Development of a rapid and efficient system for CR genes identification based on hairy root transformation in Brassicaceae

Many economically important crops and vegetables belonging to the cruciferous family are heavily endangered by clubroot disease caused by Plasmodiophora brassicae infection.Breeding of clubroot resistant cultivars based on mapping and cloning of resistant genes is commonly regarded as the most cost-effective and efficient way to fight against this disease.The traditional way of R gene functional validation requires stable transformation that is both time-and labor-consuming.In this study,a rapid and efficient hairy-root transgenic protocol mediated by Agrobacterium rhizogenes was developed.The transformation positive rate was over 80%in Brassica napus showed by GUS reporter gene and this transformation only took 1/6 of the time compared with stable transformation.The system was applicable to different B.napus varieties and other cruciferous crops including Brassica rapa and Brassica oleracea.In particular,two known CR genes,CRA3.7.1 and CRA8.2.4 were used respectively,as example to show that the system works well for CR gene study combined with subsequent P.brassicae infection in B.napus.Most importantly,it works both in over-expression that led to disease resistance,as well as in RNAi which led to disease susceptible phenotype.Therefore,this system can be used in batch-wise identification of CR genes,and also offered the possibility of manipulating key genes within the P.brassicae genome that could improve our knowledge on host-pathogen interaction.

In Vitro and in Silico Insights on the Biological Activities, Phenolic Compounds Composition of Hypericum perforatum L. Hairy Root Cultures

Three Hypericum perforatum hairy root lines(HR B,HR F and HR H)along with non-transformed roots were analyzed for phenolic compounds composition and in vitro enzyme inhibitory properties.In silico molecular modeling was performed to predict the interactions of the most representative phenolic compounds in HR clones with enzymes related to depression,neurodegeneration and diabetes.Chromatographic analyses revealed that HR clones represent an efficient source of quinic acid and hydroxybenzoic acids,epicatechin and procyanidin derivatives,quercetin and kaempferol glycosides,as well numerous xanthones.In vitro antidepressant activity of HR extracts through monoamine oxidase A(MAO-A)inhibition was attributed to the production of oxygenated and prenylated xanthones.The neuroprotective potential of HR extracts was related to the accumulation of quercetin 6-C-glucoside,epicatechin,procyanidins andγ-mangostin isomers as potential inhibitors of acetylcholinesterase(AChE)and butyrylcholinesterase(BChE).Vanillic acid and prenylated xanthones in HR clones as promising inhibitors of tyrosinase additionally contributed to the neuroprotective activity.Five preeminent xanthones in HR(γ-mangostin,mangiferin,garcinone C,garcinone E and 1,3,7-trihydroxy-6-metoxy-8-prenyl xanthone)along with the flavonol quercetin 6-C-glucoside effectively inhibitedα-amylase andα-glucosidase indicating the antidiabetic properties of HR extracts.Transgenic roots of H.perforatum can be exploited for the preparation of novel phytoproducts with multi-biological activities.

Gm NAC15 overexpression in hairy roots enhances salt tolerance in soybean

The NAC （NAM, ATAF1/2 and CUC2） transcription factor family plays a key role in plant development and responses to abiotic stress. GmNAC15 （Glyma 15g40510.1）, a member of the NAC transcription factor family in soybean, was functionally characterized, especially with regard to its role in salt tolerance. In the present study, qRT-PCR （quantitative reverse transcription PCR） analysis indicated that GmNAC15 was induced by salt, drought, low temperature stress, and ABA treatment in roots and leaves. GmNAC15 overexpression in soybean （Glycine max） hairy roots enhanced salt tolerance. Transgenic hairy roots improved the survival of wild leaves; however, overexpression of GmNAC15 in hairy root couldn＇t influnce the expression level of GmNAC15 in leaf. GmNAC15 regulates the expression levels of genes responsive to salt stress. Altogether, these results provide experimental evidence of the positive effect of GmNAC15 on salt tolerance in soybean and the potential application of genetic manipulation to enhance the salt tolerance of important crops.

