Alkaloid production in Vernonia cinerea: Callus, cell suspension and root cultures

Fast-growing callus, cell suspension and root cultures of Vernonia cinerea, a medicinal plant, were analyzed for the presence of alkaloids. Callus and root cultures were established from young leaf explants in Murashige and Skoog (MS) basal media supplemented with combinations of auxins and cytokinins, whereas cell suspension cultures were established from callus cultures. Maximum biomass of callus, cell suspension and root cultures were obtained in the medium supplemented with 1 mg/L alpha-naphthaleneacetic acid (NAA) and 5 mg/L benzylaminopurine (BA), 1.0 mg/L NAA and 0.1 mg/L BA and 1.5 mg/L NAA, respectively. The 5-week-old callus cultures resulted in maximum biomass and alkaloid contents (750 microg/g). Cell suspension growth and alkaloid contents were maximal in 20-day-old cultures and alkaloid contents were 1.15 mg/g. A 0.2-g sample of root tissue regenerated in semi-solid medium upon transfer to liquid MS medium containing 1.5 mg/L NAA regenerated a maximum increase in biomass of 6.3-fold over a period of 5 weeks. The highest root growth and alkaloid contents of 2 mg/g dry weight were obtained in 5-week-old cultures. Maximum alkaloid contents were obtained in root cultures in vitro compared to all others including the alkaloid content of in vivo obtained with aerial parts and roots (800 microg/g and 1.2 mg/g dry weight, respectively) of V. cinerea.     

Establishment of hairy root cultures ofDatura stramonium. Characterization and stability of tropane alkaloid production during long periods of subculturing

The aim of this paper was the screening of the variability of growth patterns, biomass and tropane alkaloid production of 500 hairy root lines ofDatura stramonium. Data on the long term stability in alkaloid production of these lines for more than 5 years are also provided. In an effort to obtain high alkaloid-producing root clones, it is demonstrated that systematic selection is necessary. Comparisons are made, mainly concerning alkaloid production and its stability, with normal root cultures initiated from the same mother plants when necessary. Hairy root cultures were found to have a hyoscyamine and scopolamine bioproductivity of 2 orders of magnitude higher than mother plants.     

Production of tropane alkaloids in diploid and tetraploid plants and in vitro hairy root cultures of Egyptian henbane (Hyoscyamus muticus L.)

In this study, the effects of ploidy level and culture medium were studied on the production of tropane alkaloids. We have successfully produced stable tetraploid hairy root lines of Hyoscyamus muticus and their ploidy stability was confirmed 30?months after transformation. Tetraploidy affected the growth rate and alkaloid accumulation in plants and transformed root cultures of Egyptian henbane. Although tetraploid plants could produce 200% higher scopolamine than their diploid counterparts, this result was not observed for corresponding induced hairy root cultures. Culture conditions did not only play an important role for biomass production, but also significantly affected tropane alkaloid accumulation in hairy root cultures. In spite of its lower biomass production, tetraploid clone could produce more scopolamine than the diploid counterpart under similar growth conditions. The highest yields of scopolamine (13.87?mg?l^?1) and hyoscyamine (107.7?mg 1^?1) were obtained when diploid clones were grown on medium consisting of either Murashige and Skoog with 60?g/l sucrose or Gamborg??s B5 with 40?g/l sucrose, respectively. Although the hyoscyamine is the main alkaloid in the H. muticus plants, manipulation of ploidy level and culture conditions successfully changed the scopolamine/hyoscyamine ratio towards scopolamine. The fact that hyoscyamine is converted to scopolamine is very important due to the higher market value of scopolamine.     

Population balance approach to modeling hairy root growth

Though numerous models have been developed to describe the growth of microbial cell cultures, far fewer models are available to describe the growth of hairy root cultures. Here a population balance model is proposed to simulate the growth of hairy roots. The model accounts for the increase in biomass due to elongation of a branch by cell division as well as the formation of new branches. The model incorporates the fact that although the likelihood of the formation of a new lateral branch is a maximum at a specific age of the parent branch, lateral branches can form over a distribution of ages of the parent branch. Model parameters are estimated using the genetic algorithm based on experimental data for batch and continuous bioreactors. The model proposed here may provide a better understanding of the increase in biomass of hairy root cultures.     

