Agrobacterium Rhizogenes Research Articles

Collection of Hairy Roots as a Basis for Fundamental and Applied Research.

Due to population growth, instability of climatic conditions, and reduction of the areas of natural ecosystems, it becomes necessary to involve modern biotechnological approaches to obtain highly productive plant material. This statement applies both to the creation of plant varieties and the production of new pharmaceutical raw materials. Genetic transformation of valuable medicinal plants using Agrobacterium rhizogenes ensures the production of stable and rapidly growing hairy roots cultures that have a number of advantages compared with cell culture and, above all, can synthesize root-specific substances at the level of the roots of the intact plant. In this regard, special attention should be paid to the collection of hairy roots of the Institute of Plant Physiology RAS, Russian Academy of Sciences, the founder of which was Dr. Kuzovkina I.N. Currently, the collection contains 38 hairy roots lines of valuable medicinal and forage plants. The review discusses the prospects of creating a hairy roots collection as a basis for fundamental research and commercial purposes.

SmDXS5, acting as a molecular valve, plays a key regulatory role in the primary and secondary metabolism of tanshinones in Salvia miltiorrhiza.

Red sage, the dry root and rhizome of the herbaceous plant Salvia miltiorrhiza Bunge, is widely used for treating various diseases. The low content of tanshinones (terpenoids) has always restricted development of the S. miltiorrhiza industry. Here, we found that SmDXS5, a rate-limiting enzyme-coding gene located at the intersection of primary and secondary metabolism, can effectively change the transcription level and secondary metabolome profile of hairy roots of S. miltiorrhiza, and significantly increase the content of tanshinones. Agrobacterium rhizogenes was used to infuse S. miltiorrhiza explants, and hairy roots of S. miltiorrhiza expressing the SmDXS5 gene were obtained successfully. We identified 39 differentially accumulated metabolites (DAMs) by metabolomics based on ultra-high performance liquid chromatography quadrupole exactive mass spectrometry and multivariate statistics. These DAMs might be key metabolites of SmDXS5 gene regulation. RNA sequencing was used to compare gene expression between the hairy roots of the SmDXS5 overexpressing group and the blank control (BC) group. Compared with the BC group, 18,646 differentially expressed genes were obtained: 8994 were upregulated and 9,652 downregulated. The combined transcriptome and metabolome analyses revealed that the mevalonate and methylerythritol phosphate pathways and synthase gene expression levels in the SmDXS5 overexpressing group were upregulated significantly, and the accumulation of tanshinone components was increased significantly, which promoted the process of glycolysis and promoted the transformation of carbohydrates to secondary metabolism. Moreover, the expression of SmPAL, the first rate-limiting enzyme gene of the phenylpropane pathway, decreased, reducing the accumulation of phenolic acid, another secondary metabolite. Therefore, SmDXS5 can be defined as a 'valve' gene, mainly responsible for regulating the distribution of primary and secondary metabolic flow of tanshinones in S. miltiorrhiza, and for other secondary metabolic pathways. The discovery of SmDXS5 and its molecular valve function in regulating primary and secondary metabolism will provide a basis for the industrial production of tanshinone components, and cultivation of high quality S. miltiorrhiza.