Two new coumarin glucosides biosynthesized by transgenic hairy roots of Polygonum multiflorum

The glycosylation of hydroxylcoumarin was investigated by using suspension cultures of hairy roots of Polygonum multiflorum. Two new coumarin glucosides （3 and 4） were biosysthesized by regioselectively glycosylation of corresponding substrates （1 and 2） in the system. The structures of two products were identified as 7-hydroxy-4-methylcoumarin 5-O-β-D-glucopyranoside and 7- hydroxy-3,4-dimethylcoumarin 5-O-β-D-glucopyranoside on the ground of chemical and spectroscopic methods, respectively. 2007 Rong Min Yu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

Overexpression of GmBIN2,a soybean glycogen synthase kinase 3 gene,enhances tolerance to salt and drought in transgenic Arabidopsis and soybean hairy roots

Glycogen synthase kinase 3 （GSK3） is a kind of sedne/threonine kinase widely found in eukaryotes. Many plant GSK3 kinases play important roles in regulating stress responses. This study investigated BRASSINOSTEROID-INSENSITIVE 2 （GmBIN2） gene, a member of the GSK3 protein kinase family in soybean and an orthologue of Arabidopsis BIN2/AtSK21. GmBIN2 expression was increased by salt and drought stresses, but was not significantly affected by the ABA treatment. To examine the function of GrnBIN2, transgenic Arabidopsis and transgenic soybean hairy roots were generated. Overexpression of GmBIN2 in Arabidopsis resulted in increased germination rate and root length compared with wild-type plants under salt and mannitol treatments. Overexpression of GmBIN2 increased cellular Ca2~ content and reduced Na~ content, enhancing salt tolerance in transgenic Arabidopsis plants. In the soybean hairy root assay, overexpression of GmBIN2 in transgenic roots also showed significantly higher relative root growth rate than the control when subjected to salt and mannitol treatments. Measurement of physiological indicators, including proline content, superoxide dismutase （SOD） activity, and relative electrical conductivity, supported this conclusion. Furthermore, we also found that GmBIN2 could up-regulate the expression of some stress-related genes in transgenic Arabidopsis and soybean hairy roots. Overall, these results indicated that GmBIN2 improved tolerance to salt and drought in transgenic Arabidopsis and soybean hairy roots.

Effects of Rare Earth Elements on Promoting Isoflavonoid Production and Release of Pueraria Lobata Hairy Roots in Vitro

The effects of various rare earth elements on growth and isoflavonoid production in hairy root cultures of Pueraria lobata (Willd.) Ohwi (strain TR2) cultured in 500 ml flasks were studied. After 32 days of culture, the biomass of hairy roots increase 15 times and reach 3.2 g dry weight. Hairy root growth was inhibited by Y_2O_3, NaSeO_3 and Sm^(3+) because of brown formation. But significant promoting effect on root growth due to callus formation was observed in La^(3+) treatments. It is the most noteworthy that the production of total isoflavonoids and puerarin was enhanced greatly by La^(3+) treatment. A major portion of increased total isoflavonoids and puerarin was released into medium in La^(3+) treatment while the hairy root viabilities were preserved. Some specific secondary metabolite release processes could be induced by La^(3+) and their possible mechanism is discussed.

Ficus carica hairy roots:In vitro anti-leishmanial activity against Leishmania major promastigotes and amastigotes

Objective:To investigate the biochemical capacity,and in vitro inhibitory effects of hairy roots from two cultivars of Ficus carica L.(Sabz and Siah)on Leishmania major promastigotes and amastigotes.Methods:In the hairy roots,the activity of antioxidant enzymes compared to normal leaves and roots,and the presence of some phenolic compounds in comparison with fruits were investigated.The IC_(50) values of hairy roots in promastigotes was determined by tetrazolium-dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and trypan blue assays.By calculating the infectivity index of peripheral blood mononuclear cells(PBMCs),the leishmanicidal activity(IC_(50) values)of hairy roots for amastigotes was estimated.The effects of hairy roots(IC_(50) values)treatment on the levels of IFN-γ and iNOS expression,intracellular reactive oxygen species,and iNOS protein expression in infected-PBMCs were determined.Results:Based on antioxidant enzyme assays and high performance liquid chromatography analysis,hairy roots exhibited high antioxidant capacity and contained high levels of phenolic compounds.According to the results of tetrazolium-dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and trypan blue assays,the hairy root extracts of both cultivars showed considerable dose-dependent inhibitory effects against Leishmania major promastigotes.Depending on the concentration and exposure time,treatment of infected-PBMCs with hairy root extracts caused the generation of a significant reactive oxygen species,up-regulation of IFN-γ and iNOS genes expression,and high value of iNOS protein compared to controls.Conclusions:The findings of this study suggest that the hairy roots of Ficus carica can be considered as a promising natural source of antileishmanial agents.

