Stimulation of asiaticoside accumulation in the whole plant cultures of <Emphasis Type="Italic">Centella asiatica</Emphasis> (L.) Urban by elicitors

The effects of a number of different elicitors on asiaticoside production in whole plant cultures of Centella asiatica were studied, including yeast extract, CdCl₂, CuCl₂ and methyl jasmonate (MJ). Only MJ and yeast extract stimulated asiaticoside production—1.53 and 1.41-fold, respectively. Maximum asiaticoside production was achieved following treatment with 0.1 mM MJ (116.8 mg/l). The highest asiaticoside production (342.72 mg/l) was obtained after 36 days of elicitation in cultures treated with 0.1 mM MJ and 0.025 mg/l 1-phenyl-3-(1,2,3-thidiazol-5-yl)urea (TDZ). Interestingly, MJ not only stimulated the production of asiaticoside but also had an important role in the senescence of C. asiatica. Although asiaticoside content did not change when TDZ was added to medium containing an elicitor, TDZ did increase shoot growth of C. asiatica. We discuss the interactive roles of MJ and TDZ in secondary metabolic production and biomass in whole plants of C. asiatica     

Elicitation of anthocyanin production in callus cultures of <Emphasis Type="Italic">Daucus carota</Emphasis> and the involvement of methyl jasmonate and salicylic acid

The effect of methyl jasmonate (MeJA) and salicylic acid (SA) on the anthocyanin accumulation, endogenous titres of polyamines and ethylene production in callus cultures of Daucus carota were studied. The interaction of these signaling molecules with elicitors from Aspergillus niger was investigated and the involvement of MeJA was elucidated through the use of the jasmonic acid (JA) biosynthetic inhibitor ibuprofen. MeJA and SA were both found to stimulate the anthocyanin production in the callus cultures. The highest levels of anthocyanin was observed in the cultures treated with 200 μM SA 0.36 % and 0.01 μM MeJA 0.37 %. The MeJA and SA treatments were also found to result in higher activity of Ca²⁺ ATPase suggesting that the enhancement of anthocyanin by SA and MeJA could be mediated through the involvement of the calcium channel. The treatment of the callus cultures with SA was found to result in marginally higher titres of endogenous polyamines (PAs) whereas MeJA resulted in lower levels of PAs as compared to the control. The SA treatment was found to result in lower ethylene production and the treatment with MeJA stimulated the ethylene production. These results suggest that the stimulation of anthocyanin production by MeJA and SA in callus cultures of D. carota is not related to the ethylene production.     

A combination of elicitation and precursor feeding leads to increased anthocyanin synthesis in cell suspension cultures of <Emphasis Type="Italic">Vitis vinifera</Emphasis>

Both elicitation and precursor feeding are effective strategies for improving secondary metabolite production in plant cell suspension cultures. In this study, cell suspension cultures of Vitis vinifera subjected to methyl jasmonate treatment resulted in a significant increase in levels of anthocyanin production. Moreover, a combination of 5 mg/L phenylalanine and 50 mg/L methyl jasmonate promoted the highest level of anthocyanin biosynthesis, resulting in 4.6- and 3.4-fold increases in anthocyanin content and yield, respectively, over the control. The optimum period for elicitation of anthocyanin synthesis was 4 days following incubation in the presence of elicitors, at the beginning of the exponential growth phase. V. vinifera cell lines of different anthocyanin-producing capabilities responded differently to elicitation and precursor feeding. Anthocyanin production of a low-producing cell line, VV06, could be enhanced with addition of elicitors and precursor feeding. Methyl jasmonate was the only elicitor that increased anthocyanin production of the high-producing cell line VV05, but contributed to moderate enhancement of anthocyanin production compared with VV06. For cell line VV06, synergistic effects were observed for all treatment combinations of methyl jasmonate along with other elicitors and precursors. In addition, 6.1- and 4.6-fold increases in anthocyanin content and yield, respectively, were obtained in the presence of 5 mg/L phenylalanine, 50 mg/L methyl jasmonate, and 1 mg/L dextran. However, none of these treatment combinations exhibited synergistic effects in cell line VV05.     

