. Additionally, the discovery of the local wound signal peptide Regeneration Factor 1 (REF1) has been shown to enhance transformation efficiency by activating wound‐induced regeneration pathways in both monocot and dicot plants. Future research may combine these advances to develop truly genotype‐independent transformation methods. [ABSTRACT FROM AUTHOR]

Plants are rich sources of specialized metabolites, such as alkaloids, terpenes, phenolic acids, flavonoids, coumarins, and volatile oils, which provide various health benefits including anticancer, anti-inflammatory, antiaging, skin-altering, and anti-diabetic properties. However, challenges such as low and inconsistent yields, environment and geographic factors, and species-specific production of some specialized metabolites limit the supply of raw plant material for the food, cosmetic, or pharmaceutical industries. Therefore, biotechnological approaches using plant in vitro systems offer an appealing alternative for the production of biologically active metabolites. Among these, hairy root cultures induced by Rhizobium rhizogenes have firmed up their position as "green cell factories" due to their genotypic and biosynthetic stability. Hairy roots are valuable platforms for producing high-value phytomolecules at a low cost, are amenable to pathway engineering, and can be scaled up in bioreactors, making them attractive for commercialization. This review explores the potential of hairy roots for specialized metabolites biosynthesis focusing on biotechnology tools to enhance their production. Aspects of morphological peculiarities of hairy roots, the diversity of bioreactors design, and process intensification technologies for maximizing biosynthetic capacity, as well as examples of patented plant-derived (green-labeled) products produced through hairy root cultivation at lab and industrial scales, are addressed and discussed. [ABSTRACT FROM AUTHOR]

The present study explored the impact of rolB gene transformation on biological activities of Nicotiana tabacum. PCR, and RT-qPCR were used to validate the integration of rolB gene and its expression. HPLC analysis was used for phytochemical profiling, and various in-vitro and in-vivo assays were used to assess pharmacological activities of wild-type and transgenic extracts using ANOVA and Tukey's multiple comparison method for data analysis. Our findings indicated increased total phenolic content (24.4 ± 0.2 mg GAE/g DW of extract) and total flavonoid content (58.8 ± 1.1 mg QE/g DW of extract) in transgenic plants. In case of antioxidant activities, an increase in reducing power (51.82 ± 0.4 AAE mg/g), and radical scavenging (IC50 188 ± 2.7 µg/mL) was observed. Similarly, transgenic lines exhibited DNA protection against damage caused by free radicals. Moreover, we detected significant antileishmanial potential (IC50 350.7 ± 8.3 µg/mL), butyrylcholinesterase (IC50 268.6 ± 4.7 µg/mL), and acetylcholinesterase inhibitions (IC50 421.6 ± 3.7 µg/mL) in transgenic plants. Similarly, compared with the non-transgenic plants, the rolB-transgenic plants showed increased analgesic (71.4%), anti-inflammatory activities (77.8%), and 35.6% decrease in depression compared to non-transgenic plants. The improved pharmacological activities in transgenic plants have been linked to the level of phytochemicals that increase the therapeutic and curative properties of N. tabacum plants. [ABSTRACT FROM AUTHOR]

Dittrichia viscosa is a ruderal plant species growing along roadsides and well adapting to extreme environmental conditions. D. viscosa plant tissues, especially leaves, are known to be a rich source of bioactive metabolites which have antioxidant, cytotoxic, antiproliferative and anticancer properties. Hairy root cultures are a suitable biotechnological system for investigating plant metabolic pathways and producing specialized metabolites in in vitro conditions. In this study, D. viscosa hairy root transformed lines induced by Agrobacterium rhizogenes ATCC15834 were obtained using leaf explants, and the integration of rolB and rolC genes in the genomes of transformed hairy roots were confirmed by PCR analysis. Three hairy root D. viscosa lines (DvHrT1, DvHrT4 and DvHrT5) having different phenotypic features were characterized in terms of total phenolics, flavonoids and antioxidant activity. Correlated with antioxidant activity, phenolic and flavonoid content of DvHrT1 was significantly higher than control roots and the other DvHrT lines. Our results suggest that D. viscosa hairy roots can be a valuable tool for producing various bioactive compounds having antioxidant activity and are to be further investigated to produce other specific molecules that could find application in agricultural or pharmaceutical fields. [ABSTRACT FROM AUTHOR]

Tetrandrine, a bioactive active compound mainly found in the roots of Stephania tetrandra, exhibits various pharmacological properties. In vitro hairy root (HR) culture may serve as a promising solution for the extraction of tetrandrine, overcoming the limitations of natural cultivation. The present study describes the consistent production of tetrandrine from S. tetrandra hairy roots induced by different strains of Agrobacterium rhizogenes. Cultivation in woody plant medium (WPM) resulted in the highest HR biomass (0.056 g/petri-dish) and tetrandrine content (7.28 mg/L) as compared to other media. The maximum HR biomass (6.95 g dw/L) and tetrandrine production (68.69 mg/L) were obtained in the fifth week of cultivation. The presence of ammonium nitrate (800 mg/L), calcium nitrate (1156 mg/L), sucrose (20 g/L) and casein (2 g/L) enhanced the tetrandrine production. Moreover, the fed-batch cultivation demonstrated that the NH 4 NO 3 (1200 mg/L) was an important growth limiting factor that yielded the highest tetrandrine amount (119.59 mg/L). The cultivation of hairy roots in a mist trickling bioreactor for eight weeks was less (26.24 mg/L) than in the flask. Despite a lower tetrandrine yield observed in bioreactors compared to flask cultures, refining the growth medium and fine-tuning bioreactor operations hold promise for boosting tetrandrine yield. [Display omitted] • S. tetrandrae hairy roots were established from leaf explant using A. rhizogenes. • Maximum hairy root and tetrandrine production were obtained in WPM medium. • Presence of NH 4 NO 3 (1200 mg/L) yielded highest tetrandrine amount (119.59 mg/L). • Hairy roots growth was attempted in bubble column and mist trickling bioreactors. [ABSTRACT FROM AUTHOR]

In order to meet growing consumer demands in terms of naturalness, the pharmaceutical, food, and cosmetic industries are looking for active molecules of plant origin. In this context, hairy roots are considered a promising biotechnological system for the sustainable production of compounds of interest. Poplars (genus Populus, family Salicaceae) are trees of ecological interest in temperate alluvial forests and are also cultivated for their industrial timber. Poplar trees also produce specialized metabolites with a wide range of bioactive properties. The present study aimed to assess the hybrid poplar hairy root extracts for antimicrobial and antibiofilm activities against four main life-threatening strains of Gram-positive (Staphylococcus aureus, Bacillus subtilis) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa) bacteria. Ethyl acetate extracts from two hairy root lines (HP15-3 and HP A4-12) showed significant antibacterial properties as confirmed by disc diffusion assay. Antibiofilm activities were found to be dose dependent with significant biofilm inhibition (75–95%) recorded at 1000 µg.mL−1 in all the bacterial strains tested. Dose-dependent enhancement in the release of exopolysaccharides was observed in response to treatment with extracts, possibly because of stress and bacterial cell death. Fluorescence microscopy confirmed loss of cell viability of treated bacterial cells concomitant with increased production of reactive oxygen species compared to the untreated control. Overall, this study demonstrates for the first time a high potential of poplar hairy root extracts as a natural and safe platform to produce antimicrobial agents in pharmaceutical, food, industrial water management, or cosmetic industries. [ABSTRACT FROM AUTHOR]