Biological control of hairy root disease using beneficial Pseudomonas strains

Hairy root disease (HRD) caused by Agrobacterium rhizogenes is a problematic disease leading to substantial losses in marketable yield in hydroponically grown crops including cucumbers (Cucumis sativus) and tomatoes (Solanum lycopersicum). Growers use strict hygiene protocols and sanitation, relying on hydrogen peroxide and other chemical-based sanitizers to clean the hydroponic system after disease outbreaks, however, this can be an expensive and time-consuming process. Biological control is emerging as a possible solution to this troubling problem. Biological control is an environmentally sound and effective means of reducing or mitigating pests and diseases using natural enemies. In this work we tested and identified numerous strains of Pseudomonas that can inhibit the growth of A. rhizogenes under in vitro and in planta conditions. In our in vitro experiments, 13 out of 49 different Pseudomonas strains tested were able to inhibit pathogen growth. Under in planta conditions we identified three different Pseudomonas strains (P. protegens 1B1, P. clororaphis 48G9, and P. brassicacearum 93G8) from the 13 in vitro-active strains that were able to reduce disease incidence up to 95% on Kalanchoe, tomato, and soybean. Pseudomonas treatments were able to reduce Agrobacterium rhizogenes numbers by nearly 1,000-fold in soybean and 100-fold in tomato. On hydroponically grown tomatoes, strains 1B1 and 93G8 were able to reduce disease incidence by 80%, and strain 48G9 was able to reduce disease incidence by 50% as compared with the control. We also screened the remaining collection on tomato (potting media) aiming to identify strains that may have been missed in the in vitro study. From this study we identified five additional strains that were effective in reducing disease incidence on potting media. We further tested strains 93F8, Wood3, and 90F12-1 in our hydroponic system and found that strain 93F8 reduced disease by 62%, Wood3 by 50%, and 90F12-1 by 55%. Our results suggest that certain Pseudomonas strains can inhibit A. rhizogenes growth and disease development under hydroponic conditions and may be suitable for use as biocontrol agent(s) for the control of HRD in hydroponically grown tomatoes.

Increasing the production of limonin 17-β-D-glucopyranoside in hairy roots of Citrus × paradisi. cv Duncan by overexpression of limonoid UDP-glucosyltransferase via Agrobacterium rhizogenes mediated transformation

Limonin 17-β-D-glucopyranoside (LG) is a terpenoid compound with a high medicinal value produced in different Citrus species. This substance has high anti-cancer and antioxidant properties and protects the liver from damage. Although LG is tasteless, many compounds related to it are very bitter, although these bitter substances also have significant medicinal properties. Usually, in most Citrus genotypes, bitter-tasting limonoids are produced more than tasteless types, which are unpleasant despite their therapeutic value. The aim of our study was the in vitro production of LG at high concentrations. For this purpose, hairy roots were induced in ‘Duncan’ grapefruit (Citrus × paradisi) leaf explants. Also, the limonoid UDP-glucosyltransferase (LGTase) gene was transferred from Citrus aurantium to hairy roots by Agrobacterium rhizogenes and forced to overexpression. Two different sets of hairy roots were obtained. The wildtype group only received the Ri plasmid, while cotransformation with the LGTase overexpression constructs in the transgenic group. The gene expression analysis showed that the transcription of the LGTase gene in transgenic hairy roots dramatically increases compared with wildtypes. The overexpression of LGTase significantly increased the total concentration of limonoids in transgenic hairy roots compared with leaves and non-transgenic roots. Evaluation of HPLC chromatograms showed that transgenic hairy roots produced more LG than other samples. In some cases, the concentration of LG increases up to 11 times compared with leaves in some lines. In addition, the concentration ratio of LG to limonin increased in transgenic lines, and transgenic types produced the most LG. Enhancing LGTase gene expression can be a suitable approach to producing high amounts of LG in hairy roots.

Expression sequences of opine synthase genes in natural GMOs based on analysis of their transcriptomes

Agrobacterium is a natural genetic material delivery system that humans use to produce genetically modified plants (GMO). In nature, GMOs also occur with the participation of agrobacteria. In 2019, the list of known natural GMOs was expanded by an order of magnitude, and facts were found in favor of the expression of agrobacterial genes in natural GMOs. The frequency of this phenomenon for dicotyledon plants has been estimated at 7 percent. Opine synthase genes turned out to be the predominant ones of agrobacterial origin in natural GMOs. They probably perform important functions in natural GMOs. In 2021, an article was published with an updated list of natural GMOs, but the list of genes expressed in natural GMOs has not been updated since 2019.The aim of this work is to update the list of opine synthase genes expressed in natural GMOs. The research methods included bioinformatic search using queries based on the sequences of opine synthase proteins from Agrobacterium rhizogenes, A. tumefaciens and A. vitis, their homologues from Ipomoea and Nicotiana plants, in the TSA database of the National Center for Biotechnology Information (NCBI) using the TBLASTN algorithm with default settings.The study resulted in the addition of another 18 species to the list of natural GMOs with expressed opine synthase genes, 12 of which belong to genera where natural GMOs were not previously described (Albizia, Cenostigma, Averrhoa, Gynostemma, Eurycoma, Gypsophila, Myosoton, Camptotheca, Gustavia, Eschweilera, Cestrum, Jasminum, and Paulownia).An analysis of the diversity of the detected sequences showed that homologues of cucumopine and mikimopine synthase predominate among them. The end products of these genes are optical isomers. In the future, it makes sense to start studying the functions of opine synthases in plants from these genes.

