Agrobacterium Rhizogenes Strain Research Articles

Effect of Yeast Extract on h6h Gene Expression and Tropane Alkaloids Production in Atropa belladonna L. Hairy Roots

Plant alkaloids, are one of the largest groups of pharmaceutical metabolites. Scopolamine and atropine are most common tropane alkaloids in Atropa belladonna L. with great application in pharmaceutical industry. Phytochemical biosynthetic pathways induction via various elicitors is an effective strategy for increasing the production of valuable substances. Therefore, in this research effect of yeast extract were studied on expression of h6h gene, antioxidant enzymes activity and production of tropane alkaloid in A. belladonna hairy roots. Hairy roots of A. belladonna were obtained from cotyledonary leaf explants by A7 strain of Agrobacterium rhizogenes. Polymerase chain reaction (PCR) analysis with specific primers confirmed the insertion of rolB in putative transgenic hairy roots genome. Different concentrations of yeast extract (YE) (0, 0.5, 1 and 1.5 mg/L) at different exposure time (24 and 48 h) were used as elicitor. Scopolamine and atropine content and activity of antioxidant enzymes such as guaiacol peroxidase, ascorbate peroxidase and catalase increased and expression of h6h (hyoscyamine 6 beta-hydroxylase) gene in elicited hairy roots were investigated. High concentration of YE decreased the growth of hairy roots but increased the atropine content compared to the control. According to the results, the highest content of scopolamine (9.21%) and atropine (43.39%) which were 9 and 5 fold than control, were obtained by 1 and 1.5 mg/L YE at 48 h exposure time, respectively. The results demonstrated that YE can be used as effective elicitor to increase tropane alkaloids production in hairy root cultures of A. belladonna.

A Stable Agrobacterium rhizogenes-Mediated Transformation of Cotton (Gossypium hirsutum L.) and Plant Regeneration From Transformed Hairy Root via Embryogenesis.

Genetic transformation is a powerful tool to study gene function, secondary metabolism pathways, and molecular breeding in crops. Cotton (Gossypium hirsutum L.) is one of the most important economic crops in the world. Current cotton transformation methods take at least seven to culture and are labor-intensive and limited to some cultivars. In this study, we first time achieved plantlet regeneration of cotton via embryogenesis from transformed hairy roots. We inoculated the cotyledon explants of a commercial cultivar Zhongmian-24 with Agrobacterium rhizogenes strain AR1193, harboring a binary vector pBI-35S::GFP that contained the NPT II (neomycin phosphotransferase) gene and the GFP (green fluorescent protein) gene as a fluorescent marker in the T-DNA region. 82.6% explants produced adventitious roots, of which 53% showed GFP expression after transformation. 82% of transformed hairy roots produced embryonic calli, 12% of which regenerated into stable transformed cotton plants after 7 months of culture. The integration of GFP in the transformed cotton genomes were confirmed by PCR (Polymerase chain reaction) and Southern blot analysis as well as the stable expression of GFP were also detected by semi-quantitative RT-PCR analysis. The resultant transformed plantlets were phenotypically, thus avoiding Ri syndrome. Here we report a stable and reproducible method for A. rhizogenes-mediated transformation of cotton using cotyledon as explants, which provides a useful and reliable platform for gene function analysis of cotton.

Induction of Hairy Roots on Somatic Embryos of Rhizoclones from Typha domingensis Seedlings.

A protocol for the induction of hairy roots on somatic embryos of rhizoclones from Typha domingensis seedlings grown in hydroponic rhizotron systems was established for the first time. Rhizogenesis was induced through the agrotransformation of somatic embryos in oblong and scutellar states of development using the K599, LBA9402, and A4 strains of Agrobacterium rhizogenes. The transfection to the embryos was performed by cocultivation of rhizoclones on a Murashige and Skoog mineral medium at 50% strength (MS0.5), in the dark, at 28 ± 2 °C for 72 h. In contrast to nontransformed embryos that did not exhibit any root tissue, transformed embryos presented hairy roots that varied in number, length, and density depending on the bacterial strain, and K599 was the most effective strain. After analysis via optical microscopy, the transformed embryos were collected and transferred to fresh culture media supplemented with 400 mg mL−1 cefotaxime and 10 mg L−1 ascorbic acid. The efficiency of transformation and survival of the oblong and scutellar embryos were similar among the three bacterial strains. The results show that agrotransformation of somatic embryos of rhizoclones from T. domingensis is a novel and viable strategy for the generation of genetic transformants of Typha that have potential applications in bioremediation technologies.