AbstractCalendula officinalis is a widespread medicinal plant with a sufficiently well-studied chemical composition. Secondary metabolites synthesized by C.officinalis plants have pharmacological value for treating numerous diseases, and various types of aseptic in vitro cultures can be used as a source of these compounds. From this perspective, hairy roots attract considerable attention for the production of bioactive chemicals, including flavonoids with antioxidant activity. This paper shows the possibility of C.officinalis hairy roots obtaining with 100% frequency by Agrobacterium rhizogenes genetic transformation. Hairy root lines differed in growth rate and flavonoid content. In particular, flavonoids were accumulated in the amount of up to 6.68 ± 0.28 mg/g of wet weight. Methyl jasmonate in the concentration of 10 µM inhibited root growth to a small extent but stimulated the synthesis of flavonoids. The antioxidant activity and the reducing power increased in the roots grown in the medium with methyl jasmonate. The strong correlation of antioxidant activity and reducing power with flavonoid content was detected. The influence of extraction conditions on the content of flavonoids in the extracts and their bioactivity was determined. The potent reducing activity of extracts from hairy roots allowed the production of silver nanoparticles, which was confirmed by transmission electron microscopy. [ABSTRACT FROM AUTHOR]

An attempt is made to create transgenic plants using rol genes from A4, 15834, and K599 strains of A. rhizogenes. Strains A4 and 15834 transformed tobacco plants are found to display better root growth than their wild types under normal conditions and at elevated concentrations of sodium chloride or cadmium acetate. At the same time, the rol genes acquired from the K599 strain negatively affect root growth under both normal and stressful conditions (e.g., salinization, hypothermia, or excess cadmium acetate). The levels of total protein, proline, total glutathione, and superoxide dismutase activity are higher in the roots of the transgenic plants with rol genes from the A4 and 15834 strains than in nontransgenic control plants under both optimum conditions and salinization. When using rol transgenes from the K599 strain, the activities of superoxide dismutase, guaiacol peroxidase, and glutathione-S-transferase are enhanced in the roots subjected to salinization. The positive effect rol genes have on the root growth of transgenic plants could therefore be explained by their influence on components of the antioxidant system. Results suggest that using rol genes from the A4 and 15834 strains of A. rhizogenes are promising for breeding plant cultivars and liness with improved parameters of root growth and stress tolerance. At the same time, rol genes from the K599 strain appear to be inadequate for this task. [ABSTRACT FROM AUTHOR]

Bread wheat (Triticum aestivum L.) is one of the most important cereals and staple food crops worldwide and is grown quite often in arid regions of the world where water shortages and salinity cause serious crop losses. The TaNAC69 gene of bread wheat encodes one of the most important transcription factors involved in the regulation of resistance to water deficit caused by drought and hypothermia, but the role of this gene under salinity is poorly understood. This study aimed to clone the TaNAC69 gene of bread wheat in a binary vector with the 35S CaMV promoter and to create hairy roots (HRs) of Nicotiana tabacum L. with constitutive expression of the TaNAC69 gene to assess the effectiveness of the target genetically engineered construct under salinity. As the result of Agrobacterium-mediated transformation of tobacco leaf discs using Agrobacterium rhizogenes, 15 lines of HR cultures were created, in six of which the presence and high level of expression of the target gene were proven. Tobacco HRs with constitutive expression of the TaNAC69 gene were characterized by faster growth rates, greater branching, and higher fresh and dry weights when exposed to 150 mM NaCl than control tobacco HRs without the target gene. Analysis of the antioxidant system revealed increased activity of the enzymes guaiacol peroxidase and glutathione-S-transferase, higher content of proline and water-soluble sugars, and higher total antioxidant capacity in HR with the TaNAC69 gene compared to the untransformed control. The TaNAC69 with the 35S CaMV promoter construct can be recommended for the genetic transformation of crops to increase their resistance to abiotic stresses. [ABSTRACT FROM AUTHOR]

Citrus is one of the world's most important and widely produced fruit crops, with over a 100 million metric tons harvested from nearly 10 million hectares in 2023. Challenges in crop maintenance, production, and fruit quality necessitate developing new traits through Agrobacterium-mediated genetic transformation. While a few Agrobacterium strains (EHA105, GV3101, LBA4404) are known to transform citrus, many wild strains remain untested. We screened forty-one wild-type Agrobacterium strains isolated from various woody species and identified five capable of DNA transfer into citrus cells. Strain 1D1416 demonstrated the highest transient transformation frequency in Carrizo epicotyl explants (88%), outperforming the control EHA105 (84%) with comparable shoot regeneration rates (32% and 42%, respectively). Notably, 1D1416 exhibited no overgrowth and had the lowest necrosis and mortality rates in transformed tissues. It efficiently transferred the DsRed gene and induced galls in mature tissues of Mexican lime (70%), lemon (48%), Washington navel orange (25%), and clementine (6%). Genome sequencing of 1D1416 allowed for the disarming of the native T-DNA and addition of GAANTRY technology. This novel strain, combined with an optimized transformation procedure, make it a valuable tool for advancing citrus transformation. [ABSTRACT FROM AUTHOR]

Aims: The study was aimed to investigate the efficiency of direct and co-cultivation methods in the genetic transformation of Brassica napus at the molecular level. Methodology and results: The seedlings' leaves were prepared from explants parts of oilseed rape and inoculated with a culture of A. rhizogenes ATCC15834 using direct injection and co-cultivation methods. Cultures of hairy roots were created, then were aggregated and placed on a solid MS medium and sub-cultured by transferring them 3-4 times on an MS solid medium containing graded concentrations (100, 200 and 250 mg/L) of cefotaxime until obtaining hairy root cultures free of bacteria. Moreover, the DNA was extracted and amplified from each type of hairy root by polymerase chain reaction (PCR). The results show that the process of the inoculation of the leaves by direct injection with the Agrobacterium rhizogenes ATCC15834 suspension at the density of 1.151 × 108 cells/mL was the most efficient way for the formation of hairy roots compared with the other densities after 30 days with 80.64% ratio and of 2.72 roots/segment average. Additionally, hairy roots were also formed on oilseed rape leaves by incubating them with co-cultivation and bacterial A. rhizogenes ATCC15834 at a density of 1.151 × 108 cells/mL for 4 h, which shows their superiority from the other incubation periods, recording 44.44% and 2.55 average. Gel electrophoresis results demonstrate that one band was amplified by PCR with a molecular weight of 590 bp, identical to the estimated size of rolC gene recovered from the genomic DNA of hairy roots using specific primers. Conclusion, significance and impact of study: It is concluded that hairy roots can be produced from oilseed rape B. napus L. seedling leaves using direct injection and co-cultivation methods by A. rhizogenes ATCC15834. [ABSTRACT FROM AUTHOR]