Establishment of Rhodiola quadrifida Hairy Roots and Callus Culture to Produce Bioactive Compounds

Rhodiola quadrifida is a rare mountain medicinal plant whose root extracts are used in traditional Chinese medicine as a hemostatic,antitussive,and tonic in the treatment of gynecological diseases.The aim of the study was to obtain R.quadrifida cultures at different degrees of differentiation in vitro and compare their growth characteristics and the content of salidroside and rosavin.Hairy roots were obtained by incubating cotyledons and hypocotyls in a suspension of Agrobacterium rhizogenes strain A4.The presence of the rolB and rolC genes was proven by polymerase chain reaction.The obtained roots were cultivated in Murashige-Skoog medium(MS).Calluses were obtained from the hairy roots in MS medium with the addition of hormones:3 mg/L 2,4 D and 0.5 mg/L BAP.The presence of the main secondary metabolites of R.quadrifida,salidroside and rosavin,in calluses and salidroside in hairy roots by HPLC/MS was confirmed.The content of salidroside in callus culture was significantly higher than in hairy roots,0.158 and 0.047%,respectively.The content of rosavin in callus culture was 0.07%.The content of rosavin and salidroside in callus culture was close to the level of these substances in the rhizomes of R.quadrifida plants growing in vivo,making this culture promising for its possible biotechnological use.

Hairy Root Induction in Helicteres isora L.and Production of Diosgenin in Hairy Roots

Mature seeds of Helicteres isora L.were collected from seven geographical locations of Maharashtra and Goa(India)and evaluated for diosgenin(a bioactive steroidal sapogenin of prime importance)extraction and quantification.Chemotypic variations were evidenced with diosgenin quantity ranging from 33 lg g^(-1)seeds(Osmanabad forests)to 138 lg g^(-1)(Khopoli region).Nodal and leaf explants from in vitro-raised seedlings were used for callus and Agrobacterium-mediated transformation,respectively.Compact,hard,whitish-green callus(2.65 g explant-1)was obtained on MS?13.32 lM BAP?2.32 lM Kin after 30 days of inoculation.Various parameters including types of explant and Agrobacterium strain,culture density,duration of infection and various medium compositions were optimized for hairy root production.A.rhizogenes strain ATCC-15834 successfully induced hairy roots from leaf explants(1 cm2)with 42%efficiency.Transgenic status of the roots was confirmed by PCR using rolB and VirD specific primers.Hairy roots showed an ability to synthesize diosgenin.Diosgenin yield was increased*8 times in hairy roots and*5 times in callus than the seeds of wild plants.Enhanced diosgenin content was associated with proline accumulation in hairy roots.This is the first report on induction of hairy roots in H.isora.

Rapid,Efficient and High-Performance Protocol for Agrobacterium rhizogenes-Mediated Hairy Root Transformation of the Common Bean Phaseolus vulgaris

A rapid, efficient and high-performance transformation protocol employing Agrobacterium rhizogenes was developed for the common bean Phaseolus vulgaris. In this study, we examined competencies of various protocols to induce and explants that respond to hairy root transformation in bean plants. Utilizing young seedlings with severed radicles/hypocotyls, we developed a highly efficient procedure for achieving hairy root transformation frequencies as high as 100% as visualized by GUS reporter gene expression system. Transgenic hairy roots in these young composite plants were susceptible to nodulation by rhizobia, and form an excellent system for high throughput genomic analysis to study root biology and endosymbiosis in common bean.

