Hairy Roots Research Articles

Genome editing of an oxalyl-CoA synthetase gene in Lathyrus sativus reveals its role in oxalate metabolism.

Established an Agrobacterium-mediated hairy root transformation system for gene function analysis in Lathyrus sativus. Arabidopsis mutant complementation and genome editing in Lathyrus confirmed role of LsOCS in the oxalate metabolism. Grass pea (Lathyrus sativus) is a resilient legume cultivated for its protein-rich seeds and fodder. However, the presence of a naturally occurring neurotoxin, β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP), which causes neurolathyrism, limits its extensive cultivation. This paper reports the in-planta characterization of oxalyl-CoA synthetase (OCS), an enzyme involved in oxalate metabolism and important in the oxalylating step leading to β-ODAP production in Lathyrus. For this, we used complementation experiments in an Arabidopsis OCS mutant. The LsOCS-complemented lines showed oxalate content similar to wild-type levels, and the analysis of seeds by field emission scanning electron microscope (FESEM) showed that the LsOCS-complemented lines were rescued from seed-coat defects found in the mutant seeds. We used genome editing of LsOCS in Lathyrus hairy roots to further characterize LsOCS function. The mutations in LsOCS resulted in the accumulation of oxalate in the hairy roots of Lathyrus, as observed in Arabidopsis mutants, but did not affect the ODAP levels. The hairy root genome editing system could serve as a rapid tool for functional studies of Lathyrus genes and optimizing the agronomic traits.

In vitro strategy to enhance the production of bioactive polyphenols and caffeoylputrescine in the hairy roots of Physalis peruviana L.

The Rhizobium rhizogene-transformed root culture from Physalis peruviana L. (P. peruviana) may be a promising and novel source of valuable phenolics, including caffeoylputrescine (CP), which is known for antioxidant, antidiabetic, insect-resistant, disease-resistant, and neuroprotective properties. In this study, to improve the production efficiency of phytochemical components in P. peruviana hairy root cultures, we optimized various culture conditions, including the inoculum size, liquid volume, culture media type, carbon source, sucrose concentration, initial pH, and application of elicitors, to enhance the total phenolic content and CP yield in these hairy root cultures. The findings indicate that the use of sucrose as carbon source resulted in the highest biomass (13.28g DW/L), total phenolic content (6.26mg/g), and CP yield (2.40mg/L). The White medium excelled in enhancing the total phenolic content (9.35mg/g), whereas the B5 medium was most effective for the biomass (13.38g DW/L) and CP yield (6.30mg/L). A sucrose concentration of 5% was best for the biomass (18.40g DW/L), whereas a sucrose concentration of 4% was ideal for the CP yield. Optimal culture conditions were as follows: an inoculum size of 0.5g/100 mL, a liquid volume of 100 mL in a 250-mL flask, B5 medium, 4% sucrose, and a pH of 5.5. Among the tested elicitors, methyl jasmonate (MeJA) at 100 µM significantly increased the biomass (21.3g/L), total phenolic content (23.34mg/g), and CP yield (141.10mg/L), which represent 0.96-, 2.12-, and 13.04-fold increases, respectively, over the control after 8 days. The optimized HR culture of P. peruviana provides a promising system to enhance the production of CP for pharmaceutical applications.

Response surface methodology-based optimization of hairy roots cultures for in vitro AM production

