Overexpression of Wild Soybean Expansin Gene GsEXLB14 Enhanced the Tolerance of Transgenic Soybean Hairy Roots to Salt and Drought Stresses.

As a type of cell-wall-relaxing protein that is widely present in plants, expansins have been shown to actively participate in the regulation of plant growth and responses to environmental stress. Wild soybeans have long existed in the wild environment and possess abundant resistance gene resources, which hold significant value for the improvement of cultivated soybean germplasm. In our previous study, we found that the wild soybean expansin gene GsEXLB14 is specifically transcribed in roots, and its transcription level significantly increases under salt and drought stress. To further identify the function of GsEXLB14, in this study, we cloned the CDS sequence of this gene. The transcription pattern of GsEXLB14 in the roots of wild soybean under salt and drought stress was analyzed by qRT-PCR. Using an Agrobacterium rhizogenes-mediated genetic transformation, we obtained soybean hairy roots overexpressing GsEXLB14. Under 150 mM NaCl- and 100 mM mannitol-simulated drought stress, the relative growth values of the number, length, and weight of transgenic soybean hairy roots were significantly higher than those of the control group. We obtained the transcriptomes of transgenic and wild-type soybean hairy roots under normal growth conditions and under salt and drought stress through RNA sequencing. A transcriptomic analysis showed that the transcription of genes encoding expansins (EXPB family), peroxidase, H+-transporting ATPase, and other genes was significantly upregulated in transgenic hairy roots under salt stress. Under drought stress, the transcription of expansin (EXPB/LB family) genes increased in transgenic hairy roots. In addition, the transcription of genes encoding peroxidases, calcium/calmodulin-dependent protein kinases, and dehydration-responsive proteins increased significantly. The results of qRT-PCR also confirmed that the transcription pattern of the above genes was consistent with the transcriptome. The differences in the transcript levels of the above genes may be the potential reason for the strong tolerance of soybean hairy roots overexpressing the GsEXLB14 gene under salt and drought stress. In conclusion, the expansin GsEXLB14 can be used as a valuable candidate gene for the molecular breeding of soybeans.

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.

An efficient genetic transformation system mediated by Rhizobium rhizogenes in fruit trees based on the transgenic hairy root to shoot conversion.

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.

Injection-based hairy root induction and plant regeneration techniques in Brassicaceae.

BACKGROUND: Hairy roots constitute a valuable tissue culture system for species that are difficult to propagate through conventional seed-based methods. Moreover, the generation of transgenic plants derived from hairy roots can be facilitated by employing carefully designed hormone-containing media. RESULTS: We initiated hairy root formation in the rare crucifer species Asperuginoides axillaris via an injection-based protocol using the Agrobacterium strain C58C1 harboring a hairy root-inducing (Ri) plasmid and successfully regenerated plants from established hairy root lines. Our study confirms the genetic stability of both hairy roots and their derived regenerants and highlights their utility as a permanent source of mitotic chromosomes for cytogenetic investigations. Additionally, we have developed an effective embryo rescue protocol to circumvent seed dormancy issues in A. axillaris seeds. By using inflorescence primary stems of Arabidopsis thaliana and Cardamine hirsuta as starting material, we also established hairy root lines that were subsequently used for regeneration studies. CONCLUSION: We developed efficient hairy root transformation and regeneration protocols for various crucifers, namely A. axillaris, A. thaliana, and C. hirsuta. Hairy roots and derived regenerants can serve as a continuous source of plant material for molecular and cytogenetic analyses.

Enhanced production of withaferin A from the hairy root culture of Withania somnifera via synergistic effect of Methyl jasmonate and ß-cyclodextrin.

