Artificial seeds as a way to produce ecologically clean herbal remedies and to preserve endangered plant species

Influence of entrapment of the segments of isolated pRi T-DNA transformed roots of skullcap (Scutellaria baicalensis Georgi) in alginate gel capsules on the morphological, anatomical, physiological, and biochemical parameters of root cultures obtained from so-called artificial seeds (ASs) has been studied. The obtained ASs remain viable even after long-term storage at 4°C. Using the encapsulation technique, we have renewed a skullcap root culture and made it healthier. During anatomical study of the renewed root culture, the formation of chloroplasts has been observed in the cells of roots cultivated under lighting. Total chlorophyll content, chlorophyll a: chlorophyll b ratio, and the flavone content in the green roots have been determined.     

The Relationship Between Endogenous β‐Glucuronidase Activity and Biologically Active Flavones‐Aglycone Contents in Hairy Roots of Baikal Skullcap

Here, we examine the relationship between contents of principal flavones in hairy roots of Scutellaria baicalensis with the activity of the β‐glucuronidase (sGUS) enzyme during a culturing cycle. Using RP‐HPLC, we show that the highest contents of aglycones, baicalin and wogonin is observed at the growth days 8, 14, and 71 and reach 45, 41, and 62% (based on the total weight of hairy roots of the Baikal skullcap), correspondingly. Their accumulation is accompanied by increase of the sGUS activity, which we determined fluorometrically. Moreover, the enzyme activity is characterized by significant and reasonable correlation only with the wogonin contents. Our results confirm a significant role of sGUS at the final steps of the metabolism in root‐specific flavones of Baikal skullcap and suggest how one can optimize the conditions of culturing the hairy roots for biotechnological production of individual flavonoids. For example, at the culturing day 71 wogonin constituted over 80% of all flavones extracted from cells.     

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.     

Effect of baikal skullcap root (Scutellaria baicalensis radix) on cholesterol level and meat quality in rabbits fed a cholesterol rich diet

The aim of this study was to evaluate the effect of the Baikal skullcap root (Scutellaria baicalensis radix) on the cholesterol level and chemical composition of the hind leg muscles of rabbits. Thirty two White New Zealand rabbits were assigned to four groups. Group C consisted of control animals which were fed a basal mixture for rabbits. Group CH received the same basal diet with a 1% (w/w) pure cholesterol supplement. Group CH+SR received the basal diet with two supplements: 1% (w/w) pure cholesterol and 9% (w/w) skullcap root. Group SR received the basal diet with a 9% (w/w) skullcap root supplement. After 6 weeks rabbits were slaughtered and, total cholesterol as well as dry matter, protein, fat, ash and pH24 were determined in samples of hind leg muscles. Using a Baikal skullcap root with hypercholesterolemic diet (group CH+SR) caused significant reduction (P < or = 0.05) in total cholesterol level in comparison with hypercholesterolemic diet (CH group). The addition of Baikal skullcap root to the food of rabbits significantly increased the muscle protein content (P < or = 0.05) in comparison with C and CH groups. Moreover, supplementation with Baikal skullcap root (CH+SR) decreased about 15.6% (P < or = 0.05) fat level in comparison to CH group. No significant effects were seen in dry matter content, ash, and pH24 value of hind leg muscles of experimental rabbits     

Enhanced secondary metabolite (tanshinone) production of <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis> hairy roots in a novel root–bacteria coculture process