Arbuscular mycorrhizal fungi can do wonders in promoting the crop growth as well maintaining soil health. In vitro root organ culture technology is an exciting avenue for AM biofertilizer production. This study aims to optimize the key influencing factors for enhancing the hairy root production used for in vitro AMF culturing using response surface methodology, a statistical and mathematical tool used for designing optimization studies. Study uses Rhizobium rhizogenes MTCC 2364 for transformation in carrot explant. Factors considered for optimization are concentration of gelling agent (phytagel), carbon source (sucrose) and pH of the Modified Strullu and Romand medium (MSR). Design Expert software uses second order polynomial regression equation for predicting the outcome of each experiment. Totally, 17 experiments were run following Box-Behnken design and average hairy root length and average side branch emergence were taken as response. ANOVA analysis reveals that the concentration of phytagel and sucrose had a strong influence on root length, while the phytagel and pH of the medium had a strong effect on side branch emergence. Overall, taking into account the both responses, concentration of phytagel had a significant impact on hairy root production. The maximum average hairy root length obtained was 1.12 cm and number of side branches emerged were 6.78 per day. Based on these results, the optimal parameters were MSR medium with 3gL-1 phytagel, 11gL-1 sucrose and a pH of 4 for boosting hairy root development. This study is a cost-effective approach and minimizes the time taken for establishing the hairy root technology.

MicroRNA858 represses the transcription factor gene SbMYB47 and regulates flavonoid biosynthesis in Scutellaria baicalensis.

MicroRNAs (miRNAs) are non-coding endogenous single-stranded RNAs that regulate target gene expression by reducing their transcription and translation. Several miRNAs in plants function in secondary metabolism. The dried root of Scutellaria baicalensis Georgi is a traditional Chinese medicine that contains flavonoids (baicalin, wogonoside, and baicalein) as its main active ingredients. Although the S. baicalensis genome sequence has been published, information regarding its miRNAs is lacking. In this study, 12 small RNA libraries of different S. baicalensis tissues were compiled, including roots, stems, leaves, and flowers. A total of 129 miRNAs were identified, including 99 miRNAs from 27 miRNA families and 30 predicted miRNAs. Furthermore, 46 reliable target genes of 15 miRNA families were revealed using psRNAtarget and confirmed by degradome sequencing. It was speculated that the microRNA858 (miR858)-SbMYB47 module might be involved in flavonoid biosynthesis. Transient assays in Nicotiana benthamiana leaves indicated that miR858 targets SbMYB47 and suppresses its expression. Artificial miRNA-mediated knockdown of miR858 and overexpression of SbMYB47 significantly increased the flavonoid content in S. baicalensis hairy roots, while SbMYB47 knockdown inhibited flavonoid accumulation. Yeast one-hybrid and dual-luciferase assays indicated that SbMYB47 directly binds to and activates the S. baicalensis phenylalanine ammonia-lyase 3 (SbPAL-3) and flavone synthase II (SbFNSⅡ-2) promoters. Our findings reveal the link between the miR858-SbMYB47 module and flavonoid biosynthesis, providing a potential strategy for the production of flavonoids with important pharmacological activities through metabolic engineering.

Epigenetic control of T-DNA during transgenesis and pathogenesis.

Mobile elements known as T-DNAs are transferred from pathogenic Agrobacterium to plants and reprogram the host cell to form hairy roots or tumors. Disarmed non-oncogenic T-DNAs are extensively used to deliver transgenes in plant genetic engineering. Such T-DNAs were the first known targets of RNA silencing mechanisms, which detect foreign RNA in plant cells and produce small RNAs that induce transcript degradation. These T-DNAs can also be transcriptionally silenced by the deposition of epigenetic marks such as DNA methylation and the dimethylation of lysine 9 (H3K9me2) in plants. Here, we review the targeting and the roles of RNA silencing and DNA methylation on T-DNAs in transgenic plants as well as during pathogenesis. In addition, we discuss the crosstalk between T-DNAs and genome-wide changes in DNA methylation during pathogenesis. We also cover recently discovered regulatory phenomena, such as T-DNA suppression and RNA silencing-independent and epigenetic-independent mechanisms that can silence T-DNAs. Finally, we discuss the implications of findings on T-DNA silencing for the improvement of plant genetic engineering.

GmSTOP1-3 Increases Soybean Manganese Accumulation Under Phosphorus Deficiency by Regulating GmMATE2/13 and GmZIP6/GmIREG3.