The RolB/RolC homolog from sweet potato promotes early flowering and triggers premature leaf senescence in transgenic Arabidopsis thaliana plants

Expression of the root oncogenic loci (rol) genes from Agrobacterium rhizogenes provokes multiple divergent effects on physiological properties in transgenic plants and cell cultures. Recently, the homolog of the rolB and rolC oncogenes, named Ib-rolB/C, has been identified in the genome of a naturally transgenic food crop, i.e. sweet potato. In this study, we revealed that the Ipomoea batatas genome contains two full-length copies of Ib-rolB/C. The expression level of Ib-rolB/C in leaves of sweet potato showed a clear age-dependent pattern and increased as leaves senesce. Moreover, dark-induced senescence strongly up-regulates transcription of the Ib-rolB/C gene. Though Ib-rolB/C shares homology with its counterparts in A. rhizogenes, this gene was not capable to induce hairy roots or tumors in kalanchoe and tobacco plants. The Ib-rolB/C gene induced early-flowering phenotype, altered leaf morphology, and promoted premature leaf senescence in transgenic Arabidopsis thaliana plants. At the same time, Ib-rolB/C did not affect root morphology and biomass. Our results suggest that Ib-RolB/RolC participates in both age- and dark-triggered leaf senescence programs.

Cut–dip–budding delivery system enables genetic modifications in plants without tissue culture

Of the more than 370 000 species of higher plants in nature, fewer than 0.1% can be genetically modified due to limitations of the current gene delivery systems. Even for those that can be genetically modified, the modification involves a tedious and costly tissue culture process. Here, we describe an extremely simple cut-dip-budding (CDB) delivery system, which uses Agrobacterium rhizogene to inoculate explants, generating transformed roots that produce transformed buds due to root suckering. We have successfully used CDB to achieve the heritable transformation of plant species in multiple plant families, including two herbaceous plants (Taraxacum kok-saghyz and Coronilla varia), a tuberous root plant (sweet potato), and three woody plant species (Ailanthus altissima, Aralia elata, and Clerodendrum chinense). These plants have previously been difficult or impossible to transform, but the CDB method enabled efficient transformation or gene editing in them using a very simple explant dipping protocol, under non-sterile conditions and without the need for tissue culture. Our work suggests that large numbers of plants could be amenable to genetic modifications using the CDB method.

AtGCS promoter-driven clustered regularly interspaced short palindromic repeats/Cas9 highly efficiently generates homozygous/biallelic mutations in the transformed roots by Agrobacterium rhizogenes-mediated transformation.

Agrobacterium rhizogenes-mediated (ARM) transformation is an efficient and powerful tool to generate transgenic roots to study root-related biology. For loss-of-function studies, transgenic-root-induced indel mutations by CRISPR/Cas9 only with homozygous/biallelic mutagenesis can exhibit mutant phenotype(s) (excluding recessive traits). However, a low frequency of homozygous mutants was produced by a constitutive promoter to drive Cas9 expression. Here, we identified a highly efficient Arabidopsis thaliana gamma-glutamylcysteine synthetase promoter, termed AtGCSpro, with strong activity in the region where the root meristem will initiate and in the whole roots in broad eudicots species. AtGCSpro achieved higher homozygous/biallelic mutation efficiency than the most widely used CaMV 35S promoter in driving Cas9 expression in soybean, Lotus japonicus, and tomato roots. Using the pAtGCSpro-Cas9 system, the average homozygous/biallelic mutation frequency is 1.7-fold and 8.3-fold higher than the p2 × 35Spro-Cas9 system for single and two target site(s) in the genome, respectively. Our results demonstrate the advantage of the pAtGCSpro-Cas9 system used in ARM transformation, especially its great potential in diploids with multiple-copy genes targeted mutations and polyploid plants with multiplex genome editing. AtGCSpro is conservatively active in various eudicots species, suggesting that AtGCSpro might be applied in a wide range of dicots species.