Methyl jasmonate increases the production of valepotriates by transformed root cultures of Valerianella locusta

Transformed roots of V. locusta (Valerianaceae) were obtained through transformation with Agrobacterium rhizogenes strains A4 and ATCC 15834. Six known valepotriates, including diavaltrate, acevaltrate, didrovaltrate, IVHD-valtrate, isovaltrate, and valtrate were the major components detected. An LC/PDA method was used in the quantitation of these compounds in the transformed root extracts. The treatment of transformed roots with biotic (methyl jasmonate, salicylic acid, yeast extract) and abiotic elicitors (CuSO₄, HgCl₂, CaCl₂) was used as a strategy to improve the production of valepotriates. Methyl jasmonate appeared to be the best elicitor for valepotriate production, yielding up to a 7-fold increase in total valepotriate content, while HgCl₂ had the most deteriorating effect on the production of valepotriates. Salicylic acid-, CuSO₄- and CaCl₂-treated roots showed significant increases in the production at a short duration of exposure; the production decreased as the time of elicitation increased. The highest total valepotriate content achieved in this study was 139 mg g^–1 DW (13.9%) from transformed roots treated for 10 days with 100 M methyl jasmonate. This amount was >50- and 12-fold higher than the values reported from the cultivated plants and callus culture, respectively, and was comparable to the amount reported from the high valepotriate-producing species Valeriana thalictroides Graebn. The production of diavaltrate, acevaltrate, didrovaltrate, and isovaltrate were significantly higher, while the production of IVHD-valtrate was lower and that of valtrate was similar to that of the control. The IVAL/VAL production ratio was affected by the treatment with methyl jasmonate but not by other elicitors. The use of transformed root cultures in combination with the treatment with biotic and abiotic elicitors offer a new route for high valepotriate production.     

Enhanced glucotropaeolin production in hairy root cultures of Tropaeolum majus L. by combining elicitation and precursor feeding

We have studied the effects of precursor amino acids (phenylalanine and cystein), elicitors (salicylic and acetylsalicylic acids, methyl jasmonate, β-aminobutyric acid, yeast extract), PAL-inhibitor (1-amino-2-phenylethylphosphonic acid) applied alone or in combination on glucotropaeolin production and myrosinase activity in hairy root cultures of Tropaeolum majus. Short, 24-h treatment, and subsequent transfer of hairy roots to the fresh medium enabled avoiding detrimental effect of studied stimulators on biomass growth. In control cultures the highest glucotropaeolin content, 58.1 ± 6.7 mg g⁻¹ DW, was detected on the 3rd day after transfer of the roots to the fresh medium but glucotropaeolin yield (mg 100 ml^–1 culture volume) had been increasing until the 9th day after transfer as a result of continuous biomass growth. Glucotropaeolin content and yield were 2-fold enhanced after treatment with precursor amino acids or PAL-inhibitor alone, but their combination additively led to 4-fold increase in glucotropaeolin production. Among the studied elicitors acetylsalicylic acid induced the highest, 3-fold increase in glucotropaeolin production, it also enhanced myrosinase activity, but to a smaller extend (by about 50%). Acetylsalicylic acid also potentiated induced by precursors, PAL-inhibitor, methyl jasmonate and yeast extract production of glucotropaeolin. The highest, 4.8-fold increase in glucotropaeolin production was found after combined acetylsalicylic acid and precursors treatment. Additive effect of acetylsalicylic acid-combined treatment on myrosinase activity was not detected. The obtained results indicate that amino acid precursors, phenylalanine and cystein, availability may be a limiting factor in the process of stimulation of glucotropaeolin production in T. majus hairy root cultures.     