Due to low amounts of withanolides produced in some plants and high demand for various applications, their biotechnological production is widely researched. The effects of two explant types (i.e., leaf and stem from the in vitro seedlings of three genotypes of Withania somnifera) and four Rhizobium strains (i.e., LBA 9402, A4, ATCC 15834, and C58C1) to improve hairy root formation efficiency was studied. Furthermore, the combined effects of ß-cyclodextrin (ß-CD) and methyl jasmonate (MeJA) on withaferin A production after 48 h exposure time was examined. Four hairy roots having the maximum percentage of induced roots and mean number of induced roots to analyze their growth kinetics and identified G3/ATCC/LEAF culture having the maximum specific growth rate (µ = 0.036 day-1) and growth index (GI = 9.18), and the shortest doubling time (Td = 18.82 day) were selected. After 48 h exposure of G3/ATCC/LEAF culture to different elicitation conditions, maximum amounts of withaferin A were produced in samples co-treated with 0.5 mM ß-CD + 100 µM MeJA (9.57 mg/g DW) and 5.0 mM ß-CD + 100 µM MeJA (17.45 mg/g DW). These outcomes represented a 6.8-fold and 12.5-fold increase, respectively, compared to the control. Similarly, combined ß-CD/MeJA elicitation increased gene expression levels of HMGR, SQS, SMT-1, and SDS/CYP710A involved in withanolides biosynthetic pathway, of which just SMT-1 had significant correlation with withaferin A production. These results demonstrated the superiority of G1-leaf explant and ATCC 15834 for hairy root induction, and revealed synergistic effect of MeJA and ß-CD on withaferin A production.

Glycyrrhizin Production in Licorice Hairy Roots Based on Metabolic Redirection of Triterpenoid Biosynthetic Pathway by Genome Editing.

Glycyrrhizin, a type of the triterpenoid saponin, is a major active ingredient contained in the roots of the medicinal plant licorice (Glycyrrhiza uralensis, G. glabra and G. inflata), and is used worldwide in diverse applications, such as herbal medicines and sweeteners. The growing demand for licorice threatens wild resources and therefore a sustainable method of supplying glycyrrhizin is required. With the goal of establishing an alternative glycyrrhizin supply method not dependent on wild plants, we attempted to produce glycyrrhizin using hairy root culture. We tried to promote glycyrrhizin production by blocking competing pathways using CRISPR/Cas9-based gene editing. CYP93E3 CYP72A566 double-knockout (KO) and CYP93E3 CYP72A566 CYP716A179 LUS1 quadruple-KO variants were generated, and a substantial amount of glycyrrhizin accumulation was confirmed in both types of hairy root. Furthermore, we evaluated the potential for promoting further glycyrrhizin production by simultaneous CYP93E3 CYP72A566 double-KO and CYP88D6-overexpression. This strategy resulted in a 3-fold increase (∼1.4 mg/g) in glycyrrhizin accumulation in double-KO/CYP88D6-overexpression hairy roots, on average, compared with that of double-KO hairy roots. These findings demonstrate that the combination of blocking competing pathways and overexpression of the biosynthetic gene is important for enhancing glycyrrhizin production in G. uralensis hairy roots. Our findings provide the foundation for sustainable glycyrrhizin production using hairy root culture. Given the widespread use of genome editing technology in hairy roots, this combined with gene knockout and overexpression could be widely applied to the production of valuable substances contained in various plant roots.

Production of Phenolic Compounds and Antioxidant Activity in Hairy Root Cultures of Salvia plebeia.

Salvia plebeia (Lamiaceae) is a medicinal plant containing diverse bioactive constituents that have biological properties. In this study, we determined the optimal conditions (media and auxin) for the hairy root culture of S. plebeia for the growth and accumulation of phenolic compounds and evaluated its antioxidant activities. Rosmarinic acid and five phenylpropanoids were detected using high-performance liquid chromatography. The hairy roots grown in 1/2 SH medium with 1 mg/L NAA had a high level of rosmarinic acid content. Hairy roots cultured in 1 mg/L NAA had the highest total content of five phenylpropanoids. Compared to wild-type roots grown in the field, hairy roots (NAA 1) expressed similar levels of rosmarinic acid but significantly enhanced phenylpropanoid accumulation. Furthermore, the total phenolic content and total flavonoid content of hairy roots (NAA 1) were 2.22 and 1.73 times higher than those of wild-type roots. In the results of DPPH, ABTS, and reducing power assays, the hairy roots (NAA 1) showed higher free radical scavenging effects and reduction potential than the wild-type roots. These results suggest that S. plebeia hairy roots cultured under optimal conditions, which exhibit enhanced phenolic compound accumulation and antioxidant activity, can potentially be used as sources of antioxidants.