Effect of EuCl_3 on Hairy Root Growth of Cassia Obtusifolia and Production of Its Active Principle

Treated with extremely low EuCl 3 (0 001, 0 01, 1 0 mg·L -1 ) in MS medium, the hairy root growth of Cassia obtusifolia was inhibited. The inhibition was strongest at the treatment with 0 001 mg·L -1 EuCl 3, and the dry weight of hairy roots was 22% lower than that of control. Treated with rather low EuCl 3 (10 mg·L -1 ) in Murashige and Skoog(MS) medium, the hairy root growth was enhanced, and the dry weight of hairy roots was 27% higher than that of control. In the each treatment with 0 001 to 1 0 mg·L -1 EuCl 3 in MS medium, the total content of six free anthraquinones was lower than that of control. While treated with 10 mg·L -1 EuCl 3, the total content of six free anthraquinones was 26% higher than that of control, 97% higher than the treatment of 1 0 g·L -1 EuCl 3. Treatment with 10 mg·L -1 EuCl 3 in MS medium enhances the growth of hairy root of Cassia obtusifolia and improves the total content and production of six free anthraquinones in them.

Hairy Root and Its Application in Plant Genetic Engineering

Agrobacterium rhizogenes Conn. causes hairy root disease In plants. Hairy root-Infected A. rhizogenes Is characterlzed by a high growth rate and genetic stability. Hairy root cultures have been proven to be an efficient means of producing secondary metabolites that are normally biosyntheslzed In roots of differentiated plants. Furthermore, a transgenlc root system offers tremendous potential for introducing additional genes along with the RI plasmld, especially with modified genes, into medicinal plant cells with A. rhizogenes vector systems. The cultures have turned out to be a valuable tool with which to study the biochemical properties and the gene expression profile of metabolic pathways. Moreover, the cultures can be used to elucidate the Intermediates and key enzymes Involved In the biosynthesis of secondary metabolites. The present article discusses various appllcations of hairy root cultures in plant genetic engineering and potential problems aseoclsted with them.

Nitric Oxide Potentiates Oligosaccharide-induced Artemisinin Production in Artemisia annua Hairy Roots

The purpose of the present study was to characterize the generation of nitric oxide （NO） in Artemisia annua roots induced by an oligosaccharide elicitor （OE） from Fusarium oxysporum mycelium and the potentiation role of NO in the elicitation of artemisinin accumulation. The OE （0.3 mg total sugar/mL） induced a rapid production of NO in cultures, which exhibited a biphasic time course, reaching the first plateau within 1.5 h and the second within 8 h of OE treatment. Artemisinin content in 20-day-old hairy roots was increased from 0.7mg/g dry wt to 1.3 mg/g dry wt by using the OE treatment for 4d. In the absence of OE, the NO donor sodium nitroprusside （SNP） at 10, 50 ~1 and 100 ~1 enhanced the growth of hairy roots, but had no effect on artemisinin synthesis, The combination of SNP with OE increased artemisinin content from 1.2 mg/g drywt to 2.2 mg/g dry wt, whereas the maximum production of artemisinin in cultures was 28.5 mg/L, a twofold increase over the OE treatment alone. The effects of SNP on the OE-induced artemisinin were suppressed strongly by the NO scavenger 2-（4- carboxyphenyl）-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide （cPTIO）. The results suggest that NO can strongly potentiate elicitor-induced artemisinin synthesis in A. annua hairy roots.

Biotransformation of 4-Hydroxybenzen Derivatives by Hairy Root Cultures of Polygonum multiflorum Thunb

The biotransformation of four 4-hydroxybenzen derivatives （1,4-benzenediol （compound 1）, 4-hydroxybenzaldehyde （compound 2）, 4-hydroxybenzyl alcohol （compound 3） and 4-hydroxybenzoic acid （compound 4）） by the hairy root cultures of Polygonum multiflorum Thunb. as a new biocatalyst was investigated. It was found that the substrates were transformed to their corresponding glucosides, 4-hydroxyphenyl β-D-glucopyranoside （arbutin, compound la）, 4-hydroxymethylphenyl β-D-glucopyranoside （gastrodin, compounds 2a, 3a） and 4-carboxyphenyl α-D- glucopyranoside （compound 4a）, respectively. In the meantime, the hairy roots of P. multiflorum were able to stereoselectively and regioselectively glucosylate phenolic hydroxyl groups of compounds 1-4, but the cultures could not glucosylate the aldehyde group of compound 2 or the benzyllc hydroxyl group of compound 3, and no glucosyl esterification of carboxyl groups of compound 4 was detected. On the other hand, the result also showed that the hairy roots of P. multiflorum were able to reduce the 4-hydroxybenzaldehyde to its corresponding alcohol. This is the first report that substrate 4 has been converted into its α-D-glucopyranoside by a plant biotransformα- tion system.

