Carrot Hairy Roots Research Articles

Production of species-specific anthocyanins through an inducible system in plant hairy roots

Anthocyanins are widely distributed pigments in flowering plants with red, purple or blue colours. Their properties in promoting heath make anthocyanins perfect natural colourants for food additives. However, anthocyanins with strong colour and stability at neutral pH, suitable as food colourants are relatively rare in nature. Acylation increases anthocyanin stability and confers bluer colour. In this study, we isolated two anthocyanin regulators SbMyb75 and SbDel from S. baicalensis, and showed that constitutive expression of the two TFs led to accumulation of anthocyanins at high levels in black carrot hairy roots. However, these hairy roots had severe growth problems. We then developed a β-estradiol inducible system using XVE and a Lex-35S promoter, to initiate expression of the anthocyanin regulators and induced this system in hairy roots of black carrot, tobacco and morning glory. Anthocyanins with various decorations were produced in these hairy roots without any accompanying side-effects on growth. We further produced highly acylated anthocyanins with blue colour in a 5 L liquid culture in a bioreactor of hairy roots from morning glory. We provide here a strategy to produce highly decorated anthocyanins without the need for additional engineering of any of the genes encoding decorating enzymes. This strategy could be transferred to other species, with considerable potential for natural colourant production for the food industries.

Carrot hairy roots (Daucus carota L.) characterisation and optimisation for high β‐carotene extraction

Hairy roots are widely known as a biological system for the production of highly diverse biomolecules. β‐carotene – a precursor for vitamin A – is known to be an anti‐oxidant and anti‐gastric cancer and protection agent against cardiovascular disease, heart disease and stroke. β‐carotene has been chemically synthesised and consumed by humans. However, the chemical process often produces a by‐product that may be harmful to human health. Therefore, this study established a protocol to induce hairy roots (HRs) from a Vietnamese carrot variety and produce natural β‐carotene. The Rhizobium rhizogenes ATCC15834 harbouring Ri plasmid and a Vietnamese carrot variety were used as materials for genetic transformation and HR induction studies. The result showed that approximately 50 HR lines were obtained. Culture medium supplemented with 30 mg/L of sucrose that gave the highest biomass of HR was shown in carrot HR line 30, which had a doubling time of 6.5 days. The highest content of β‐carotene extraction, at 128 mg/100g hairy roots, was achieved with a ratio volume (v/v) of 2‐propanol and plant samples of 20:1, followed by two hours’ incubation with 2‐propanol at 60 °C. Our study reveals a highly efficient protocol for Vietnamese carrot hairy root establishment and multiplication. A very efficient protocol for β‐carotene extraction from the hairy root was established to produce natural β‐carotene that achieves the same β‐carotene quantity as that produced by normal roots. This study provides new insight into the production of high‐content and natural β‐carotene for therapeutic application.

Transgenic plants-immunomodulators for animal husbandry

In modern pharmacology, there is a tendency to move from low molecular weight drugs to protein drugs. Interferon is among them. Interferon is used in animal husbandry to prevent and treat viral and bacterial diseases, as a single drug and as an adjuvant for vaccines and antibiotics to increase their efficiency and reduce the amount of use. After interferon therapy, meat, milk and eggs can be consumed without restrictions. Currently, interferon for veterinary purposes is obtained using genetically modified organisms (mainly bacteria). The limiting factor is the high cost of the active substance isolating and purifying, which is up to 8090% of the product cost. Edible plants-producers can solve this problem. Plants have the lowest price per unit of biomass, are not infected by mammalian pathogens, and have an eukaryotic system of protein synthesis.
 Transgenic tobacco plants (Nicotiana tabacum L.) for the production of bovine gamma-interferon were obtained by agrobacterium mediated transformation at the Department of Genetics and Biotechnology, Saint Petersburg State University. Testing of this model showed that interferon is successfully produced in plants and retains biological activity, including oral administration. Problems were also identified: tobacco toxicity and low levels of protein accumulation. At the next stage of the experiment, it was decided to transform edible carrot (Daucus carota) plants, modify the target protein (shorten) to increase its resistance to proteolytic degradation, and use the pSRD1 root-specific promoter. At present, the corresponding transgenic constructs have been obtained, the effectiveness of which will be determined first on the carrot hairy roots, and then on whole carrot plants.
 The research was supported by the Ministry of Science and Higher Education of the Russian Federation in accordance with agreement No. 075-15-2020-922 date 16.11.2020 on providing a grant in the form of subsidies from the Federal budget of Russian Federation. The grant was provided for state support for the creation and development of a World-class Scientific Center Future Agrotechnologies.

