Putrescine influences growth and production of coumarins in transformed and untransformed root cultures of witloof chicory (Cichorium intybus L. cv. Lucknow local)

The effect of putrescine on growth and production of two coumarins, esculin, and esculetin in the transformed and untransformed roots of chicory (Cichorium intybus L. cv. Lucknow local) was examined. To study the role of putrescine (Put) on growth and production of coumarins, polyamine inhibitors namely α-DL-difluromethylornithine (DFMO) and α-L-difluromethylarginine (DFMA) were used at 1 mM levels. Treatment with 1.5 mM of putrescine (Put) produced 1.96 - fold and 4.0 - fold increase in the growth of transformed and untransformed roots of chicory, respectively. The treatment with polyamine inhibitors showed much lower growth, as well as production compared with both 1.5 mM putrescine treatment and control in both transformed and untransformed chicory roots. The endogenous polyamines, both free and conjugated, were studied over the whole culture period, and it was seen that conjugated titers of all three polyamines viz., putrescine, spermidine and spermine were higher than level of free polyamines, throughout the culture period in both transformed and untransformed roots of chicory. Treatment in which polyamine inhibitors were used showed lower level of endogenous polyamines as compared with the 1.5 mM putrescine treated sample in both the systems. The treatment wherein putrescine was added at 1.5 mM level showed maximum accumulation of endogenous conjugated putrescine (2098.86±157.6 nmoles g⁻¹ FW; 896.8±67.2 nmoles·g⁻¹ FW), on the 14^th day in both transformed and untransformed roots respectively. The production of esculin and esculetin was strictly correlated with growth in every treatment in both systems. Putrescine at 1.5 mM resulted in greater length of primary root in transformed (18.3±1.4 cm) and untransformed (6.86±0.51 cm) as compared with their respective controls (11±0.9 cm; 2.9±0.1cm) and greater number of secondary and tertiary roots. This study suggests that putrescine influences plant root development and differentiation, and it also provides insight into the morphological changes that occur in roots in response to the external supply of polyamines.     

Influence of exogenous hormones on growth and secondary metabolite production in hairy root cultures of Cichorium intybus L. CV. Lucknow local

Summary The effect of exogenously fed hormones on hairy root cultures of Cichorium intybus L. ev. Lucknow Local was studied. It was seen that auxin in the presence of low levels of kinetin induces rapid disorganization in hairy root cultures of C. intybus, ultimately to form suspension cultures, and this process was associated with the decrease in coumarin content in the cells. Of various treatments, it was observed that with an increase in the auxin: cytokinin ratio, the biomass decreased with the increase in disorganization index during the culture period of 28 d. The disorganization index was less when the inoculum size was enhanced to 10-fold. The total endogenous indole-3-acetic acid titers and indole-3-acetic acid oxidase activity also decreased with an increase in disorganization index, and was independent of initial inoculum size, with only a magnitude difference. The total coumarin content strictly correlated with growth in all the treatments. In contrast, exogenously supplied gibberellic acid at the 0.5 mg l⁻¹ level enhanced growth, coumarin content, and branching patterns over the control and other treatments on day 28. The exogenously fed growth regulators had an effect on growth, auxin and coumarin biosyntheses, wherein transformed roots treated with increasing concentration of auxin to cytokinin ratios lost their ability for coumarin biosynthesis. The behavior of hairy roots from an Indian cultivar of chicory upon growth regulator treatment is discussed in terms of growth, coumarin and auxin biosyntheses.     

Chicory extracts from Cichorium intybus L. as potential antifungals

In this work extracts from roots of the common vegetable Cichorium intybus L., highly appreciated for its bitter taste, were studied to investigate their possible biological activity on fungi from a variety of ecological environments: some are parasites on plants (phytopathogens) or of animals and humans (zoophilic and anthropophilic dermatophytes), others live on the soil and only seldom parasitize animals (geophilic dermatophytes). The extracts were ineffective on geophilic species and on tested phytopathogens, with the exception of Pythium ultimum, whereas they inhibited the growth of zoophilic and anthropophilic dermatophytes, in particular Trichophyton tonsurans var. sulfureum, whose treatment caused morphological anomalies, here observed by scanning electron microscopy. This behaviour is discussed on the basis of the presence in the chicory extract of the two main sesquiterpene lactones, 8-deoxylactucin and 11β,13-dihydrolactucin.     

Secondary metabolism of hairy root cultures in bioreactors

Summary In vitro cultures are being considered as an alternative to agricultural processes for producing valuable secondary metabolites. Most efforts that use differentiated cultures instead of cell suspension cultures have focused on transformed (hairy) roots. Bioreactors used to culture hairy roots can be roughly divided into three types: liquid-phase, gas-phase, or hybrid reactors that are a combination of both. The growth and productivity of hairy root cultures are reviewed with an emphasis on successful bioreactors and important culture considerations. The latter include strain selection, production of product in relation to growth phase, media composition, the gas regime, use of elicitors, the role of light, and apparent product loss. Together with genetic engineering and process optimization, proper reactor design plays a key role in the development of successful large scale production of secondary metabolites from plant cultures.     