Enhancement of lignan biosynthesis in suspension cultures of <Emphasis Type="Italic">Linum nodiflorum</Emphasis> by coronalon,indanoyl-isoleucine and methyl jasmonate

The effect of the two synthetic elicitors coronalon and indanoyl-isoleucine and of methyl jasmonate (MeJA) on the accumulation and biosynthesis of lignans by cell suspension cultures of Linum nodiflorum (Linaceae) was investigated. The production of 6-methoxypodophyllotoxin (MPTOX) could be increased more than tenfold, the maximal content reaching up to over 2.5% of the cell dry weight. The highest yield was achieved by administering 50 μM of the synthetic elicitors on the fourth day and extracting the products on the tenth day of the culture period. An additional lignan accumulated in elicitor-treated cultures. Its structure was elucidated by extensive 1D and 2D NMR measurements, revealing its identity as 5′-demethoxy-MPTOX (5′-dMPTOX). Its average content amounted up to over 5% of the cell dry weight. Growth was only slightly affected by the addition of the elicitors. Methyl jasmonate exerted a moderate stimulating effect on the L. nodiflorum cells with MPTOX and 5′-dMPTOX contents going up to 1.4 and 2.1% of the cell dry weight, respectively. The activities of deoxypodophyllotoxin 6-hydroxylase and β-peltatin 6-O-methyltransferase, two enzymes involved in MPTOX biosynthesis, were increased up to 21.9-fold and 14.6-fold, respectively, in the treated cultures.     

Elicitor-enhanced production of gymnemic acid in cell suspension cultures of <Emphasis Type="Italic">Gymnema sylvestre</Emphasis> R. Br.

Cell suspension cultures of Gymnema sylvestre treated with four different elicitors, methyl jasmonate (MJ), yeast extract, chitin and pectin were studied for the production of gymnemic acid as gymnemagenin equivalent, that was analyzed by high performance liquid chromatography (HPLC). All the four tested elicitors induced gymnemic acid production in cell suspension cultures. Highest gymnemic acid content was achieved following treatment with yeast extract (100.47 ± 0.28 mg/l), this was followed by MJ (70.43 ± 0.26 mg/l), pectin (64.19 ± 0.23 mg/l) and chitin (62.72 ± 0.13 mg/l). The addition of elicitors has shown a significant influence on cell growth that affected cell growth compared to respective controls. The highest gymnemic acid production was obtained after 20 days of elicitation in cultures treated with 0.5 g l^−l yeast extract, it was 5.25-folds greater than in control. These results suggest that the addition of an elicitor to Gymnema sylvestre cell suspension cultures could stimulate and enhance gymnemic acid production. In our present study we could able to overproduce gymnemic acid up to 51.97 ± 0.26 mg l^−l (dry weight basis) in yeast extract treated cell suspension cultures.     

Effect of salicylic acid and methyl jasmonate on antioxidant systems of <Emphasis Type="Italic">Haematococcus pluvialis</Emphasis>

The influence of phytohormones, salicylic acid (SA) and methyl jasmonate (MJ) on the antioxidant systems in Haematococcus pluvialis was investigated. Both SA and MJ at 500 μM concentration reduced the growth of alga with salicylic acid, having more pronounced effect. Carotenoid and chlorophyll contents were decreased by SA and increased by MJ. Salicylic acid (100 μM) increased astaxanthin content to 6.8-fold under low light (30 μmol m⁻² s⁻¹), while MJ (10 μM) showed marginal increase in astaxanthin. Salicylic acid (500 μM) increased superoxide dismutase activity to 4.5- and 3.3-fold and ascorbate peroxidase (APX) activity to 15.5- and 7.1-fold under low and high light, respectively. Methyl jasmonate increased catalase activity (1.4-fold) under high light and APX activity (5.4-fold) under low light. Different mechanism of oxidative stress induced antioxidant production may be the plausible reason for this varied response for salicylic acid and methyl jasmonate. Higher concentrations of SA and MJ inhibited astaxanthin accumulation by different mechanisms either by scavenging the free radicals or by increasing primary carotenoids production. At lower concentrations, these phytohormones could be used for elicitation of secondary carotenoid production.     