Transgenic lines engineered through wild type Rhizobium rhizogenes display an altered phenotype known as the Ri phenotype. This phenotype includes a more compact plant habit, which has proved useful to obtain more compact varieties that require less chemical growth regulation. Here, we develop a method for the molecular and cytogenetic characterization of Cape daisy (Osteospermum fruticosum Norl.) Ri lines in order to predict segregation of pRi T-DNA genes. Analysis of copy number variation (CNV) by means of digital PCR indicated large variation in the copy number of the inserted root oncogenic loci (rol) genes, ranging from 1 to more than 15 copies. In addition, up to 9 copies of the auxin biosynthesis genes (aux) were present in a single Ri line. Visualization of pRiA4 and pRi1724 rol and aux insertion in 4 Ri lines was performed through Fluorescence In Situ Hybridization. The number of rol integrated loci varied from 1 to 3 loci. In contrast, the different TR-gene copies were confined to a single locus which consistently co-localized with a TL locus, this was demonstrated for the first time. Based on CNV and FISH a single Ri line, harboring 7 pRi1724 rol gene copies dispersed over 3 integration loci, was selected for breeding. Copy number segregation in R1 progeny of 2, 3, 4 and 5 pRi1724 copies was confirmed, indicating that the evaluation of the breeding value of first generation Ri lines is possible through CNV and FISH. [ABSTRACT FROM AUTHOR]

Key message: Natural transformation with R. rhizogenes enhances osmotic stress tolerance in oilseed rape through increasing osmoregulation capacity, enhancing maintenance of hydraulic integrity and total antioxidant capacity. Transformation of plants using wild strains of agrobacteria is termed natural transformation and is not covered by GMO legislation in, e.g., European Union and Japan. In this study, offspring lines of Rhizobium rhizogenes naturally transformed oilseed rape (Brassica napus), i.e., A11 and B3 (termed root-inducing (Ri) lines), were investigated for osmotic stress resilience. Under polyethylene glycol 6000 (PEG) 10% (w/v)-induced osmotic stress, the Ri lines, particularly A11, had less severe leaf wilting, higher stomatal conductance (8.2 times more than WT), and a stable leaf transpiration rate (about 2.9 mmol m−2 s−1). Although the leaf relative water content and leaf water potential responded similarly to PEG treatment between the Ri lines and WT, a significant reduction of the turgid weight to dry weight ratio in A11 and B3 indicated a greater capacity of osmoregulation in the Ri lines. Moreover, the upregulation of plasma membrane intrinsic proteins genes (PIPs) in roots and downregulation of these genes in leaves of the Ri lines implied a better maintenance of hydraulic integrity in relation to the WT. Furthermore, the Ri lines had greater total antioxidant capacity (TAC) than the WT under PEG stress. Collectively, the enhanced tolerance of the Ri lines to PEG-induced osmotic stress could be attributed to the greater osmoregulation capacity, better maintenance of hydraulic integrity, and greater TAC than the WT. In addition, Ri-genes (particularly rolA and rolD) play roles in response to osmotic stress in Ri oilseed rape. This study reveals the potential of R. rhizogenes transformation for application in plant drought resilience. [ABSTRACT FROM AUTHOR]

In current work, we studied hairy root induction in Trigonella foenum graecum, which is an important medicinal plant, and examined the impact of different elicitors on some phytochemical characteristics and metabolites production in hairy root cultures. Accordingly, some factors such as five strain types of Agrobacterium rhizogenes (1724, 15834, A4, A13 and MSU) and three different explants, namely leaf, cotyledon and hypocotyl were studied. The results showed that different A. rhizogenes strains exhibited different infection efficiency. MSU and 15834 had highest efficiency of hairy root induction than other strains. Also, hairy root induction frequency in leaf explants was higher than in other explants. Salicylic acid (SA), nitric oxide (NO), CaCl2 and penconazole (PEN) were used in elicitation process. Hairy roots were treated with SA (0.1 and 0.5 mM), NO (10 and 50 µM), CaCl2 (5 and 10 mM) and PEN (5 and 10 mg/L). Applied elicitors enhanced antioxidant enzymes activities and reduced oxidative stress markers; this observation might be ascribed to regulation of the oxidative status of the elicited cells. Significant increase of antioxidant metabolites (total phenol, flavonoid and anthocyanin) in PEN-treated hairy roots was associated to phenylalanine ammonia lyase activity, indicating an up-regulation of phenylpropanoid/flavonoid metabolism. PEN and CaCl2 treatment enhanced steroidal sapogenin in hairy root cultures. These results suggested that use of elicitors can enhance the production of secondary metabolites in transformed hairy roots. Among the elicitors applied, CaCl2 and PEN were the most effective in increasing secondary metabolite production in transformed hairy roots of T. foenum graecum. [ABSTRACT FROM AUTHOR]

Hypericum perforatum transformed shoot lines (TSL) regenerated from corresponding hairy roots and non-transformed shoots (NTS) were comparatively evaluated for their phenolic compound contents and in vitro inhibitory capacity against target enzymes (monoamine oxidase-A, cholinesterases, tyrosinase, α-amylase, α-glucosidase, lipase, and cholesterol esterase). Molecular docking was conducted to assess the contribution of dominant phenolic compounds to the enzyme-inhibitory properties of TSL samples. The TSL extracts represent a rich source of chlorogenic acid, epicatechin and procyanidins, quercetin aglycone and glycosides, anthocyanins, naphthodianthrones, acyl-phloroglucinols, and xanthones. Concerning in vitro bioactivity assays, TSL displayed significantly higher acetylcholinesterase, tyrosinase, α-amylase, pancreatic lipase, and cholesterol esterase inhibitory properties compared to NTS, implying their neuroprotective, antidiabetic, and antiobesity potential. The docking data revealed that pseudohypericin, hyperforin, cadensin G, epicatechin, and chlorogenic acid are superior inhibitors of selected enzymes, exhibiting the lowest binding energy of ligand–receptor complexes. Present data indicate that H. perforatum transformed shoots might be recognized as an excellent biotechnological system for producing phenolic compounds with multiple health benefits. [ABSTRACT FROM AUTHOR]