H2O2-Elicitation of Black Carrot Hairy Roots Induces a Controlled Oxidative Burst Leading to Increased Anthocyanin Production.

Hairy roots (HRs) grown in vitro are a powerful platform for plant biotechnological advances and for the bio-based production of metabolites of interest. In this work, black carrot HRs able to accumulate anthocyanin as major secondary metabolite were used. Biomass and anthocyanin accumulation were improved by modulating growth medium composition—different Murashige & Skoog (MS)-based media—and H2O2-elicitation, and the level of the main antioxidant enzymes on elicited HRs was measured. Higher growth was obtained on liquid 1/2 MS medium supplemented with 60 g/L sucrose for HRs grown over 20 days. In this medium, 200 µM H2O2 applied on day 12 induced anthocyanin accumulation by 20%. The activity of superoxide dismutase (SOD)—which generates H2O2 from O2•−—increased by over 50%, whereas the activity of H2O2-scavenging enzymes was not enhanced. Elicitation in the HRs can result in a controlled oxidative burst, in which SOD activity increased H2O2 levels, whereas anthocyanins, as effective reactive oxygen species scavengers, could be induced to modulate the oxidative burst generated. Moreover, given the proven stability of the HR lines used and their remarkable productivity, this system appears as suitable for elucidating the interplay between antioxidant and secondary metabolism.

Carrot hairy roots: factories for secondary metabolite production

This article comments on:Barba-Espín G, Chen S-T, Agnolet S, Hegelund JN, Stanstrup J, Christensen JH, Müller R, Lütken H. 2020. Ethephon-induced changes in antioxidants and phenolic compounds in anthocyanin-producing black carrot hairy root cultures. Journal of Experimental Botany 71, 7030–7045.

Expression of Heat-labile Enterotoxin of Escherichia coli in Biolistic Transformed Hairy Roots of Daucus carota L.

Expression and secretion of recombination proteins in transgenic hairy roots have opened door to simple, feasible and economic option compared to animal and bacterial systems. Here, we report a feasibility study for producing the nontoxic B subunit of Escherichia coli heatlabile enterotoxin (LTB) in Daucus carota L hairy root where transformation is achieved by biolistic gene gun method. Agrobacterium rhizogenes strain (R-1000) cells containing synthetic LTB gene in pMYO51T plant expression vector under the CaMV 35S promoter introduced to Daucus carota L roots by biolistic gene gun method. PCR amplification confirmed the integration of synthetic LTB gene in carrot hairy roots. Western blot analysis confirmed production of LTB pentamer in similar banding pattern to the native bacterial derived LTB. 0.50-0.70% of LTB protein estimated in carrot hairy roots. GM1-ganglioside assay indicated formation of biological active pentamers. LTB protein with biochemical properties identical to native LTB protein in the hairy roots of edible carrot roots opens the way for inexpensive, safe, and effective plant-based edible vaccines for humans and animals.