Comparison of morphogenesis and anatomy between hairy roots and adventitious roots of Ngoc Linh ginseng (Panax vietnamensis Ha et Grushv.)

In this study, morphogenesis and anatomy of adventitious roots and hairy roots of Ngoc Linh ginseng (Panax vietnamensis Ha et Grushv.) were compared. Adventitious roots were derived from four different samples (leaf, petiole, root and callus) in vitro cultured on medium SH supplemented with 5 mg/L IBA. Hairy roots were derived from callus infected with Agrobacterium rhizogenes strain ATCC 15834. The results showed that there were significant differences in morphogenesis and anatomy, the pathway of secondary roots formation, culture medium between adventitous roots and hairy roots. The hairy roots strongly grew and branched on the free-plant growth regulator medium. The secondary root of the hairy root was formed in two pathways: (1) The secondary root formation began at the vesicles system of main root; (2) The secondary root formation was independent of the vesicles system of main root; it kept developing in length and connected to the vesicles system of main root. On the other hand, growing adventitous roots only grew on the environment with exogenous auxin supplementation; also their branching ability were low. The secondary root formation of the adventitous root was started at the vesicles of the main roots and there was only one secondary root that was formed at an location of adventitous root. The results that obtained in this study will be the reference to identify two types of root.

The first report on the induction of hairy roots in Trapa natans, a unique aquatic plant with photosynthesizing roots

Hairy root cultures are often used to produce valuable metabolites. They are grown on sucrose-rich medium, which is highly susceptible to contamination. Trapa natans is a unique plant with photosynthesizing roots. It is a promising object to obtain photoautotrophic hairy root culture. Protocols for transformation of this species are yet unknown. We report that hairy roots can be induced in aquarium and in vitro cultures of T. natans by agrobacterium-mediated and biolistic transformation. 64 roots were induced by Agrobacterium rhizogenes strain 15834, two roots were obtained using strain K599. Strain A4 was not effective. Biolistics with either amplicons of rol genes and 1301 pCAMBIA plasmid carrying rol genes resulted in the formation of six roots. All these roots contained chloroplasts. This achievement opens a prospect for genetic transformation of T. natans and use of its green photosynthesizing hairy root cultures in production of bioactive substances and in phytoremediation. Protocols for the Agrobacterium-mediated and biolistic transformation of Trapa natans to induce hairy roots have been developed for the first time. All induced roots contained chloroplasts.

Bioactive diterpenoid metabolism and cytotoxic activities of genetically transformed Euphorbia lathyris roots

Plants in the genus Euphorbia produce a wide variety of pharmacologically active diterpenoids with anticancer, multidrug resistance reversal, and antiviral properties. Some are the primary industrial source of ingenol mebutate, which is approved for treatment of the precancerous skin condition actinic keratosis. Similar to other high value phytochemicals, Euphorbia diterpenoids accumulate at low concentrations in planta and chemical synthesis produces similarly low yields. We established genetically transformed root cultures of Euphorbia lathryis as a strategy to gain greater access to diterpenoids from this genus. Transformed roots produced via stem explant infection with Agrobacterium rhizogenes strain 15834 recapitulated the metabolite profiles of field-grown plant roots and aerial tissues. Several putative diterpenoids were present in transformed roots, including ingenol and closely related structures, indicating that root cultures are a promising approach to Euphorbia-specific diterpenoid production. Treatment with methyl jasmonate led to a significant, albeit transient increase in mRNA levels of early diterpenoid biosynthetic enzymes (farnesyl pyrophosphate synthase, geranylgeranyl pyrophosphate synthase, and casbene synthase), suggesting that elicitation could prove useful in future pathway characterization and metabolic engineering efforts. We also show the potential of transformed E. lathyris root cultures for natural product drug discovery applications by measuring their cytotoxic activities using a panel of human carcinoma cell lines derived from prostate, cervix, breast, and lung.