Tropane alkaloid production by hairy roots of Atropa belladonna obtained after transformation with Agrobacterium rhizogenes 15834 and Agrobacterium tumefaciens containing rol A, B, C genes only

Atropa belladonna leaf disks were infected by a wild strain Agrobacterium rhizogenes 15834 harboring the Ri-TL-DNA and by a disarmed Agrobacterium tumefaciens strain harboring a construction with only rol ABC and npt II genes. Thirteen root lines were established and examined for their growth rate and alkaloid productivity to evaluate the possible role of rol genes in morphological differentiation and in tropane alkaloid formation. A great diversity has been observed in the growth rate of these 13 root lines. The root biomass increased up to 75 times. The total alkaloid contents were similar in the root lines obtained by infection with A. rhizogenes 15834 and A. tumefaciens rol ABC. The last ones accumulated between 4 (1.1 mg g(-1) DW) and 27 (8 mg g(-1) DW) times more alkaloids than the intact roots (0.3 mg g(-1) DW). This work has shown that the rol ABC genes were sufficient to increase tropane alkaloid production in A. belladonna hairy root cultures.     

Establishment and characterization of photosynthetic hairy root cultures of Datura stramonium

Twelve different lines of Datura stramonium (normal and hairy) root cultures were subjected to conditions which induce photoautotrophy. Two of the hairy root lines responded to induction, showing clearly a diminished growth rate when compared to heterotrophic cultures, an increase in chlorophyll, a net O₂ evolution, CO₂ fixation, and de novo synthesis of the ribulose 1,5 biphosphate carboxylase enzyme. A time course of growth and tropane alkaloid levels in the tissue and medium, revealed a correlation between the development of the photosynthetic apparatus and the increase in scopolamine. Although normal cultures did not grow photosynthetically, they showed some greening response under the first step of the induction. The correlation between development of photosynthesis and increase in scopolamine synthesis were corroborated with normal root cultures. This experimental model is used for the basic study of the regulatory enzymes involved in the biosynthesis of tropane alkaloids, as well as for the study of their mechanisms of transport.     

Indole alkaloid production by transformed and non-transformed root cultures ofCatharanthus roseus

Summary Ten transformed and two non-transformed root lines ofCatharanthus roseus were established. A systematic study of the growth kinetics and alkaloid content was performed over a culture cycle and showed significant differences between transformed and non-transformed cultures. Mean doubling times for transformed and normal root lines were 2.8 and 19.5 days, respectively. Alkaloid content in hairy roots was from two- to threefold higher than in the non-transformed tissues. The established transformed root lines produced a wide variety of indole alkaloids as can be observed from their complex thin layer chromatography patterns. A large quantity of serpentine was determined in two of the transformed root cultures. Alkaloid content, both quantitatively and qualitatively, has been stable in the hairy root cultures for more than 2 yr of subculturing.     

Product enhancement and recovery from transformed root cultures of Nicotiana glauca

Transformed roots of Nicotiana glauce synthesize the alkaloids nicotine and anabasine at levels reflecting the parent plants. Media composition, strength, and pH were evaluated with respect to biomass yield and productivity. Full-strength Gamborg's B5 medium proved the best for biomass yield while half-strength, or low-salt, medium enhanced alkaloid accumulation. A detailed investigation of media nitrate levels demonstrated how these may be manipulated to promote growth and intracellular or extracellular alkaloid levels. High nitrate concentrations were found to significantly enhance media alkaloid levels at the end of the growth phase. Media pH is also important, although transformed roots will grow in Gamborg's B5 medium between pH 3 and 9, root biomass is favored by an increase in medium alkalinity, while alkaloid release is encouraged by mildly acidic pH.Transformed roots release a proportion of their secondary metabolites into the growth medium. By continually removing root products, any feedback inhibition on enzymatic reactions is reduced, as are the toxic effects resulting from product accumulation. In this article we describe the use of Amberlite resins (XAD-2 and XAD-4) to enhance alkaloid levels (nicotine and anabasine) of hairy root cultures of Nicotiana glauca by a factor of 10 with no adverse effect on root growth. The performance of the Amberlite columns was subsequently investigated with respect to alkaloid adsorption and desorption, including an evaluation of the effects of pH and loading capacity. The resins also adsorb media constituents which are identified and quantified as part of this work. Resulting nutritional stresses are thought to be partly responsible for enhancing secondary metabolism at the expense of biomass yield. However, the net effects of using Amberlite resins as a means of product removal significantly increases the overall product yield and the extent to which products are released into the growth medium.     