Mineral nutrient deficiencies and metal ion toxicities coexist on acid soils. Phosphorus (P) deficiency in plants is generally accompanied with significant levels of leaf manganese (Mn) accumulation. However, the molecular regulatory mechanisms underpinning remain unclear. The present study found that P-deficient soybean plants accumulated more Mn compared to P-sufficient ones on acid soils in both field and greenhouse experiments. Meanwhile, both P deficiency and Mn toxicity enhanced the expression of GmSTOP1-3. Over-expressing GmSTOP1-3 enhanced Mn accumulation in transgenic soybean hairy roots, but RNA-interference did not show obvious differences. Moreover, transgenic soybeans with GmSTOP1-3-overexpression showed enhanced root citrate exudation and augmented Mn accumulation. RNA-sequence identified four downstream genes of GmSTOP1-3, including multidrug and toxic compound extrusion (GmMATE2/13) and metal transporter genes (GmZIP6/GmIREG3), which encode plasma membrane proteins. GmSTOP1-3 activated the transcription of these four genes by directly binding to their promoter regions. In addition, both GmZIP6 and GmIREG3 functioned in Mn uptake as manifested by the higher Mn concentration and decreased growth of soybean hairy roots with their overexpression. Taken together, it is suggested that upregulation of GmSTOP1-3 by low P stress on acid soils activates transcripts of GmMATE2/13 and GmZIP6/GmIREG3, which consequently result in enhanced Mn accumulation in soybean.

GmTRAB1, a Basic Leucine Zipper Transcription Factor, Positively Regulates Drought Tolerance in Soybean (Glycine max. L).

The basic leucine zipper (bZIP) transcription factors play crucial roles in plant resistance to environmental challenges, but the biological functions of soybean bZIP members are still unclear. In this study, a drought-related soybean bZIP gene, GmTRAB1, was analyzed. The transcript of GmTRAB1 was upregulated under drought, ABA, and oxidative stresses. Overexpression of GmTRAB1 improved the osmotic stress tolerance of transgenic Arabidopsis and soybean hairy roots associated with increased proline content and activity of antioxidant enzymes and reduced accumulations of malonaldehyde and reactive oxide species. However, RNA interference silencing of GmTRAB1 in the soybean hairy roots improved drought sensitivity. Furthermore, GmTRAB1 increased the sensitivity of transgenic plants to ABA and participated in modulating ABA-regulated stomatal closure upon drought stress. In addition, GmTRAB1 stimulated the transcript accumulation of drought-, ABA-, and antioxidant-related genes to respond to drought. Collectively, this research will contribute to understanding the molecular mechanisms of bZIP transcription factors in soybean's resistance to drought.

Combined transcriptomic, metabolomic and physiological analysis reveals the key role of nitrogen, but not phosphate and potassium in regulating anthocyanin biosynthesis induced by nutrient deficiency in Eucalyptus.

Anthocyanin biosynthesis in Eucalyptus plants can be flexibly and rapidly modulated in response to hormones or environmental stimuli, including nutrient deprivation (ND). However, the underlying mechanism of ND in inducing anthocyanin biosynthesis in plants remains largely elusive. In this study, we discovered that anthocyanin levels in leaves and stems could well reflect nitrogen availability in Eucalyptus. Supplementation with nitrogen, but not with phosphate or potassium, effectively inhibited ND-induced anthocyanin biosynthesis. To further investigate how nitrogen regulates this process, comprehensive time-resolved transcriptomic and targeted metabolomic analyses were conducted. The results revealed that 3405, 2706, and 3153 genes were differentially regulated by nitrogen treatment at 1, 2, and 3 d, respectively. Pathway analysis indicated the majority of the enriched KEGG pathways were associated with cellular metabolism, such as amino acid and flavonoid biosynthesis. Moreover, metabolomic analysis showed that the most abundantly accumulated anthocyanins, cyanidin-3-O-galactoside and cyanidin-3-O-glucoside, along with key intermediates in the flavonoid pathway, were downregulated by nitrogen treatment. Furthermore, nitrogen-responsive MYB-bHLH-WDR complex genes were identified, including six EgrMYB113 family members. Overexpression of one of the EgrMYB113-like genes induced anthocyanin biosynthesis in the transgenic hairy roots of Eucalyptus. Interestingly, mutation of AtMYB113 inhibited nitrogen deficiency-induced anthocyanin biosynthesis in Arabidopsis, suggesting that MYB113 is a novel N-responsive MYB transcriptional regulator of anthocyanin biosynthesis. Additionally, nitrogen treatment upregulated a few GA biosynthesis genes while simultaneously downregulated the expression of several GA2ox genes. Exogenous application of GA3 decreased ND-induced anthocyanin biosynthesis. Conclusively, this study provides novel insights into the molecular mechanism of nitrogen in the regulation of anthocyanin biosynthesis in Eucalyptus.

