Statistical design of the effect of inoculum conditions on growth of hairy root cultures of Catharanthus roseus

Summary The factors in the inoculum conditions that influence the growth rate of hairy roots of Catharanthus roseus in liquid culture were investigated in a randomized two-level, three-factor statistical design. The three variables were the number of root tips, the length of root tips, and the initial volume of media. The experiments and analysis demonstrated the effect of inoculum conditions on hairy root growth, with the length of hairy root tips being the dominant variable without any clonal variability. The best growth rates (doubling times) were obtained with an inoculum of 5 hairy root tips, each 35–40 mm long in 50 mL media: 3.7 and 3.1 days for C. roseus hairy root clones LBE-6-1 and LBE-4-2, respectively.     

Use of Chenopodium murale L. transgenic hairy root in vitro culture system as a new tool for allelopathic assays

We investigated Chenopodium murale transgenic hairy root in vitro culture system as a new tool for allelopathic assays. Transgenic hairy roots were induced by Agrobacterium rhizogenes A4M70GUS from roots, cotyledons, leaves, and internodes of C. murale seedlings. Roots were found to be the best target explants, providing transformation efficiency of up to 11.1%. Established hairy root clones differed in their morphology and growth potential. Molecular characterization of these clones was carried out by PCR, RT-PCR and histochemical GUS analyses. No differences in rol gene expression were observed. Liquid culture system of characterized hairy root clones was maintained for over 2 years. Six hairy root clones were selected for assaying the allelopathic effect of their growth medium against germination and seedling elongation of wheat and lettuce test plants. The inhibitory potential varied depending on the hairy root clone. Some transgenic clones showed significantly higher inhibition compared to wild-type roots. These results revealed that hairy roots as an independent system synthesize some bioactive substances with allelopathic activity and exude them into the growth medium. Concentrations of caffeic, ferulic and p-coumaric acids (0.07-2.85 μmol/L) identified by HPLC analysis in the growth media were at least 1000 times lower than the inhibitory active concentration (5 mmol/L) of pure grade phenolic acids, suggesting that they have a limited role in the allelopathic phenomena of C. murale. The presented hairy root system appears to be a suitable tool for further investigation of the potential and nature of root-mediated allelopathic interference of C. murale.     

Traits of Panax ginseng hairy roots after cold storage and cryopreservation

Summary Panax ginseng hairy root cultures were established by infecting petiole segments with Agrobacterium rhizogenes strain 15834. Hairy root segments including root tips placed onto phytohormone-free 1/2 Murashige and Skoog solid medium and stored at 4 °C in the dark for 4 months, resumed elongation when the temperature was raised to 25 °C in the dark. For cryopreservation, a vitrification method was applied. Root tips precultured with 0.1 mg/l 2,4-D for 3 days and dehydrated with PVS2 solution for 8 minutes prior to immersion into liquid nitrogen had a survival rate of 60 % and could regenerate. The hairy roots regenerated from cryopreserved root tips grew well and showed the same ginsenoside productivity and patterns as those of the control hairy roots cultured continuously at 25 °C. The conservation of T-DNAs in the regenerated hairy roots was proved by PCR analysis.Abbreviations 1/2 MS a half strength Murashige and Skoog (1962) - B5 Gamborg B5 (Gamborg et al. 1968) - WP woody plant (Lloyd and McCown 1980) - RC root culture (Thomas and Davey 1982) - RCI root culture medium containing 100 mg/l myoinositol - HF phytohormone-free - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - TIBA 2,3,5-triiodobenzoic acid - PCR polymerase chain reaction - PVS2 plant vitrification solution 2 (Sakai et al., 1990) - FDA fluorecein diacetate     

Production of Tropane Alkaloids by Hairy Root Cultures of Scopolia japonica

Hairy root clones of Scopolia japonica were established by selection of adventitious roots formed on the root segments inoculated with Agrobacterium rhizogenes strain 15834. Twenty-nine isolated hairy root clones displayed various phenotypes characterized by growth rate, opine production and tropane alkaloid production. Of these, two highly alkaloid productive clones SI and S22 were examined for their growth rate and alkaloid productivity under various cultural conditions. When the most scopolamine-productive clone SI was cultured for 4 weeks at 25°C in the dark, the weight of the root tissue was increased by 40 times and the content of scopolamine reached a level of 0.5% on a dry weight basis in each optimum medium. On culture of the most hyoscyamine-productive clone S22 under the same conditions as with S1, the weight was increased by 102 times and the content of hyoscyamine was 1.3% on a dry weight basis in each optimum medium.     