Indole alkaloid production by hairy root cultures ofCatharanthus roseus: Growth kinetics and fermentation

Summary Growth kinetics and indole alkaloid production ofCatharanthus roseus hairy root cultures were studied in shake flasks and in a small scale fermenter. A logistic growth model commonly used for microbes described well the growth of hairy roots. Of the several parameters analyzed during the cultivation of hairy roots, a linear relationship between sucrose consumption and dry weight increase was obtained. This suggests the validity of sugar analysis as a means in monitoring the growth of hairy roots in fermenters.     

Stability of alkaloid production in cell suspension cultures of Tabernaemontana divaricata during long-term subculture

Two cell lines of Tabernaemontana divaricata cell suspension culture with different growth and alkaloid production profiles were transferred to the same medium. During 30 subcultures the changes in growth and alkaloid production were followed and compared to those of the original cell lines. The presence of NAA and BAP in the medium resulted in an increase of biomass and alkaloid yield. The effect on the growth proved to be stable during these 30 subcultures. Alkaloid production showed a maximum in the 4th subculture after the change of the medium, and stabilized on a higher level than found in the original cell lines. During some growth cycles also the activities of tryptophan decarboxylase (TDC), strictosidine synthase (SSS), and phenylalanineammonia-lyase (PAL) were measured. In both the original cell lines and the derived cell lines, growth and alkaloid production proved to be stable all through the experiment, although the derived cell lines had a period of adaptation to the new medium with increased productivity.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - BAP benzylaminopurine - DW dry weight - TDC tryptophan decarboxylase - SSS strictosidine synthase - PAL phenylalanineammonia-lyase - PAT phenylalanineammonia-transaminase     

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     

Tropane alkaloid production inDatura stramonium root cultures

Summary Tropane alkaloid production was studied in different root cultures ofDatura stramonium. Cultured roots were obtained with 10⁻⁶ M of indolbutyric acid. Their doubling times were from 6 to 19 days. Hyoscyamine content varied from 0.17 to 0.62% dry weight, and scopolamine content from 0.08 to 0.33% dry weight, depending on the lines. A comparison of the bioproductivity of these compounds in the pot-grown plant roots showed that it was two to three orders lower than cultured roots, and it increased one order of magnitude considering the productivity on the whole plant. Bioproductivity, growth capacity and alkaloid production stability during subsequent transfers (more than 2 yr) are reported. Only one root line (N5) showed excretion of the alkaloids to the culture medium. Characterization of three selected lines (N1, N5, and N9) showed that the highest alkaloid production is reached at the stationary phase of growth, with the exception of line N9.     

Production of solasodine by hairy root,callus, and cell suspension cultures of Solanum aviculare Forst.

The production of the steroidal alkaloid solasodine, an alternative to diosgenin as a precursor for the commercial production of steroid drugs, was studied in hairy root, callus, and cell suspension cultures of Solanum aviculare Forst. through manipulation of culture medium. The individual and combined effects of medium components on the growth index and the production of solasodine were analyzed using factorial analysis of variance. Solasodine content was optimized to 6.2 mg g⁻¹in the hairy root, 1.4 mg g⁻¹callus, and 0.7 mg g⁻¹in cell suspension cultures (dry weight). An improved isocratic reversed phase high performance liquid chromatographic method provided selective determination of the solasodine content of these samples. Analysis of growth and solasodine content of hairy root cultures and callus cultures demonstrated that the production of solasodine was shown to be growth-dependent in hairy root cultures but not in callus cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

The effect of plant growth regulators on biomass formation and lobeline production of Lobelia inflata L. hairy root cultures

