Effect of light and nitrates on nitrate reductase activity and stability in seedling leaves of selected barley genotypes

Parental genotypes (cv. Aramir and line R567) and the selected doubled haploid (DH) lines C23, C47/1, C41, C55 did not differ in NR activity when they grew on a nutrient solution containing 10 mM KNO₃ and were illuminated with light at 124 μmol·m⁻²·s⁻¹ intensity. A decrease of nitrate content in the nutrient medium to 0.5 mM at 44 μmol·m⁻²·s⁻¹ light intensity caused a significant reduction of NR activity in the parental genotypes as well as in the lines C41 and C55. An increase in light intensity to 124 μmol·m⁻²·s⁻¹ raised NR activity in the leaf extracts of these genotypes. However, independently of light intensity, a high level of this enzyme activity was maintained in the line C23 growing on the nutrient medium with 10 mM and 0.5 mM KNO₃. The NR activity in that line dropped only when nitrate content in the medium decreased to 0.1 mM. NR in the leaves of the line C23, as compared to C41, was characterized by a higher thermal stability in all experimental combinations. An increase in light intensity had no significant influence on NR thermal stability in the leaves of the line C41, but induced a significant increase of this enzyme stability in the line C23. The lines C23 and C41 growing on the nutrient medium with 0.5 mM KNO₃ differed appreciably by nitrate concentration in leaves. A higher accumulation of nitrates was detected in the leaves of the line C41.     

Iron Mediated Nitrate Reductase Activity in Different Parts of Young Maize Seedlings

Incubation of 5-d-old maize seedlings in the half-strength Hoagland's nutrient solution containing 10 mM KNO₃ with FeCl₃ or FeSO₄ (0.5 or 2.0 mM) caused a significant increase in nitrate reductase (NR) activity and slightly increased total protein content in root, shoot and scutellum. In case of root, NADPH:NR activity was inhibited contrary to the NADH:NR activity. In spite of NR activity, nitrate uptake was inhibited from 13 to 37 % by the iron. The results presented demonstrate an isoform specific, organ specific, and to some extent salt specific responses of NR to iron.     

Optimized nitrate reductase assay predicts the rate of nitrate utilization in the halotolerant microalga Dunaliella viridis

An in situ method for measuring nitrate reductase (NR) activity in Dunaliella viridis was optimized in terms of incubation time, concentration of KNO₃, permeabilisers (1-propanol and toluene), pH, salinity, and reducing power (glucose and NADH). NR activity was measured by following nitrite production and was best assayed with 50 mM KNO₃, 1.2 mM NADH, 5% 1-propanol (v/v), at pH 8.5. The estimated half-saturation constant (K_s) for KNO₃ was 5 mM. Glucose had no effect as external reducing power source, and NADH concentrations >1.2 mM inhibited NR activity. Nitrite production was linear up to 20 min; longer incubation did not lead to higher nitrate reduction. The use of the optimized assay predicted the rate of NO ₃ ⁻ removal from the external medium by D. viridis with high degree of precision. This revised version was published online in September 2006 with corrections to the Cover Date.     

Influence of fruit load and environmental factors on nitrate reductase activity and on concentration of nitrate and carbohydrates in leaves of eggplant (Solanum melongena)

Both the in vivo (+ nitrate) nitrate reductase (NR) activity (leaf disks incubated in the presence of KNO₃) and the in vivo (? nitrate) NR activity (leaf disks incubated without KNO₃) in leaves of eggplant (Solanum melongena L. cv. Bonica) were affected by rapidly growing fruits. Plants with a fruit load showed more pronounced diurnal variation in (+ nitrate) NR activity and higher (? nitrate) NR activity than plants without fruit. The higher (? nitrate) NR activity was accompanied by higher nitrate and lower sucrose and starch contents of leaves. The more pronounced diurnal changes in (+ nitrate) NR activity were paralleled by more pronounced diurnal variation in carbohydrate content of leaves. Fruit removal led to a decrease in both (? nitrate) NR activity and nitrate concentration in leaves, while the carbohydrate content increased. Plants supplied with ammonium instead of nitrate showed only slightly lower (+ nitrate) but no (? nitrate) NR activity. As for plants treated with nitrate, diurnal changes in (+ nitrate) NR activity were most pronounced in leaves of plants with fruit and this again was paralleled by a more pronounced diurnal variation in the carbohydrate concentration in the leaves. Increasing the oxygen level of the atmosphere to 50% led to a dramatic decrease in the (+ nitrate) NR activity and to an increase in both (? nitrate) NR activity and nitrate concentration, which was accompanied by decreasing carbohydrate contents of the leaves. Low light intensities and extended dark periods caused similar changes in NR activity and nitrate and carbohydrate concentrations in leaves. Increasing the nitrate concentration in the nutrient solution led to a rise in (+ nitrate) and (? nitrate) NR activity, but only the (? nitrate) NR activity paralleled the nitrate concentration in the leaves. This increase in the nitrate concentration was accompanied by a decrease in the carbohydrate content of the leaves. It is concluded that the level of and the diurnal changes in both (+ nitrate) and (? nitrate) NR activity and the concentration of nitrate in the leaves are dependent upon their carbohydrate status.     