Salvia miltiorrhiza Bunge (Lamiaceae) hairy root cultures were inoculated (at 0.02 and 0.2% v/v) and co-cultured with Bacillus cereus bacteria. The root biomass growth was inhibited significantly by the bacteria inoculated to the root culture on the first day (day 0) but not by the bacteria inoculated on days 14 or 21 (in a 28-day overall period). On the other hand, the growth of the bacteria in the hairy root culture was also strongly inhibited by the hairy roots, partially because of the antibacterial activity of the secondary compounds produced by the roots. Most interestingly, the tanshinone production was promoted by the inoculation of bacteria at any of these days but more significantly by an earlier bacteria inoculation. With 0.2% bacteria inoculated on day 0, for example, the total tanshinone content of roots was increased by more than 12-fold (from 0.20 to 2.67 mg g⁻¹ dry weight), and the volumetric tanshinone yield increased by more than sixfold (from 1.40 to 10.4 mg l⁻¹). The tanshinone production was also stimulated by bacterial water extract and bacterial culture supernatant but less significantly than by the inoculation of live bacteria. The results suggest that the stimulation of tanshinone production by live bacteria in the root cultures may be attributed to the elicitor compounds originating from the bacteria, and the hairy root–bacteria coculture may be an effective strategy for improving secondary metabolite production in plant tissue cultures.     

Establishment of transformed root cultures of Perezia cuernavacana producing the sesquiterpene quinone perezone

Summary The sesquiterpene quinone currently known as perezone is abundantly produced by the roots of Perezia cuernavacana. This compound is of biotechnological interest since it may be used as a pigment and has several pharmacological properties. In this work we demonstrate that perezone is also produced in transformed root cultures of P. cuernavacana. Hairy roots were induced by inoculation of internodal segments of sterile plants of P. cuernavacana with Agrobacterium rhizogenes AR12 strain. The axenic liquid MS medium cultures of the hairy roots isolated from the internodes showed active growth in the absence of growth regulators. The transformed nature of the tissue was confirmed by genomic integration (PCR and slot blot hybridization) and expression (enzyme activity) of the marker gus-gene. The production of perezone by a transformed root culture was evidenced by IR spectroscopy. Our results offer an alternative for enhanced production of perezone and represent an advantage over its extraction from natural plant populations which present problems in their agronomic culture.     

Performance of hairy root cultures of Cichorium intybus L. In bioreactors of different configurations

Summary A transformed root culture of Cichorium intybus L. cv. Lucknow Local grown in different configurations of bioreactors was examined. The roots grown in an acoustic mist bioreactor showed the best performance in terms of increased specific growth rate (0.072d⁻¹) and esculin content (18.5gl⁻¹), the latter of which was comparable to that of shake flask data. C. intybus hairy root cultures grown in an acoustic mist bioreactor produced nearly twice as much esculin as compared to roots grown in bubble column and nutrient sprinkle bioreactors. Studies relating to on-line estimation of conductivity and osmolarity to predict the growth of hairy root cultures are also discussed. The results demonstrate the efficacy and the advantages of an acoustic mist bioreactor for the cultivation of hairy root cultures, especially with reference to C. intybus hairy roots.     

Formation of biologically active substances by rhizosphere bacteria and their effect on plant growth

Nine out of seventeen strains of bacteria with a pronounced effect on seed germination and on seedling growth, isolated from root surfaces and rhizosphere soil of maize, were selected for a study on the formation of biologically active substances. β-Indole acetic acid (45–72 μg/1.000 ml) was produced by four strains, gibberelline-like substances (1.0–60.0 μg/1.000ml) by all strains, biotin and pantothenic acid by the majority of strains and nicotinic acid by five strains. Amino acids were formed by all strains but in low amounts. Four strains produced growth inhibitors. The highest amounts of biologically active substances were found in cultures ofPseudomonas fluorescens andBacillus brevis. The various cultures ofPseudomonas fluorescens differed in their capability to produce biologically active substances. The majority of bacterial cultures or their supernatants significantly stimulated the germination of seeds and some of them significantly affected the growth of plants. Inoculation of maize seeds with strainsPseudomonas fluorescens andChromobacterium violaceum significantly increased the yield of dry matter of plants.     