We have studied the biomass and alkaloid production of geneticallytransformed hairy root cultures of Lobelia inflata L. Thehairy root clone 8009/h7 transformed with Agrobacteriumrhizogenes strain R 1601 was cultivated on B5 solid media containingdifferent amounts of the growth regulators KIN, IAA or NAA. KIN significantlydecreased growth and lobeline production and strongly inhibited biomassformation at 5 mg/l. IAA and NAA had characteristic morphologicaleffects on growth, in increasing the number of the lateral roots. However theyrestricted linear growth. Addition of IAA or NAA into the culture mediumincreased the biomass formation and lobeline production of hairy roots. It wasfound that the greatest amount of lobeline was obtained at the 0.2mg/l IAA concentration, similar to the effect of NAA.     

Combining enhanced root and shoot growth reveals cross talk between pathways that control plant organ size in Arabidopsis

Functionally distinct Arabidopsis (Arabidopsis thaliana) genes that positively affect root or shoot growth when ectopically expressed were combined to explore the feasibility of enhanced biomass production. Enhanced root growth resulting from cytokinin deficiency was obtained by overexpressing CYTOKININ OXIDASE/DEHYDROGENASE3 (CKX3) under the control of the root-specific PYK10 promoter. Plants harboring the PYK10-CKX3 construct were crossed with four different transgenic lines showing enhanced leaf growth. For all combinations, the phenotypic traits of the individual lines could be combined, resulting in an overall growth increase. Unexpectedly, three out of four combinations had more than additive effects. Both leaf and root growth were synergistically enhanced in plants ectopically expressing CKX3 and BRASSINOSTEROID INSENSITIVE1, indicating cross talk between cytokinins and brassinosteroids. In agreement, treatment of PYK10-CKX3 plants with brassinolide resulted in a dramatic increase in lateral root growth that could not be observed in wild-type plants. Coexpression of CKX3 and the GROWTH-REGULATING FACTOR5 (GRF5) antagonized the effects of GRF5 overexpression, revealing an interplay between cytokinins and GRF5 during leaf cell proliferation. The combined overexpression of CKX3 and GIBBERELLIN 20-OXIDASE1 led to a synergistic increase in leaf growth, suggesting an antagonistic growth control by cytokinins and gibberellins. Only additive effects on root and shoot growth were visible in plants ectopically expressing both CKX3 and ARABIDOPSIS VACUOLAR PYROPHOSPHATASE1, hinting at an independent action mode. Our results show new interactions and contribute to the molecular and physiological understanding of biomass production at the whole plant level.     

Studies on the optimization of growth and indole alkaloid production by hairy root cultures of Catharanthus roseus

Catharanthus roseus hairy root cultures, genetically transformed with Agrobacterium rhizogenes, produce a wide variety of indole alkaloids. The effect of sucrose, phosphate, nitrate, and ammonia concentrations on growth and indole alkaloid production of C. roseus hairy root cultures were studied by using statistical experimental designs and linear regression analysis. Contradictory effects of these nutrients on growth and indole alkaloid production were found. The maximal growth was obtained by having 77. 8 mg NaH(2)PO(4) . H(2)O/L and 1. 311 g KNO(3)/L in the medium, whereas the specific production of alkaloids was highest at the lowest levels of all the nutrients studied. The maximal dry weight was obtained with high values of sucrose and ammonia, but clear optimum concentrations could not be found. When having enough nutrients to support reasonable growth, it appeared difficult to affect the specific alkaloid production rates considerably. The growth (dry wt.) with the optimized nutrient concentrations in the medium was more than 50% better than in the control medium with about the same alkaloid production.     