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.

Bio-interaction of Agrobacterium rhizogenes with Capsicum annuum L. (sweet variety) and Establishment Hairy Roots Cultures

Genetic transformation using Agrobacterium is one of the techniques used to transfer desired genes to plants. This protocol is considered a short – cut to get transformed plants which could be an alternative method and suitable system compared with the classical methods. This study aimed to investigate the response of C. annuum seedlings (sweet variety) to the formation transformed hairy roots induced by A. rhizogenes strain R1601. Sterilized seeds of Capsicum annuum were inoculated with the Agrobacterium rhizogenes inoculum. The samples were then transferred to the surface of solidified MS medium. Hairy roots were developed at the inoculation sites and were enucleated 1.0-1.5 cm length and placed in 9.0 cm Petri-dishes containing 15 ml of agar solidified MS medium. Agropine test was performed according to the standard method. The inoculated seedlings showed a good response 90%. Transformed hairy roots were established at the injection sites within 10 days and these roots were easily grown on agar-solidified MS medium. The results are confirmed that these roots were transformed roots it in terms of positive agropine detection. The current study concluded that the biological interaction between Agrobacterium rhizogenes strain R1601 and Capsicum anuum L. seedlings, was successful. This study encourages future research to improve this plant by continuing and applying modern technologies to obtain genetically modified plants.

Bio-interaction of Agrobacterium rhizogenes with Capsicum annuum L. (sweet variety) and Establishment Hairy Roots Cultures

Genetic transformation using Agrobacterium is one of the techniques used to transfer desired genes to plants. This protocol is considered a short – cut to get transformed plants which could be an alternative method and suitable system compared with the classical methods. This study aimed to investigate the response of C. annuum seedlings (sweet variety) to the formation transformed hairy roots induced by A. rhizogenes strain R1601. Sterilized seeds of Capsicum annuum were inoculated with the Agrobacterium rhizogenes inoculum. The samples were then transferred to the surface of solidified MS medium. Hairy roots were developed at the inoculation sites and were enucleated 1.0-1.5 cm length and placed in 9.0 cm Petri-dishes containing 15 ml of agar solidified MS medium. Agropine test was performed according to the standard method. The inoculated seedlings showed a good response 90%. Transformed hairy roots were established at the injection sites within 10 days and these roots were easily grown on agar-solidified MS medium. The results are confirmed that these roots were transformed roots it in terms of positive agropine detection. The current study concluded that the biological interaction between Agrobacterium rhizogenes strain R1601 and Capsicum anuum L. seedlings, was successful. This study encourages future research to improve this plant by continuing and applying modern technologies to obtain genetically modified plants.

Establishment of the hairy root culture of Echium plantagineum L. and its shikonin production.

Echium plantagineum L. (Boraginaceae) is an invasive species in Australia and contains medicinal shikonins in its roots. In this study, the hairy root lines of E. plantagineum were established using Agrobacterium rhizogenes strain ATCC15834 and confirmed by the amplification of the rolB gene. Results showed significant difference in shikonin production between the hairy root lines in the 1/2B5 and M9 media. The biomass of the lines in the 1/2B5 medium was fivefold of that in the M9 medium. However, the components of detected shikonins were similar in these two liquid media. By contrast, different accumulation profiles appeared in the hairy root lines. HPLC analysis revealed the presence of nine possible related compounds, including shikonins, and acetylshikonin was the most abundant shikonin derivative. The content of acetylshikonin in the 1/2B5 medium (36.25mg/L on average) was twofold of that in the M9 medium. Our results showed that the hairy root cultures of E. plantagineum can be used in enhancing the production of potential pharmaceutical compounds, such as acetylshikonin.