Ajmalicine production in methyl jasmonate-induced Catharanthus roseus cell cultures depends on Ca2+ level

Cytosolic Ca²⁺ and jasmonate mediate signals that induce defense responses in plants. In this study, the interaction between Ca²⁺ and methyl jasmonate (MJ) in modulating defense responses was investigated by monitoring ajmalicine production in Catharanthus roseus suspension cultures. C. roseus suspensions were treated with nine combinations of CaCl₂ (3, 23, and 43 mM) and MJ (0, 10, and 100 μM) on day 6 of growth. Increased Ca²⁺ influx through the addition of extracellular CaCl₂ suppressed ajmalicine production in MJ-induced cultures. The highest ajmalicine production (4.75 mg/l) was observed when cells were treated with a low level of calcium (3 mM) combined with a high level of MJ (100 μM). In the presence of 3 mM CaCl₂ in the medium, the addition of Ca²⁺ chelator EGTA (1, 2.5, and 5 mM) or Ca²⁺ channel blocker verapamil (1, 10, and 50 μM) to MJ-induced (100 μM) cultures on day 6 also inhibited ajmalicine production at higher levels of the Ca²⁺ inhibitors. Hence, ajmalicine production in MJ-induced C. roseus cultures depended on the intracellular Ca²⁺ concentration and a low extracellular Ca²⁺ concentration (3 mM) enhanced MJ-induced ajmalicine production.     

Effects of methyl jasmonate and salicylic acid on the production of bilobalide and ginkgolides in cell cultures of Ginkgo biloba

Summary In an attempt to increase productivity, the effects of the elicitors methyl jasmonate (MJ) and salicylic acid (SA) on the production of bilobalide (B), ginkgolide A (GA), and ginkgolide B (GB) were studied in cell suspension cultures of Ginkgo biloba. MJ treatments increased the amounts of B, GA, and GB, concomitant with a slight decrease in cell growth. After treatment of 0.01 mM MJ, levels of GA and GB increased 4.3-and 8.2-fold over controls by 12 h and declined after 24h. The 1.0mM MJ treatment produced a maximal release of B after 12h of exposure and increased the concentration of B in the culture medium up to 6.25-fold compared with the controls. Treatment with 1.0mM SA transiently enhanced GA and GB production up to 3.1-and 6.1-fold, respectively, compared with the control. However, treatment 1.0 mM SA did not have a significant effect on B production. When treated with 0.01 mM SA, the level of B in the cells was increased 5.4-fold over controls by 12h and declined after 24h. The concentrations and exposure times of both MJ and SA were factors that strongly affected the production of B, GA, and GB. The results from this study suggest that MJ and SA directly or indirectly increased the production of B, GA, and GB in cells, and stimulated the release of these metabolites into the culture medium.     

Stimulation of betacyanin synthesis through exogenous methyl jasmonate and other elicitors in suspension-cultured cells of Portulaca