MicroRNAs play crucial roles in plant defense responses. However, the underlying mechanism by which miR398b contributes to soybean responses to soybean cyst nematode (Heterodem glycines) remains elusive In this study, by using Agrobacterium rhizogenes - mediated transformation of soybean hairy roots, we observed that miR398b and target genes GMC.CS and GniCSD/b played vital functions in soybean-H. glycines interaction. The study revealed that the abundance of miR398b was downregulated by H. glycines infection, and overexpression of miR398b enhanced the susceptibility of soybean to H. glycines. Conversely, silencing of miR398b improved soybean resistance to H. glycines. Detection assays revealed that miR398b rapidly senses stress-induced reactive oxygen species, leading to the repression of target genes GmCCS and GinCSDIb and regulating the accumulation of plant defense genes against nematode infection. Moreover, exogenous synthetic ds-miR398b enhanced soybean sensitivity to H. glycines by modulating 11202 and 02-levels. Functional analysis demonstrated that overexpression of GmCCS and GmCSDlb in soybean en - hanced resistance to H. glycines, RNA interference-mediated repression of GmCCS and GmCSDI b in soybean increased susceptibility to H. glycines. RNA sequencing revealed that a majority of differentially expressed genes in overexpressed GmCCS were associated with oxidative stress. Overall, the results indicate that miR398b targets superoxide dismutase genes, which negatively regulate soybean resistance to H. glycines via modulating reactive oxygen species levels and defense signals. [ABSTRACT FROM AUTHOR]

Jute (Corchorus capsularis L.) is the second most important natural plant fiber source after cotton. However, developing an efficient gene editing system for jute remains a challenge. In this study, the transgenic hairy root system mediated by Agrobacterium rhizogenes strain K599 was developed for Meifeng 4, an elite jute variety widely cultivated in China. The transgenic hairy root system for jute was verified by subcellular localization and bimolecular fluorescence complementation (BiFC) assays. The CHLOROPLASTOS ALTERADOS 1 (CcCLA1) gene, which is involved in the development of chloroplasts, was targeted for editing at two sites in Meifeng 4. Based on this hairy root transformation, the gRNA scaffold was placed under the control of cotton ubiquitin GhU6.7 and -GhU6.9 promoters, respectively, to assess the efficiency of gene editing. Results indicated the 50.0% (GhU6.7) and 38.5% (GhU6.9) editing events in the target 2 alleles (gRNA2), but no mutation was detected in the target 1 allele (gRNA1) in transgenic-positive hairy roots. CcCLA1 gene editing at gRNA2 under the control of GhU6.7 in Meifeng 4 was also carried out by Agrobacterium tumefaciens-mediated transformation. Two CcCLA1 mutants were albinic, with a gene editing efficiency of 5.3%. These findings confirm that the CRISPR/Cas9 system, incorporating promoter GhU6.7, can be used as a gene editing tool for jute. [ABSTRACT FROM AUTHOR]

Summary: Genetic transformation is a critical tool for gene editing and genetic improvement of plants. Although many model plants and crops can be genetically manipulated, genetic transformation systems for fruit trees are either lacking or perform poorly. We used Rhizobium rhizogenes to transfer the target gene into the hairy roots of Malus domestica and Actinidia chinensis. Transgenic roots were generated within 3 weeks, with a transgenic efficiency of 78.8%. Root to shoot conversion of transgenic hairy roots was achieved within 11 weeks, with a regeneration efficiency of 3.3%. Finally, the regulatory genes involved in stem cell activity were used to improve shoot regeneration efficiency. MdWOX5 exhibited the most significant effects, as it led to an improved regeneration efficiency of 20.6% and a reduced regeneration time of 9 weeks. Phenotypes of the overexpression of RUBY system mediated red roots and overexpression of MdRGF5 mediated longer root hairs were observed within 3 weeks, suggesting that the method can be used to quickly screen genes that influence root phenotype scores through root performance, such as root colour, root hair, and lateral root. Obtaining whole plants of the RUBY system and MdRGF5 overexpression lines highlights the convenience of this technology for studying gene functions in whole plants. Overall, we developed an optimized method to improve the transformation efficiency and stability of transformants in fruit trees. [ABSTRACT FROM AUTHOR]

Summary: Succulents, valued for their drought tolerance and ornamental appeal, are important in the floriculture market. However, only a handful of succulent species can be genetically transformed, making it difficult to improve these plants through genetic engineering. In this study, we adapted the recently developed cut‐dip‐budding (CDB) gene delivery system to transform three previously recalcitrant succulent varieties – the dicotyledonous Kalanchoe blossfeldiana and Crassula arborescens and the monocotyledonous Sansevieria trifasciata. Capitalizing on the robust ability of cut leaves to regenerate shoots, these plants were successfully transformed by directly infecting cut leaf segments with the Agrobacterium rhizogenes strain K599. The transformation efficiencies were approximately 74%, 5% and 3.9%–7.8%, respectively, for K. blossfeldiana and C. arborescens and S. trifasciata. Using this modified CDB method to deliver the CRISPR/Cas9 construct, gene editing efficiency in K. blossfeldiana at the PDS locus was approximately 70%. Our findings suggest that succulents with shoot regeneration ability from cut leaves can be genetically transformed using the CDB method, thus opening up an avenue for genetic engineering of these plants. [ABSTRACT FROM AUTHOR]

Stress tolerance in apple (Malus domestica) can be improved by grafting to a stress-tolerant rootstock, such as 'SH6' (Malus honanensis × M. domestica ' Ralls Genet'). However, the mechanisms of stress tolerance in this rootstock are unclear. In Arabidopsis (Arabidopsis thaliana), the transcription factor ZINC FINGER OF ARABIDOPSIS THALIANA 10 is a key component of plant tolerance to multiple abiotic stresses and positively regulates antioxidant enzymes. However, how reactive oxygen species are eliminated upon activation of ZINC FINGER OF ARABIDOPSIS THALIANA 10 in response to abiotic stress remains elusive. Here, we report that MhZAT10 in the rootstock SH6 directly activates the transcription of three genes encoding the antioxidant enzymes MANGANESE SUPEROXIDE DISMUTASE 1 (MhMSD1), ASCORBATE PEROXIDASE 3A (MhAPX3a) and CATALASE 1 (MhCAT1) by binding to their promoters. Heterologous expression in Arabidopsis protoplasts showed that MhMSD1, MhAPX3a and MhCAT1 localize in multiple subcellular compartments. Overexpressing MhMSD1 , MhAPX3a or MhCAT1 in SH6 fruit calli resulted in higher superoxide dismutase, ascorbate peroxidase and catalase enzyme activities in their respective overexpressing calli than in those overexpressing MhZAT10. Notably, the calli overexpressing MhZAT10 exhibited better growth and lower reactive oxygen species levels under simulated osmotic stress. Apple SH6 plants overexpressing MhZAT10 in their roots via Agrobacterium rhizogenes –mediated transformation also showed enhanced tolerance to osmotic stress, with higher leaf photosynthetic capacity, relative water content in roots and antioxidant enzyme activity, as well as less reactive oxygen species accumulation. Overall, our study demonstrates that the transcription factor MhZAT10 synergistically regulates the transcription of multiple antioxidant-related genes and elevates reactive oxygen species detoxification. [ABSTRACT FROM AUTHOR]