Effect of different Agrobacterium rhizogenes strains for in-vitro hairy root induction, total phenolic, flavonoids contents, antibacterial and antioxidant activity of (Cucumis anguria L.)

The present study aimed to investigate the effect of different Agrobacterium rhizogenes on the induction of hairy root of Cucumis anguria and determine its total phenolic, flavonoids contents, antibacterial and antioxidant activity. In this investigation A. rhizogenes strains such as, 15834, 13333, A4, R1200, R1000, LBA9402, R1301 and R1601 are all investigated, were developing hairy root conception in cotyledon and leaf tissue explants. Polymerase chain response (PCR) and the converse transcription-PCR are transgenic clones of hairy roots has been utilized rolA and rolB particular primers. In the middle of the different attention of better regulators the extreme transformation frequency was achieved in (IBA + NAA) cotyledon explant. Transgenic hairy roots increase in MS liquid medium added to with IBA + NAA (2.46 + 1.07) displayed the maximum accumulation of biomass 0.68 g/l dry weight (DW) and 6.52 ± 0.49 g/l fresh weight (FW) were obtained at the 21 days of cotyledon explant. The flavonoid and total phenolic contents were estimated using aluminium chloride method and Folin-Ciocalteu method. The amount of phenolic compounds in Cucumis anguria L non transformed root (124.46 ± 6.13 mg GA/g) was lower than that in the methanol extracts of Cucumis anguria L. hairy roots (160.38 ± 5.0 mg GA/g), being was Cucumis anguria L non transformed root lower (42.93 ± 1.58 mg rutin/g) than that in the concentration of total flavonoids in Cucumis anguria L. hairy root (16.26 ± 1.84 mg rutin/g). Additionally, transgenic hairy roots professionally produced various phenolic and flavonoid composites. The total antimicrobial activity, phenolics, flavonoids content and antioxidant were more in the hairy roots related to non-transformed roots. In our discovery, the A. rhizogenes R1000 is promising candidate for hairy root initiation of C. anguria from cotyledone explants were realized large number of hairy roots compared with leaf explants. The antioxidant potential of methanol extracts of flavonoid and phenolic compounds from the hairy roots have great potential to treat various diseases.

Genetic transfection, hairy root induction and solasodine accumulation in elicited hairy root clone of Solanum erianthum D. Don

An efficient genetic transfection technique has been established using A4 strain of Agrobacterium rhizogenes for the first time in a medicinally valuable plant Solanum erianthum D. Don. The explants were randomly pricked with sterile needle, inoculated with bacterial suspension. The infected leaves were then washed and transferred to MS basal medium fortified with cefotaxime for hairy root induction. A maximum transformation efficiency of 72 % has been recorded after two days of co-cultivation period. The transfer of rolA and rolB genes from the bacterium to the plant genome has been confirmed in five transformed hairy rootlines by standard Polymerase Chain Reaction technique. On the basis of growth analysis and secondary metabolite study two potential rhizoclones (A4-HR-A and A4-HR-B) were selected. Rhizoclone A4-HR-A can produce highest amount of alkaloid, phenolic and flavonoid, whereas A4-HR-B was observed to be highest tannin producer. Alkaloid like solasodine, commercially important for steroidal drug synthesis, was quantified from leaf and A4-HR-A clone by an improved High Performance Liquid Chromatography method. This showed a sustainable increase (1.33 fold) in production of solasodine in hairy rootline.