Furofuran lignans from a callus culture of Cichorium intybus

Three new and one known furofuran lignans—syringaresinol derivatives—along with the known phenylpropanoids cichoriin and syringin were isolated from a callus tissue of Cichorium intybus. The compounds were characterised by spectral methods. This is the first report on the presence of furofuran lignans in Cichorium species.     

Comparative evaluation of modified bioreactors for enhancement of growth and secondary metabolite biosynthesis usingPanax ginseng hairy roots

Hairy root cultures have demonstrated great promise in terms of their biosynthetic capability toward the production of secondary metabolites, but continue to constitute a major challenge with regard to large-scale cultures. In order to assess the possibility of conducting mass production of biomass, and the extraction of useful metabolites fromPanax ginseng. P. ginseng hairy roots, transformed byRhizobium rhizogenes KCTC 2744, were used in bioreactors of different types and sizes. The most effective mass production of hairy roots was achieved in several differently sized air bubble bioreactors compared to all other bioreactor types. Hairy root growth was enhanced by aeration, and the production increased with increasing aeration rate in a 1 L bioreactor culture. It was determined that the hairy root growth rate could be substantially enhanced by increases in the aeration rate upto 0.5 wm, but at aeration rates above 0.5 wm, only slight promotions in growth rates were observed. In 20 L air bubble bioreactors, with a variety of inoculum sizes, the hairy roots exhibited the most robust growth rates with an inoculum size of 0.1% (w/v), within the range 0.1 to 0.7% (w/v). The specific growth rates of the hairy roots decreased with increases in the inoculum size.     

Bioreactor production of camptothecin by hairy root cultures of Ophiorrhiza pumila

A large-scale culture of hairy root of Ophiorrhiza pumila using a modified 3 l bioreactor was established. The hairy roots, incited by infection of Agrobacterium rhizogenes were grown in the bioreactor equipped with a stainless net. The final concentration of camptothecin was 0.0085% fresh wt of tissue, and the total production of camptothecin, an anti-neoplastic quinoline alkaloid, reached 22 mg over 8 weeks' culture in the reactor. Approx. 17% (3.6 mg) of the total camptothecin produced was excreted into the culture medium.     

Development of a nutrient mist bioreactor for growth of hairy roots

Summary Hairy root cultures of Artemisia annua L. were cultivated in three different mist bioreactors, each fitted with three stainless steel meshes. The growth rates in the three 2.3-L mist bioreactors differed. After 25 d, the growth index (final dry weight/initial dry weight) of the roots was 42 in a nutrient mist bioreactor, 61 in an inner-loop nutrient mist bioreactor, and 68 in a modified inner-loop nutrient mist bioreactor. Under a misting cycle of 3/30 (ON 3 min/OFF 30 min) for 25 d, dry weight reached 13.6 g/L of medium in the modified inner-loop nutrient mist bioreactor in which nutrient could be supplied without dilution of mist by air flow.     

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     

Callus induction and plantlet regeneration inCichorium intybus L.: II. Effect of different hormonal treatments

Summary Expiants ofCichorium intybus L. storage roots were grownin vitro on a modified Heller's medium lacking auxins and cytokinins, or supplemented with auxins (either 2,4-D or NAA) alone or with a cytokinin (kinetin) or auxin and kinetin combinations in different concentrations. The morphogenetic responses of root explants varied with the different hormonal treatments. The best response for callus growth was obtained in presence of 2,4-D. On the contrary, kinetin alone was very effective for shoot induction, increasing the formation of adventitious buds (up to 100% of the explants) in respect to control (hormone-free medium). NAA induced either shoot differentiation (in a medium frequency) or root formation. Expiants excised from root zones near to apex, which showed on hormone-free medium a very low regenerative capacity (lower than proximal zones of the root), responded to kinetin by increasing significantly the number of shoots from adventitious buds.Cytological analyses in developing primary calli showed, in all media, high incidence of amitotic phenomena confirmed by DNA cytophotometry in calli at different growth stages. The histological analysis demonstrated the formation of meristematic growth centers on the organogenesis inducing media and the subsequent development of these meristemoids as shoot (or root) apices in the callus mass.The results are discussed in comparison with previous observations of the authors inCichorium intybus (Caffaro et al. 1982) and in relation to the action of hormonal treatments on callus formation and organogenesis. The cytological and histological results are also discussed in relation to the hormonal composition of the medium.     

Agrobacterium mediated transfer of a mutant Arabidopsis acetolactate synthase gene confers resistance to chlorsulfuron in chicory (Cichorium intybus L.)