Idesia polycarpa is a promising woody oilseed species because of its high oil yield. However, its use is greatly limited due to the lack of varieties with good qualities; additionally, gene function has been less studied in this plant because an efficient transformation method has not been established yet. In this study, we established a rapid and efficient hairy root transformation method by infecting the whole seedling, the rootless seedling, and the leaf petiole with Agrobacterium rhizogenes using different infection methods. Among these transformation methods, a higher transformation efficiency was obtained using the whole seedling, which could reach up to 71.91%. Furthermore, we found that the seedling age significantly affected the transformation efficiency, either using whole or rootless seedlings. Additionally, we found that the transgenic roots could regenerate transgenic shoots. Taken together, our study lays the foundation for future study and for genetically modifying wood traits in the future. [ABSTRACT FROM AUTHOR]

Withania somnifera (L.) Dunal is an important indigenous medicinal plant with extensive pharmaceutical potential. The root is the main source of major bioactive compounds of this plant species including withanolides, withanine, phenolic acids, etc. Hairy root culture (HRC) is a crucial method for low-cost production of active compounds on a large scale. Four different Agrobacterium rhizogenes strains have been used for the hairy root induction. Maximum transformation efficiency (87.34 ± 2.13%) was achieved with A4 bacterial strain-mediated transformed culture. The genetic transformation was confirmed by using specific primers of seven different genes. Seven HR (Hairy root) lines were selected after screening 29 HR lines based on their fast growth rate and high accumulation of withanolides and phenolic acids content. Two biotic and three abiotic elicitors were applied to the elite root line to trigger more accumulation of withanolides and phenolic acids. While all the elicitors effectively increased withanolides and phenolic acids production, among the five different elicitors, salicylic acid (4.14 mg l–1) induced 11.49 -fold increase in withanolides (89.07 ± 2.75 mg g–1 DW) and 5.34- fold increase in phenolic acids (83.69 ± 3.11 mg g− 1 DW) after 5 days of elicitation compared to the non-elicited culture (7.75 ± 0.63 mg g–1 DW of withanolides and 15.66 ± 0.92 mg g–1 DW of phenolic acids). These results suggest that elicitors can tremendously increase the biosynthesis of active compounds in this system; thus, the HRC of W. somnifera is cost-effective and can be efficiently used for the industrial production of withanolides and phenolic acids. • Establish Agrobacterium rhizogenes mediated hairy root culture in Withania somnifera. • Selection of elite hairy root clone for elicitor treatment. • Exogenous application of biotic and abiotic elicitors in hairy root culture. • Enhance production of withanolides and phenolic acids. [ABSTRACT FROM AUTHOR]

Lotus corniculatus is an important perennial legume forage species and has been widely used to study the relationships between plants and rhizobia or arbuscular mycorrhizal fungi. Composite plants (wild-type shoots with transgenic hairy roots) can be produced via genetic transformation mediated by Agrobacterium rhizogenes. The A. rhizogenes puncture method and a method of cutting embryonic roots and coating the hypocotyl incision with A. rhizogenes were used to induce hairy roots from seedlings. The identification of transgenic hairy roots from composite plants is also an important task. Here, young branches from adult plants were used as explants to produce composite plants instead of seedlings via the one-step cutting method in the natural environment. The results showed that the explants of young branches had higher transformation frequency than those of seedlings, and the composite plants obtained from the young branches had greater growth and were also more robust than the plants obtained from the seedlings. In this study, a system for the biosynthesis of betalains named RUBY was integrated into an expression vector as a reporter gene. It is very easy to distinguish transgenic roots from nontransgenic roots based on the red color of the hairy roots. Young branches were used as explants for hairy root transformation with higher transformation frequency than the seedlings. RUBY was used as a reporter gene to distinguish transgenic roots from nontransgenic roots based on the red color of the hairy roots. [ABSTRACT FROM AUTHOR]

Strobilanthes cusia is an indigo-yielding plant and scarcity of both wild and cultivated plants has increased the supply pressure of the natural dye. Since hairy root cultures are suitable for the in vitro production of secondary metabolites, the hairy root culture of Strobilanthes cusia was established using two Rhizobium rhizogenes strains (ATCC 15834 and MTCC 532). Polymerase chain reaction (PCR) using rol B primer confirmed the molecular evidence of hairy root transformation. The differential transformation frequency was studied based on factors like explants types (leaves, shoot tips, and stem); infection techniques, infection period, co-cultivation period, bacterial strain types, acetosyringone concentration, and antibiotic treatment conditions. Leaf explants, infected with ATCC 15834 exhibited the highest induction rate (58.67%) among the three explants. The most efficient hairy root transformation was achieved with the immersion technique, involving 10 min of infection and 5 days of co-cultivation using 100 µM acetosyringone during infection. When the indigo content of the hairy roots and different parts of in vitro plantlets were compared, the hairy roots produced 0.314 mg/g fresh weight (FW) of indigo, slightly more than the roots (0.21 mg/g FW), but less than the leaves (1.08 mg/g FW) of in vitro plantlets. Further, the indigo content of the hairy root cultures treated with 200 µM indican and 200 µM salicylic acid were enhanced up to 0.78 mg/g FW and 0.71 mg/g FW, respectively. Thus, this study reveals the potential of hairy roots of S. cusia for indigo biosynthesis, which may serve as a potential alternative source for natural indigo.Key message: First report of hairy root induction of Strobilanthes cusia and elicitation of the indigo content by using different elicitors and precursors feeding. [ABSTRACT FROM AUTHOR]

Confronting the environmental threat posed by textile dyes, this study highlights bioremediation as a pivotal solution to mitigate the impacts of Crystal Violet, a widely-utilized triphenylmethane dye known for its mutagenic and mitotic toxicity. We isolated and identified several bacterial strains capable of degrading Crystal Violet under various environmental conditions. Newly identified strains, including Mycolicibacterium nivoides, Chryseobacterium sp., Agrobacterium rhizogenes, Pseudomonas crudilactis, and Pseudomonas koreensis demonstrated significant decolorization activity of Crystal Violet, complementing the already known capabilities of Stenotrophomonas maltophilia. Initial experiments using crude extracts confirmed their degradation potential, followed by detailed studies that investigated the impact of different pH levels and temperatures on some strains' degradation efficiency. Depending on the bacteria, the degree of activity change according to pH and temperature was different. At 37 °C, Chryseobacterium sp. and Stenotrophomonas maltophilia exhibited higher degradation activity compared to 25 °C, while Pseudomonas crudilactis and Mycolicibacterium nivoides did not exhibit a statistically significant difference between the two temperatures. Mycolicibacterium nivoides performed optimally at pH 8, while Pseudomonas crudilactis showed high activity at pH 5. Stenotrophomonas maltophilia's activity remained consistent across the pH range. These findings not only underscore the effectiveness of these bacteria as agents for Crystal Violet degradation but also pave the way for their application in large-scale bioremediation processes for the treatment of textile effluents, marking them as vital to environmental sustainability efforts. [ABSTRACT FROM AUTHOR]

