An improved protocol for inducing hairy roots in Vitex negundo using Agrobacterium rhizogenes

Fenugreek(Trigonella foenum-graecum L.) is a rich source of important medicinal metabolites. This plant belongs to the Fabaceae family. Induced hairy roots by Agrobacterium rhizogenes are a suitable tissue for the production of secondary metabolites, due to the stability and high production of roots without phytohormone in a short time. Different strains of Agrobacterium rhizogenes (A4, ATCC11325 and ATCC15834) were evaluated for induction of transformed hairy roots in T. foenum-graecum L. using seedling explants. The application of hairy root culture may become an alternative method for increase secondary metabolites. Transgenic status of the roots was confirmed by PCR using rolB specific primers. All of the A. rhizogenes strains led to hairy roots induction. The maximum frequency of transformation (97.87%) was obtained using A4 strain in 7-days-old seedling. The 7-days-old explants were inoculated using A4 strain result in highest fresh (0.166 g) and dry (0.080 g) weight of roots. The explants were inoculated by ATCC11325 strain produced hairy roots with highest amount of total phenol (8.113 mg/g DW) and flavonoid content (3.215 µg/g DW).

This article presents the abilities and efficiencies of five different strains of Agrobacterium rhizogenes (strain ATCC 31798, ATCC 43057, AR12, A4 and A13) to induce hairy roots on Solanum mammosum through genetic transformation. There is significant difference in the transformation efficiency (average number of days of hairy root induction) and transformation frequency for all strains of A. rhizogenes (P < 0.05). Both A. rhizogenes strain AR12 and A13 were able to induce hairy root at 6 days of co-cultivation, which were the fastest among those tested. However, the transformation frequencies of all five strains were below 30 %, with A. rhizogenes strain A4 and A13 showing the highest, which were 21.41 ± 10.60 % and 21.43 ± 8.13 % respectively. Subsequently, the cultures for five different hairy root lines generated by five different strains of bacteria were established. However, different hairy root lines showed different growth index under the same culture condition, with the hairy root lines induced by A. rhizogenes strain ATCC 31798 exhibited largest increase in fresh biomass at 45 days of culture under 16 h light/8 h dark photoperiod in half-strength MS medium. The slowest growing hairy root line, which was previously induced by A. rhizogenes strain A13, when cultured in optimized half-strength MS medium containing 1.5 times the standard amount of ammonium nitrate and potassium nitrate and 5 % (w/v) sucrose, had exhibited improvement in growth index, that is, the fresh biomass was almost double as compared to its initial growth in unmodified half-strength MS medium.

Vitex negundo L. is an aromatic, woody, blooming shrub in the Verbenaceae family which can grow into a small tree. Traditionally, V. negundo root has been used to cure diabetes, colic, boils, leprosy, and rheumatism. Keeping the importance of its roots in mind, an attempt has been taken for development of a protocol for efficient hairy root proliferation system. The Agrobacterium rhizogenes strains (MTCC 532 and MTCC 2364) were used for hairy root induction. For A. rhizogenes infection, both in vitro and in vivo leaves as well as internodes were used as explant. In vitro leaves and internodal explants were obtained by the inoculation of matured nodal segments on the optimum medium [MS + 2.0 mg/L N6-Benzylaminopurine (BAP)] with c.a. 91.6% shoot regeneration and an average of 8.1 shoots per explants. In vitro leaf showed best hairy root induction followed by in vitro internode on ½ MS medium augmented with acetosyringone. Highest transformation efficiency was achieved using MTCC 2364 strain, while no transformation was observed in MTCC 532 strain. Different factors affecting transformation including co-cultivation period, infection time and optical density (O.D.) value were standardized. The highest efficacy, 88.8% hairy root induction was observed in in vitro leaves infected by MTCC 2364 for 60 minutes infection time with an O.D. value of 0.29 maintained over a 44-48 hours of co-cultivation period. The prescribed protocol may be used as a reference for development of industrial scale hairy root production for bioactive compound located in root of V. negundo.