Agrobacterium rhizogenes: paving the road to research and breeding for woody plants.

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.

Induction and metabolomic analysis of hairy roots of Atractylodes lancea.

Atractylodes lancea is an important source of traditional Chinese medicines. Sesquiterpenoids are the key active compounds in A. lancea, and their presence determines the quality of the material. Hairy hoot (HR) culture is a potential method to produce medicinally active compounds industrially; however, the induction and metabolic profiling of A. lancea HR have not been reported. We found that optimal induction of A. lancea HR was achieved by Agrobacterium rhizogenes strain C58C1 using the young leaves of tissue culture seedlings in the rooting stage as explants. Ultra-performance liquid chromatography-tandem mass spectrometric analyses of the chemical compositions of HR and normal root (NR) led to the annotation of 1046 metabolites. Over 200 differentially accumulated metabolites were identified, with 41 found to be up-regulated in HR relative to NR and 179 down-regulated in HR. Specifically, atractylodin levels were higher in HR, while the levels of ß-eudesmol and hinesol were higher in NR. Metabolic pathway analyses showed a significant difference in metabolites of the shikimate acid pathway between HR and NR. Five A. lancea compounds are potential biomarkers for evaluation of HR and NR quality. This study provides an important reference for the application of HR for the production of medicinally active compounds. KEY POINTS: • We established an efficient protocol for the induction of HR in A. lancea • HR was found to have a significantly higher amount of atractylodin than did NRs • Metabolic pathway analyses showed a significant difference in metabolites of the shikimate acid pathway between HR and NR.

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

BACKGROUND: The 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. RESULTS: Here, 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. CONCLUSION: We 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.

Functional Characterisation of the Transcription Factor GsWRKY23 Gene from Glycine soja in Overexpressed Soybean Composite Plants and Arabidopsis under Salt Stress.

WRKY proteins are a superfamily of transcription factors (TFs) that play multiple roles in plants' growth, development, and environmental stress response. In this study, a novel WRKY gene called GsWRKY23 that is specifically upregulated in salt-tolerant Glycine soja accession BB52 seedlings was identified by transcriptomic analysis under salt stress. How the physiological functions and mechanisms of the GsWRKY23 gene affect salt tolerance was investigated using transformations of soybean hairy roots and Arabidopsis, including wild-type (WT) and atwrky23-mutant plants. The results showed that GsWRKY23 in the roots, stems, and leaves of BB52, along with its promoter in the cotyledons and root tips of GsWRKY23pro::GUS Arabidopsis seedlings, displayed enhanced induction under salt stress. GsWRKY23 localises to the nucleus and shows transcriptional activation ability in yeast cells. Compared to GsWRKY23-RNAi wild soybean hairy-root composite plants under salt stress, obvious improvements, such as superior growth appearance, plant height and fresh weight (FW), and leaf chlorophyll and relative water content (RWC), were displayed by GsWRKY23-overexpressing (OE) composite plants. Moreover, their relative electrolytic leakage (REL) values and malondialdehyde (MDA) contents in the roots and leaves declined significantly. Most of the contents of Na+ and Cl- in the roots, stems, and leaves of GsWRKY23-OE plants decreased significantly, while the content of K+ in the roots increased, and the content of NO3- displayed no obvious change. Ultimately, the Na+/K+ ratios of roots, stems, and leaves, along with the Cl-/NO3- ratios of roots and stems, decreased significantly. In the transgenic WT-GsWRKY23 and atwrky23-GsWRKY23 Arabidopsis seedlings, the salt-induced reduction in seed germination rate and seedling growth was markedly ameliorated; plant FW, leaf chlorophyll content, and RWC increased, and the REL value and MDA content in shoots decreased significantly. In addition, the accumulation of Na+ and Cl- decreased, and the K+ and NO3- levels increased markedly to maintain lower Na+/K+ and Cl-/NO3- ratios in the roots and shoots. Taken together, these results highlight the role of GsWRKY23 in regulating ionic homeostasis in NaCl-stressed overexpressed soybean composite plants and Arabidopsis seedlings to maintain lower Na+/K+ and Cl-/NO3- ratios in the roots and shoots, thus conferring improved salt tolerance.