Summary: Gibberellins (GA) have a profound influence on the formation of lateral root organs. However, the precise role this hormone plays in the cell‐specific events during lateral root formation, rhizobial infection and nodule organogenesis, including interactions with auxin and cytokinin (CK), is not clear.We performed epidermal‐ and endodermal‐specific complementation of the severely GA‐deficient na pea (Pisum sativum) mutant with Agrobacterium rhizogenes. Gibberellin mutants were used to examine the spatial expression pattern of CK (TCSn)‐ and auxin (DR5)‐responsive promoters and hormone levels.We found that GA produced in the endodermis promote lateral root and nodule organogenesis and can induce a mobile signal(s) that suppresses rhizobial infection. By contrast, epidermal‐derived GA suppress infection but have little influence on root or nodule development. GA suppress the CK‐responsive TCSn promoter in the cortex and are required for normal auxin activation during nodule primordia formation.Our findings indicate that GA regulate the checkpoints between infection thread (IT) penetration of the cortex and invasion of nodule primordial cells and promote the subsequent progression of nodule development. It appears that GA limit the progression and branching of IT in the cortex by restricting CK response and activate auxin response to promote nodule primordia development. [ABSTRACT FROM AUTHOR]

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The genus Verbascum, belonging to the family Scrophulariaceae, has a significant center of diversity in Iran. Two of its species, V. erianthum and V. stachydiforme, originate in the Iranian-Turanian region, but no studies have been conducted on the induction of their hairy roots. This genus is a valuable source of biologically active compounds such as iridoid glycosides and flavonoids. Hairy root culture is a suitable technique for the production and accumulation of secondary metabolites. Three different studies were conducted to optimize the induction and establishment of hairy roots. In the first experiment, the influence of explant type (leaf and hypocotyl), six infection methods, and co-culture time (48 and 72 h) on the efficiency of hairy root induction was investigated. The results showed that the highest hairy root induction (68.18%) was observed in the leaf explants inoculated by direct infection with three wounds within 72 h co-culture time. In the second experiment, the effect of four Agrobacterium rhizogenes strains (ATCC 15834, A4, A7, and A13) and leaf age (14, 21, and 28 days) on transformation efficiency and some morphological traits examined in both species were studied. The high transformation efficiency of hairy root (80.55%) was detected in the 21-day-old leaf explant of V. erianthum species that was inoculated with the A13 strain. The transformed hairy root colons were confirmed by PCR using rolB gene-specific primers. To optimize hairy root growth and avoid tissue browning, hairy roots were cultured in various media containing different antioxidants and improver agents (including ascorbic acid, citric acid, and NAA). The results showed that the highest fresh growth index (20.42) and the lowest tissue browning (9%) as well as total phenol (8.51 mg GA/g DW), and total flavonoid content (4.42 mg QUE/g DW) were obtained in medium B5 with 1.5 mg/l NAA. [ABSTRACT FROM AUTHOR]

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%e51.09%, 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. [ABSTRACT FROM AUTHOR]

Rhizobacteria are capable of inducing defense responses via the expression of pathogenesis-related proteins (PR-proteins) such as chitinases, and many studies have validated the functions of plant chitinases in defense responses. Soybean (Glycine max) is an economically important crop worldwide, but the functional validation of soybean chitinase in defense responses remains limited. In this study, genome-wide characterization of soybean chitinases was conducted, and the defense contribution of three chitinases (GmChi01, GmChi02, or GmChi16) was validated in Arabidopsis transgenic lines against the soil-borne pathogen Fusarium oxysporum. Compared to the Arabidopsis Col-0 and empty vector controls, the transgenic lines with GmChi02 or GmChi16 exhibited fewer chlorosis symptoms and wilting. While GmChi02 and GmChi16 enhanced defense to F. oxysporum, GmChi02 was the only one significantly induced by Burkholderia ambifaria. The observation indicated that plant chitinases may be induced by different rhizobacteria for defense responses. The survey of 37 soybean chitinase gene expressions in response to six rhizobacteria observed diverse inducibility, where only 10 genes were significantly upregulated by at least one rhizobacterium and 9 genes did not respond to any of the rhizobacteria. Motif analysis on soybean promoters further identified not only consensus but also rhizobacterium-specific transcription factor-binding sites for the inducible chitinase genes. Collectively, these results confirmed the involvement of GmChi02 and GmChi16 in defense enhancement and highlighted the diverse inducibility of 37 soybean chitinases encountering F. oxysporum and six rhizobacteria. [ABSTRACT FROM AUTHOR]

Safеd musli, also known as Chlorophytum borivilianum , is gaining recognition in thе hеrbal industry due to its rich phytochеmical contеnt, extracted from the rhizome. In an attempt to produce these phytochemicals via an alternative method, optimization of proliferation of safed musli hairy roots were conducted. The primary objective was to identify and optimize the conditions that promote the growth of hairy roots in Safеd musli such as the type and concentrations of auxin, carbon sourcеs, and dark-light regimes. Thе hairy roots wеrе cultured in mеdia with varying concеntrations of IBA and NAA (ranging from 0 to 0.8 mg/L), both individually and in combinations. Thе combination of 0.8 mg/L IBA and 0.6 mg/L NAA proved to be most еffеctivе resulting in 100 % branching within 12 days. This combination yiеldеd 25.2 branchеs with an avеragе root length of 16.23 cm, whereas in control no branching occurred. Following this, carbon sources (sucrose and fructose) under light and dark conditions were studied. Significant enhancement on proliferation of hairy roots was obtained in treatment with 3 % sucrose giving a fresh weight of 3.68 g, root length of 242.4 cm, root diameter of 0.82 mm, and higher surface area (49.7 cm2) under light (16 h, 35 μmol m−2 s−1). This study on optimization of auxin, carbon sources and light-dark regimes has demonstrated the potential for enhancing hairy root proliferation, offering a robust and efficient method to produce bioactive compounds which can be upscaled for industrial bioreactor production. [Display omitted] • Hairy roots induced from safed musli and showed proliferation with auxin in the media. • NAA,IBA alone or combination triggered the lateral branching growth. • Media with a combination of 0.6 mg/L NAA + 0.8 mg/L IBA showed higher response. • Shorter time, higher branches initiated after 12 days of culture. • 3 % sucrosе and light incubation was effective in inducing hairy roots. [ABSTRACT FROM AUTHOR]