Effects of various Agrobacterium rhizogenes strains on hairy root induction and analyses of primary and secondary metabolites in Ocimum basilicum.

The hairy root (HR) culture system is an excellent alternative strategy to the whole plant system for producing valuable compounds. However, selection of suitable Agrobacterium strain for the successful induction of HR is an essential step for enhanced production of beneficial secondary metabolites. In this study, we examined the transformation efficiency of various A. rhizogenes strains (ATCC 13333, ATCC 15834, A4, R1000, R1200, and R1601) for transgenic HRs induction in Ocimum basilicum. Among the tested strains, the R1601 was found to be one of the most promising strain for mass production of HR in terms of transformation efficiency (94%) and the number and length of HR (8.4 ± 0.52 and 1.68 ± 0.14 cm). The HR induced by the same strain exhibited highest levels of rosmarinic acid level (62.05 ± 4.94 µg/g DW) and total phenolic content (62.3 ± 4.95 µg/g DW). A total of 55 metabolites were identified using high-performance liquid chromatography (HPLC) and gas chromatography-time-of-flight mass spectrometry (GC-TOFMS). The PCA and PLS-DA plot of the identified metabolites showed that HR induced by A4 and ATCC 15834 displayed variation in primary and secondary metabolite contents. Analysis of the metabolic pathway identified a total of 56 pathways, among which 35 were found to be impacted. A heat map and hierarchical clustering analysis indicated that HR induced by different Agrobacterium strains exhibited differential metabolites profiles. In conclusion, Agrobacterium strains R1601 is one of the best and most promising strains for inducing mass HR production and enhanced levels of secondary metabolites in O. basilicum.

An Efficient Agrobacterium rhizogenes-Mediated Hairy Root Transformation Method in a Soybean Root Biology Study.

The stable genetic transformation of soybean is time-consuming and inefficient. As a simple and practical alternative method, hairy root transformation mediated by Agrobacterium rhizogenes is widely applied in studying root-specific processes, nodulation, biochemical and molecular functions of genes of interest, gene editing efficiency of CRISPR/Cas9, and biological reactors and producers. Therefore, many laboratories have developed unique protocols to obtain hairy roots in composite plants composed of transgenic roots and wild-type shoots. However, these protocols still suffer from the shortcomings of low efficiency and time, space, and cost consumption. To address this issue, we developed a new protocol efficient regeneration and transformation of hairy roots (eR&T) in soybean, by integrating and optimizing the main current methods to achieve high efficiency in both hairy root regeneration and transformation within a shorter period and using less space. By this eR&T method, we obtained 100% regeneration of hairy roots for all explants, with an average 63.7% of transformation frequency, which promoted the simultaneous and comparative analysis of the function of several genes. The eR&T was experimentally verified Promoter:GUS reporters, protein subcellular localization, and CRISPR/Cas9 gene editing experiments. Employing this approach, we identified several novel potential regulators of nodulation, and nucleoporins of the Nup107-160 sub-complex, which showed development-dependent and tissue-dependent expression patterns, indicating their important roles in nodulation in soybean. Thus, the new eR&T method is an efficient and economical approach for investigating not only root and nodule biology, but also gene function.