Hairy root culture: a potent method for improved secondary metabolite production of Solanaceous plants.

Secondary metabolites synthesized by the Solanaceous plants are of major therapeutic and pharmaceutical importance, many of which are commonly obtained from the roots of these plants. 'Hairy roots', mirroring the same phytochemical pattern of the corresponding root of the parent plant with higher growth rate and productivity, are therefore extensively studied as an effective alternative for the in vitro production of these metabolites. Hairy roots are the transformed roots, generated from the infection site of the wounded plants with Agrobacterium rhizogenes. With their fast growth, being free from pathogen and herbicide contamination, genetic stability, and autotrophic nature for plant hormones, hairy roots are considered as useful bioproduction systems for specialized metabolites. Lately, several elicitation methods have been employed to enhance the accumulation of these compounds in the hairy root cultures for both small and large-scale production. Nevertheless, in the latter case, the cultivation of hairy roots in bioreactors should still be optimized. Hairy roots can also be utilized for metabolic engineering of the regulatory genes in the metabolic pathways leading to enhanced production of metabolites. The present study summarizes the updated and modern biotechnological aspects for enhanced production of secondary metabolites in the hairy root cultures of the plants of Solanaceae and their respective importance.

Elicitation enhances the production of friedelin and epifriedelanol in hairy root cultures of Cannabis sativa L.

Cannabis sativa L. (hemp) has a global distribution and social impact, and it is widely used as a medicinal plant, food ingredient, and textile fiber. Its roots have received less attention than other parts, especially the inflorescence, leaves, and shoots. Triterpenoids, including friedelin and epifriedelanol, have been found in hemp roots, and their anti-inflammatory effects have been reported. In this study, the potential enhancement of triterpenoid accumulation in the roots of C. sativa by elicitation was examined. Hairy roots were successfully established, and they contained 2.02-fold higher triterpenoid levels than natural roots. Furthermore, hairy roots treated with 75 µM salicylic acid had 1.95-fold higher friedelin levels (0.963 mg/g DW) and 1.4-fold higher epifriedelanol levels (0.685 mg/g DW) than untreated hairy roots. These results suggested that the elucidation of hairy root cultures using an optimized elicitor could represent an alternative strategy to produce the valuable triterpenoids friedelin and epifriedelanol.

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

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

Production of secondary metabolites using tissue culture-based biotechnological applications.

Plants are the sources of many bioactive secondary metabolites which are present in plant organs including leaves, stems, roots, and flowers. Although they provide advantages to the plants in many cases, they are not necessary for metabolisms related to growth, development, and reproduction. They are specific to plant species and are precursor substances, which can be modified for generations of various compounds in different plant species. Secondary metabolites are used in many industries, including dye, food processing and cosmetic industries, and in agricultural control as well as being used as pharmaceutical raw materials by humans. For this reason, the demand is high; therefore, they are needed to be obtained in large volumes and the large productions can be achieved using biotechnological methods in addition to production, being done with classical methods. For this, plant biotechnology can be put in action through using different methods. The most important of these methods include tissue culture and gene transfer. The genetically modified plants are agriculturally more productive and are commercially more effective and are valuable tools for industrial and medical purposes as well as being the sources of many secondary metabolites of therapeutic importance. With plant tissue culture applications, which are also the first step in obtaining transgenic plants with having desirable characteristics, it is possible to produce specific secondary metabolites in large-scale through using whole plants or using specific tissues of these plants in laboratory conditions. Currently, many studies are going on this subject, and some of them receiving attention are found to be taken place in plant biotechnology and having promising applications. In this work, particularly benefits of secondary metabolites, and their productions through tissue culture-based biotechnological applications are discussed using literature with presence of current studies.