Sanitizing long-term micropropagated grapes from covert and endophytic bacteria and preliminary field testing of plants after 8 years <Emphasis Type="Italic">in vitro</Emphasis>

Summary Micropropagated grape (Vitis vinifera L.) cv. Arka Neelamani cultures showed a decline in root and shoot growth performance after 6–7 yr of continuous in vitro culture. Indexing the culture medium using nutrient agar or 523 bacteriological medium (Viss et al., 1991) revealed covert bacteria in 75–100% cultures. Testing the tissue from different parts of in vitro plantlets on nutrient agar showed bacteria comprising of six or more morphotypes in 100% of root and collar tissue samples but less frequently in stem segments. The shoot tips had the lowest incidence of bacterial association. The whole shoots treated with NaOCl (4% chlorine) or HgCl₂ (0.1%) showed endophytic bacterial survival. Culturing the HgCl₂-treated (5 min) shoot tips on antibiotic overlaid medium (1 ml of 50 mg l⁻¹ gentamycin and/or cefazolin) in culture tubes (150×25 mm) for 1 mo. facilitated the cleansing of cultures with 75% recovery of contaminant-free shoots as monitored through indexing for the next 2 yr. Repeated indexing of medium and tissue from various plant parts during the first two to four subculture cycles following antibiotic treatment was instrumental in reliably identifying clean cultures and preventing bacterial re-emergence. Antibiotic incorporation in the medium was detrimental to grape microcuttings. Bacteria-freed cultures showed 80–100% rooting and a high number of plantlets that could be acclimatized. The plants put in the field after 8 yr of active micropropagation showed some juvenile characteristics initially, which disappeared in 6–8 mo., and the pruned shoots showed flowering and bunch development within 1 yr of field planting. This indicated the feasibility of keeping grape plants in vitro for long periods if covert microbes were eliminated.     

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.     

Flavones in genetically transformed <Emphasis Type="Italic">Scutellaria baicalensis</Emphasis> roots and induction of their synthesis by elicitation with methyl jasmonate

The composition and content of flavones were estimated in pRi T-DNA-transformed skullcap (Scutellaria baicalensis Georgi) roots obtained by the inoculation of axenically grown seedlings with a wild A4 strain of the soil bacterium Agrobacterium rhizogenes. It was elucidated by analytical and preparative HPLC of phenolic compounds in the extracts from the pRi T-DNA-transformed roots and also by ultraviolet spectroscopy and ¹H and ¹³C NMR that cultured skullcap roots contained similar basic flavones as intact roots of this plant species, i.e., baicalein and wogonin and corresponding glucuronides, baicalin and wogonoside. The content of these flavones in cultured roots was threefold lower than in the roots of intact five-year-old plants. When skullcap roots were cultured on B5 or Murashige and Skoog medium, the ratios between major flavones changed but their total content remained unchanged. The treatment of three-week-old cultured roots with methyl ether of jasmonic acid (MeJa) doubled the total concentration of major flavones in roots; the content of aglycons, baicalein and wogonin, increased to a greater degree, e.g., by 2.3 and 3.3 times, respectively. The induction of flavone production by elicitors indicates that flavones behave as phytoanticipins because major flavones of skullcap manifest a distinct antimicrobial activity. The results of the short-term treatment of skullcap roots with MeJa show that stress biotic factors can considerably increase the content of physiologically active flavones.Translated from Fiziologiya Rastenii, Vol. 52, No. 1, 2005, pp. 90–96.Original Russian Text Copyright © 2005 by Kuzovkina, Guseva, Kovács, Szöke, Vdovitchenko.     