Characterization of Carnivorous Plants Sarracenia purpurea L. Transformed with Agrobacterium rhizogenes

People have used carnivorous plants of the genus Sarracenia in folk medicine for centuries due to the biochemical composition of Sarracenia plants, which are rich in numerous bioactive compounds with anti-inflammatory, antioxidant, antiviral and antibacterial properties. The subject of this study was the genetic transformation of Sarracenia purpurea L. with Agrobacterium rhizogenes strains 15834, 9402 and A4 using two different methods: bacterial injection or co-culture of the bacteria with plant explants. This study confirmed the possibility of hairy root induction in S. purpurea using A. rhizogenes strain 15834 and the injection method. Seven lines of transformed plants, exhibiting the integration of the rolB gene, were obtained. The hairy roots formed showed morphological differences in comparison to the roots of unmodified plants. A mathematical model was used to optimize the conditions for the extraction of bioactive compounds. Extracts isolated under optimal conditions from the transformed plants showed biochemical changes, i.e., an increase in the accumulation of total polyphenols (line 7#1 in hairy roots: 71.048 mg GAE g−1 DW; in leaves: 9.662 mg GAE g−1 DW) and triterpenes (line 7#1 in hairy roots: 1.248 mg BA g−1 DW; in leaves: 0.463 mg BA g−1 DW) in comparison to untransformed plants (polyphenols in roots: 7.957 mg GAE g−1 DW and in leaves: 5.091 mg GAE g−1 DW; triterpenes in roots: 0.298 mg BA g−1 DW and in leaves: 0.296 mg BA g−1 DW), especially when induced roots were analyzed. HPLC analysis showed an increase in the level of betulinic acid in some transformed Sarracenia lines. Betulinic acid is a pentacyclic triterpenoid compound with high pharmacological significance.

Anthocyanin pigmentation as a quantitative visual marker for arbuscular mycorrhizal fungal colonization of Medicago truncatula roots.

Visualization of root colonization by arbuscular mycorrhizal fungi (AMF) is the most elementary experiment in the field of mycorrhizal symbiosis. The most widely used approach for evaluating levels of AMF colonization is staining with trypan blue or ink, which is scored using the time-consuming grid intersection method. Here we demonstrate the use of an anthocyanin-based visual marker system for visualizing AMF colonization of Medicago truncatula roots. Expression of MtLAP1, a transcription factor which regulates the production of anthocyanins, from the AMF-induced Kunitz Protease Inhibitor 106 promoter, allowed the visualization of arbuscules in live plant tissues without microscopy or staining. This marker system allowed straightforward qualitative evaluation of the ram1, vpy and dmi3 AMF phenotypes using Agrobacterium rhizogenes hairy-root transformation. For the strigolactone biosynthesis mutant carotenoid cleavage dioxygenase 8a and a novel mutant scooby, which show quantitative AMF symbiotic phenotypes, the amount of anthocyanins in the roots estimated by spectrophotometry correlated very well with colonization levels estimated by staining and scoring using the grid intersection method. The LAP1-based marker system therefore provides a highly efficient approach for mutant screening and monitoring of AMF colonization in live tissues by eye, or for quantitative assessment using a simple and quick photometric assay.

Genome-wide identification, expression, and characterization of CaLysM-RLKs in chickpea root nodule symbiosis

Legumes establish a nitrogen-fixing symbiosis with rhizobia which depends on the exchange of chemical signals between the two partners. Successful interaction between legumes and rhizobia depends on the perception of Nod factors (NFs) by the receptor-like kinases. A few cell surface receptors have been identified that perceive the rhizobial NFs and activate the downstream signaling pathway for nodulation in model legumes like Lotus japonicus and Medicago truncatula . However, crop legume such as chickpea lacks detailed analysis of LysM-RLKs genes for NFs perception. Here, we identified and characterized CaLysM-RLK genes and analyzed their gene structure, motif composition, chromosomal localization, phylogenetic relationship with Arabidopsis, Oryza sativa , and Medicago truncatula . To understand the spatiotemporal expression patterns, we performed transcript profiling of Ca LysM-RLKs genes at a different time interval of the chickpea-rhizobia symbiotic interaction that revealed CaNFP and CaLYK3 can act as candidate genes for NF perception. Down-regulation of CaNFP and CaLYK3 in chickpea by agrobacterium rhizogenes mediated hairy root sysytem leads to signifificant reduction in nodule numbers. This study will be useful for improving root nodule symbiosis in chickpea. • Seventeen CaLysM-RLKs genes were identified in chickpea. • Based on the structure CaLysM-RLKs family members were classified into three main groups LYS-I, LYS-II, and LYS-III. • Analysis of paralogous gene pair (PGP) leads to the identification of a total of 6 PGPs. • CaLYK3 , and CaNFP regulate root nodule symbiosis in chickpea.