Development of NAD(P)H: and NADH:Nitrate Reductase Activities in Soybean Cotyledons

The cotyledons of soybean begin to develop photosynthetic capacity shortly after emergence. The cotyledons develop nitrate reductase (NR) activity in parallel with an increase in chlorophyll and a decrease in protein. In crude extracts of 5- to 8-day-old cotyledons, NR activity is greatest with NADH as electron donor. In extracts of older cotyledons, NR activity is greatest with NADPH. Blue-Sepharose was used to purify and separate the NR activities into two fractions. When the blue-Sepharose was eluted with NADPH, NR activity was obtained which was most active with NADPH as electron donor. Assays of the NADPH-eluted NR with different concentrations of nitrate revealed that the highest activity was obtained in 80 millimolar KNO₃. Thus, this fraction has properties similar to the low nitrate affinity NAD(P)H:NR of soybean leaves. When 5- to 8-day-old cotyledons were extracted and purified, further elution of the blue-Sepharose with KNO₃, subsequent to the NADPH elution, yielded an NR fraction most active with NADH. Assays of this fraction with different nitrate concentrations revealed that this NR had a higher nitrate affinity and was similar to the NADH:NR of soybean leaves. The KNO₃-eluted NR fraction which was purified from the extracts of 9- to 14-day-old cotyledons, was most active with NADPH. The analysis of these fractions prepared from the extracts of older cotyledons indicated that residual NAD(P)H:NR contaminated the NADH:NR. Despite this complication, the pattern of development of the purified NR fractions was consistent with the changes observed in the crude extract NR activities. It was concluded that NADH:NR was most active in young cotyledons and that as the cotyledons aged the NAD(P)H:NR became more active.     

Symbiotic nitrogen fixation and nitrate reduction in two peanut cultivars with different growth habit and branching pattern structures

We have investigated the response of two peanut cultivars (TEGUA and UTRE) with different growth habits and branching pattern structures to different nitrogen (N) sources, namely, N-fertilizer or N₂ made available by symbiotic fixation, and analysed the pattern of nitrate reductase (NR) activity in these cultivars. Nitrate and amino acid contents were also examined under these growth conditions. In terms of nitrogen source, cv. TEGUA showed a better response to inoculation with Bradyrhizobium sp. SEMIA 6144 at 40 days after planting, while cv. UTRE responded better to N-fertilizer (5 mM KNO₃). Both cultivars showed different patterns of NR activity in the analyzed plant organs (leaves, roots, and nodules), which were dependent on the N source. When nitrogen became available to the plant through symbiotic N₂ fixation, the patterns of NR activity distribution were different in the two cultivars, with cv. TEGUA showing a higher NR activity in the nodules than in the leaves and roots, and cv. UTRE showing no difference in terms of NR activity among organs. The nitrate and amino acid contents showed a similar trend between the two cultivars, with the highest nitrate content in the leaves of fertilized plants and the highest amino acid content in the nodules. The high nitrate content of the leaves of cv. UTRE indicated the better response of this cultivar to N-fertilizer.     

Nitrogenase activity and nitrogen assimilation in Anabaena flos-aquae growing in continuous culture

Summary Anabaena flos-aquae is grown in chemostats under phosphate and urea-limited conditions. Nitrogenase activity in phosphate-limited cells has a maximum activity at a dilution rate of 0.025 h^-1 and is repressed 24-fold by 15 mM KNO₃. Cultures growing on 1.5 mM nitrate obtain 1/2–2/3 of cell nitrogen from N₂. Cells form inducible nitrite assimilating enzymes when grown on nitrate. Algae growing under A or He on limiting urea or phosphate-limited with nitrate have active nitrogenase. The ratio of nitrogenase activity to heterocyst numbers varied 90-fold depending on source of nitrogen, 15 mM KNO₃ gave the smallest ratio. The regulatory mechanisms controlling the activity of nitrogenase in blue-green algae is discussed.     