A novel life cycle arising from leaf segments in plants regenerated from horseradish hairy roots

Summary Horseradish (Armoracia rusticana) hairy root clones were established from hairy roots which were transformed with the Ri plasmid in Agrobacterium rhizogenes 15834. The transformed plants, which were regenerated from hairy root clones, had thicker roots with extensive lateral branches and thicker stems, and grew faster compared with non-transformed horseradish plants. Small sections of leaves of the transformed plants generated adventitious roots in phytohormone-free G (modified Gamborg's) medium. Root proliferation was followed by adventitious shoot formation and plant regeneration. Approximately twenty plants were regenerated per square centimeter of leaf. The transformed plants were easily transferable from sterile conditions to soil. When leaf segments of the transformed plants were cultured in a liquid fertilizer under non-sterile conditions, adventitious roots were generated at the cut ends of the leaves. Adventitious shoots were generated at the boundary between the leaf and the adventitious roots and developed into complete plants. This novel life cycle arising from leaf segments is a unique property of the transformed plants derived from hairy root clones.     

Transformed root cultures for biotechnology

This review is concerned with the application of hairy roots, i.e. plant roots formed from plant cells after transformation by Agrobacterium rhizogenes for the production of bioactive compounds. Transformed root cultures have been established from numerous species of dicotyledonous plants. The plants, as well as the main products accumulated in hairy root cultures derived from these plants, are listed in this paper. Data are presented on novel compounds, hitherto detected only in transformed roots but not occurring in the corresponding intact plants. The possible use of hairy root cultures for the over-production of secondary metabolites and biotransformation of chemicals is discussed. In order to enhance the productivity of hairy root cultures, various methods have been derived, and optimized procedures are proposed. They include selection of high-producing clones, elicitation, composition of growth media, culture conditions and genetic approach. Hairy roots usually store secondary metabolites in vacuoles inside the cells. Therefore, several methods have been used to increase the amount of products released into the medium. Unfortunately, no general procedure is known that works in all cases, and the excretion behaviour of hairy root cultures varies from one species to another and even within one species from one clone to another. Special attention is given to the cultivation methods and bioreactor systems for hairy root cultures. Hairy roots are cultivated usually in shake flasks; however, shake flask culture is not suitable for the complex optimization and continuous control of the culture conditions. In this paper, we are going to present bioreactors proposed for the cultivation of hairy roots under more or less controlled conditions. Modifications of typical bacterial bioreactors, i.e. stirred tanks, airlift loop reactors and other constructions, are presented. A very special type of bioreactor providing good conditions for loose root mass multiplication without oxygen or substrate limitations, is the mist bioreactor. Nowadays, it is practically impossible to select the one best bioreactor type for hairy root culture.     

Baicalin production in transformed hairy root clones ofScutellaria baicalensis

A transformed hairy root clone ofScutellaria baicalensis was established following infection withAgrobacterium rhizogenes ATCC15834. Three root clones ofS baicalensis were selected by growth habit and baicalin content. The most active strain-the SR-03 clone-was examined for its growth and baicalin content under various culture conditions. The root growth and baicalin content were maximized in a Schenk and Hildebrandt medium supplemented with 4 and 6% sucrose, respectively. The accumulation of baicalin in transformed hairy roots was enhanced through exposure to various elicitors. Elicitation was attained by the addition of methyl jasmonate, salicylic acid, and various concentrations of fungal cell wasll elicitors to the medium. The accumulation of baicalin in the elicited cultures ranged from 10.5 to 18.3 mg/g dry weight of the roots, which was 1.5-to 3-fold the amount attained in controls.     