Cataloguing Protein Complexes In Planta Using TurboID-Catalyzed Proximity Labeling.

Mapping protein-protein interactions is crucial to understand protein function. Recent advances in proximity-dependent biotinylation (BioID) coupled to mass spectrometry (MS) allow the characterization of protein complexes in diverse plant models. Here, we describe the use of BioID in hairy root cultures of tomato and provide detailed information on how to analyze the data obtained by MS.

Investigating the Transformation Products of Selected Antibiotics and 17 α-Ethinylestradiol under Three In Vitro Biotransformation Models for Anticipating Their Relevance in Bioaugmented Constructed Wetlands.

The degradation of three antibiotics (sulfamethoxazole, trimethoprim, and ofloxacin) and one synthetic hormone (17 α-ethinylestradiol) was investigated in three in-vitro biotransformation models (i.e., pure enzymes, hairy root, and Trichoderma asperellum cultures) for anticipating the relevance of the formation of transformation products (TPs) in constructed wetlands (CWs) bioaugmented with T. asperellum fungus. The identification of TPs was carried out employing high-resolution mass spectrometry, using databases, or by interpreting MS/MS spectra. An enzymatic reaction with ß-glucosidase was also used to confirm the presence of glycosyl-conjugates. The results showed synergies in the transformation mechanisms between these three models. Phase II conjugation reactions and overall glycosylation reactions predominated in hairy root cultures, while phase I metabolization reactions (e.g., hydroxylation and N-dealkylation) predominated in T. asperellum cultures. Following their accumulation/degradation kinetic profiles helped in determining the most relevant TPs. Identified TPs contributed to the overall residual antimicrobial activity because phase I metabolites can be more reactive and glucose-conjugated TPs can be transformed back into parent compounds. Similar to other biological treatments, the formation of TPs in CWs is of concern and deserves to be investigated with simple in vitro models to avoid the complexity of field-scale studies. This paper brings new findings on the emerging pollutants metabolic pathways established between T. asperellum and model plants, including extracellular enzymes.

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

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

Blue LED light promoting the growth, accumulation of high-value isoflavonoids and astragalosides, antioxidant response, and biosynthesis gene expression in Astragalus membranaceus (Fisch.) Bunge hairy root cultures.

The root of Astragalus membranaceus (Fisch.) Bunge is one of the most frequently used herbs in traditional Chinese medicine (TCM) formulae for fighting COVID-19 infections, due to the presence of isoflavonoids and astragalosides associated with antiviral and immune-enhancing activities. For the first time, the exposure of A. membranaceus hairy root cultures (AMHRCs) to different colors of LED lights i.e., red, green, blue, red/green/blue (1/1/1, RGB), and white, was conducted to promote the root growth and accumulation of isoflavonoids and astragalosides. LED light treatment regardless of colors was found beneficial for root growth, which might be a result of the formation of more root hairs upon light stimulation. Blue LED light was found most effective for enhancing phytochemical accumulation. Results showed that the productivity of root biomass in blue-light grown AMHRCs with an initial inoculum size of 0.6% for 55 days was 1.40-fold higher than that in dark (control), and yields of high-value isoflavonoids and astragalosides including calycosin, formononetin, astragaloside IV, and astragaloside I increased by 3.17-fold, 2.66-fold, 1.78-fold, and 1.52-fold relative to control, respectively. Moreover, the photooxidative stress together with transcriptional activation of biosynthesis genes might contribute to the enhanced accumulation of isoflavonoids and astragalosides in blue-light grown AMHRCs. Overall, this work offered a feasible approach for obtaining higher yields of root biomass and medicinally important compounds in AMHRCs via the simple supplementation of blue LED light, which made blue-light grown AMHRCs industrially attractive as plant factory in controlled growing systems. Supplementary Information: The online version contains supplementary material available at 10.1007/s11240-023-02486-7.