Role of nitrogen metabolism in the development of salt tolerance in barley plants

The 7- to 8-day-old barley (Hordeum vulgare L.) seedlings grown in KNO3 solutions (1-40 mM) were characterized by the substrate activation of nitrate reductase (NR) in the apical leaf segments (1–2 cm in length), as well as by stimulated growth, broadened leaf blades, and by vigorously developed system of shortened roots. When the seedlings were grown in the presence of 20 mM KNO₃, the ability of leaf segments to generate superoxide anion radical remained at the level typical of control plants grown in water. The content of 5-aminolevulinic acid (ALA) in plants grown in the presence of 20 mM KNO₃ was 2.2–2.4 times higher than in control plants. The plants grown in the presence of nitrate had an elevated content of chlorophylls a and b, heme, and protein (by 42%). At the same time, the proline content was almost twofold lower than in control plants, which was due to substantial reduction (by 40%) in activity of Δ¹-pyrroline-5-carboxylate synthetase (P5CS). It is concluded that the substrate activation of NR by KNO₃ under normal growth conditions results in predominant utilization of glutamic acid (the primary product of inorganic nitrogen assimilation) for biosynthesis of tetrapyrroles and protein amino acids at the expense of inhibition of proline synthesis. When barley seedlings were grown in 150 mM NaCl solution, the plant growth and the root system development were suppressed to the levels of 63 ± 6% and 61 ± 11% of the control values, respectively. In the apical leaf tissues of plants adapted to NaCl, there was a slight decrease in the total NR activity (by 10%), a significant reduction in protein content (by 32%), and a parallel increase in the content of ALA (by a factor of 4.3), chlorophylls, heme, carotenoids, proline (2.2-fold) and P5CS (1.6-fold) with respect to the control values. It is proposed that the accumulation of ALA and proline under salinity-induced suppression of nitrogen assimilation results from the predominant allocation of glutamate for biosyntheses of ALA and proline at the expense of inhibition of growth-related processes requiring intense protein synthesis. The substrate activation of NR by KNO₃ under salinity conditions was associated with prevailing allocation of the assimilated nitrogen for synthesis of proline and protein amino acids, which reinforced plant cell protection against salinity and stimulated plant growth.     

Nitrate reductase activity of free-living and symbiotic uptake hydrogenase-positive and uptake hydrogenase-negative strains of Bradyrhizobium japonicum

The nitrate reductase activity (NR) of selected uptake hydrogenase-positive (hup ⁺) and uptake hydrogenase-negative (hup ^-) strains of Bradyrhizobium japonicum were examined both in free-living cells and in symbioses with Glycine max L. (Marr.) cv. Williams. Bacteria were cultured in a defined medium containing either 10 mM glutamate or nitrate as the sole nitrogen source. Nodules and bacteriods were isolated from plants that were only N₂-dependent or grown in the presence of 2 mM KNO₃. Rates of activity in nodules were determined by an in vivo assay, and those of cultured cells and bacteriods were assayed after permeabilization of the cells with alkyltrimethyl ammonium bromide. All seven strains examined expressed NR activity as free-living cells and as symbiotic forms, regardless of the hup genotype of the strain used for inoculation. Although the presence of nitrate increased nitrate reduction by cultures cells and nodules, no differences in NR activity were observed between bacteroids isolated from nodules of plants fed with nitrate or grown on N₂-fixation exclusively. Cultured cells, nodules and bacteriods of strains with hup ^- genotype (USDA 138, L-236, 3. 15B3 and PJ17) had higher rates of NR activity than those with hup ⁺ genotype (USDA 110, USDA 122 DES and CB1003). These results suggest that NR activity is reduced in the presence of a genetic determinant associated with the hup region of B. japonicum.Abbreviations EDTA ethylene-diamine tetraacetic acid - Hup hydrogen uptake - MOPS 3-(N-morpholino)-propane sulfonic acid - NR nitrate reductase - PVP polyvinyl-polypyrrolidone - Tris Tris(hydroxymethyl)-aminomethane     

Effect of nitrate on pea sucrose synthase

The in vivo and in vitro nitrate effects on pea (Pisum sativum L.) sucrose synthase (SS) were studied. At the period of plant transition from heterotrophic to autotrophic nutrition, exogenous nitrate (14.2 mM) absorbed in the form of KNO₃ and Ca(NO₃)₂ during 10–20 days activated SS in the roots by 22–100% as compared with plants grown on nitrogen-free medium. Such effect was observed only at plant growing under high light (natural illumination up to 25 klx) and thus their sufficient supplement with sucrose. Under low light (climate-controlled chamber, 2.5 klx), nitrate could not activate SS. In the in vitro experiments, nitrate activated SS exponentially by a dose-dependent mode with the plateau at 3–5 mM, where its activity was increased by 50%. It is supposed that there is a second constituent in SS activation by nitrate, and it carries information about plant carbohydrate status. Possible mechanisms of nitrate-induced SS activation are discussed.     