Combining GWAS and RNA-Seq approaches identifies the FtADH1 gene for drought resistance in Tartary buckwheat

Drought is one of the major environmental constraints that significantly affects seedling emergence, yield, and quality of Tartary buckwheat, thereby hindering the development of its industry. However, the molecular mechanisms underlying drought tolerance genes in Tartary buckwheat remain largely unexplored. Alcohol dehydrogenase (ADH), one of the essential plant proteins, plays a crucial role in growth, development, and stress responses, but its specific role in drought resistance is still unclear. In this study, we identified an ADH gene FtADH1, using a membership function value of drought tolerance (MFVD) combined with a genome-wide association study (GWAS) and transcriptomic profiles that confers drought tolerance in Tartary buckwheat. Our findings demonstrated that the overexpression of FtADH1 in Arabidopsis and Tartary buckwheat hairy roots enhances drought tolerance by promoting root elongation and mitigating elevated levels of reactive oxygen species (ROS). Our findings demonstrated that FtADH1 can enhanced tolerance to drought stresses in both Tartary buckwheat and Arabidopsis. This study identifies the FtADH1 as a new player in affecting ROS level and the stress response of Tartary buckwheat by regulating protective enzyme activities at a high level to scavenge ROS and modulating root growth under drought stress. Further, we identified proteins interacting with FtADH1 through a prokaryotic expression pull-down assay combined with mass spectrometry, revealing that FtADH1 specifically interacts with the S-adenosyl-L-methionine (SAM) synthetase protein, FtSAMS1. Overexpression of FtSAMS1 was found to enhance ADH enzymatic activity, leading to increased SAM content in overexpressing Tartary buckwheat hairy roots under water-deficit conditions. Additionally, FtSAMS1 overexpression induced a drought-resistant phenotype in Arabidopsis and Tartary buckwheat hairy roots under drought stress, revealing the biological function of FtADH1. Evolutionary analysis indicates that ADH1 in Fagopyrum species has undergone significant evolutionary events, including duplication and purifying selection, which may contribute to functional diversification and adaptive advantages such as drought resistance in cultivated buckwheat. In summary, this study proposes that FtADH1 is a key contributor to drought tolerance, and its interaction with FtSAMS1 holds potential for the development of drought-resistant varieties in Tartary buckwheat and its relative species.

Genome-wide characterization of the DIR gene family in sesame reveals the function of SiDIR21 in lignan biosynthesis

Furofuran-type lignans, mainly sesamin and sesamolin, are the most representative functional active ingredients in sesame (Sesamum indicum L.). Their exceptional antioxidant properties, medicinal benefits, and health-promoting functions have garnered significant attention. Dirigent (DIR) proteins, found in vascular plants, are crucial for the biosynthesis of secondary metabolites, like lignans, and essential for responding to abiotic and biotic stresses. Despite their importance, they have yet to be systematically analyzed, especially those involved in lignan synthesis in sesame. This study unveiled 44 DIR genes in sesame. Phylogenetic analysis categorized these SiDIRs into five subgroups (DIR-a, DIR-b/d, DIR-e, DIR-f, and DIR-g), aligning with conserved motifs and gene structures analyses. Expression analysis unveiled distinct tissue-specific and hormone-responsive expression patterns among the SiDIR gene family members. Particularly, SiDIR21, a member of the DIR-a subgroup, exhibited robust expression in lignan-accumulating tissues and consistently high expression levels in germplasm during seed development with high sesamin content. Furthermore, SiDIR21 overexpression in hairy roots significantly increased sesamin and sesamolin contents, confirming its role in lignan synthesis. Overall, our study offers a valuable resource for exploring SiDIRs’ functions and the lignan biosynthesis pathway in sesame.