Genetic transformation of the tropical vine Pentalinon andrieuxii (Apocynaceae) via Agrobacterium rhizogenes produces plants with an increased capacity of terpenoid production

Genetic modification using Agrobacterium rhizogenes has been an important biotechnological tool for the improvement of secondary metabolite production in plant species over the past four decades. The tropical vine Pentalinon andrieuxii, a native plant from the Yucatan Peninsula, is known to produce the triterpene betulinic acid and the novel tri-nor-sesquiterpene urechitol A and is used in the Mayan traditional medicine to treat leishmaniasis-derived lesions. Agrobacterium rhizogenes strain ATCC 15834 was used to infect leaf and hypocotyl explants of P. andrieuxii to generate 14 transformed plant lines with increased production of the terpenoids betulinic acid and urechitol A. Insertion of the transgenes was confirmed through PCR amplification of the rolA, rolB, and rolC genes. Betulinic acid and urechitol A were detected and quantified using standardized high-performance liquid chromatography and gas chromatography analyses, respectively. A significant increase in the terpenoid content was observed in some of the transgenic plants with up to 11.37 and 3.35 times more betulinic acid and urechitol A, respectively, when compared with the wild-type control. These new transgenic lines are promising tools to further the studies and knowledge about the biosynthesis of terpenoids in P. andrieuxii, especially regarding the biosynthetic origin of the rare urechitols.

Establishment of in vitro genetically engineered cultures in Scutellaria orientalis and S. araxensis

Scutellaria orientalis and S. araxensis are perennial species of genus Scutellaria (Lamiaceae) which produce some important pharmaceutical secondary metabolites. Four strains of Agrobacterium rhizogenes (A4, A13, ATCC15834 and MSU440), three types of explant (stem, petiole and leaf), six concentrations of acetosyringone (25–150 μM), two different co-cultivation methods (light and dark condition) and two co-cultivation media (full and half-strength MS) were used for optimization of A. rhizogenes mediated transformation efficiency. Moreover, two inoculation approaches including immersion and injection as well as three infection period (5, 7 and 10 min) were examined. The molecular confirmation analysis of putative transgenic roots was performed by PCR using specific primers of the rol B gene. The results revealed that the transformation efficiency was affected by bacterial strain, co-cultivation medium and type of explants. The highest transformation frequency of 93.3% was achieved on stem explants of S. orientalis with ATCC15834 strain, followed by MSU440, A13 and A4 with the frequency of 63.3%, 46.6% and 30%, respectively. In S. araxensis, the best transformation frequency of 76.6% and 43.3% were obtained on stem and petiole explants with MSU440 strain. 5 and 7 min for infection period significantly improved the transformation efficiency in S. orientalis and S. araxensis respectively. Transformation rates of stem explants induced by ATCC15834, A4 and A13 strains were 53.3%, 36.6% and 20% respectively. Transformation frequency was significantly higher in half-strength MS, regardless of A. rhizogenes strains.

Comparison of Profiling of Hairy Root of Two Tibetan Medicinal Plants Przewalskia tangutica Maxim. and Anisodus tanguticus Maxim.

Tropane Alkaloids (TAs) are important drugs for curing many diseases in the medical industry. To sustainably exploit TA resources in endangered traditional Tibetan herbs, the hairy root (HR) systems of Przewalskia tangutica Maxim. and Anisodus tanguticus Maxim. were compared under the same culture conditions. The results indicated that both the Agrobacterium rhizogenes strains and explants affected the HR induction frequency, MSU440, A4 and LBA9402 strains could induce hairy roots following infection of cotyledon and hypocotyl of A. tanguticus while LBA9402 could not induce HR on either explants of P. tangutica. The efficiency of LBA9402 was higher than A4 and MSU440 on A. tanguticus and A4 was better strain than MSU440 on P. tangutica. The hypocotyl explant was more suitable for P.tangutica and cotyledon explant was better for A.tangutica with a transformation frequency of 33.3% (P. tangutica) and 82.5% (A. tanguticus), respectively. In a flask reactor system, both the growth curves of HR for two species both appeared to be "S" curve; however, the HR of P. tangutica grew more rapidly than that of A. tanguticus, and the latter accumulated more biomass than the former. As the culture volume increased, the HR proliferation coefficient of both the species increased. HPLC analysis results showed that the content of TAs in the HR of P. tangutica was 257.24mg/100g·DW, which was more than that of A. tanguticus HR (251.08mg/100g·DW), and the anisodamine in the Pt- HR was significantly higher than that in At-HR. Moreover, tropane alkaloids in the HR of the two species were all significantly higher than that of the roots of aseptic seedlings. Our results suggest that HR of P. tangutica and A. tanguticus both could provide a useful platform for sustainable utilization of two Tibetan medicinal plants in the Qinghai-Tibetan Plateau in the future.