An efficient protocol for hairy root induction in Berberis aristata DC. was established using two different strains of Agrobacterium rhizogenes, MTCC 532 and 2364 from IMTECH (Institute of Microbial Technology), Chandigarh, India. The strain 532 was more effective than strain 2364 in hairy root induction and in vitro grown callus (61.11 ± 1.60 % transformation frequency) was found to be suitable explant in comparison to leaves (42.59 ± 0.92 % transformation frequency) and nodal segments (34.25 ± 0.92 % transformation frequency) of in vitro grown microshoots for hairy root induction. The presence of rol A and rol B genes during amplification confirmed the transgenic nature of hairy roots and transformed callus. Transformation frequency of callus was further enhanced (from 61.11 ± 1.60 % to 72.22 ± 1.60 %; when infection time was 1 h) by using acetosyringone (100 µM) during co-cultivation period (48 h) on semisolid MS (Murashige and Skoog) medium. In conclusion, this study describes the protocol for hairy root induction which could further be useful for the production of berberin and may reduce the overharvesting of this endangered species from its natural habitat.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-015-1222-1) contains supplementary material, which is available to authorized users.

The development of an efficient protocol for successful hairy root induction by Agrobacterium rhizogenes is the key step toward an in vitro culturing method for the mass production of secondary metabolites. The selection of an effective Agrobacterium strain for the production of hairy roots is highly plant species dependent and must be determined empirically. Therefore, our goal was to investigate the transformation efficiency of different A. rhizogenes strains for the induction of transgenic hairy roots in Fagopyrum tataricum ‘Hokkai T10’ cultivar; to determine the expression levels of the polypropanoid biosynthetic pathway genes, such as ftpAL, FtC4H, Ft4CL, FrCHS, FrCH1, FrF3H, FtFLS1, FtFLS2, FtF3, H1, FtF3′H2, FtANS, and FtDFR; and to quantify the in vitro synthesis of phenolic compounds and anthocyanins. Among different strains, R1000 was the most promising candidate for hairy root stimulation because it induced the highest growth rate, root number, root length, transformation efficiency, and total anthocyanin and rutin content. The R1000, 15834, and A4 strains provided higher transcript levels for most metabolic pathway genes for the synthesis of rutin (22.31, 15.48, and 13.04 μg/mg DW, respectively), cyanidin 3-O-glucoside (800, 750, and 650 μg/g DW, respectively), and cyanidin 3-O-rutinoside (2410, 1530, and 1170 μg/g DW, respectively). A suitable A. rhizogenes strain could play a vital role in the fast growth of the bulk amount of hairy roots and secondary metabolites. Overall, R1000 was the most promising strain for hairy root induction in buckwheat.

Plants are the important sources of drugs. Secondary metabolites are responsible for therapeutic propertiesin plants. Three compounds including (-)-hyoscyamine, its racemate atropine, and scopolamine (hyoscine) are the most famous tropane alkaloids in the Solanaceae familly. Nowadays, attempts to developthese alkaloids in biotechnological procedures which are principally based on the hairy root cultures using Agrobacterium rhizogenes. In the present study, we showed the percent of induced hairy roots percent in the leaf and root explants of four plants from the Solanaceae family (Atropa belladonna L., Hyoscyamus niger L., Datura stramonium L. and Datura metel L.), that infected with the six strains of Agrobacterium rhizogenes (A4, A7, Ar15834, Ar9534, Ar9402, and Ar318). Hairy roots were appeared from the leaf and roots explants on ½MS medium culture. The presence of T-DNA in the supposedly transformal lines was shown by PCR. The highest transformation yield of 93% was accomplished using leaf explants of D. metel infected by AR15834 and A4 strains. One fastest growing clone of transforming D. metel roots line (induced by AR15834)was selected and the biomass of hairy and natural roots were measured and compared after 0, 2, 4, 6, 8 and 10 days. The results showed that, the fresh and dry weight of hairy roots was 4.44 and 4.92 times higher than the weights of non-transformed roots respectively after 8- 10 days. These roots are hormone-autotrophic and have the great lateral branches. Therefore, the hairy roots of D. metel and D. stramonium can be used to increase tropane alkaloids production yield in the pharmaceutical industry.

