Improved shoot regeneration protocol for hairy roots of the legume Astragalus sinicus

An improved protocol for shoot regeneration from hairy roots transformed by Agrobacterium rhizogenes of the legume species Astragalus sinicus (Chinese milk vetch) has been developed. The A. rhizogenes strain DC-AR2 harboring the binary vector pBI121 which carries the uidAgene encoding -glucuronidase activity and the kanamycin resistance gene nptII, was used to transform cut ends of plantlet hypocotyls. Transformed hairy roots were selected on medium containing 75 g ml^–1 kanamycin, and transformation was monitored by detection of the opine mikimopine, histochemical -glucuronidase activity, the polymerase chain reaction, and Southern blot analysis. The cytokinins benzylaminopurine, kinetin, and thidiazuron suppressed the growth of 8-month and 3-year-old hairy roots, but were necessary for adventitious shoot formation that could occur with some lines. Putative somatic embryos developed from transformed roots on medium with 7.5-10.0 mg l^–1 2,4-dichlorophenoxyacetic acid. Light did not affect shoot regeneration from transformed hairy roots. This transformation and shoot regeneration system should be useful for testing gene expression quickly and be amenable to studies of shoot morphogenesis and interactions with rol gene expression.     

Cryopreservation of encapsulated shoot primordia induced in horseradish (Armoracia rusticana) hairy root cultures

Shoot primordia induced inArmoracia rusticana Gaertn. Mey. et Scherb. (horseradish) hairy root cultures were successfully cryopreserved by two cryogenic procedures. Encapsulated shoot primordia were precultured on solidified Murashige-Skoog medium supplemented with 0.5M sucrose for 1 day and then dehydrated with a highly concentrated vitrification solution (PVS2) for 4 h at 0°C prior to a plunge into liquid nitrogen. The survival rate of encapsulated vitrified primordia amounted to 69%. In a revised encapsulation-dehydration technique, the encapsulated shoot primordia were precultured with a mixture of 0.5M sucrose and 1M or 1.5M glycerol for 1 day to induce dehydration tolerance and then subjected to air-drying prior to a plunge into liquid nitrogen. The survival rate of encapsulated dried primordia was more than 90%, and the revived primordia produced shoots within 2 weeks after plating. A long-term preservation of shoot primordia was also achieved by the technique. Thus, this revised encapsulation-dehydration technique appears promising as a routine method for the cryopreservation of shoot primordia of hairy roots.Abbreviations PVS2 Vitrification solution - LN liquid nitrogen - BA 6-benzyladenine - NAA -naphthalene-acetic acid - MS Murashige and Skoog (1962) medium     

Improvement of adventitious shoot formation from carnation leaf explants

Adventitious shoot formation from leaf explants of carnation (Dianthus caryophyllus L.) was investigated. The two leaves from one node of in vitro-grown plants showed different shoot-forming potential, depending on the order in which the leaves were removed from the stem. The leaf removed second formed more shoots and also had a large amount of adhering stem tissue. Explants with equal amounts of adhering stem tissue were obtained by making two incisions through the fused leaf bases, prior to their removal, resulting in an improved shoot formation. The procedure developed for leaf explants from in vitro-grown plants was also applied to leaf explants from greenhousegrown plants. Shoot formation from leaf explants taken from greenhouse-grown plants was further improved by cutting the leaf explant longitudinally into two parts.Abbreviations BA benzyladenine - NAA -naphthaleneacetic acid     

Indole alkaloid production by hairy root cultures of Catharanthus roseus

Hairy root cultures of Catharanthus roseus were established by infection with six different Agrobacterium rhizogenes strains. Two plant varieties were used and found to exhibit significantly different responses to infection. Forty-seven hairy root clones derived from normal plants and two derived from the flowerless variety were screened for their growth and indole alkaloid production. The growth rate and morphological appearance showed wide variations between the clones. The alkaloid spectra observed were qualitatively but not quantitatively very similar to that of the corresponding normal plant roots. No vindoline or deacetyltransferase activity could be detected in any of the cultures studied. O-acetylval-lesamine, an alkaloid which has not been previously observed in C. roseus was identified from extracts of hairy root clone No. 8. Two root clones were examined for their growth and alkaloid accumulation during a 26-day culture period. Alkaloid accumulation parallelled growth in both clones with ca. 2 mg ajmalicine and catharanthine per g dry weight being observed.Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