Nitrate and Nitrite Reduction in Relation to Nitrogenase Activity in Soybean Nodules and Rhizobium japonicum Bacteroids

Soybean (Glycine max L. cv Williams) seeds were sown in pots containing a 1:1 perlite-vermiculite mixture and grown under greenhouse conditions. Nodules were initiated with a nitrate reductase expressing strain of Rhizobium japonicum, USDA 110, or with nitrate reductase nonexpressing mutants (NR⁻ 108, NR⁻ 303) derived from USDA 110. Nodules initiated with either type of strain were normal in appearance and demonstrated nitrogenase activity (acetylene reduction). The in vivo nitrate reductase activity of N₂-grown nodules initiated with nitrate reductase-negative mutant strains was less than 10% of the activity shown by nodules initiated with the wild-type strain. Regardless of the bacterial strain used for inoculation, the nodule cytosol and the cell-free extracts of the leaves contained both nitrate reductase and nitrite reductase activities. The wild-type bacteroids contained nitrate reductase but not nitrite reductase activity while the bacteroids of strains NR⁻ 108 and NR⁻ 303 contained neither nitrate reductase nor nitrite reductase activities.

Addition of 20 millimolar KNO₃ to bacteroids of the wild-type strain caused a decrease in nitrogenase activity by more than 50%, but the nitrate reductase-negative strains were insensitive to nitrate. The nitrogenase activity of detached nodules initiated with the nitrate reductase-negative mutant strains was less affected by the KNO₃ treatment as compared to the wild-type strain; however, the results were less conclusive than those obtained with the isolated bacteroids.

The addition of either KNO₃ or KNO₂ to detached nodules (wild type) suspended in a semisolid agar nutrient medium caused an inhibition of nitrogenase activity of 50% and 65% as compared to the minus N controls, and provided direct evidence for a localized effect of nitrate and nitrite at the nodule level. Addition of 0.1 millimolar sucrose stimulated nitrogenase activity in the presence or absence of nitrate or nitrite. The sucrose treatment also helped to decrease the level of nitrite accumulated within the nodules.

     

Relations between carbohydrate accumulation in leaves, sucrose phosphate synthase activity and photoassimilate transport in chilling treated maize seedlings

Sucrose and starch concentration, sucrose phosphate synthase (SPS) activity in leaves, and long distance transport were studied in maize seedlings treated with moderate chilling (14 °C/12 °C - day/night). Two inbred lines were tested: chilling-tolerant KW1074 and chilling-sensitive CM109. Seedlings were grown in phytotrone on water nutrient until the 4-th leaf appearance. The estimations were done on fully developed 2-nd leaf. Six days after the temperature was lowered, leaves of line KW 1074 plants contained 5-fold more sucrose and starch than the control ones. The same treatment of CM 109 seedlings resulted in accumulation of sucrose and starch by 2-fold and 8.5-fold, respectively. As the result of chilling-treatment, ¹⁴C assimilation rate (P_a), transport speed in the leaf blade (TS₁) and along the plant (TS_m) decreased by about 50 % in both lines. On the other hand, time necessary for radiolabel movement into the phloem loading region (AT) increased strongly, especially in chilling-sensitive line CM 109. It was also noted, that the radioactivity exported from leaves (R₁) and imported by roots (R_m) decreased in line CM 109, and increased slightly in line KW 1074. The activity of SPS extracted from leaves of both lines decreased by about 3.3 when temperature was lowered form 30°C to 10°C. There was no effect of 6 day treatment of chilling on SPS activity. Changes in sucrose and starch concentration, SPS activity as well as differences in transport parameters observed in KW1074 and CM109 seedlings at moderate low temperatures are discussed in terms of mechanism of maize chilling-sensitivity.     