Ethephon-induced changes in antioxidants and phenolic compounds in anthocyanin-producing black carrot hairy root cultures

Hairy root (HR) cultures are quickly evolving as a fundamental research tool and as a bio-based production system for secondary metabolites. In this study, an efficient protocol for establishment and elicitation of anthocyanin-producing HR cultures from black carrot was established. Taproot and hypocotyl explants of four carrot cultivars were transformed using wild-type Rhizobium rhizogenes. HR growth performance on plates was monitored to identify three fast-growing HR lines, two originating from root explants (lines NB-R and 43-R) and one from a hypocotyl explant (line 43-H). The HR biomass accumulated 25- to 30-fold in liquid media over a 4 week period. Nine anthocyanins and 24 hydroxycinnamic acid derivatives were identified and monitored using UPLC-PDA-TOF during HR growth. Adding ethephon, an ethylene-releasing compound, to the HR culture substantially increased the anthocyanin content by up to 82% in line 43-R and hydroxycinnamic acid concentrations by >20% in line NB-R. Moreover, the activities of superoxide dismutase and glutathione S-transferase increased in the HRs in response to ethephon, which could be related to the functionality and compartmentalization of anthocyanins. These findings present black carrot HR cultures as a platform for the in vitro production of anthocyanins and antioxidants, and provide new insight into the regulation of secondary metabolism in black carrot.

Rational design of biosafe crop resistance to a range of nematodes using RNA interference.

SummaryDouble‐stranded RNA (dsRNA) molecules targeting two genes have been identified that suppress economically important parasitic nematode species of banana. Proteasomal alpha subunit 4 (pas‐4) and Actin‐4 (act‐4) were identified from a survey of sequence databases and cloned sequences for genes conserved across four pests of banana, Radopholus similis, Pratylenchus coffeae, Meloidogyne incognita and Helicotylenchus multicinctus. These four species were targeted with dsRNAs containing exact 21 nucleotide matches to the conserved regions. Potential off‐target effects were limited by comparison with Caenorhabditis, Drosophila, rat, rice and Arabidopsis genomes. In vitro act‐4 dsRNA treatment of R. similis suppressed target gene expression by 2.3‐fold, nematode locomotion by 66 ± 4% and nematode multiplication on carrot discs by 49 ± 5%. The best transgenic carrot hairy root lines expressing act‐4 or pas‐4 dsRNA reduced transcript message abundance of target genes in R. similis by 7.9‐fold and fourfold and nematode multiplication by 94 ± 2% and 69 ± 3%, respectively. The same act‐4 and pas‐4 lines reduced P. coffeae target transcripts by 1.7‐ and twofold and multiplication by 50 ± 6% and 73 ± 8%. Multiplication of M. incognita on the pas‐4 lines was reduced by 97 ± 1% and 99 ± 1% while target transcript abundance was suppressed 4.9‐ and 5.6‐fold. There was no detectable RNAi effect on nontarget nematodes exposed to dsRNAs targeting parasitic nematodes. This work defines a framework for development of a range of nonprotein defences to provide broad resistance to pests and pathogens of crops.

English

Carrot hairy root induction by two different strains of Agrobacterium rhizogenes- MTCC- 532 and MTCC- 2364 grown in yeast extract mannitol agar medium (YEMA) was studied in carrot (Daucus carota). The maximum hairy root induction was observed when carrot was simmered with 48 h old culture of A. rhizogenes MTCC-532. With different concentrations of acetosyringone (AS) tested, addition of 150 µM acetosyringone was found to enhance the transformation frequency up to 75(±2.60)a and 60 (±2.08)a percentage by A. rhizogenes MTCC-532 and MTCC 2364 strains, respectively. Transformation efficiency was highly dependent on the acetosyringone concentrations, type of bacterial strains and carrot genotype. Transfer of Ri Ti-DNA was confirmed by polymerase chain reaction (PCR) analysis, the detection of ags gene in transformed carrot hairy root. A. rhizogenes transformed hairy roots had the ability to form copious lateral roots as well as a negative geotropic growth habit in a shorter period of time. The Murashige and Skoog (MS) medium was found to be the best medium for hairy root mass multiplication, which induced high root biomass production and rapid root tip elongation.  Key words: Carrot, Agrobacterium rhizogenes, acetosyringone, hairy roots, Murashige and Skoog (MS) medium, arbuscular mycorrhizae.