Rosmarinic acid production in hairy root cultures of Salvia nemorosa L. (Lamiaceae)

Rosmarinic acid (RA) is a widely known and interesting medicinal phenolic acid produced in plant, cell, tissue, and organ in vitro cultures. In this study, hairy roots were induced using various strains of Agrobacterium rhizogenes (ATCC15834, A4, R1000, and GM1534). Furthermore, the effect of sucrose, IBA anddifferent in vitro basal medium (MS, ½ MS) on the hairy root generation and RA production was studied. ATCC15834 strain showed the maximum transformation efficiency (76%). Visible growth of the roots was observed in liquid MS medium containing 0.1 mg/L IBA. Based on a time-course experiment, the highest biomass production and RA content were observed after 2 weeks. The highest biomass (9.28 g/L DW) and RA content (15.9 mg/g DW) from the hairy root culturs were obtained in ½ MS medium supplemented with 1% and 3% sucrose, respectively. The results demonstrated high possible potential of S. nemorosa hairy root cultures on RA production.

Using machine learning to link the influence of transferred Agrobacterium rhizogenes genes to the hormone profile and morphological traits in Centella asiatica hairy roots.

Hairy roots are made after the integration of a small set of genes from Agrobacterium rhizogenes in the plant genome. Little is known about how this small set is linked to their hormone profile, which determines development, morphology, and levels of secondary metabolite production. We used C. asiatica hairy root line cultures to determine the putative links between the rol and aux gene expressions with morphological traits, a hormone profile, and centelloside production. The results obtained after 14 and 28 days of culture were processed via multivariate analysis and machine-learning processes such as random forest, supported vector machines, linear discriminant analysis, and neural networks. This allowed us to obtain models capable of discriminating highly productive root lines from their levels of genetic expression (rol and aux genes) or from their hormone profile. In total, 12 hormones were evaluated, resulting in 10 being satisfactorily detected. Within this set of hormones, abscisic acid (ABA) and cytokinin isopentenyl adenosine (IPA) were found to be critical in defining the morphological traits and centelloside content. The results showed that IPA brings more benefits to the biotechnological platform. Additionally, we determined the degree of influence of each of the evaluated genes on the individual hormone profile, finding that aux1 has a significant influence on the IPA profile, while the rol genes are closely linked to the ABA profile. Finally, we effectively verified the gene influence on these two specific hormones through feeding experiments that aimed to reverse the effect on root morphology and centelloside content.

In vitro and In silico Studies on Leonotis nepetifolia (L.) R. Br. Root Extract against Cancer Cells.