An unusual root tip formation in hairy root culture of Hyoscyamus muticus

Summary Hairy root cultures of Hyoscyamus muticus were established using Agrobacterium rhizogenes ATCC 15834. In one out of 8 clones established, an unusual root tip formation was observed after transfer of cultures from half-strength Murashige and Skoog (1962) to White's medium (1939). This phenomenon was associated with the production of a fine brownish cell suspension culture. Hairy root development resumed after transfer of the root tips from White to half-strength Murashige and Skoog medium. After plating the isolated brownish cells on hormone-free half-strength Murashige and Skoog or White solid medium, callus proliferation was observed, and then redifferentiation of hairy roots occurred. The polymerase chain reaction analysis of the H. muticus hairy root (clone Z2) revealed that only the tl region of the T-DNA was integrated. The growth and the production of five tropane alkaloids by this clone were examined.Abbreviations PCR Polymerase Chain Reaction - MS medium Murashige and Skoog Medium - 1/2 MS medium half-strength MS medium - WP medium Woody Plant medium - RC medium Root Culture medium - WH medium White medium - HPLC High Performance Liquid Chromatography - wt. weight     

Agrobacterium rhizogenes mediated transformation of the medicinal plant Decalepis arayalpathra and production of 2-hydroxy-4-methoxy benzaldehyde

Agrobacterium rhizogenes mediated transformation of Decalepis arayalpathra, an ethnomedicinal plant, was achieved by infecting juvenile hypocotyl explants with different strains, including A4, MTCC 532, TR105 and LBA 5402. Hypocotyl explants induced hairy roots at a higher frequency (53.2 ± 0.3 %) than cotyledons (32.1 ± 0.2 %) when infected with the most virulent strain TR105. The explants co-cultivated 48 h in half-strength salts and vitamins of Murashige and Skoog basal medium (half-MSB) induced hairy roots either directly from the wounds or followed by the formation of gall like structures. Irrespective of the explants, the strain MTCC 532 induced callus alone. The root initials on the galls proliferated vigorously and elongated more rapidly when they were segmented and subcultured on half-MSB medium than the proliferation and elongation of directly emerged roots. The established hairy roots showed intermittent gall formation which was the active sites for hairy roots induction. The molecular evidence of rol A and rol C gene integration was confirmed by PCR amplification and southern blot hybridization. Growth of the hairy roots was undertaken by measuring root growth unit after culturing root tips in half-MSB solid medium and determined fresh weight/dry weight/conductivity during time-course study in shake flask cultures. The maximum biomass and accumulation of the root specific compound, 2-hydroxy-4-methoxy benzaldehyde (MBALD) (0.22 % dry weight), was recorded at the 6th week of growth, which was more than that observed in normal root cultures (0.16 % dry weight).     

Plantlet regeneration enhances solasodine productivity in hairy root cultures of <Emphasis Type="Italic">Solanum khasianum</Emphasis> Clarke

Summary Shoot regeneration in hairy root cultures of Solanum khasianum Clarke influences root growth, solasodine production. and permeabilization of solasodine into the medium. These parameters are dependent on exogenously supplied auxin and cytokinin: the effect being both concentration-and clone-dependent. Hairy root cultures with no shoot regeneration showed high permeabilization of solasodine into the medium by the sixth week of incubation, suggesting the medium acts as a sink for the solasodine synthesized by the roots. Solasodine in the culture medium was toxic to the transformed roots and caused browning of root tips. In a separate set of experiments, the hairy root cultures showed regeneration of approximately 50–70 mm long shoots after treatment with indole-3-acetic acid and kinetin. These hairy root cultures had inereased levels of solasodine production, compared to cultures without shoot regeneration. The plantlets formed in the hairy root cultures accumulated some of the solasodine, thereby reducing its permcabilization into the medium. Transport of solasodine from root to shoot reduced the toxic effect of solasodine in the root zone and extended the exponential growth phase by 8-10d.     