Betacyanin production in suspension-cultured cells of Portulaca was significantly enhanced by both abiotic and biotic elicitors. Betacyanin levels increased 1.3 and 1.5-fold over the controls in the presence of two abiotic elicitors (20 mumol/L CuSO4 and 100 mumol/L FeEDTA) and increased 1.8 and 1.6-fold in the presence of two biotic elicitors (0.5 mg/L beta-glucan and 0.5 mg/L chitosan). Maximum betacyanin synthesis with the two most effective elicitors was obtained when cultures were treated on day 1 and day 0 by beta-glucan and FeEDTA, respectively. A concentration-dependent response was exhibited by cultures treated with exogenous methyl jasmonate (MJ). MJ alone at 0.1 mumol/L caused a 2.6-fold increase in betacyanin synthesis when administered to the suspension culture on day 3. However, no additive effect on betacyanin accumulation was observed in treatments, which combined MJ and beta-glucan or FeEDTA. Treatment with ibuprofen (IB), an inhibitor of jasmonate biosynthesis, reduced the level of betacyanin in cells cultured in standard medium at all concentrations tested (25, 50, 100 mumol/L). The effect of IB on betacyanin synthesis in the cells treated with MJ or beta-glucan, however, differed with the IB concentration applied. The two higher concentrations (50 and 100 mumol/L) of IB significantly reduced the betacyanin content while the lower concentration (25 mumol/L) did not show an adverse effect on the betacyanin enhancement triggered by MJ or beta-glucan. Our findings suggest that, in suspension-cultured cells of Portulaca, an MJ-mediated signal transduction pathway prominently exists in betacyanin synthesis. This pathway seems to act antagonistically towards beta-glucan-mediated signaling. As far as we know this is the first report on the elevation of betacyanin level by jasmonate or other elicitors in cell suspension cultures.     

Improved cardenolide production in Calotropis gigantea hairy roots using mechanical wounding and elicitation

A hairy root culture system of Calotropis gigantea was established and effects of mechanical wounding (MW) and elicitors [methyl jasmonate (MJ), yeast extract (YE) and chitosan (CS)] on cardenolide production were investigated. All treatments stimulated the production of cardenolide in hairy root cultures of C. gigantea. CS was the most effective elicitor, followed by MJ. YE and MW also improved cardenolide yield in individual treatments. The highest cardenolide yield (1,050 ± 55 mg/l) was obtained after adding 50 mg CS/l for 20 days, which was 2.7-fold higher than the control.     

Influence of growth regulators and elicitors on cell growth and α-tocopherol and pigment productions in cell cultures of <Emphasis Type="Italic">Carthamus tinctorius</Emphasis> L.

The present study examined the effects of plant growth hormones, incubation period, biotic (Trametes versicolor, Mucor sp., Penicillium notatum, Rhizopus stolonifer, and Fusarium oxysporum) and abiotic (NaCl, MgSO₄, FeSO₄, ZnSO₄, and FeCl₃) elicitors on cell growth and α-tocopherol and pigment (red and yellow) productions in Carthamus tinctorius cell cultures. The cell growth and α-tocopherol and pigment contents improved significantly on Murashige and Skoog (MS) liquid medium containing 50.0 μM α-naphthalene acetic acid (NAA) and 2.5 μM 6-Benzyladenine (BA) at 28 days of incubation period. Incorporation of T. versicolor (50 mg l⁻¹) significantly enhanced the production of α-tocopherol (12.7-fold) and red pigment (4.24-fold). Similarly, supplementation of 30 mg l⁻¹ T. versicolor (7.54-fold) and 70 mg l⁻¹ Mucor sp. (7.40-fold) significantly increased the production of yellow pigment. Among abiotic elicitors, NaCl (50–70 mg l⁻¹) and MgSO₄ (10–30 mg l⁻¹) significantly improved production of α-tocopherol (1.24-fold) and red pigment (20-fold), whereas yellow pigment content increased considerably by all the abiotic elicitor treatments. Taken together, the present study reports improved productions of α-tocopherol and the pigment as a stress response of safflower cell cultures exposed to these elicitors.     

Precursor limitations in methyl jasmonate-induced Catharanthus roseus cell cultures

Jasmonates enhance the expression of various genes involved in terpenoid indole alkaloid (TIA) biosynthesis in Catharanthus roseus. We applied precursor feeding to our C. roseus suspensions to determine how methyl jasmonate (MJ) alters the precursor availability for TIA biosynthesis. C. roseus suspensions were induced with MJ (100 μM) on day 6 and fed loganin (0.30 mM), tryptamine (0.15 mM), loganin plus tryptamine, or geraniol (0.1–1.0 mM) on day 7. While MJ increased ajmalicine production by 3-fold, induced cultures were still limited by terpenoid precursors. However, both induced and non-induced cultures became tryptamine-limited with excess loganin. Geraniol feeding also increased ajmalicine production in non-induced cultures. But MJ appeared to increase geraniol availability in induced cultures, due presumably to the increased expression of Dxs with MJ addition.     