Establishing monoxenic culture of arbuscular mycorrhizal fungus Glomus intraradices through root organ culture

Arbuscular mycorrhizal fungi are soil fungi distributed worldwide, forming symbiosis with most of the vascular plants for their growth and survival, which is used for sustainable agriculture and ecosystem management. This study investigated the establishment of monoxenic cultures of Glomus intraradices in association with transformed carrot hairy root. The G.intraradices spores were isolated from sugarcane rhizosphere by wet sieving and decanting technique and propagated in open pot culture. Transformation in to carrot hairy root was done using Agrobacterium rhizogenes. Surface sterilization of G.intraradices spores co-cultured with transformed carrot hairy root in Modified Strulla and Romand (MSR) medium was found the host root growth as well as for germination AM spores. After three months of incubation in dark condition, significant production of extensive hyphal growth on MSR medium and an average of 8500-9000 spores per petri dish was observed. The in vitro inoculum exhibited higher potential of root colonization due to numerous intraradices mycelium with extensive spore load. The produced monoxenic inoculum can be used in place of traditional system where it has a advantage of producing contaminant free propagulas. Thus the monoxenic culture system, a powerful tool, of AM sporulation, can be used for the mass production of monoxenic inoculum of AM fungi besides studying its biology.

Effects of prometryn and acetochlor on arbuscular mycorrhizal fungi and symbiotic system

Prometryn and acetochlor are common herbicides widely used to control weeds in agricultural systems. The impacts of the two herbicides on spore germination, hyphal elongation, the biomass and malondialdehyde content of carrot hairy roots were investigated using a strict in vitro cultivation system associating the Ri T-DNA-transferred carrot hairy roots with Glomus etunicatum. Alternatively, root colonization, daughter spore production and the proportion of hyphae with succinate dehydrogenase (SDH) and alkaline phosphatase (ALP) activities were also investigated. No significant impact on spore germination was noted in the presence of acetochlor at all three concentrations tested, while a significant decrease was observed with prometryn only at the highest concentration. Moreover, an inverse correlation was identified between herbicides concentrations and G. etunicatum root colonization and spore production as well as hyphal SDH and ALP activity, with a positive correlation identified among these four factors. Both herbicides exerted negative effects on the arbuscular mycorrhizal (AM) fungus and symbiosis at increasing concentrations, with prometryn apparently more toxic than acetochlor. Furthermore, the AM symbiotic system was shown to improve biomass, reduce malondialdehyde accumulation and ease lipid peroxidation in carrot hairy roots and decrease damage in host plants, thus enhancing plant tolerance to adverse conditions. In this study, the effect of prometryn and acetochlor on the physiology and metabolic activities of the AM fungus Glomus etunicatum were investigated. Our findings demonstrate for the first time, the impact of the two herbicides at three concentrations (0.1, 1 and 10 mg l(-1)) on transformed carrot hairy roots/AM fungus association under strict in vitro culture conditions, which may guide the application of the two herbicides in modern agriculture.

Polyamines stimulate hyphal branching and infection in the early stage of Glomus etunicatum colonization

Polyamines are known to strongly stimulate hyphal growth in arbuscular mycorrhizal fungi. The effect of the polyamines putrescine, spermidine and spermine on spore germination, hyphal elongation and branching by the AM fungus Glomus etunicatum was investigated in this study. The effect of spermine on infection and the development of the host and of daughter spores was further investigated using the dual monoaxenic culture system comprised of Gl. etunicatum fungal cultures in Ri T-DNA transformed carrot hairy roots. Spermidine and spermine showed positive effects on germination and all three polyamines significantly promoted hyphal growth. Hyphal branching was also strongly stimulated by treatment with polyamines, such as an increase in the number of branches. Infection during the early stages of the in vitro co-culture life cycle was enhanced in the presence of spermine, and daughter spores appeared at earlier timepoints compared to the control. Our results demonstrate that polyamines stimulate germination and hyphal branching in the early stage of AM fungal colonization. Moreover, results from the investigations conducted in the fungus-root co-culture suggest that polyamines may be involved in establishing the symbiotic relationship between root and fungus.