多效唑(PP333)对美洲商陆毛状根生长和商陆皂苷甲产生的影响

为了探究植物生长调节剂多效唑(PP333)调控药用植物美洲商陆(Phytolacca americana)毛状根生长和次生代谢的可能性, 设计实验并探讨PP333对美洲商陆毛状根生长及其商陆皂苷甲含量的影响。结果表明, 与对照相比, PP333使毛状根根尖及侧根表面呈浅红色, 侧根变得短而粗, 且随着培养基内PP333浓度的升高, 根表面颜色加深。培养基中添加0.5-5.0 mg·L^-1 PP333能促进毛状根中商陆皂苷甲的产生, 其中以1.0 mg·L^-1 PP333的效果最好, 其商陆皂苷甲含量达6.22 mg·g^-1 DW, 约为对照的1.94倍。PP333能提高毛状根苯丙氨酸裂解酶(PAL)的活性, 并可能通过对PAL酶活性的调节来促进毛状根中商陆皂苷甲的产生。     

Azadirachtin production by hairy root cultivation of Azadirachta indica in a modified stirred tank reactor

Present investigation involves hairy root cultivation of Azadirachta indica in a modified stirred tank reactor under optimized culture conditions for maximum volumetric productivity of azadirachtin. The selected hairy root line (Az-35) was induced via Agrobacterium rhizogenes LBA 920-mediated transformation of A. indica leaf explants (Coimbatore variety, India). Liquid culture of the hairy roots was developed in a modified Murashige and Skoog medium (MM2). To further enhance the productivity of azadirachtin, selected growth regulators (1.0?mg/l IAA and 0.025?mg/l GA₃), permeabilizing agent (0.5?% v/v DNBP), a biotic elicitor (1?% v/v Curvularia (culture filtrate)) and an indirectly linked biosynthetic precursor (50?mg/l cholesterol) were added in the growth medium on 15th day of the hairy root cultivation period in shake flask. Highest azadirachtin production (113?mg/l) was obtained on 25th day of the growth cycle with a biomass of 21?g/l DW. Further, batch cultivation of hairy roots was carried out in a novel liquid-phase bioreactor configuration (modified stirred tank reactor with polyurethane foam as root support) to investigate the possible scale-up of the established A. indica hairy root culture. A biomass production of 15.2?g/l with azadirachtin accumulation in the hairy roots of 6.4?mg/g (97.28?mg/l) could be achieved after 25?days of the batch cultivation period, which was ~27 and ~14?% less biomass and azadirachtin concentration obtained respectively, in shake flasks. An overall volumetric productivity of 3.89?mg/(l?day) of azadirachtin was obtained in the bioreactor.     

滇黄芩毛状根的诱导及其黄芩苷含量测定

本文利用发根农杆菌A grobacterizum rhizogenes1.2556感染滇黄芩再生苗的茎段和叶片,建立了毛状根培养及其植株再生体系。毛状根可直接从受伤的茎、叶外植体表面产生,在无外源激素的MS固体和液体培养基上自主生长,表现出典型的发根特征。毛状根茎段的诱导率较叶片高,最高可达到14.44%;经rolB基因PCR分析和甘露碱纸电泳检测,证明Ri质粒T-DNA已整合到滇黄芩基因组中并表达;毛状根在附加6-BA2mg/L和NAA0.2mg/L的MS固体培养基上直接诱导不定芽,并在MS培养基上生根,形成再生植株。获得的毛状根系经MS液体培养基培养30d后通过HPLC都能检测到黄芩苷,其中1个转化系黄芩苷含量为2.59%,是药材黄芩的0.20倍,而从3年单位时间黄芩苷生成量计算,毛状根是药材黄芩的7.18倍。本研究建立的毛状根培养体系,将对滇黄芩转基因技术的完善和利用毛状根生产黄芩苷的生物转化提供了实验基础。     

Characterization of root clones obtained after transformation of monohaploid and diploid potato genotypes with hairy root inducing strains of Agrobacterium