Nitrate reductase activity and nitrate accumulation in in vitro produced axillary shoots,plantlets and seedlings of Pinus pinaster

The initial and induced in vivo Nitrate Reductase Activity, the nitrate accumulation by in vitro-produced axillary shoots and plantlets of Pinus pinaster were compared respectively with those of shoots collected from seedlings and whole plants.The usefulness of the nitrate of the medium used for in vitro axillary shoot formation is demonstrated by the occurrence of initial NR activity in the explants. When fed in a non in vitro situation with a 50 mM KNO₃ solution, they have the same induced capacity to reduce nitrate as do shoots from seedlings, even though the latter accumulate less nitrate. Plants regenerated in vitro exhibit an ability to reduce nitrate similar to that of seedlings. In both types of plants, the Nitrate Reductase potential is greater in roots than in shoots.Abbreviation NR Nitrate Reductase - BA 6-Benzyladenine     

Role of potassium and malate in nitrate uptake and translocation by wheat seedlings

Wheat seedlings (Triticum vulgare) treated with 1 mm KNO₃ or NaNO₃, in the presence of 0.2 mm CaSO₄, were compared during a 48-hour period with respect to nitrate uptake, translocation, accumulation and reduction; cation uptake and accumulation; and malate accumulation. Seedlings treated with KNO₃ absorbed and accumulated more nitrate, had higher nitrate reductase levels in leaves but less in roots, accumulated 17 times more malate in leaves, and accumulated more of the accompanying cation than seedlings treated with NaNO₃. Within seedlings of each treatment, changes in nitrate reductase activity and malate accumulation were parallel in leaves and in roots. Despite the great difference in malate accumulation, leaves of the KNO₃-treated seedlings had only slightly greater levels of phosphoenolpyruvate carboxylase than leaves of NaNO₃-treated seedlings. NADP-malic enzyme levels increased only slightly in leaves and roots of both KNO₃- and NaNO₃-treated seedlings. The effects of K⁺ and Na⁺ on all of these parameters can best be explained by their effects on nitrate translocation, which in turn affects the other parameters. In a separate experiment, we confirmed that phosphoenolpyruvate carboxylase activity increased about 2-fold during 36 hours of KNO₃ treatment, and increased only slightly in the KCl control.     

Effects of NaCI stress on nitrogen and phosphorous metabolism in a true mangrove Bruguiera parviflora grown under hydroponic culture

The influence of varying levels of salinity (0, 100, 200 and 400 mM) on the activities of nitrate reductase (NR, E.C. 1.6.6.1), acid phosphatase (ACP, E.C. 3.1.3.2), and alkaline phosphatase (ALP, EC 3.1. 3.1) as well as on nitrate and phosphate uptake and total nitrogen levels in leaves of a true mangrove Bruguiera parviflora was investigated under hydroponic culture conditions. NR activity increased in 100 mM NaCl treated plants, whereas it decreased gradually in 200 and 400 mM treated plants, relative to the controls. Decreased activity of NR by NaCl stress was also accompanied by a decrease in total nitrogen level and nitrate uptake. Decreases in NR activity, nitrate (NO₃⁻), and total nitrogen level due to high salinity may be responsible for a decrease in growth and biomass production in this plant. However, salinity caused an increase in both ACP and ALP activity. Activity staining of ACP by native polyacrylamide gel electrophoresis revealed three isoforms: ACP-1, ACP-2, and ACP-3. We observed a preferential enhancement in the ACP-3 isoform by salinity. In order to understand whether the salinity-induced increase in phosphatase activity was due to inhibition in phosphate uptake, we monitored phosphate (Pi) levels in leaves and noted that phosphate levels decreased significantly under salinity. These results suggest that the induction of acid and ALP under salt stress may be due to a phosphorous deficiency.     

NUTRITIONAL REQUIREMENTS FOR SEXUAL REPRODUCTION IN MESOTAENIUM KRAMSTAI (CHLOROPHYTA) 1

Optimum conditions for conjugation in the heterothallic saccoderm desmid Mesotaenium kramstai Lemmer-mann have been determined. In culture, cells acquired the ability to form gamete pairs just prior to the onset of stationary phase after sufficient nitrate had been depleted from the medium. The appearance of potential gametes was delayed by increasing the concentration of KNO₃ When cells of both mating types were harvested from 15 to 18 day old cultures, washed, resuspended in fresh medium, and mixed, approximately 50 percent of the cells paired (measured three days after mixing) in a medium containing 0.13 mM or less KNO₃. At greater concentrations, fewer pairs formed; no pairs formed in medium containing 0.5 mM KNO₃. Conjugation was not inhibited by other macronutrients. Calcium and magnesium were essential for maximum conjugation. Although Ca²⁺ and Mg²⁺ contentrations of 0.05 mM and 0. I mM, respectively, were sufficient for optimum growth, maximum conjugation required more than 10 times these values. Few gamete pairs formed when either Ca²⁺ or Mg²⁺ was omitted from the medium, no pairing occurred when both Ca²⁺ and Mg²⁺ were omitted.     