Acid-induced wall loosening is confined to the accelerating region of the root growing zone

The aims of this study were to quantify developmental differences in acid growth along the root axis and to determine whether these differences were due to alterations in cell turgor or cell wall properties. The apoplast pH of maize roots growing in hydroponics was altered from pH 7.0 to pH 3.4 using 2 mol m^-3 citrate-phosphate buffer or unbuffered solutions. Whole root elongation rate rapidly increased and measurement of the local growth profile indicated that this increase in growth occurred in young cells in the accelerating zone (apical 0-4 mm) while more proximal growing cells were unaffected. Unbuffered solutions of identical pH produced qualitatively similar results. Single cell turgor pressures were unchanged between pH treatments both longitudinally and radially in the root tip. This suggests that the rapid acid-induced changes in growth rate were due to an increase in cell wall loosening. Single cell osmotic pressure and water potential were not significantly different between pH treatments. Acid pH caused net solute import at the root tip to increase 3- to 4-fold, which, coupled with the maintenance of turgor and osmotic pressure, indicated that solute import was not limiting expansion. Thus, acidic solutions cause an increase in growth in accelerating but not decelerating regions. It has been shown for the first time that acid growth in intact, growing roots is not due to differences in turgor, assigning these changes to cell wall properties. Possible cell wall biochemical alterations are discussed.     

Heterologous expression of Vitreoscilla hemoglobin (VHb) and cultivation conditions affect the alkaloid profile of Hyoscyamus muticus hairy roots

Fast-growing hairy root cultures of Hyoscyamus muticus induced by Agrobacterium rhizogenes offer a potential production system for tropane alkaloids. Oxygen deficiency has been shown to limit growth and biomass accumulation of hairy roots, whereas little experimental data is available on the effect of oxygen on alkaloid production. We have investigated the effect of Vitreoscilla hemoglobin (VHb) expression and cultivation conditions on the complete alkaloid profile of H. muticus hairy roots in shake flasks and in a laboratory scale bioreactor. We optimized the growth medium composition and studied the effects of sucrose, ammonium, nitrate, and phosphate on growth and alkaloid production. Maximum biomass accumulation was achieved with the highest and maximum hyoscyamine content with the lowest sucrose concentration. The optimum nitrate concentration for growth was higher for the VHb line than the control. Neither VHb expression nor aeration improved the hyoscyamine content significantly, thus suggesting that hyoscyamine biosynthesis is not limited by oxygen availability. Interestingly, the effect of VHb expression on the alkaloid profile was slightly different from that of aeration. VHb expression did not affect the concentrations of cuscohygrine, which was increased by aeration. Therefore, the effect of VHb is probably not related only to its ability to increase the intracellular effective oxygen concentration.     

Opportunities for regulation of sugar beet storage root growth

The percentage of sucrose in sugar beet storage root fresh and dry matter is closely related to root structure. It has been suggested that the sucrose content might be increased by using plant growth regulators to modify storage root structure through control of cambial development, cell division and cell expansion. During storage root development correlations were found between the changing phytohormone profiles and the formation of secondary cambia and their subsequent cell division and expansion. Sugar beet root derived cell suspension cultures were used for detailed studies of the roles of endogenous phytohormones. The gibberellin synthesis inhibitor paclobutrazol was tested in cell cultures and whole plants. The observations provide a basis for development of plant growth regulator regimes to optimise sucrose yield from sugar beet.     

Correction for non-linear relationships between root size and short term Pi uptake in genotype comparisons

Variation in phosphate (Pi) absorption rates, root length and root mass was investigated in 25 genotypes or inbred lines of Arabidopsis thaliana (L.) Heynh. All variables differed among lines (P<0.0001), and larger plants with larger roots and shoots had faster Pi uptake rates (influx) (P<0.0001). Within genotypes, influx was non-linearly related to root size. Increases in influx were not directly proportional to increases in root size; with a doubling in root length or biomass, there was only a 1.30 or 1.27 concomitant increase in influx, respectively. Because inbred lines are genetically homogeneous, this within-genotype pattern was random, and not genetically based.Influx has traditionally been standardized for variation in plant size by dividing by root length or biomass. This assumes a linear relationship between influx and root size. As uptake rate was non-linearly dependent on root size in A. thaliana, this resulted in a spurious negative correlation between influx per cm or mg root and root length or biomass. We present a general method that standardizes influx for variation in root size within genetically homogenous genotypes. This method determines and corrects for random non-linear relationships between influx and root size. In A. thaliana, influx per unit root length or biomass differed among lines (P<0.0001), but these differences did not correlate with root length or biomass. These data contradict the common interpretation in previous literature that species or cultivars with small roots have evolved greater influx per unit size of root.