Leonotis nepetifolia (L.) R. Br. (Lamiaceae) is a shrub traditionally used to alleviate inflammatory conditions. The present study aimed at investigating the biological activity of methanolic nontransformed and transformed Rhizobium rhizogenes root extracts from L. nepetifolia against human melanoma cells. Cytotoxicity and genotoxicity properties, the impact on topoisomerase I activity, and proapoptotic activity were evaluated by the MTT test, comet assay, topoisomerase I assay, and fluorescence-activated cell sorting analysis. Moreover, the expressions of p53 were examined by qPCR and Western blot analysis. Docking studies were conducted to assess the potential interactions of the identified phytochemicals with the p53 binding protein Mdm-2, and computational analyses exhibited their antioxidant potential. Both extracts showed cytotoxic potential against human melanoma cells, but generally the activity was more potent for transformed roots than untransformed (IC50 760 μg/mL and 980 μg/mL, respectively). A similar effect was revealed during the evaluation of genotoxic and proapoptotic properties. Moreover, the expression of p53 was also found to be increased after extract treatment. The most dominant identified compounds in both extracts were as follows: (+)- catechin, p-coumaric acid, m-coumaric acid, and (+)-rosmarinic acid. Docking studies and computational analysis showed that (+)-rosmarinic acid possesses the highest binding affinity to the p53 binding protein, Mdm-2, and exhibits the best antioxidant property from the most commonly identified phytochemicals. Our findings revealed the potential of L. nepetifolia transformed root extract as a source of bioactive compounds with cytotoxic, genotoxic, and proapoptotic activity against human melanoma cells as well as antioxidant properties.

Transcriptome analysis of genes related to glucoraphanin and sulforaphane synthesis in methyl jasmonate treated broccoli (Brassica oleracea var. italica) hairy roots.

Hairy roots obtained by infecting broccoli (Brassica oleracea var. italica) leaves with Agrobacterium rhizogenes (ATCC15834) have the characteristics of phytohormone autonomy, genetic stability and can produce a large amount of the anti-cancer substance Sulforaphane (SF) and the biosynthetic precursor Glucoraphanin (GRA). Under the induction of the exogenous signaling molecule methyl jasmonate (MeJA), the production of SF in broccoli hairy roots was significantly increased. However, the molecular mechanism of GRA and SF synthesis in hairy roots of broccoli treated with MeJA has not been reported. In this study, according to the yield of GRA and SF, the best concentration of MeJA treatment for hairy roots of broccoli was selected. After 18days of growth, broccoli hairy roots were treated with 10mmol L-1 MeJA for 0, 3, 6, 9 and 12h. Compared with 0h, the yield of GRA and SF increased under other treatments. The highest yield of GRA and SF occurred at 9h, which were 2.22-fold and 1.74-fold higher than those at 0h. Brassica oleracea var. botrytis was used as reference genome, and 5,757 differentially expressed genes (DEG) were observed at 0, 3, 6, 9 and 12h under 10mmol L-1 MeJA treatment, of which 4,673 were down-regulated and 1084 were up-regulated. The key genes regulating GRA synthesis, CYP79F1, CYP83A1, UGT74B1, FMOGS-OX5 and GSL-OH, were up-regulated at 0 and 3h, and down-regulated the rest of the time; BCAT2 was up-regulated at 6, 9, 12h, and at 0, 3h expression was down-regulated, transcription factors MYB28 and MYB29 were down-regulated by exogenous MeJA treatment. A pathway of GRA biosynthesis and transformation pathways in MeJA-treated broccoli hairy roots was simulated and the molecular mechanism of GRA biosynthesis and SF accumulation in broccoli hairy roots under MeJA treatment was revealed.

Glucoraphanin and sulforaphane biosynthesis by melatonin mediating nitric oxide in hairy roots of broccoli (Brassica oleracea L. var. italica Planch): insights from transcriptome data