Expression of transferred genes during hairy root development in pea

Root border cell development and expression of reporter genes were evaluated in transgenic pea hairy roots. Successful induction of hairy roots in pea is conditioned by bacterial strain and plant genotype, as well as by developmental and environmental factors. Morphological changes sometimes occur when hairy roots are transferred from infected plants to tissue culture media, but such changes are confined to specific clones. Expression of reporter genes under the control of promoters from bean (Phaseolus vulgaris L.) stress genes encoding phenylalanine ammonia lyase and chalcone synthase were evaluated. Expression patterns vary between hairy roots taken directly from infected plants, and those grown in culture; most hairy roots taken from infected plants exhibit expression throughout all tissues, whereas expression in cultured hairy roots is most often localized to specific tissues. Patterns of expression that occur during different stages of hairy root development are very similar to those observed in transgenic plants expressing the same fusion genes. Border cell separation and release in hairy roots is normal, and expression of glucuronidase in border cells of some transgenic roots resulted in development of bright blue single cells. Cultured hairy roots should provide a very useful model for studying the effect of defined changes in root border cells on microbial associations with roots of this important legume.Abbreviations YEM yeast extract-mannitol - GUS glucuronidase - PAL phenylalanine ammonium lyase - CHS chalcone syntase     

Nickel tolerance and hyperaccumulation in shoot cultures regenerated from hairy root cultures of <Emphasis Type="Italic">Alyssum murale</Emphasis> Waldst et Kit

Shoot cultures of nickel hyperaccumulating Alyssum murale were established from epicotyl explants of seedlings aseptically germinated on hormone-free MS medium. They were further maintained on media with 0–0.92 μM kinetin. Optimal shoot multiplication was at 0.46 μM kinetin. Inoculation by shoot wounding was performed with overnight suspension of A. rhizogenes A4M70GUS which contains GUS gene cointegrated in pRiA4. After 30 days hairy roots were produced at the wounding site in 31 explant (25% out of 124). Hairy roots were excised and further propagated on hormone-free medium as separate clones. In the first passage clones 3 and 6 could be distinguished by fast growth and spontaneous shoot regeneration. In other clones (12, 23 and 25) shoot regeneration required presence of cytokinins. The five shoot culture clones regenerated from hairy roots were further cultured on media with 0.46 μM kinetin. These shoots were characterized by good elongation and lateral shoot branching, short internodes, minute slightly curled leaves and well developed plagiotropic root system spreading over the surface of media. Thus all plants regenerated from hairy root cultures manifested the characteristic Ri syndrome phenotype. They all had a strong positive GUS reaction. PCR analysis confirmed presence of uidA sequence from the gus construct. They were also tolerant to nickel accumulating up to 24,700 μg g⁻¹ dry weight.     

Establishment of hairy root cultures of Psoralea species

Eight Psoralea species (Leguminosae) were inoculated with Agrobacterium rhizogenes, strains 8196 and 9402. Hairy roots were only induced by strain 9402. Attention was focussed on Psoralea lachnostachys. Transformed roots grew very rapidly in Gamborg B5 liquid medium with a doubling time of the culture of 38 hours. Whatever the culture conditions, the two furanocoumarins usually found in roots of Psoralea plants, psoralen and angelicin, were not detected in cultured transformed and non transformed roots even when some chitosan was added to the medium. However, 669 g.g^–1 dry matter of psoralen and 215 g.g^–1 dry matter of angelicin were found in roots from soil grown plants. A possible translocation of these compounds from the aerial parts to the roots is suggested.Abbreviations B5 medium Gamborg's medium (Flow laboratories's formulation) - NAA Naphthaleneacetic acid     

Cytogenetic analysis of hairy root cultures from a number of plant species transformed by Agrobacterium rhizogenes

Hairy root cultures, obtained after transformation of seven plant species by A. rhizogenes, were examined cytologically to assess their chromosome number. All species had the correct 2n diploid number of chromosomes in root tip cells. Free cell suspensions of two of the species were also examined and were found to be variable with polyploids or aneuploids predominating. The DNA from the hairy root cultures was isolated and the presence of the Agrobacterium T-DNA confirmed by Southern blotting. The relevance of these observations to the use of hairy roots in the study of plant secondary metabolite production is discussed.     