A Novel Solid Media-Free In-Planta Soybean (Glycine max. (L) Merr.) Transformation Approach

Soybean’s lengthy protocols for transgenic plant production are a bottleneck in the transgenic breeding of this crop. Explants cultured on a medium for an extended duration exhibit unanticipated modifications. Stress-induced somaclonal variations and in vitro contaminations also cause substantial losses of transgenic plants. This effect could potentially be mitigated by direct shoot regeneration without solid media or in-planta transformation. The current study focused primarily on developing a rapid and effective media-free in-planta transformation technique for three soybean genotypes (Wm82) and our newly developed two hybrids, designated as ZX-16 and ZX-3. The whole procedure for a transgenic plant takes the same time as a stable grown seedling. Multiple axillary shoots were regenerated on stable-grown soybean seedlings without the ectopic expression of developmental regulatory genes. An approximate amount of 200 µL medium with a growth regulator was employed for shoot organogenesis and growth. The maximal shoot regeneration percentages in the Wm82 and ZX-3 genotypes were 87.1% and 84.5%, respectively. The stable transformation ranged from 3% to 8.0%, with an average of 5.5%. This approach seems to be the opposite of the hairy root transformation method, which allowed transgenic shoots to be regenerated on normal roots. Further improvement regarding an increase in the transformation efficiency and of this technique for a broad range of soybean genotypes and other dicot species would be extremely beneficial in achieving increased stable transformation.

Multiomics analysis reveals the involvement of OnDIVARICATA 3 in controlling dynamic flower coloring of Oncidium hybridum

Flower color is one of the main quality and economic traits of ornamental plants, and a large amount of research on flower color mainly focuses on the differences between varieties, while there were few reports on the change of flower color at different developmental stages. In this study, the metabolome and transcriptome of a new strain 'XM-1' with dynamic color changes of Oncidium were analyzed. The results showed that rutin, quercetin and carotenoids metabolism decreased significantly during the change of color from yellow to white. Analyzing the correlation network between metabolites and differential expressed genes, 25 key structural genes were detected and regulated by multiple MYB-related transcription factors. The MYB-related transcription factor Cluster-100966.1_OnDIVARICATA 3 was selected for further analysis. The phylogenetic tree of DIVARICATA in different species of Orchidaceae and Arabidopsis thaliana was constructed and the most closely related members were selected for sequence comparison. The results showed that OnDIVARICATA 3 contained MYB-like conserved domains. Subcellular localization results showed that OnDIVARICATA 3 was located in the nucleus. In overexpressing OnDIVARICATA 3 transgenic hairy roots, the expression of flower color related genes FLS, ZEP, and CHYB were significantly up-regulated. In summary, this study characterized the key metabolic pathways in the formation of the dynamic flower color of Oncidium hybridum, and constructed the regulatory network of the MYB-related. These results laid a theoretical foundation for the subsequent research on flower color and genetic engineering technology breeding of Oncidium hybridum.

The WRKY Family Transcription Factor GmWRKY72 Represses Glyceollin Phytoalexin Biosynthesis in Soybean.