Transgenic aequorin monitors cytosolic calcium transients in soybean cells challenged with β-glucan or chitin elicitors

Transgenic soybean (Glycine max L.) cells expressing aequorin were used to monitor changes in cytosolic Ca²⁺ concentrations in response to treatment with fungal elicitors. After an apparent lag phase of about 60 s, both chitin fragments and β-glucan elicitors caused a rapid increase in cytosolic Ca²⁺ concentration, which peaked within 2–2.5 min of treatment. The Ca²⁺ concentration then decreased and reached the basal level after about 5 min in the case of the treatment with chitin fragments, while a second rise in the Ca²⁺ concentration with a maximum occurring after about 7–8 min was observed in the case of β-glucan treatment. Calibration of the signals showed that the elicitors enhanced the cytosolic Ca²⁺ concentration from resting concentrations as low as 0.1 lM to highest levels of about 2 lM. Dose-response experiments showed that the concentration of elicitors giving a Ca²⁺ response at the 50% level was 0.4 nM for the chitin fragment and 28 lM and 72 lM, respectively, for a synthetic hepta-β-glucoside and a fungal β-glucan fraction. The β-glucan- or N,N′,N′′,N′′′-tetraacetyl chitotetratose (CH4)-induced Ca²⁺ signals were inhibited by both the Ca²⁺ chelator 1,2-bis-(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA) and by the Ca²⁺-channel inhibitor La³⁺. Neomycin, whose target in plant cells has not yet been clearly identified, reduced predominantly the expression of the second peak of the biphasic Ca²⁺ curve following β-glucan treatment. Bacterial cyclic β-glucans known to suppress β-glucan-induced phytoalexin production were also found to function as a suppressor for the Ca²⁺ response that was elicited by the fungal β-glucans. The results clearly show that the increase in the cytosolic Ca²⁺ concentration is an early and rapid event in the elicitor-sensing mechanism of soybean cells, and is probably connected with the subsequent activation of defence responses. Received: 23 July 1998 / Accepted: 16 October 1998     

β-Cyclodextrins enhance artemisinin production in Artemisia annua suspension cell cultures

Artemisinin is a sesquiterpene antimalarial compound produced, though at low levels (0.1–1% dry weight), in Artemisia annua in which it accumulates in the glandular trichomes of the plant. Due to its antimalarial properties and short supply, efforts are being made to improve our understanding of artemisinin biosynthesis and its production. Native β-cyclodextrins, as well as the chemically modified heptakis(2,6-di-O-methyl)-β-cyclodextrin (DIMEB) and 2-hydroxypropyl-β-cyclodextrins, were added to the culture medium of A. annua suspension cultures, and their effects on artemisinin production were analysed. The effects of a joint cyclodextrin and methyl jasmonate treatment were also investigated. Fifty millimolar DIMEB, as well as a combination of 50 mM DIMEB and 100 μM methyl jasmonate, was highly effective in increasing the artemisinin levels in the culture medium. The observed artemisinin level (27 μmol g⁻¹ dry weight) was about 300-fold higher than that observed in untreated suspensions. The influence of β-cyclodextrins and methyl jasmonate on the expression of artemisinin biosynthetic genes was also investigated.     