Glucoraphanin (GRA) is present in the seeds and nutrient organs of broccoli and is the precursor of the anti-cancer compound sulforaphane (SF). The hairy roots obtained by infecting broccoli (Brassica oleracea L. var. Italic Planch) leaves with Agrobacterium rhizogenes (ATCC15834) are phytohormonally autonomous, genetically stable, and can produce large amounts of the anti-cancer substance SF. Melatonin (MT) is a natural hormone widely found in plants. Studies have shown that melatonin can regulate the synthesis of secondary metabolites of downstream targets by mediating the synthesis of signal molecules. However, whether MT regulates the synthesis of NO and H2O2 and mediates the synthesis mechanism of secondary metabolites, GRA and SF, is not yet clear. In this study, the hairy roots of broccoli were treated with 500 μmol/L MT, and the genome of broccoli (Brassica oleracea L. var. botrytis L) was used as the reference genome for transcriptome analysis. By this approach, we found that MT regulates the synthesis of NO and H2O2 and mediates the synthesis of secondary metabolites GRA and SF. GO annotations indicated that DEGs involved in the MT treatment of broccoli hairy roots were mainly related to catalytic activity, cells, and metabolic processes; the KEGG pathway analysis indicated that MT treatment likely affects the hormone signal transduction process in broccoli hairy roots; broccoli hairy roots were treated with 500 μmol/L MT for 0, 6, 12, 20, and 32 h, respectively; compared with 0 h, the yield of GRA and SF increased under the other treatments. The highest yields of GRA and SF occurred at 12 h. The NO content was the highest at 12 h, and the H2O2 content was positively correlated with MT concentration. The content of NO and H2O2 were regulated, and the content of GRA and SF was increased under MT treatment. NO synthase inhibitor (L-NAME and TUN) could effectively inhibit the content of NO in broccoli hairy roots and reduce GRA and SF yield; MT could regulate NO levels by regulating NO synthesis-related enzymes and could alleviate the reduction of NO content in tissue cells caused by NO synthase inhibitor and promote NO synthesis. These results have important theoretical implications for understanding the regulation of GRA and SF synthesis events by NO and H2O2.

Novel Small Antimicrobial Peptides Extracted from Agricultural Wastes Act against Phytopathogens but Not Rhizobacteria

Nonedible materials such as agricultural wastes can serve as sources of antimicrobial peptides (AMPs) effective against bacterial plant pathogens. In this study, thirteen agricultural samples were collected and their protein hydrolysates obtained using pepsin. Peptides smaller than 3 kDa were purified by reverse-phase chromatography, cation exchange chromatography, and pI-based fractionation and tested for activity against plant pathogenic bacteria at each step. Active peptides were then analyzed for putative mechanisms using nanoLC–MS/MS and the Mascot program. Ultimately, eight candidate peptides originating from bagasse were selected and chemically synthesized for a comparative study of growth inhibition in plant pathogenic bacteria and plant growth-promoting rhizobacteria (PGPRs). Three synthesized peptides exhibited a potent activity against plant pathogenic bacteria while also supporting the growth of PGPRs. Proteomics analysis revealed the peptides PQLAVF (Pro-Gln-Leu-Ala-Val-Phe) and MDRFL (Met-Asp-Arg-Phe-Leu) to act against Xanthomonas oryzae pv. oryzae via membrane-active mechanisms, while peptide VQLMNSL (Val-Gln-Leu-Met-Asn-Ser-Leu) acted against Pectobacterium carotovorum and Agrobacterium rhizogenes through intracellular-active mechanisms. Further study remains necessary to customize peptides by amino acid substitution not only for a higher effective activity against these and other critical pathogens, but also for a higher stability of peptides in critical condition when applied in industrial processes in the future.

A high yield method for protoplast isolation and ease detection of rol B and C genes in the hairy roots of cauliflower (Brassica oleracea L.) inoculated with Agrobacterium rhizogenes

Protoplasts represent a unique experimental system for the circulation and formation of genetically modified plants. Here, protoplasts were isolated from genetically modified hairy root tissues of Brassica oleracea L. induced by the Agrobacterium rhizogenes strain (ATCC13332). The concentration of enzyme solutions utilized for protoplast isolation was 1.5 % Cellulase YC and 0.1 % Pectolyase Y23 in 13% mannitol solution, which resulted in high efficiency of isolation within 8 hours, in which the protoplast yield was 2 x 104 cells ml-1 and the percentage of viability was 72%. Each protoplast has one nucleus with a nucleation of 48%. A polymerase chain reaction (PCR) assay verified the presence of rol B and rol C genes in hairy root tissues by detaching a single bundle of DNA replication from these roots using a specific pair of primers. The current study demonstrated that A. rhizogenes strain (ATCC13332) is a vector for the incorporation of T-DNA genes into cauliflower plants, as well as the success of the hairy roots retention of rol B and rol C genes transferred to it.