Hairy roots induced by Agrobacterium rhizogenes and production of regenerative plants in hairy root cultures in maize

Hairy roots of maize were induced by infecting 15-d calli with Agrobacterium rhizogenes. The hairy roots cultured in hormone-free media showed the vigorous growth and typical hairy root features. The regenerated plants were produced from hairy roots in MS media supplemented with 1.6 mg/L ZT and 0.4 mg/L NAA. The PCR-Southern hybridization demonstrated that T-DNA had been integrated into the chromosome of regenerated plants.

Transformation of Ginkgo Hairy Root and Establishment of Its Suspension Culture Clone

Tender leaves,buds and stems of ginkgo biloba L .were transformed successfully by Agrobacterium rhizogenes with Ri plasmid,and its suspension cultured clone was eastablished.The result of detection of opines suggested that A.rhizogenes had integrated the T DNA of the Ri plasmid into genomic DNA of ginkgo cells.A new way is offered to exploit ginkgo resources with biological technology.

Kinetics of Growth and Nutrient Consumption in the Culture of Trichosanthes kirilowiiHairy Root

Ribosome-inactivating proteins in Trichosanthes kirilowii having high anti-HIV activity can be efficiently obtained by culturing Trichosanthes kirilowii hairy root. A hairy root line from Trichosanthes kirilowii was cultivated in flasks and in a 5-L bioreactor. The results show that a logistic equation can be used to describe the relationship between the hairy root biomass and the culture time. The medium conductivity decrease is linearly related to the biomass amount. In the suspension culture, the hairy root growth is closely related to the nutrient consumption. The biomass to nitrate yield is 0.352 g/mmol. The growth rate of the hairy root in the bioreactor is higher than that in the flasks, and after a 12-day culture its growth rate enters a high-speed period when the growth rate is 0.738 g/( L·d).

New Sesquiterpene Glycosides from Culture Hairy Roots of Catharanthus roseus

Two new compounds cadin-2-en-1β-ol-1β-D-glucuronopyranoside （1）, guaia-l,7-dien-3β,13-diol-13α-D- glucofuranoside （2） along with three known compounds have been isolated from the culture hairy roots of Catharanthus roseus. Their structures have been elucidated with the help of 500 MHz NMR using 1D and 2D spectral methods： viz： ^1H and ^13C NMR, ^1H-^1H COSY, ^1H-^13C HETCOR and DEPT aided by ELMS, FAB-MS, HR-FABMS and IR spectroscopy.

Studies on Biological Characteristics of Ginkgo Hairy Root Culture

The comparative studies on properties of growth and cultivated conditions of seven transformed ginkgo hairy root clones were reported. Different clones display various phenotypes characterized by growth rate.The results show that the suitable inoculum is benefical to the growth of ginkgo hairy root.NH + 4/NO - 3, pH ,sucrose, and inositol have important effects on the growth of ginkgo hairy root.

Optimizing hairy root production from explants of Phyllanthus hainanensis,a shrub used for traditional herbal medicine

Phyllanthus hainanensis is a shrub that has been used in traditional herbal medicine.It has great pharmaceutical potential for treating diseases such as cancer and diabetes.As a prerequisite for propagation of this species on a large scale,hairy roots in P.hainanensis were induced using Rhizobium rhizogenes and various factors affecting hairy root induction and growth evaluated.Seven factors were tested:(1)type of explant,(2)type of culture medium,(3)duration of pre-culture,(4)R.rhizogenes inoculum cell density,(5)duration of infection,(6)acetosyringone concentration in the culture medium,and(7)duration of incubation.The optimal protocol for hairy root induction and growth was:young shoots,pre-cultured in Y1 for 2 d,inoculated with R.rhizogenes broth with an OD600 of 0.6 for 20 min,and incubated for 3 d.Putative transgenic hairy roots were initially identified by morphology and then confirmed by polymerase chain reaction.Successful and optimal production of hairy roots is a critical prerequisite for industrial scale clonal propagation of P.hainanensis.Being able to cultivate the plant on a large scale will provide rapid and ready supply of the plant materials that can be used in herbal medicine and in scientific and industrial exploitation.