Polysaccharide elicitors enhance anthocyanin and phenolic acid accumulation in cell suspension cultures of <Emphasis Type="Italic">Vitis vinifera</Emphasis>

The effects of yeast extract and selected polysaccharide elicitors on secondary metabolite production, particularly of anthocyanin and phenolic acid, in cell suspension cultures of Vitis vinifera were investigated. All elicitors either maintained or promoted cell growth in culture. Overall, secondary metabolite production in V. vinifera cell suspension cultures responded differently to different elicitors. Chitosan, pectin, and alginate enhanced production of anthocyanin within 13 days of culture with levels of 2.5-, 2.5-, and 2.6-fold increase, respectively, over that of control. Chitosan, alginate, and gum arabic significantly promoted accumulation of phenolic acids, particularly 3-O-glucosyl-resveratrol, in V. vinifera cultures, as well as in the culture medium. Intracellular phenolic acid production was significantly enhanced by alginate and chitosan, with 1.7- and 1.5-fold levels, respectively, of that of control. Extracellular phenolic acid production was also significantly increased in the presence of chitosan and gum arabic, with levels of 3.3- and 1.7-fold higher, respectively, than those of control. In addition, DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity was enhanced in the presence of elicitors, and this was positively correlated with increased accumulation of anthocyanin in V. vinifera cell suspension cultures.     

Inorganic nutrient manipulation for highly improved <Emphasis Type="Italic">in vitro</Emphasis> plant regeneration in finger millet—<Emphasis Type="Italic">Eleusine coracana</Emphasis> (L.) Gaertn.

Summary Growth and morphogenesis of plant tissues under in vitro conditions are largely influenced by the composition of the culture media. In this study, effects of different inorganic nutrients (ZnSO₄ and CuSO₄) on callus induction and plant regeneration of Eleusine coracana in vitro were examined. Primary callus induction without ZnSO₄ resulted in improved shoot formation upon transfer of calluses to normal regeneration medium. CuSO₄ increased to 5x the normal concentration in the media for primary seed callus induction and plant regeneration resulted in a 4-fold increase in number of regnnerated shoots. For long-term callus cultures, 2x KNO₃ or 4x Fe-EDTA could replace the requirement for α-naphthaleneacetic acid in the regeneration medium, while 60 μM ZnSO₄ or 0.5 μM CuSO₄ was optimal for plant regeneration from callus cultures.     

Characterization of urease from Sporosarcina ureae

Alkaline stable (pH 7.75–12.5) urease from Sporosarcina ureae was purified over 400-fold by ion exchange and hydrophobic interaction chromatography. The cytoplasmic enzyme was remarkably active with a specific activity of greater than 9300 μmol urea degraded min^-1 mg protein^-1 at pH 7.5, where it has optimal activity. Although S. ureae is closely related to Bacillus pasteurii, known to posses a homopolymeric urease containing 1 nickel per subunit [M _r=65000], the S. ureae enzyme is comprised of three subunits [apparent M _r=63100 (α), 14500 (β), and 8500 (γ)] in an estimated ∝βγ stoichiometry and contains 2.1±0.6 nickel ions per ∝βγ unit as measured by atomic absorption spectrometry. Stationary phase cultures sometimes possessed low levels of urease activity, but the specific activity of cell extracts of partially purified urease preparations from such cultures could be elevated by heat treatment, dilution, or dialysis to values comparable to those observed in samples from exponentially grown cells.     

Combination effect of lactoalbumin hydrolysate and methyl jasmonate on ginsenoside and polysaccharide production in <Emphasis Type="Italic">Panax quinquefolium</Emphasis> L. cells cultures

Lactoalbumin hydrolysate (LH) at 100 mg L⁻¹ with methyl jasmonate (MJ) at 2 mg L⁻¹ synergistically stimulated ginsenoside accumulation in Panax quinquefolium cells compared with 100 mg L⁻¹ LH. Combination elicitors led to higher ginsenoside productivity (45.93 mg L⁻¹) than single treatment of 100 mg L⁻¹ LH (31.37 mg L⁻¹). This present result will be helpful in providing a tool for enhancing the productivity of ginsenoside by Panax quinquefolium cell cultures on a commercial scale.