This article discusses the optimization of an ex vitro one-step hairy root transformation method in drug- and hemp-type cannabis using the RUBY system. Cannabis is a versatile plant species that has gained attention in medical research and industry due to recent legalization. The concentration of the cannabinoid Δ9‐tetrahydrocannabinol (THC) determines the legal categorization of cannabis as hemp-type or drug-type. The study presents a new method for transforming cannabis roots using the RUBY system, which allows for the visual identification of transformed roots. The results show that the transformation efficiency varied based on the bacterial strain and seed type, with drug-type cannabis exhibiting higher transformation rates. This ex vitro method offers simplicity, speed, and reduced contamination risk, making it suitable for the efficient production of secondary metabolites in cannabis. [Extracted from the article]

The root oncogenic loci (rol) C oncogene has been identified as a key player during the plant‐Rhizobium rhizogenes interaction and is considered to confer resistance to plant abiotic stresses, yet the underlying mechanisms remain largely elusive. In this study, Arabidopsis plants constitutively overexpressing rolC were produced. Biometric analysis showed that rolC induced diverse phenotypic modifications, including dwarfism, increased number of stem branches (1.3 times more than WT), weak root growth, early flowering (1.6 days earlier than WT) and premature leaf senescence. In addition, senescence stimulus (exogenous ethylene: 10 or 100 μL L−1) and 10% (w/v) polyethylene glycol 6000 (PEG) treatments demonstrated that rolC mediated leaf abscisic acid (ABA) enhancement in Arabidopsis, and this might be involved in rolC‐induced premature leaf senescence and resistance enhancement to osmotic stress. It is concluded that rolC‐induced premature leaf senescence may be involved in an abiotic stress escape mechanism in Arabidopsis, which is closely related to the increase of endogenous ABA levels. These findings provide new insights into the role of rolC in plant‐bacterial interaction and uncover the potential of biotechnological application associated with R. rhizogenes/rol genes in plant drought defense. [ABSTRACT FROM AUTHOR]

Background: Common bean (Phaseolus vulgaris L.) is a major food legume with high nutritional value and economic importance globally. A remarkable attribute of this plant is its propensity to generate adventitious roots from petioles of detached leaves when moisture is appropriate. This distinctive feature presents promising prospects for harnessing petioles in the facilitation of transgenic hairy root production. Methods: We achieved the successful induction of transgenic hairy roots from common bean petioles through the utilization of Agrobacterium rhizogenes strain K599. Our experimentation encompassed a diverse array of common bean varieties and leaves of varying ages. Subsequently, we refined and optimized the procedure for hairy root induction. Result: Our investigations have revealed that the most conducive conditions for hairy root transformation with K599 are attained using 5-day-old leaves from the cultivar ‘Honghuaqingjia’, exhibiting a remarkable induction efficiency of 59%. The usefulness of this system was demonstrated through a subcellular localization analysis of the transcription factor PvTCP2 protein in combination with GFP (Green Fluorescent Protein). [ABSTRACT FROM AUTHOR]

Kalanchoë gastonis-bonnieri Raym.-Hamet & Perrier is a plant used for medicinal purposes in the treatment of several ailments. The aim of this study was to analyze the chemical profile of extracts from K. gastonis-bonnieri embryogenic calli, generated from genetically transformed roots by Agrobacterium rhizogenes. Putative transformants were verified by PCR. Hydroalcoholic extracts were obtained and the chemical profile was analyzed by LC-ESI-MS/MS. Root formation was obtained from 80% of infected seedlings. Fifteen root lines were isolated, and two lines showed prominent longitudinal growth and profuse branching in the B5 semi-solid medium. In all lines, the formation of nodules and later embryogenic callus was observed. Putative transgenic root lines were cultivated in free-plant growth regulators B5 medium. In the two selected lines, the PCR amplification of rolA, rolB, rolC, rolD, and aux1 genes was detected. The extract of embryogenic calli showed 60 chemical compounds tentatively identified, such as ferulic acid, quinic acid, neobaisoflavone, and malic acid, among others, and the chemical profile was different in comparison to wild-type extracts. This is the first study reporting the analysis of the chemical profile of hairy root extracts derived from Kalanchoë gastonis-bonnieri. This work displays the great potential for obtaining chemical compounds of pharmacological importance from hairy roots and facilitates the identification of new useful drugs against human chronic-degenerative diseases. [ABSTRACT FROM AUTHOR]

Summary: Paclitaxel is one of the most effective anticancer drugs ever developed. Although the most sustainable approach to its production is provided by plant cell cultures, the yield is limited by bottleneck enzymes in the taxane biosynthetic pathway: baccatin‐aminophenylpropanoyl‐13‐O‐transferase (BAPT) and 3′‐N‐debenzoyltaxol N‐benzoyltransferase (DBTNBT). With the aim of enhancing paclitaxel production by overcoming this bottleneck, we obtained distinct lines of Taxus baccata in vitro roots, each independently overexpressing either of the two flux‐limiting genes, BAPT or DBTNBT, through a Rhizobium rhizogenes A4‐mediated transformation. Due to the slow growth rate of the transgenic Taxus roots, they were dedifferentiated to obtain callus lines and establish cell suspensions. The transgenic cells were cultured in a two‐stage system and stimulated for taxane production by a dual elicitation treatment with 1 μm coronatine plus 50 mm of randomly methylated‐β‐cyclodextrins. A high overexpression of BAPT (59.72‐fold higher at 48 h) and DBTNBT (61.93‐fold higher at 72 h) genes was observed in the transgenic cell cultures, as well as an improved taxane production. Compared to the wild type line (71.01 mg/L), the DBTNBT line produced more than four times higher amounts of paclitaxel (310 mg/L), while the content of this taxane was almost doubled in the BAPT line (135 mg/L). A transcriptional profiling of taxane biosynthetic genes revealed that GGPPS, TXS and DBAT genes were the most reactive to DBTNBT overexpression and the dual elicitation, their expression increasing gradually and constantly. The same genes exhibited a pattern of isolated peaks of expression in the elicited BAPT‐overexpressing line. [ABSTRACT FROM AUTHOR]