Induction of hairy root cultures from Gossypium hirsutum and Gossypium barbadense to produce gossypol and related compounds

Hairy root cultures were induced by inoculating cotyledonary leaves and hypocotyl segments from two cotton species, Gossypium hirsutum and Gossypium barbadense, with Rhizobium rhizogenes 15834. For both species, more hairy roots formed on inoculation sites on cotyledonary leaves than on hypocotyls. The addition of sucrose to basal Murashige–Skoog media increased the frequency of hairy root formation, whereas the addition of naphthalene acetic acid (0.54 μM) did not. After transfer to a liquid culture, hairy root growth was very rapid. After 3 wk in liquid culture, both cotton species produced gossypol, a di-sesquiterpene secondary metabolite with known anticancer activity, and two related methylated derivatives. Most (60–95%) gossypol produced by cultures was retained within the hairy root tissues, but some was found in the media. The average gossypol level observed among 96 different cultures was 15 mg/g of dry culture mass; however, some cultures produced >40 mg/g of dry culture mass. Variation in gossypol levels was greater for cultures arising from different transformation events than for multiple subclones of a single transformant. The high level of gossypol production attained by most of these cultures suggests that they will be valuable for studying the biochemical and molecular aspects of gossypol biosynthesis, capable of producing large amounts of gossypol and related compounds, and useful for generating modified forms of gossypol (e.g., radio-labeled gossypol) for understanding bioactivity mechanisms. Mention of trade names or commercial products or vendors in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

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     

Action spectrum for light-induced formation of adventitious shoots in hairy roots of horseradish

An action spectrum was determined for lightinduced formation of adventitious shoots in hairy roots of horseradish (Armoracia rusticana Gaert., Mey. et Scherb.) cultured in vitro. Near ultraviolet (350–400 nm), blue (440–460 nm) and red light (600–680 nm) were most effective in inducing formation of adventitious buds. Farred light (730 nm) inhibited the promotive effect of all three wavelength regions. These results are consistent with induction by phytochrome(s) of adventitious shoots in hairy roots of horseradish.     

Indigo production in hairy root cultures of Polygonum tinctorium Lour.

The transformed root culture of Polygonum tinctorium Lour. was established by infecting leaf explants with Agrobacterium rhizogenes A4. These cultures were examined for their growth and indigo content under various culture conditions. Among the four different culture media tested, SH medium showed the highest yield for root growth (28 mg dry wt/30 ml) and indigo production (152 g/dry wt). In SH medium, 30 g sucrose l^–1, 2500 mg KNO₃ l^–1, 300 mg NH₄H₂PO₄ l^–1 were the best conditions for indigo production at pH 5.7. The production of indigo in hairy roots slightly increased with the addition of 200 mg chitosan l^–1 (186 g/dry wt) and 20 U pectinase l^–1 (181 g/dry wt).     

Rosmarinic acid production in hairy root cultures of Agastache rugosa Kuntze

Rosmarinic acid, an important phenolic active compound, is one of the main active constituents of Agastache rugosa Kuntze and has astringent properties, antioxidant capacity, anti-inflammatory activity, antimutagenic ability, antimicrobial capacity, and antiviral properties. To investigate in vitro production of rosmarinic acid, we established a hairy root culture of A. rugosa by infecting leaf and stem explants with Agrobacterium rhizogenes R1000, and tested the growth and rosmarinic acid production of these cultures. Hairy roots were cultured in Murashige and Skoog liquid medium and maximum growth (14.1 g dry wt/l) was attained after 14 days of culture, at which time the content of rosmarinic acid was 116 mg/g dry wt. The present results demonstrate that hairy root culture of A. rugosa is a valuable alternative approach for the production of rosmarinic acid.     

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.     

Initiation of and solasodine production in hairy root cultures of Solanum mauritianum Scop.

Initiation and establishment of hairy root cultures from leaf or seedling hypocotyl explants of Solanum mauritianum Scop., using six strains of Agrobacterium rhizogenes was attempted. Success was only achieved following hypocotyl inoculation with strain LBA 9402. Transformation frequency was very low, with only one instance out of a possible 90 being recorded. Resultant hairy root cultures grew rapidly and could be maintained using a Murashige and Skoog (1962) medium supplemented with 0.1 g L^–1 myo-inositol and 3% sucrose, either as a solid or liquid culture. Under these conditions, the roots had a solasodine content of 126 g g^–1 DW. Lower levels of solasodine and decreased root growth rates were recorded when the medium strength was reduced by half or 3% glucose substituted for the 3% sucrose.Abbreviations MS Murashige and Skoog's (1962) medium     

Transformation of homozygous diploid potato with an Agrobacterium tumefaciens binary vector system by adventitious shoot regeneration on leaf and stem segments

Transformed potato (Solanum tuberosum) plants were obtained from homozygous diploid potato by using a transformation procedure in combination with an adventitious shoot regeneration method. Leaf and stem explants were inoculated with an Agrobacterium tumefaciens strain which contained a binary vector (pVU 1011) carrying the neomycin phosphotransferase gene. Shoot regeneration most effectively on stem explants, occurred within six weeks directly from the explants without introducing a callus phase. A strong seasonal influence on transformation efficiencies was observed. Analysis of a number of randomly selected regenerated shoots for their ability to root and form shoots on kanamycin-containing medium shows that over 90% of the regenerated shoots obtained are transformed. In a number of shoots transformation was confirmed by a test for the presence and expression of the NPT-II gene.     