Production Enhancement of Medicinally Active Coumarin and Phenolic Compounds in Hairy Root Cultures of Pelargonium sidoides: The Effect of Elicitation and Sucrose

African geranium (Pelargonium sidoides DC., Geraniaceae) is a herbaceous perennial plant with well-known medicinal properties. In the present study, the effect of elicitation by methyl jasmonate (MeJ) (25, 50, and 100 µM) and sucrose (15 and 30 g/L) on the fresh weight (FW), dry weight (DW) and the production of pharmaceutically active coumarin (umckalin) and phenolics (gallic, vanillic, and p-coumaric acids) in P. sidoides hairy root cultures was investigated. Hairy roots were induced from the leaves of in vitro sterile P. sidoides seedlings inoculated with Agrobacterium rhizogenes strains (LBA-9402, C58C1) and Agrobacterium tumefaciens C58C1 (pRiA4). Among the fifteen evaluated hairy root lines (HRLs), HRL4 showed the highest accumulation of biomass (9.6 g FW and 0.56 DW) and was selected for elicitation and sucrose management for 50 days in a shaking system. The treated HRs, developed in Murashige and Skoog liquid media, demonstrated the highest FW (32.68 g) and DW (1.1 g) 50 days after elicitation with 100 µM of MeJ. However, a 1.4-fold enhancement of gallic and vanillic acids was achieved with 50 and 25 µM of MeJ, respectively, in HRs under control conditions. The highest accumulation of umckalin (1.61-fold increase compared to the control) was obtained with 100 µM of MeJ. These findings can be useful to develop commercial production of umckalin and phenolics in P. sidoides HR cultures in scaled-up systems and bioreactors.

Production of phenolic acids in hairy root cultures of medicinal plant Mentha spicata L. in response to elicitors.

In this study, hairy root induction in leaf and stem explants of Mentha spicata using various Agrobacterium rhizogenes strains was established for the first time. Although inoculation of explants by immersion method resulted in tissue necrosis, direct injection of explants by all examined strains (A13,R318,A4,GMI 9534 and ATCC15834) was effective. All different parts of the stem were susceptible to A. rhizogenes infection. However, the middle and lower internodes showed a higher rate of transformation. Among the different strains, the strain A13 exhibited the highest infection efficiency (almost 75% of the explants). A13 and R318-infected hairy roots showed the highest biomass production (close to 60 mg/flask), while infection with GMI 9534 produced the highest content of phenolic acids. Finally, the effect of phytohormone elicitation on hairy root growth and phenolic acid biosynthesis was investigated. A substantial increase in root growth and phenolic acids accumulation was obtained followed by 0.3 mg L-1 IBA and 100 µM MeJA treatment, respectively.

The Effect of Different Elicitors on Hairy Root Biomass and Resveratrol Production in Wild Vitis vinifera