Enzymes of Nitrogen Metabolism and Proteases Activity in Hairy Roots of Clover and Carrots, with and without Arbuscular Mycorrhizal Fungi

ABSTRACT Hairy roots can be used for metabolic studies and also as a substrate for arbuscular mycorrhizal fungi (AMF). However, little is known about the behavior of these roots when infected with AMF. The metabolism of hairy roots grown with or without AMF, on two culture media, was investigated. An increase in protease activity was observed when the roots were cultured on medium with high nitrogen (N)-content. Glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH) activities increased along the development in both carrot and clover hairy roots. With the advancement of root senescence (at 132 d) a greater degradation of root tissue occurred, which was characterized by an increase in catabolic enzymes activity (proteases and GDH deamination). The results indicate that the glutamine synthetase/GOGAT pathway, characteristic of plant tissues, ceases to act in synchronism and is replaced by the GDH/GOGAT system under conditions of stress or senescence when excess ammonium is present.

Calcium-dependent protein phosphorylation in hairy roots of Daucus carota

In vitro protein phosphorylation in soluble fractions (85,000×g supernatant) prepared from carrot hairy roots, which were induced by inoculation with Agrobacterium rhizogenes harboring the Ri plasmid, was analyzed by SDS-PAGE and autoradiography. The autoradiograms indicated that at least four proteins, with molecular masses of 65, 57.5, 52, and 38 kDa, were phosphorylated. The presence of Ca2+ enhanced the phosphorylation of the 65, 57.5, and 52 kDa proteins, but the presence of both Ca2+ and calmodulin did not enhance phosphorylation as much as Ca2+ alone. W-7 strongly inhibited the phosphorylation of the 65, 57.5, and 52 kDa proteins while W-5 mildly inhibited the phosphorylation of these proteins. The extent of phosphorylation of these proteins and the growth rate of the hairy roots became higher with increasing incubation temperature. W-7 had a considerable inhibitory effect on the growth of hairy roots, W-5 had a weak inhibitory effect on the growth of the roots, and a high concentration of TMB-8 inhibited growth. These results suggest that the 65, 57.5, and 52 kDa proteins are phosphorylated by CDPK and that the phosphorylation is connected with the increase in growth of carrot hairy roots.

A limiting source of organic nitrogen induces specific transcriptional responses in the extraradical structures of the endomycorrhizal fungus Glomus intraradices

The molecular bases of organic nitrogen (N) metabolism in arbuscular mycorrhizal (AM) fungi remain so far largely unexplored. To isolate genes responsive to low versus high organic N concentrations, the techniques of suppressive subtractive hybridization (SSH) and reverse Northern dot blot were performed on extraradical structures of the AM fungus Glomus intraradices grown on carrot hairy roots. This approach allowed the identification of 32 up-regulated and 2 down-regulated genes following a 48-h treatment with 2 microM of an amino acid pool (leucine, alanine, asparagine, lysine, tyrosine). The expression profile of eight genes was further confirmed by semi-quantitative and real-time RT-PCR. The majority of the sequences showed no significant similarity to proteins in databases. The other responsive genes code for putative glyoxal oxidases, transcription factors, a subunit of the 20S proteasome, a protein kinase and a Ras protein. This novel set of data indicates that G. intraradices extraradical structures perceive organic N limitation in the surrounding environment leading to a response at transcriptional level and supports the role of N as signalling molecule in AM fungi.

Optimising production of carrot hairy roots

This research describes the production of carrot hairy roots for monoxenic culture of New Zealand arbuscular mycorrhizal fungi (AMF) Induction of hairy roots on mature carrot root sections was carried out using Agrobacterium rhizogenes isolate A4T that had been grown in either LauriaBertani (/ acetosyringone) or Yeast Mannitol medium (24 or 48 h incubation) Results showed both methods could initiate hairy root production The source of the carrots was one of the most important factors with mature freshly harvested carrots showing better hairy root production compared to coolstored carrots Dissection of the cortex to expose the cambium had a positive effect on the least optimal treatment