Protective effect of exogenous nitrate on the mitochondrial ultrastructure ofOryza sativa coleoptiles under strict anoxia

Summary The effect of exogenous KNO₃, the terminal acceptor of electrons in oxygen-free medium, on mitochondrial ultrastructure and on the growth rate of 4-day-old rice coleoptiles under strictly anoxic conditions was studied. Exogenous nitrate (10 mM) did not exert any significant effect on the growth rate of coleoptiles of intact seedlings compared to their growth in KNO₃-free medium. Anaerobic incubation of detached coleoptiles in KNO₃-free medium for 48 h resulted in the complete destruction of mitochondrial and other cell membranes. In the presence of KNO₃, no mitochondrial-membrane destruction was observed even after 48 h anoxia although the mitochondrial ultrastructure was modifed. Cristae were arranged in parallel rows and elongated dumbbell-shaped mitochondria appeared in some cells. The data obtained indicate a protective role of exogenous nitrate as electron acceptors in oxygen-free medium. The results of the present investigation are discussed and compared with reports of either markedly damaging or favorable effects of exogenous nitrate on the growth, metabolism, and energetics of rice and other plants under hypoxic and anoxic conditions.     

The effect of chloride on nitrate reductase level,on anaerobic nitrite production,and on nitrate content in excisedPisum sativum L. roots

The effect was studied of chloride ions, added in the form of different salts, on nitrate reductase (NR) level in excised pea roots, on anaerobic nitrite production in an assay medium lacking both nitrate and n-propanol, on nitrate content in the roots, and on in vivo NR activity determined in an assay medium containing 5% n-propanol. The presence of Cl in nitrate containing nutrient solutions resulted in lower NR levels, however counterions supplied together with Cl tended to modify slightly this general trend. The negative effect of Cl ions was also apparent, when Cl ions were applied before nitrate ions. Anaerobic nitrite production in the medium lacking both nitrate and n-propanol was not influenced by chloride ions. Nitrate content in the roots was reduced in the presence of chloride both at 3 mM and 15 mM NO₃ in nutrient solutions; however, at 16 mM NO₃, nitrate content in the roots exoeeded even in the presence of 15 mM Cl nitrate content in those root segments which were cultivated in a nutrient solution with 6 mM nitrate, which is the concentration at which NR reaches the level of saturation in excised pea roots. The results obtained suggest that a special induction nitrate pool exists in plant cells besides the storage and metabolic nitrate pools.     

Culture medium optimization for camptothecin production in cell suspension cultures of Nothapodytes nimmoniana (J. Grah.) Mabberley

The effect of culture medium nutrients on growth and alkaloid production by plant cell cultures of Nothapodytes nimmoniana (J. Grah.) Mabberley (Icacinaceae) was studied with a view to increasing the production of the alkaloid camptothecin, a key therapeutic drug used for its anticancer properties. Amongst the various sugars tested with Murashige and Skoog (MS) medium, such as glucose, fructose, maltose, and sucrose, maximum accumulation of camptothecin was observed with sucrose. High nitrate in the media supports the biomass, while high ammonium enhances the camptothecin content. Selective feeding of 60 mM total nitrogen with a NH₄ ⁺/NO₃ ^? balance of 5/1 on day 15 of the culture cycle results in a 2.4-fold enhancement in the camptothecin content over the control culture (28.5 μg/g DW). Furthermore, the sucrose feeding strategy greatly stimulated cell biomass and camptothecin production. A modified MS medium was developed in the present study, which contained 0.5 mM phosphate, a nitrogen source feeding ratio of 50/10 mM NH₄ ⁺/NO₃ ^? and 3 % sucrose with additional 2 % sucrose feeding (added on day 12 of the cell culture cycle) with 10.74 μM naphthaleneacetic acid and 0.93 μM kinetin. Finally, the selective medium has 1.7- and 2.3-fold higher intracellular and extracellular camptothecin content over the control culture (29.2 and 8.2 μg/g DW), respectively.