Introduction: Citrullus colocynthis is considered as a precious herb due to its medicinal and nutritional values and also for its ability to produce valuable bioactive compounds such as cucurbitacin E and quercetin. The hairy root systems are valuable tools for scaling-up secondary metabolites and for introducing new beneficial traits into herbs. The present research has aimed to develop a protocol for hairy root culture of C. colocynthis using Agrobacterium rhizogenes. Materials and Methods: After the establishment of the hairy root system, factors such as explant type, bacterial strain, pre-culture period, co-cultivation period, and the use of acetosyringone that affect the efficient transformation of the herb were optimized. Four A. rhizogenes strains (MSU440, A4, A13 and ATCC 15834) and three types of explant (leaf, excised shoot and hypocotyl) were tested. Furthermore, the insertion of transgene into the genome of C. colocynthis was confirmed by polymerase chain reaction analysis. Results: The highest transformation frequency was obtained after the infection of excised shoots by MSU440. Co-cultivation for 48 hours resulted in enhanced transformation frequency, while the results of this research showed that the protocol is better not to include the pre-culturing step. In addition, the presence of acetosyringone in bacterial culture and co-cultivation medium significantly increased the success of C. colocynthis transformation. Conclusions: This study describes an efficient protocol for hairy roots culture of C. colocynthis which can be used for scaling-up the plant active phytochemicals or for genetic manipulations of the plant.

Sahendian savory (Satureja sahendica Bornm) is a medicinal plant species which produces valuable secondary metabolites including thymol, p-cymene and γ-terpinene. In this study, optimization of hairy roots culture in S. sahendica using Agrobacterium rhizogenes was efficiently established. High frequency of genetic transformation was obtained using node and internode explants and three bacterial strains (ATCC 15834, A4, and LBA 9402). However, leaf explants showed no susceptibility to induce hairy roots with the A. rhizogenes strains. The node explants and LBA 9402 strain were the best combination for root induction. Infection time of 20 min caused greater transformation occurrence compared to 10 and 30 min, respectively. Node explants infected with LBA 9402 were found to produce more transformants upon co-cultivation for 2 days. The maximum induction of hairy roots was obtained from bacterial infection at a density of OD600 = 0.8. Polymerase chain reaction by gene-specific primers from rol B gene confirmed the positive transformation events. S. sahendica efficiently provided and produced a large number of hairy roots which could be used for the production of secondary metabolites or other molecular and biotechnological applications. Production of thymol an important secondary metabolite of S. sahendica from hairy root cultures was confirmed by TLC and GC analysis. Elicitor-treated hairy root cultures with methyl jasmonate and ammonium nitrate produced more amount of thymol compared to untreated roots. These results demonstrate that secondary metabolite production such as thymol in hairy root culture of Sahendian savory could be used to increase low secondary metabolite production in this valuable medicinal plant.

Biomedical Applications and Secondary Metabolite Profiling of Hyoscyamus niger and Sesamum indicum Seed, Root and Hairy Root Cultures

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.

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.

BackgroundCapsicum annuum and Capsicum frutescens, also known as “chilies”, belong to the Solanaceae family and have tremendous beneficial properties. The application of hairy root culture may become an alternative method for future development of these species by adding value, such as by increasing secondary metabolites and improving genetic and biochemical stability compared with normal Capsicum plants. Therefore, in this research, different types of explants of both species were infected with various Agrobacterium rhizogenes strains to provide more information about the morphology and induction efficiency of hairy roots. After 2 weeks of in vitro seed germination, young seedling explants were cut into three segments; the cotyledon, hypocotyl, and radical. Then, the explants were co-cultured with four isolated A. rhizogenes strains in Murashige & Skoog culture media (MS) containing decreasing carbenicillin disodium concentrations for one month.ResultsIn this experiment, thick and short hairy roots were induced at all induction sites of C. annuum while thin, elongated hairy roots appeared mostly at wound sites of C. frutescens. Overall, the hairy root induction percentages of C. frutescens were higher compared with C. annuum. Hairy root initiation was observed earliest using radicles (1st week), followed by cotyledons (2nd week), and hypocotyls (3rd week). Cotyledon explants of both species had the highest induction frequency with all strains compared with the other explants types. Strains ATCC 13333 and ATCC 15834 were the most favourable for C. frutescens while ATCC 43056 and ATCC 43057 were the most favourable for C. annuum. The interactions between the different explants and strains showed significant differences with p-values < 0.0001 in both Capsicum species.ConclusionsBoth Capsicum species were amenable to A. rhizogenes infection and hairy root induction is recommended for use as an alternative explants in future plant-based studies.