Genetic transformation of <Emphasis Type="Italic">Harpagophytum procumbens</Emphasis> by <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>: iridoid and phenylethanoid glycoside accumulation in hairy root cultures

A genetic transformation method using Agrobacterium rhizogenes was developed for Harpagophytum procumbens. The influence of three factors on hairy root formation was tested: bacterial strains (A4 and ATCC 15834), various types of explants and acetosyringone (AS) (200 μM). The highest frequency of transformation (over 50% of explants forming roots at the infected sites after 6 weeks of culture on Lloyd and McCown (WP) medium) was achieved using a combination of nodal stem explants and A. rhizogenes strain A4. The addition of 200 μM AS to root induction medium was found to enhance hairy root induction but its effect varied depending on bacterial strain and explant type. Three of the most vigorously growing hairy root clones of H. procumbens were chosen and analyzed for accumulation of iridoid and phenylethanoid glycosides. The transgenic nature of these root clones was confirmed by PCR amplification; they were positive for rolB and rolC genes. Harpagoside, verbascoside and isoverbascoside were identified by HPLC and LC–ESI-MS as the major compounds from all analyzed hairy root clones. The Hp-3 root clone showed the higher harpagoside content (0.32 mg g⁻¹ dry wt.) compared with other analyzed transformed and non-tuberized untransformed roots of H. procumbens. However, the level of the compound in the hairy root clone was lower than that detected in a sample of commercially available root tubers of H. procumbens. The Hp-3 root clone also produced high amounts of verbascoside and isoverbascoside (8.12 mg g⁻¹ dry wt. and 9.97 mg g⁻¹ dry wt., respectively) comparable to those found in root tubers.     

Transforming growth factor-beta inhibition of mineralization by neonatal rat osteoblasts in monolayer and collagen gel culture

Summary The latent form of transforming growth factor-beta (TGF-β) is a component of the extracellular matrix of bone. The active form, when locally injected in vivo, stimulates both inflammation and ectopic bone formation. The present study was undertaken to determine if TGF-β also stimulated mineralization by isolated rat calvarial osteoblasts cultured in collagen gels. Gels were used because they should mimic in vivo conditions better than classical monolayer culture. Compared to cells in monolayers, osteoblasts cultured in collagen gels exhibited slower growth, but higher alkaline phosphatase activity and mineral deposition. Cultured cells also synthesized the osteoblast-specific marker, osteocalcin. The increase in osteocalcin in cell layers was parallel to the increase in mineral deposition. In the presence of TGF-β, neither cell growth nor alkaline phosphatase activity increased. Instead, a small decrease occurred in both parameters when compared to untreated cultures. Accumulation of collagen, the major component of the extracellular matrix where mineralization occurs, was similar in untreated and TGF-β1-treated cultures. However, 8 pM TGF-β1 dramatically suppressed mineral deposition in both types of cultures. Despite TGF-β1 stimulating a fourfold increase in lactic acid, the consequent increase in culture medium acidity did not account for the inhibitory effects of TGF-β1 on mineralization. These results demonstrate that collagen gel culture is an improved technique over conventional monolayer culture for demonstrating differentiated osteoblast function and sensitivity to TGF-β1. TGF-β1, at a concentration that has little effect on cell growth, alkaline phosphatase activity, or collagen accumulation, is a potent inhibitor of mineralization. The mechanism by which TGF-β1 inhibits mineralization remains to be determined.     

Biochemical characterization of laccase from hairy root culture of <Emphasis Type="Italic">Brassica juncea</Emphasis> L. and role of redox mediators to enhance its potential for the decolorization of textile dyes

In vitro transgenic hairy root cultures provide a rapid system for physiological, biochemical studies and screening of plants for their phytoremediation potential. The hairy root cultures of Brassica juncea L. showed 92% decolorization of Methyl orange within 4 days. Out of the different redox mediators that were used to achieve enhanced decolorization, 2, 2′-Azinobis, 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) was found to be the most efficient. Laccase activity of 4.5 U mg⁻¹ of protein was observed in hairy root cultures of Brassica juncea L., after the decolorization of Methyl orange. Intracellular laccase produced by B. juncea root cultures grown in MS basal medium was purified up to 2.0 fold with 6.62 U mg⁻¹ specific activity using anion-exchange chromatography. Molecular weight of the purified laccase was estimated to be 148 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme efficiently oxidized ABTS which was also required for oxidation of the other tested substrates. The pH and temperature optimum for laccase activity were 4.0 and 40°C, respectively. The purified enzyme was stable up to 50°C and was stable in the pH range of 4.0–6.0. Laccase activity was strongly inhibited by sodium azide, EDTA, dithiothreitol and l-cysteine. The purified enzyme decolorized various textile dyes in the presence of ABTS as an efficient redox mediator. These findings contribute to a better understanding of the enzymatic process involved in phytoremediation of textile dyes by using hairy roots.     