Taxus, a vital source of the anticancer drug paclitaxel, grapples with a pronounced supply–demand gap. Current efforts to alleviate the paclitaxel shortage involve expanding Taxus cultivation through cutting propagation. However, traditional cutting propagation of Taxus is difficult to root and time-consuming. Obtaining the roots with high paclitaxel content will cause tree death and resource destruction, which is not conducive to the development of the Taxus industry. To address this, establishing rapid and efficient stem rooting systems emerges as a key solution for Taxus propagation, facilitating direct and continuous root utilization. In this study, Agrobacterium rhizogenes were induced in the 1–3-year-old branches of Taxus × media Rehder, which has the highest paclitaxel content. The research delves into the rooting efficiency induced by different A. rhizogenes strains, with MSU440 and C58 exhibiting superior effects. Transcriptome and metabolome analyses revealed A. rhizogenes' impact on hormone signal transduction, amino acid metabolism, zeatin synthesis, and secondary metabolite synthesis pathways in roots. LC-MS-targeted quantitative detection showed no significant difference in paclitaxel and baccatin III content between naturally formed and induced roots. These findings underpin the theoretical framework for T. media rapid propagation, contributing to the sustainable advancement of the Taxus industry. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Rhizobium rhizogenes-mediated hairy root induction and transformation have been developed and applied in various plant species. In this study, an efficient procedure for hairy root induction was established for Melia azedarach, an economically important woody plant worldwide. The highest hairy root induction efficacy (91.8%) was achieved using optimal transformation parameters including R. rhizogenes strain ATCC 15834 at density of OD650 = 0.6, hypocotyl segments as initial explants, and 3 days of co-cultivation. Different hairy root phenotypes including hairy roots with limited branching (type 1), green compact callus formation (type 2), shoot induction (type 3), and white fragile callus formation (type 4) were found as results from the presences of TL and TR-DNA in hairy root lines. Among them, the abundance of the TL-DNA insertion was confirmed in most of the tested hairy roots. The two hairy root phenotypes (type 1 and type 2) showed potential to induce whole regenerated plants at the average rates from 29.13 to 76.40%. The established procedure was successfully used to validate the activity of the GUS reporter gene construct in hairy roots and directly regenerate transgenic shoots. This is the first report in development of the hairy root induction and transformation system for Melia azedarach and provides a potential approach for further fundamental and applied research in this woody plant. Key message: A sufficient procedure for Melia azedarach hairy root induction, transformation as well as direct generation of transgenic plants via Rhizobium rhizogenes-mediated method. [ABSTRACT FROM AUTHOR]

Woody plants play a vital role in global ecosystems and serve as valuable resources for various industries and human needs. While many woody plant genomes have been fully sequenced, gene function research and biotechnological breeding advances have lagged behind. As a result, only a limited number of genes have been elucidated, making it difficult to use newer tools such as CRISPR-Cas9 for biotechnological breeding purposes. The use of Agrobacterium rhizogenes as a transformative tool in plant biotechnology has received considerable attention in recent years, particularly in the research field on woody plants. Over the past three decades, numerous woody plants have been effectively transformed using A. rhizogenes-mediated techniques. Some of these transformed plants have successfully regenerated. Recent research on A. rhizogenes-mediated transformation of woody plants has demonstrated its potential for various applications, including gene function analysis, gene expression profiling, gene interaction studies, and gene regulation analysis. The introduction of the Ri plasmid has resulted in the emergence of several Ri phenotypes, such as compact plant types, which can be exploited for Ri breeding purposes. This review paper presents recent advances in A. rhizogenes-mediated basic research and Ri breeding in woody plants. This study highlights various aspects of A. rhizogenes-mediated transformation, its multiple applications in gene function analysis, and the potential of Ri lines as valuable breeding materials. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Recent advancements in plant biotechnology have highlighted the potential of hairy roots as a biotechnological platform, primarily due to their rapid growth and ability to produce specialized metabolites. This study aimed to delve deeper into hairy root development in C. asiatica and explore the optimization of genetic transformation for enhanced bioactive compound production. Previously established hairy root lines of C. asiatica were categorized based on their centelloside production capacity into HIGH, MID, or LOW groups. These lines were then subjected to a meticulous label-free proteomic analysis to identify and quantify proteins. Subsequent multivariate and protein network analyses were conducted to discern proteome differences and commonalities. Additionally, the quantification of rol gene copy numbers was undertaken using qPCR, followed by gene expression measurements. From the proteomic analysis, 213 proteins were identified. Distinct proteome differences, especially between the LOW line and other lines, were observed. Key proteins related to essential processes like photosynthesis and specialized metabolism were identified. Notably, potential biomarkers, such as the Tr-type G domain-containing protein and alcohol dehydrogenase, were found in the HIGH group. The presence of ornithine cyclodeaminase in the hairy roots emerged as a significant biomarker linked with centelloside production capacity lines, indicating successful Rhizobium-mediated genetic transformation. However, qPCR results showed an inconsistency with rol gene expression levels, with the HIGH line displaying notably higher expression, particularly of the rolD gene. The study unveiled the importance of ornithine cyclodeaminase as a traceable biomarker for centelloside production capacity. The strong correlation between this biomarker and the rolD gene emphasizes its potential role in optimizing genetic transformation processes in C. asiatica. [ABSTRACT FROM AUTHOR]

The stable transformation of common bean is a challenging and time-consuming process. Although CRISPR/Cas9 has revolutionized gene editing with its high efficiency and specificity, the performance of the system can be affected by multiple factors, such as sgRNA specificity and effectiveness, and the choice of promoter used to drive Cas9 expression. The use of a hairy root transformation system to initially check the efficiency of sgRNAs and the impact of different promoters could speed up this process and increase the chances of success. We initially tested three different transformation methods to induce hairy roots and selected a preferred method suitable for a variety of different common bean genotypes. This method involved inoculating a severed radicle with Rhizobium rhizogenes K599 and was fast, had a high transformation frequency of 42-48%, and resulted in numerous hairy roots. This method was further used for the transformation of explants using R. rhizogenes harboring different CRISPR/Cas9 constructs and evaluated the on-target activity of sgRNAs targeting raffinose family oligosaccharides biosynthetic genes and the impact of different promoters driving Cas9on the gene editing efficiency. Additionally, we evaluated the reliability of the in silico tools, CRISPOR, CRISPR RGEN, and inDelphi to predict the sgRNA efficiencies and resulting mutations. Our results showed that the hairy root transformation system allows for rapid evaluation of multiple sgRNAs and promoters. We also identified several highly efficient sgRNAs that induced frameshift mutations at rates of up to 70% when a parsley ubiquitin promoter was driving Cas9 expression, providing valuable information for the selection of the most effective sgRNAs and promoters for future transformation experiments. Although most of the computational models used to predict the sgRNA efficiency did not match the in planta results, the Lindel model proved to be the most reliable for P. vulgaris, accurately predicting the sgRNA efficiency and the type of induced mutation in most hairy roots. Furthermore, the inDelphi algorithm could correctly predict deletions and single nucleotide insertions resulting from DNA double-strand breaks in common bean. These results offer promising implications for enhancing precise editing in plants because they provide the possibility of predicting repair outcomes. [ABSTRACT FROM AUTHOR]