Summary Leaf discs of C. intybus were inoculated with an Agrobacterium tumefaciens strain harboring a neomycin phosphotransferase (neo) gene for kanamycin resistance and a mutant acetolactate synthase gene (csr1-1) from Arabidopsis thaliana conferring resistance to sulfonylurea herbicides. A regeneration medium was optimized which permitted an efficient shoot regeneration from leaf discs. Transgenic shoots were selected on rooting medium containing 100 mg/l kanamycin sulfate. Integration of the csr1-1 gene into genomic DNA of kanamycin resistant chicory plants was confirmed by Southern blot hybridizations. Analysis of the selfed progenies (S1 and S2) of two independent transformed clones showed that kanamycin and chlorsulfuron resistances were inherited as dominant Mendelian traits. The method described here for producing transformed plants will allow new opportunities for chicory breeding.     

Fed-batch hairy root cultures within situ separation

Fed-batch cultures ofL. erythrorhizon hairy root were carried out by controlling sucrose concentration and media conductivity in a shake flask and a modified stirred tank reactor. For the efficient product recovery from the culture,in situ adsorption by XAD-2 was also conducted. When sucrose was used as a carbon source, the highest shikonin production and hairy root growth were obtained. When glucose or fructose was used instead, the growth was severely inhibited. In addition, it was found that alternating feeding of sucrose could be used as an effective strategy for enhancing the productivity of shikonin derivatives., As the XAD-2 amount was increased up to 1.5 g/L, shikonin production was enhanced by removing shikonin produced and other products which might be inhibitory to cell growth. Most amount of shikonin produced was successfully recovered in XAD-2 (Over 99%). Using hairy root culture in a modified stirred tank reactor, the shikonin productivity and hairy root growth rate on the average were 9.34 mg/L day and 0.49 g DCW/L · day, respectively.     

Solasodine glycoside production by hairy root cultures of Physalis minima Linn.

Hairy root cultures of Physalis minima L. were developed using Agrobacterium rhizogenes, strain ATCC 15834 mediated transformation and grown in half strength of Murashige and Skoog medium containing 8% (w/v) sucrose. Media supplementation with 1 mg naphthalenacetic acid l(-1) and 1 mg benzyladenine increased solasodine glycoside up to 900 g dry wt, which was 20 times higher than that in the native root.     

Development of Linum flavum hairy root cultures for production of coniferin

Linum flavum hairy roots were initiated from leaf discs using Agrobacterium rhizogenes strains LBA9402 and TR105 though two other strains, 15834 and A4, were relatively ineffective for induction. Significant variation in coniferin accumulation was observed between hairy root lines originating from different L. flavum seedlings and/or A. rhizogenes strains. Coniferin reached 58 mg g^–1 dry wt by culturing the roots in Linsmaier and Skoog (LS) medium with 2,4-dichlorophenoxyacetic acid and naphthaleneacetic acid as growth regulators.     

Lobeline production by hairy root culture of Lobelia inflata L.

Hairy roots were obtained following inoculation of the stems of Lobelia inflata L. with Agrobacterium rhizogenes strain ATCC 15834. These hairy roots contained agropine and mannopine. In addition, lobeline was detected by HPLC and confirmed by mass spectrometry. Various media were tested for the growth of hairy roots as well as for the content of lobeline in hairy roots. The growth rate of hairy roots cultured in Nitsch and Nitsch's medium was approximately one third of those cultured in other media. The lobeline content of hairy roots (18–54 g/g dry weight) cultured in these media was the same order of magnitude compared with that of roots of L. inflata (24 g/g dry weight) cultivated in pots. The hairy roots cultured in Nitsch and Nitsch's medium were morphologically different from those cultured in other media.Abbreviations MS medium Murashige and Skoog's medium - 1/2 MS medium one-half strength of the standard Murashige and Skoog's medium - B5 medium Gamborg's B5 medium - NN medium Nitsch and Nitsch's medium - FW fresh weight - DW 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     

Cryopreservation of hairy root cultures of Vinca minor (L.) by encapsulation-dehydration

Hairy root cultures of Vinca minor and Ajuga reptans var. atropurpurea could be cryopreserved when the roots were precultured and encapsulated in 2% (w/v) alginate beads with 0.3 M sucrose and 0.5 M glycerol and dehydrated until the bead weight reached 25% of the initial weight before cooling in liquid nitrogen. Preculture and encapsulation of the roots with abscisic acid was effective in increasing the survival rates. For V. minor root tips moreover a sufficiently high survival rate of more than 70% was attained by eliminating glycerol from the preculture medium and dehydration of beads until 23% of the initial weight was reached instead of 25%.     

Scopolamine and hyoscyamine production by hairy root cultures of Brugmansia candida: influence of calcium chloride, hemicellulase and theophylline

CaCl₂ (50 mM) and hemicellulase (0.5 U mg^–1) increased the intracellular accumulation (60–250%), release (60–200%) and production (45–200%) of hyoscyamine and scopolamine in hairy roots of Brugmansia candida. Theophylline (0.25 mM), alone or in combination with hemicellulase, was ineffective in increasing hyoscyamine and scopolamine production.     

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).