Hairy root cultures of Gentiana macrophylla were established by infecting the different explants four Agrobacterium rhizogenes strains namely A(4)GUS, R1000, LBA 9402 and ATCC11325, and hairy root lines were established with A. rhizogenes strain R1000 in 1/2 MS + B(5) medium. Initially, 42 independent hairy root clones were maintained and seven clones belongs to different category were evaluated for growth, morphology, integration and expression of Ri T-DNA genes, and alkaloid contents in dry root samples. On the basis of total root elongation, lateral root density and biomass accumulation on solid media, hairy root clones were separated into three categories. PCR and Southern hybridization analysis revealed both left and right T-DNA integration in the root clones and RT-PCR analysis confirmed the expression of hairy root inducible gene. GUS assay was also performed to confirm the integration of left T-DNA. The accumulation of considerable amounts of the root-specific secoiridoid glucosides gentiopicroside was observed in GM1 (T +/L and T +/R) and the GM2 (T +/L and T -/R DNA) type clones in considerably higher amount whether as two T -/L but T +/R callus-type clones (GM3) accumulated much less or only very negligible amounts of gentiopicroside. Out of four media composition the 1/2 MS + B(5) vitamin media was found most suitable. We found that initial establishment of root cultures largely depends on root:media ratio. Maximum growth rate was recorded in 1:50 root:media ratio. The maximum biomass in terms of fresh weight (33-fold) was achieved in 1/2 MS + B(5) media composition after 35 days in comparison to sixfold increase in control. The biomass increase was most abundant maximum from 15 to 30 days. Influence of A. rhizogenes strains and Ri plasmid of hairy root induction, the possible role of the T(L)-DNA and T(R)-DNA genes on growth pattern of hairy root, initial root inoculum:media ratio and effect of media composition is discussed.

Using different explants of in vitro seed grown Scutellaria baicalensis Georgi plantlets, hairy roots were induced following inoculation of Agrobacterium rhizogenes strains A4GUS, R1000 LBA 9402 and ATCC11325. The A4GUS proved to be more competent than other strains and the highest transformation rates were observed in cotyledonary leaf explant (42.6 %). The transformed roots appeared after 15–20 d of incubation on hormone free Murashige and Skoog medium. Growth of hairy roots was assessed on the basis of total root elongation, lateral root density and biomass accumulation. Maximum growth rate was recorded in root:medium ratio 1:100 (m/v). Hairy root lines were further established in Gamborg B5 medium and the biomass increase was maximum from 15 to 30 d. PCR, Southern hybridization and RT-PCR confirmed integration and expression of left and right termini-linked Ri T-DNA fragment of the Ri plasmid from A4GUS into the genome of Scutellaria baicalensis hairy roots. GUS assay was also performed for further integration and expression. All the clones showed higher growth rate them non-transformed root and accumulated considerable amounts of the root-specific flavonoids. Baicalin content was 14.1–30.0 % of dry root mass which was significantly higher then that of control field grown roots (18 %). The wogonin content varies from 0.08 to 0.18 % among the hairy root clones which was also higher than in non-transformed roots (0.07 %).

Induced hairy roots by Agrobacterium rhizogenes are suitable organs for the production of secondary metabolites, due to the stability and high production of roots without phyhormones in a short time. Fenugreek (Trigonella foenum– graecum L.) is one of the most important medicinal plants with valuable medicinal compounds. For optimizing the hairy root induction of T. foenum-graecum L., six strains of A. rhizogenes ATCC15834, ATCC11325, K599, A7, A4 and A13 (MAFF-02-10266) and three explant ages (7, 14 and 21 day old seedlings) were tested. The explants were cultured in B5 in a completely randomized design with 4 replications. Hairy roots appeared after 3 to 4 weeks. Transgenic status of the roots was confirmed by PCR using rolB specific primers. The results revealed that all strains were able to induce hairy roots after 3-4 weeks. The maximum percentage of hairy root induction (87.5%) was obtained from A7 and ATCC11325 strains and 7-day-old explants. Among the strains, ATCC11325 strain showed the highest hairy root number (4.25) and root length (2.1 cm) using 14-day-old explant. Also, the highest fresh (0.203 g) and dry weights (0.029 g) were observed in the A4, A7 and ATCC11325 strains. The highest total phenolic and flavonoid contents were found at 74.05 mg g-1 DW and 11.80 µg g-1 DW in hairy roots induced by A7 strain.