Phytoalexins are plant defense metabolites that are biosynthesized transiently in response to pathogens. Despite that their biosynthesis is highly restricted in plant tissues, the transcription factors that negatively regulate phytoalexin biosynthesis remain largely unknown. Glyceollins are isoflavonoid-derived phytoalexins that have critical roles in protecting soybean crops from the oomycete pathogen Phytophthora sojae. To identify regulators of glyceollin biosynthesis, we used a transcriptomics approach to search for transcription factors that are co-expressed with glyceollin biosynthesis in soybean and stilbene synthase phytoalexin genes in grapevine. We identified and functionally characterized the WRKY family protein GmWRKY72, which is one of four WRKY72-type transcription factors of soybean. Overexpressing and RNA interference silencing of GmWRKY72 in the soybean hairy root system decreased and increased expression of glyceollin biosynthetic genes and metabolites, respectively, in response to wall glucan elicitor from P. sojae. A translational fusion with green fluorescent protein demonstrated that GFP-GmWRKY72 localizes mainly to the nucleus of soybean cells. The GmWRKY72 protein directly interacts with several glyceollin biosynthetic gene promoters and the glyceollin transcription factor proteins GmNAC42-1 and GmMYB29A1 in yeast hybrid systems. The results show that GmWRKY72 is a negative regulator of glyceollin biosynthesis that may repress biosynthetic gene expression by interacting with transcription factor proteins and the DNA of glyceollin biosynthetic genes.

Discovery of ElABCG39: a key player in ingenol transmembrane efflux identified through genome-wide analysis of ABC transporters in Euphorbia lathyris L.

Based on transport inhibition and genome-wide analysis, 123 ABC transporters of Euphorbia lathyris were identified, and it was found that the PDR family members ElABCG39 mediated ingenol efflux. Identification of ingenol biosynthetic enzymes and transporters in plant is fundamental to realize its biosynthesis in chassis cells. At present, several key enzymes of the ingenol biosynthesis pathway have been identified, while the mechanisms governing the accumulation or transport of ingenol to distinct plant tissue compartments remain elusive. In this study, transport inhibition analyses were performed, along with genome-wide identification of 123 genes encoding ABC proteins in Euphorbia lathyris L., eventually discovering that a PDR transporter ElABCG39 mediates ingenol transmembrane transport and is localized on the plasma membrane. Expression of this protein in yeast AD1-8 promoted the transmembrane efflux of ingenol with strong substrate specificity. Furthermore, in ElABCG39 RNAi transgenic hairy roots, ingenol transmembrane efflux was significantly reduced and hairy root growth was inhibited. The discovery of the first Euphorbia macrocyclic diterpene transporter ElABCG39 has not only further improved the ingenane diterpenoid biosynthesis regulatory network, but also provided a new key element for ingenol production in chassis cells.

A Rapid Method for Obtaining the Transgenic Roots of Crassulaceae Plants.

Crassulaceae plants are valued for their horticultural, ecological, and economic significance, but their genetic improvement is hindered by the absence of efficient and stable genetic transformation methods. Therefore, the development of a tailored genetic transformation method is crucial for enhancing the progress of the genetic improvement of Crassulaceae plants. The results indicate that, in the transformation experiments conducted on Kalanchoe tetraphylla, the K599 strain exhibited the highest transformation efficiency (76.67%), while C58C1 was least efficient (21.43%). An acetosyringone concentration of 100 μM was optimal for the hairy root transformation, and the immersion method yielded the highest efficiency. Additionally, the Silwet L-77 concentration significantly influenced the transformation efficiency, with 0.05% leading to a decrease. Upon four Crassulaceae species, notable differences were observed, with K. tetraphylla exhibiting the highest efficiency of 100%, and Sedum alfredii displaying the lowest efficiency of 5%. The RUBY reporter gene offers a more distinct advantage over GFP in observing the transformation effects. This study developed a simple, feasible, and cost-effective method for obtaining transgenic roots from leaves of Crassulaceae. The methodology provides technical support for the genetic improvement and gene function research of Crassulaceae plants.