Segregation of T-DNA copies in the progeny of a regenerant plant from a mannopine-positive hairy root line

We have analyzed opine content, T-DNA content, and developmental features of one hairy root culture line and its progeny. Opine-positive progeny still have the hairy root phenotype and have essentially the same T-DNA structure as the parental plant. Opineless progeny do not exhibit the hairy root phenotype. Some of them do not contain any T-DNA sequences, whereas others contain only the left part of T-DNA. These results are compatible with the hypothesis that, in the hairy root line analyzed, the left part of T-DNA is integrated independently from the core T-DNA and can therefore segregate during sexual reproduction.     

In vitro regeneration of shoot buds and plantlets from seedling root segments of Brassica napus L.

Root segments obtained from aseptically germinated seedlings of Brassica napus cv. Westar were used to optimize conditions for high-frequency shoot bud differentiation. The presence of low kinetin (0.5 M) and relatively high indole-butyric acid (1.0 M) levels facilitated optimum shoot bud differentiation. Modified MS medium (MMS) was superior to the other three basal media tested (MS, B5 and White's). Elevated sodium dihydrogen phosphate levels increased the differentiation of shoot buds. Increasing or decreasing the level of sucrose from 3% reduced the frequency of explants forming shoot buds. Addition of glutamine enhanced both the frequency of responding explants, as well as the number of shoots per responding explant. Root segments from 13-day-old seedlings produced the highest response (58%) in the presence of 100 mg l^-1 glutamine. The position of the segment on the main root, size, and the presence or absence of lateral roots altered the morphogenic response. Sealing of the donor seedling cultures with Parafilm^® instead of Stretch' n seal^® resulted in a higher production of shoot buds, although root segment cultures were not affected by the type of sealing. Spontaneous rooting occurred on all developed shoots.Dedicated to Dr. Friedrch Constabel on the occasion of his 60th birthday     

Enhanced production of antimicrobial sesquiterpenes and lipoxygenase metabolites in elicitor-treated hairy root cultures of Solanum tuberosum

Potato (Solanum tuberosum) hairy root cultures, established by infecting potato tuber discs with Agrobacterium rhizogenes, were used as a model system for the production of antimicrobial sesquiterpenes and lipoxygenase (LOX) metabolites. Of the four sesquiterpene phytoalexins (rishitin, lubimin, phytuberin and phytuberol) detected in elicitor-treated hairy root cultures, rishitin (213 g g^–1 dry wt) was the most predominant followed by lubimin (171 g g^–1 dry wt). The elicitors also induced LOX activity (25-fold increase) and LOX metabolites, mainly 9-hydroxyoctadecadienoic acid and 9-hydroxyoctadecatrienoic acid, in potato hairy root cultures. The combination of fungal elicitor plus cyclodextrin was the most effective elicitor treatment, followed by methyl jasmonate plus cyclodextrin in inducing sesquiterpenes and LOX metabolites.     

Improvement of ginsenoside production by jasmonic acid and some other elicitors in hairy root culture of ginseng (Panax ginseng C. A. Meyer)

Summary Hairy root cultures of Panax ginseng, established after the infection of root sections with Agrobacterium rhizogenes KCTC 2703, were cultured in phytohormone-free Murashige and Skoog (MS) liquid medium containing different concentrations of jasmonic acid and some other elicitors, in order to promote ginsenoside accumulation. Jasmonic acid in the range 1.0−5.0 mg l⁻¹ (4.8–23.8 μM) strongly improved total ginsenoside production in ginseng hairy roots. Peptone (300 mg l⁻¹) also showed some effect on ginsenoside improvement; however its effect was much weaker than that of jasmonic acid. Ginsenoside content and productivity were 58.65 and 504.39 mg g⁻¹, respectively. The Rb group of ginsenoside content was increased remarkably by jasmonic acid, while Rg group ginsenoside content changed only slightly compared to controls. However, jasmonic acid also strongly inhibited ginseng hairy root growth.