A procedure for transformation of monohaploid and diploid potato genotypes through infection of stem internodes with hairy root inducing strains of Agrobacterium is described. Hairy roots induced by A. rhizogenes strain LBA9402 and A. tumefaciens strain LBA1020, both containing the Ri1855 plasmid, were analysed for phenotype, growth and development, and opine expression. The ploidy level of the hairy roots was determined by measurements of the nuclear DNA content and the chromosome number. The genotypes of potato (8 monohaploids, 2 diploids) greatly differed in their response to transformation, i.e. the frequency of stem internodes with primary hairy roots, the number of roots per internode and their phenotype. Transformation efficiency was lower in most of the monohaploid genotypes as compared to that in diploid genotypes. Hairy root clones could be established in 4 of the 8 monohaploid genotypes and in both diploid genotypes after subculturing of primary hairy roots. Hormone autotrophy was observed in all the root clones. The root clones varied in their phenotype and opine expression; opine expression was found in only 50% of the clones. Twenty-five of the 26 hairy root clones of the diploid genotypes showed only parental (diploid) chromosome number, even after 6 months of culture, suggesting genetic stability during the transformation and in the resulting hairy roots. However, in monohaploid genotypes the hairy root clones were either diploid or tetraploid. The transformation of monohaploid and diploid potato genotypes can be an efficient system for the establishment of a series of genetic marker lines for gene mapping.     

Agrobacterium rhizogenes-mediated transformation of Picrorhiza kurroa Royle ex Benth.: establishment and selection of superior hairy root clone

A protocol for induction and establishment of Agrobacterium rhizogenes-mediated hairy root cultures of Picrorhiza kurroa was developed through optimization of the explant type and the most suitable bacterial strain. The infection of leaf explants with the LBA9402 strain resulted in the emergence of hairy roots at 66.7% relative transformation frequency. Nine independent, opine and TL-positive hairy root clones were studied for their growth and specific glycoside (i.e., kutkoside and picroside I) productivities at different growth phases. Biosynthetic potentials for the commercially desirable active constituents have been expressed by all the tested hairy root clones, although distinct inter-clonal variations could be noted in terms of their quantity. The yield potentials of the 14-P clone, both in terms of biomass as well as individual glycoside contents (i.e., kutkoside and picroside I), superseded that of all other hairy root clones along with the non-transformed, in vitro-grown control roots of P. kurroa. The present communication reports the first successful establishment, maintenance, growth and selection of superior hairy root clone of Picrorhiza kurroa with desired phyto-molecule production potential, which can serve as an effective substitute to its roots and thereby prevent the indiscriminate up-rooting and exploitation of this commercially important, endangered medicinal plant species. CIMAP Publication No.: 2007-28J     

Mycorrhizal associations between Tuber melanosporum mycelia and transformed roots of Cistus incanus

We set out to establish root cultures of a host plant with the aim of obtaining dual cultures of Tuber melanosporum mycorrhiza on transformed roots. Seedlings of Cistus incanus germinated under sterile conditions from seeds collected in the wild were treated with Agrobacterium rhizogenes. Nine hairy roots collected from different seedlings were cultured individually by repeated subculturing. The hairy root clones differed in growth rates and in morphology (branching frequency and distance between side roots). Root growth in a liquid medium exhibited a lag phase of about 2 weeks and an exponential phase lasting about 12 days before the start of the stationary phase. Hairy roots could be kept alive on medium M, a special solid minimal medium (low in Fe²⁺, BO₄^3-, Ca²⁺, Cu²⁺ and Zn²⁺, very low in PO₄^3- and lacking MoO₄^2-, NH₄⁺ and Co²⁺), for more than 7 months. T. melanosporum could be grown on the same medium for long periods only by subculturing the fungus with the roots. A mycorrhizal association developed between the roots and the T. melanosporum mycelium within 3 months. The association consisted of elongated roots with a mantle and a Hartig net surrounding two to three layers of cortical cells. Swollen, club-like root tips were discernible 5 months after inoculation. The mycorrhized roots could be subcultured and propagated on medium M and maintain the mycorrhizal association.     