Factors influencing efficiency of somatic embryogenesis of Gentiana kurroo (Royle) cell suspension

In this paper, we would like to show unexpected morphogenic potential of cell suspensions derived from seedling explants of Gentiana kurroo (Royle). Suspension cultures were established with the use of embryogenic callus derived from seedling explants (root, hypocotyl and cotyledons). Proembryogenic mass proliferated in liquid MS medium supplemented with 0.5 mg l⁻¹ 2,4-D and 1.0 mg l⁻¹ Kin. The highest growth coefficient was achieved for root derived cell suspensions. The microscopic analysis showed differences in aggregate structure depending on their size. To assess the embryogenic capability of the particular culture, 100 mg of cell aggregates was implanted on MS agar medium supplemented with Kin (0.0–2.0 mg l⁻¹), GA₃ (0.0–2.0 mg l⁻¹) and AS (80.0 mg l⁻¹). The highest number of somatic embryos was obtained for cotyledon-derived cell suspension on GA₃-free medium, but the best morphological quality of embryos was observed in the presence of 0.5–1.0 mg l⁻¹ Kin, 0.5 mg l⁻¹ GA₃ and 80.0 mg l⁻¹ AS. The morphogenic competence of cultures also depended on the size of the aggregate fraction and was lower when size of aggregates decreased. Flow cytometry analysis reveled luck of uniformity of regenerants derived from hypocotyl suspension and 100% of uniformity for cotyledon suspension.     

Cyclic lipopeptide profile of three <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strains; antagonists of <Emphasis Type="Italic">Fusarium</Emphasis> head blight

The objective of the study was to identify the lipopetides associated with three Bacillus subtilis strains. The strains are antagonists of Gibberella zeae, and have been shown to be effective in reducing Fusarium head blight in wheat. The lipopeptide profile of three B. subtilis strains (AS43.3, AS43.4, and OH131.1) was determined using mass spectroscopy. Strains AS43.3 and AS43.4 produced the anti-fungal lipopeptides from the iturin and fengycin family during the stationary growth phase. All three strains produced the lipopeptide surfactin at different growth times. Strain OH131.1 only produced surfactin under these conditions. The antifungal activity of the culture supernatant and individual lipopeptides was determined by the inhibition of G. zeae. Cell-free supernatant from strains AS43.3 and AS43.4 demonstrated strong antibiosis of G. zeae, while strain OH131.1 had no antibiosis activity. These results suggest a different mechanism of antagonism for strain OH131.1, relative to AS43.3 and AS43.4.     

Growth, ageing and death of a photoautotrophic plant cell culture

 Batch cultures of photoautotrophic cell suspensions of Chenopodiumrubrum L., growing in an inorganic medium on CO₂ under a daily balanced light–dark regime of 16 : 8 h could be maintained for approximately 100 d without subcultivation. The long-lived cultures showed an initial cell division phase of 4 weeks, followed by a stationary phase of another 4 weeks, after which ageing and progressive cell death reduced the number of living cells and the cultures usually expired after another 3–4 weeks. These developmental phases of the cell culture were characterised with respect to photosynthetic performance, dark respiration, content of phytohormones and capacity of cell division. Cell division of the majority of the cells finished in the G1- or G0-phase of the cell cycle, caused by a pronounced decline in the endogenous levels of auxin and cytokinins. Supply of these growth factors to resting cells resulted in resumption of cytokinesis, at least by some of the cells. However, responsiveness to the phytohomones declined during the stationary phase, and subcultivation was no longer possible beyond day 60 when the phases of ageing and death commenced. Ageing was characterised by a further decline in the photosynthetic capacity of the cells, by a climacteric enhancement of dark respiration, but also by a slight increase in the level of IAA and cytokinins concomitant with a decrease in ethylene. Similarities and differences between the development of batch-cultured photoautotrophic cells of C. rubrum and that of a leaf are discussed with respect to using the cell culture as a model for a leaf. Received: 30 April 1999 / Accepted: 21 August 1999     