Arbuscular mycorrhizal responsiveness of in vitro tomato root lines is not related to growth and nutrient uptake rates

Variability in growth and nutritional dynamics of in vitro tomato hairy root lines and their relationship with responsiveness to mycorrhizal colonization were studied. Four tomato cultivars were transformed with three Agrobacterium rhizogenes strains to obtain several hairy root lines, which were compared for growth and receptivity to Glomus intraradices. Four transformed hairy root lines were further characterized and compared with excised roots of the nontransformed tomato cultivar 'Cobra' and with Ri-T-DNA carrot hairy roots. Lines were compared during 4 months on minimal medium in terms of growth, nutrient uptake, and mycorrhizal colonization. In a subexperiment, the cultures were grown on a modified minimal medium to assess the contribution of initial inorganic phosphate concentration in mycorrhizal susceptibility of the three initially nonreceptive lines. On minimal medium, growth and nutrient uptake rates were highly correlated, but both were unrelated to mycorrhizal receptiveness. All the lines successfully established the symbiosis when the initial phosphate concentration was significantly reduced. No association was found between the origin of lines from the different tomato cultivar – bacterial strain combinations and the absence of symbiosis establishment on minimal medium. Decrease of inorganic phosphate concentration at the beginning of the culture was a key factor involved in precolonization steps of mycorrhizal symbiosis.Key words: Glomus intraradices, hairy roots, Lycopersicon esculentum, mycorrhizal responsiveness, root nutrition, inorganic phosphate.

Development of a Dual Biokinetic-Metabolic Model for Secondary Metabolites Production by Plant Cells

A dual biokinetic-metabolic model linking primary and secondary metabolisms forin vitro plant cells is under development. A first model (A) describing the primary metabolism was developed using carrot hairy roots as model system. This model A relates cells' growth to their nutritional status in inorganicphosphate (Pi), NO3- and carbohydrates.Catharanthus roseus(Madagascar periwinkle) hairy roots which produce valuable secondary metabolites such as theantimitotics vinblastine and vincristine compounds used to cure breast and leukemia cancers has been considered for the development of a structured model B describing secondary metabolis. An approach is presented on a potential way to link the cells' nutritional status to the production of major secondary metabolites.

Expressed genes in the extraradical hyphae of an arbuscular mycorrhizal fungus, Glomus intraradices, in the symbiotic phase

To collect extraradical hyphae of arbuscular mycorrhizal (AM) fungi for RNA isolation, a PVDF membrane was laid on the hyphal compartment of a two-compartment culture system of transformed carrot hairy roots and Glomus intraradices. Extraradical hyphae free from host tissue were easily collected, and their RNA was rapidly extracted with a modified acid guanidinium thiocyanate-phenol-chloroform method. A 3′-RACE (rapid amplification of cDNA ends) of a known gene indicated that this protocol enabled the isolation of mRNA molecules as small as 2.3 kb. The cDNA libraries of an AM fungus from the aseptic extraradical hyphae in a symbiotic state were constructed for the first time. Three-fourth of 150 ESTs (expressed sequence tags) indicated low or no similarities to known sequences from other organisms.

Expressed genes in the extraradical hyphae of an arbuscular mycorrhizal fungus, Glomus intraradices, in the symbiotic phase.

To collect extraradical hyphae of arbuscular mycorrhizal (AM) fungi for RNA isolation, a PVDF membrane was laid on the hyphal compartment of a two-compartment culture system of transformed carrot hairy roots and Glomus intraradices. Extraradical hyphae free from host tissue were easily collected, and their RNA was rapidly extracted with a modified acid guanidinium thiocyanate-phenol-chloroform method. A 3'-RACE (rapid amplification of cDNA ends) of a known gene indicated that this protocol enabled the isolation of mRNA molecules as small as 2.3 kb. The cDNA libraries of an AM fungus from the aseptic extraradical hyphae in a symbiotic state were constructed for the first time. Three-fourth of 150 ESTs (expressed sequence tags) indicated low or no similarities to known sequences from other organisms.