悬浮培养中不同理化因子对烟草发状根生长及烟碱合成的影响

悬浮培养中烟草(Nicotiana tabacum)发状根的生长及烟碱合成受到基本培养基浓度、初始pH值、激素种类和浓度等因素的影响。一个5 cm长的根尖, 悬浮在40 mL、pH值为6.0、附加1.0 mg.L－1IAA的1/2 MS液体培养基中, 28 ℃、散光条件下培养30天, 烟碱产量可达0.241 mg.mL－1。     

Effect of plant growth regulators on growth and biosynthesis of phenolic compounds in genetically transformed hairy roots of<Emphasis Type="Italic">Panax ginseng</Emphasis> C. A. Meyer

In this study, morphological alterations, biomass growth, and secondary metabolite production of genetically transformed hairy roots ofPanax ginseng C. A. Meyer, were evaluated after administration of plant growth regulators. The addition of benzylamino purine and kinetin to the culture media increased biomass formation and phenolic compound biosynthesis in the hairy roots. α-Naphthaleneacetic acid and indole-3-butyric acid inhibited hairy root growth, however, low concentrations of indole-3-acetic acid slightly increased hairy root growth. Low concentrations of 2,4-Dichlorophenoxyacetic acid profoundly inhibited growth of hairy roots. The addition of plant growth regulators, such as auxin, did not increase total phenolic compounds in hairy roots that did not contain gibberellic acid and cytokinins. Callus formation was induced in cultures suspended in liquid medium amended with benzylamino purine and kinetin. Hairy roots regenerated from these calluses exhibited an active growth pattern with extensive lateral branching in non-amended medium, similar to the growth pattern of the original hairy roots.     

Agrobacterium rhizogenes and A. tumefaciens co-transformation to obtain grapevine hairy roots producing the coat protein of grapevine chrome mosaic nepovirus

Hairy root cultures of grapevine were obtained from plantlets co-inoculated by virulent Agrobacterium rhizogenes strains and disarmed A. tumefaciens strains harbouring the binary vectors pKHG4 and pKVHG 2+. These plasmids contain the nptII, hpt and gus genes and differ for the presence of the gene encoding for the grapevine chrome mosaic virus coat protein. For the cultivar ‘Gravesac’, 72% of the excised root tips initiated hairy root cultures on growth regulator-free media. According to the nature of the strains used in co-inoculation, co-transformation frequencies of the hairy root clones ranged from 4 to 16%. Co-transformed roots showed resistance to kanamycin and hygromycin but responses varied from clone to clone. Fluorometric GUS expression and GCMV coat protein production showed a large variability among hairy root clones co-transformed by pKHVG2+. Though the presence of gus, nptII and GCMV coat protein genes was checked by polymerase chain reaction and Southern blotting, it was difficult to establish a clear relationship between expression of the different transgenes. The regeneration of plants was not achieved, but the possibility to graft in vitro transgenic roots to non transformed shoot systems could permit rapid testing of the resistance induced by nepovirus coat protein in roots of cultivars that are recalcitrant to A. tumefaciens-mediated transformation. This revised version was published online in June 2006 with corrections to the Cover Date.     

The effect of yeast extract and methyl jasmonate on rosmarinic acid accumulation in <Emphasis Type="Italic">Coleus blumei</Emphasis> hairy roots

The leaves of axenically grown Coleus blumei were inoculated with Agrobacterium rhizogenes strain A4 and hairy root were established. PCR and Southern hybridization analysis confirmed transgenic nature of hairy root clones. Cultures of normal roots, induced by α-naphthaleneacetic acid on leaf explants, and hairy roots were evaluated for growth and rosmarinic acid content. Significantly better growth and up to 2.8 higher amount of rosmarinic acid was detected by HPLC analysis in hairy root clones. Methyl jasmonate stimulated rosmarinic acid accumulation in 6 out of 11 tested clones, while yeast extract induced RA accumulation in two and diminished it in 5 out of 11 tested hairy root clones.