Induction of ajmalicine formation and related enzyme activities in Catharanthus roseus cells: effect of inoculum density

In Catharanthus roseus cell cultures the time courses of four enzyme activities, tryptophan decarboxylase (TDC), strictosidine synthase (SSS), geraniol-10-hydroxylase (G10H) and anthranilate synthase (AS), and alkaloid accumulation were compared under two different culture conditions (low-inoculum density and high-inoculum density on induction medium) and a control on growth medium. In growth medium a transient increase in TDC activity was first observed after which G10H reached its maximum activity; only tryptamine accumulated, no ajmalicine could be detected. Apparently, a concerted induction of enzyme activities is required for ajmalicine formation. Cells inoculated in induction medium showed such a concerted induction of AS, TDC and G10H activities. After 30 days the low-density culture had accumulated six times more ajmalicine (in moles/g) than the high-density culture. Thus, increase in biomass concentration (high-density cultures) did not enhance the total alkaloid production. The major differences observed in enzyme levels between high-and low-density cultures were in the AS and TDC activities, which were two to three times higher in the low-density culture, indicating that there is a positive correlation between ajmalicine formation and AS and TDC activities.Biotechnology Delft Leiden, Project Group Plant Cell Biotechnology Correspondence to: R. Verpoorte     

A comparison between hairy root cultures and wild plants of Saussurea involucrata in phenylpropanoids production

Saussurea involucrata is an important medicinal plant that produces a few bioactive secondary metabolites, such as hispidulin, rutin, and syringin. Previously, we established a hairy root culture system for this species through Agrobacterium-mediated transformation. The present study addressed the issue as how hairy root cultures perform in phenylpronoid accumulation. From the ethanolic extract of a hairy root culture established for Saussurea involucrata, syringin, rutin and hispidulin, were isolated and their chemical structures were confirmed by HPLC-ESI-MS. A quantitative study of the compounds showed great levels of syringin and hispidulin (being 43.5±1.13 and 0.34±0.023 mg g⁻¹ dry weight, respectively), about 40 and 3 times, respectively, higher than those from wild plants. But, the levels of rutin from hairy roots were much lower (0.71±0.043 vs. 6.59±0.56 mg g⁻¹ dry weight). Compared with untransformed root cultures, syringin and hispidulin levels were also higher. An experiment on culture media showed that MS was superior to others for phenylpropanoids accumulation in hairy roots, a 28-day culture produced 405 mg l⁻¹ syringin.     

Five year maintenance of the inducible expression of anthranilate synthase in Catharanthus roseus hairy roots

Transgenic hairy root cultures have the potential to be an industrial production platform for a variety of chemicals. This report demonstrates the long‐term stability of a transgenic Catharanthus roseus hairy root line containing the inducible expression of a feedback‐insensitive anthranilate synthase (AS). After 5 years in liquid culture, the presence of the inserted AS gene was confirmed by genomic PCR. The inducible expression of AS was confirmed by enzyme assay and by changes in terpenoid indole alkaloid concentrations. This report also demonstrates that it may take as long as 2 years for the metabolite profile to stabilize. Biotechnol. Bioeng. 2009;102: 1521–1525. © 2008 Wiley Periodicals, Inc.