Development of an efficient selection procedure for high imperatorin-yielding hairy root clones of Urena lobata L. using morphological and molecular markers

Development of an efficient selection procedure for high imperatorin-yielding hairy root clones of Urena lobata L. using morphological and molecular markers

Biomass and Phenolic Acid Accumulation in Salvia austriaca Hairy Roots Grown in Temporary Immersion and Mist-Trickling Bioreactors

Transformed roots of Salvia austriaca were cultivated for 45 days in various systems, including Erlenmeyer flasks, a temporary immersion system (TIS) bioreactor, and a mist-trickling bioreactor, under controlled light conditions. The mist-trickling bioreactor yielded the highest biomass, with fresh and dry weights of 155.4 g/L and 10.2 g/L, respectively. Quantitative UHPLC analysis of hydromethanolic extracts revealed the biosynthesis of significant phenolic acids: caffeic acid, rosmarinic acid, and salvianolic acid A. Among these, rosmarinic acid was the most abundant, with its concentration varying based on the cultivation system. The highest total phenolic acid content, 165 mg/L, was obtained in the mist-trickling bioreactor, demonstrating its superiority in both biomass production and phenolic acid biosynthesis. This study highlights the potential of mist-trickling bioreactors for optimizing growth and metabolite production in S. austriaca transformed root cultures.

Enhancing Agrobacterium-mediated soybean transformation efficiency with an auxiliary solution

Soybean is a crucial source of oil, protein, and biofuel, necessitating efficient transformation systems for advancing research. Agrobacterium-mediated transformation is currently the primary method used in the soybean transformation industry and scientific research. However, the low efficiency and genotype dependency of this technology leave significant room for improvement. This study aimed to enhance soybean transformation efficiency by generating and validating three reporter vectors (ZsGreen, TdTomato, and Ruby) and using Agrobacterium Auxiliary Solution (AAS) containing Silwet L-77 and hormone mixtures. Our findings demonstrate that AAS significantly improves hairy root transformation rates. Specifically, this combination increased total root and cotyledon transformation efficiencies compared to the control. We also found that larger vectors like Ruby reduced transformation efficiency compared to smaller markers like GFP and RFP. Furthermore, AAS slightly reduced the co-transformation rate of two separate vectors compared to single vector transformations. Additionally, AAS enhanced soybean hypocotyl transformation rates across various varieties, consistently increasing positive root and explant efficiencies. Notably, transformation rates varied significantly between varieties, with Forrest differing from Williams 82 and Dongnong 50. This research highlights the importance of auxiliary agents and vector size in optimizing soybean transformation, providing insights for future advancements in genetic modification and biotechnology.

Border-like cell formation mediated by SgPG1 confers aluminum resistance in Stylosanthes guianensis.

Stylosanthes is an important forage legume in tropical areas with strong resistance to aluminum (Al) toxicity, though knowledge of mechanisms underlying this resistance remains fragmentary. We found that border-like cells (BLCs) were constitutively produced surrounding the root tips of all 54 examined Stylosanthes guianensis genotypes, but not the Stylosanthes viscose genotype TF0140. In genotypic comparisons under Al conditions, the S. guianensis genotype RY#2 retained significantly more Al in BLCs and thereby showed higher relative root growth than TF0140. Formation of BLCs accompanied changes in cell wall pectin epitopes and differential expression of genes involved in pectin metabolism, including a polygalacturonase (SgPG1). The expression pattern of SgPG1 was consistent with the formation of BLCs in both RY#2 and TF0140. SgPG1 was localized in cell walls and exhibited high activities mediating demethyl-esterified homogalacturonan degradation. Overexpressing SgPG1 changed cell wall pectin epitopes, enhanced BLCs production, and Al resistance in both Arabidopsis and Stylosanthes hairy roots. Furthermore, combining protein-DNA binding assays in vitro and in vivo, a bHLH transcription factor SgbHLH19 was demonstrated to be the upstream regulator of SgPG1. Our study demonstrates that S. guianensis Al resistance mainly relies on BLCs, whose formation involves cell wall pectin epitope modification by SgPG1.

Variability in growth and biosynthetic activity of Calendula officinalis hairy roots.

Calendula 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.