Introduction: Resveratrol is an antioxidant secondary metabolite which belongs to a specific phytoalexins called stilbenes. Grape has been considered as the main source of resveratrol in the human diet. Many studies have been conducted on genus Vitis due to the presence of high levels of polyphenolic compounds in different tissues. In recent years, a lot of effort has been made to increase resveratrol yield in Vitis vinifera via tissue and hairy roots culture. Materials and Methods: In the present study, hairy roots have been produced by Agrobacterium rhizogenes strain ATCC15834 and successfully produced resveratrol in internode of grape genotype W16. Transformations of hairy roots were recognized by PCR using specific primers of rolB gene. After stimulation, the effect of abiotic elicitors with different concentrations including methyl jasmonate, sodium acetate, acetic acid and ammonium nitrate were tested on the production of hairy root biomass and resveratrol. Resveratrol content was measured by TLC and HPLC methods. Results: Results showed that the capacity of hairy roots for resveratrol production is higher than natural roots. A significant difference was observed between different elicitors in terms of hairy root biomass and resveratrol amount. Findings revealed that treatment with 3 mM acetic acid and 50 μM methyl jasmonate led to the highest and lowest amount of hairy roots biomass and resveratrol content, respectively. Conclusions: According to the fidings of the present study it can be stated that both hairy roots and different elicitors are effective in biomass and resveratrol production. This method can be used to increase the yield of resveratrol for large scale production via tissue culture.

Novel method for improving ardicrenin content in hairy roots of Ardisia crenata Sims plants

To develop an alternative medicine related with taxol/camptothecin, a hairy roots induction system for measuring triterpenoid saponin ardicrenin was established. In the current study, mature and healthy seeds of Ardisia crenata plants were selected for obtaining aseptic seedlings. Two Agrobacterium rhizogenes strains ATCC 15834 and A4 were used to infect aseptic euphylla for inducing hairy roots of A. crenata plants. For the best combination of seeds germination, a Murashige-Skoog medium containing 1.0 mg L−1 6-benzylaminopurine and 1.0 mg L−1 naphthalene acetic acid was made, which reached a rate of 92.4 %. Results showed that ATCC 15834 and A4 both induced hairy roots of A. crenata for improving ardicrenin production. The PCR analysis demonstrated that ATCC 15834 and A4 Ri plasmid T-DNA had been successfully transferred and integrated into the genome of leaf cell nuclei, however the Vir region was not. Further, ardicrenin content in hairy roots ACHR 15834 8.2 %) induced by ATCC 15834 was quantified by the RP-HPLC, which was also 1.8-, 2.7-, 9.4- and 2.6-fold greater than those of ACHR 4 induced by A4 (4.5 %), ACR C formed by tissue culture (3.1 %), euphylla (0.8 %) and NR C formed nature (3.2 %), respectively. Taken together, hairy root lines of A. crenata obtained were able to express naturally more ardicrenin than natural plants.

Agrobacterium rhizogenes mediated transformation of Ficus carica L. for the efficient production of secondary metabolites.

Ficus carica L., an ancient source of food and medicines, is rich in valuable nutritional and secondary compounds with antioxidant, antimicrobial, and anticancer effects. The present study is the first attempt to examine hairy root (HR) induction of F. carica (Sabz and Siah) by inoculating the 3-week-old shoots and leaves with different strains of Agrobacterium rhizogenes and also to investigate methyl jasmonate (MeJA) elicitation of HRs to produce a fast and high-yield production method for secondary metabolites. The maximum transformation rate (100%) was achieved by inoculating the shoots with Agrobacterium rhizogenes strain A7. Siah HRs elicited with 100 and 200 μmol L-1 MeJA and Sabz HRs with 100 μmol L-1 MeJA showed the highest total phenolic content. The highest flavonoid content was 3.935 mg QE g-1 DW in Siah HRs treated with 200 μmol L-1 MeJA and 2.762 mg QE g-1 DW in Sabz HRs treated with 300 μmol L-1 MeJA. The 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity and ferric reducing antioxidant power (FRAP) value of HRs were affected by MeJA treatments. Methyl jasmonate elicitation also significantly enhanced the content of six phenolic acids (gallic acid, caffeic acid, chlorogenic acid, coumaric acid, rosmarinic acid, and cinnamic acid) and three flavonoids (rutin, quercetin, and apigenin). Thymol, a monoterpene phenol, was the main HR compound detected in gas chromatography mass spectrometry (GC-MS) analysis of the essential oils. Induction of HRs and elicitation of F. carica HRs by MeJA resulted in a significant increase in the production of important phenolic compounds and a significant increase in antioxidant capacity. © 2020 Society of Chemical Industry.