Influences of different nitrogen sources on nitrogen- and water-use efficiency, and carbon isotope discrimination, in C3 Triticum aestivum L. and C4 Zea mays L. plants

The impacts of various nitrogen sources, i.e. NO⁻ ₃, NH₄ ⁺ or NH₄NO₃ in combination with gaseous NH₃, on nitrogen-, carbon- and water-use efficiency and ¹³C discrimination (δ¹³C) by plants of the C₃ species Triticum aestivum L. (wheat) and the C₄ species Zea mays L. (maize) were studied. Triticum aestivum and Z. mays were hydroponically grown with 2 mol · m⁻³ of N supplied as NO⁻ ₃, NH₄ ⁺ or NH₄NO₃ for 21 and 18 d, respectively, and thereafter exposed to gaseous NH₃ at 320 μg · m⁻³ or to ambient air for 7 d. In T. aestivum and Z. mays over a 7-d growth period, nitrogen-use efficiency (NUE) values were influenced by N-sources in the decreasing order NH₄NO₃-N > NO⁻ ₃-N > NH₄ ⁺-N and NO⁻ ₃-N > NH₄NO₃-N > NH₄ ⁺-N, respectively. Fumigation with NH₃ decreased the NUE values of plants grown with any of the N-forms. During 28- and 7-d growth periods, N-sources affected water-use efficiency (WUE) values in the decreasing order of NH₄ ⁺-N > NO⁻ ₃-N≈NH₄NO₃-N in non-fumigated T. aestivum, while fumigation with NH₃ increased the WUE of NO⁻ ₃-grown plants. There were insignificant effects of N-sources on WUE values of Z. mays over 25- and 7-d growth periods. Furthermore, δ¹³C values in plant tissues (leaves, stubble and roots) were higher (less negative) in NH₄ ⁺-grown plants of T. aestivum and Z. mays than in those supplied with NH₄NO₃ or NO⁻ ₃. Regardless of the N-form supplied to the roots of the plant species, exposure to NH₃ caused more-positive δ¹³C values in the plant tissues. These results indicate that the variations in N-source were associated with small but significant variations in δ¹³C values in plants of T. aestivum and Z. mays. These differences in δ¹³C values are in the direction expected from differences in WUE values over long or short growth periods and with differences in the extent of non-Rubisco (ribulose-1,5-bisphosphate carboxylase-oxygenase, EC 4.1.1.39) carboxylate contribution to net C acquisition, as a function of N-source. Received: 12 September 1997 / Accepted: 13 January 1998     

Reduction of the nitrogen and carbon content in swine waste with algae and bacteria

Animal waste causes environmental problems like eutrophication of ground and surface water or the pollution of the atmosphere because of its high NH₄ ⁺ content. The aim of our study was to fix the nitrogen of swine waste as biomass. Therefore, an isolated alga, Chlorella sp., and bacteria naturally living in liquid manure were grown in batch cultures (containing diluted swine waste supplied with a nutrient solution) and continuous cultures (undiluted liquid manure) to achieve reduction of NH₄ ⁺ and total organic carbon (TOC) contents. For continuous cultivation, a photobioreactor of our own design was used. The batch cultivation of Chlorella sp. and bacteria in swine waste resulted in good growth of both groups of organisms and in a reduction of 25% NH₄ ⁺ and 80% TOC. In the continuous cultivation a steady state was not achieved owing to a change in the composition of the bacterial population. NH₄ ⁺ was totally removed, but NO₂ ⁻ (up to 100 mM) was transiently released. NO₃ ⁻ was not detected. These effects might be explained by the presence of heterotrophic nitrifiers, which are able to oxidize NH₄ ⁺ to NO₂ ⁻ and to reduce NO₂ ⁻ to gaseous compounds. Received: 21 January 1999 / Received revision: 9 March 1999 / Accepted: 14 March 1999     

Induction,maturation and germination of holm oak (Quercus ilex L.) somatic embryos

Cotyledon explants of Panax ginseng produced somatic embryos directly on solid hormone-free MS medium containing 3% (w/v) sucrose while high concentration of NH₄NO₃ (60 mM) induced embryogenic callus. Ten subcultures of the embryogenic callus on hormone-free MS medium with 40 mM NH₄NO₃ gave hormone-independent proliferation of callus, which exhibited proliferation potential even on MS medium with a standard level of NH₄NO₃ (20 mM). Pulse treatment of callus with exogenous auxin or cytokinin (1.0 mg 1^–1 2,4-D, 1.0 mg 1^–1 kinetin) resulted in the loss of the hormone-independent characteristic and caused the callus to brown. For the suspension culture, embryogenic callus was transferred to MS liquid medium containing 3% (w/v) sucrose in an 500 ml Erlenmyer flask. Embryogenic cell clumps in full-strength MS liquid medium discharged toxic substances, resulting in strong suppression of cell growth. In 1/3-strength MS medium, exudation of toxic material did not occur. Embryogenic cell clumps were mass-grown on a large-scale in a bioreactor (20-1), showing a 7.1 increase of fresh weight in 1/3-strength MS medium with 3% (w/ v) sucrose after 5 weeks of culture. Total ginsenoside content of cultured embryogenic cell clumps was low and 6 times below naturally-cultivated ginseng roots.     

Sampling spatial and temporal variation in soil nitrogen availability

There are few studies in natural ecosystems on how spatial maps of soil attributes change within a growing season. In part, this is due to methodological difficulties associated with sampling the same spatial locations repeatedly over time. We describe the use of ion exchange membrane spikes, a relatively nondestructive way to measure how soil resources at a given point in space fluctuate over time. We used this method to examine spatial patterns of soil ammonium (NH⁺ ₄) and nitrate (NO⁻ ₃) availability in a mid-successional coastal dune for four periods of time during the growing season. For a single point in time, we also measured soil NH⁺ ₄ and NO⁻ ₃ concentrations from soil cores collected from the mid-successional dune and from an early and a late successional dune. Soil nitrogen concentrations were low and highly variable in dunes of all ages. Mean NH⁺ ₄ and NO⁻ ₃ concentrations increased with the age of the dune, whereas coefficients of variation for NH⁺ ₄ and NO⁻ ₃ concentrations decreased with the age of the dune. Soil NO⁻ ₃ concentration showed strong spatial structure, but soil NH⁺ ₄ concentration was not spatially structured. Plant-available NH⁺ ₄ and NO⁻ ₃ showed relatively little spatial structure: only NO⁻ ₃ availability in the second sampling period had significant patch structure. Spatial maps of NH⁺ ₄ and NO⁻ ₃ availability changed greatly over time, and there were few significant correlations among soil nitrogen availability at different points in time. NO⁻ ₃ availability in the second sampling period was highly correlated (r = 0.90) with the initial soil NO⁻ ₃ concentrations, providing some evidence that patches of plant-available NO⁻ ₃ may reappear at the same spatial locations at irregular points in time. Received: 20 February 1998 / Accepted: 23 November 1998     

Differential growth response of <Emphasis Type="Italic">Ulva lactuca</Emphasis> to ammonium and nitrate assimilation

Controlled cultivation of marine macroalgal biomass such as Ulva species, notably Ulva lactuca, is currently studied for production of biofuels or functional food ingredients. In a eutrophic environment, this macrophyte is exposed to varying types of nutrient supply, including different and fluctuating levels of nitrogen sources. Our understanding of the influences of this varying condition on the uptake and growth responses of U. lactuca is limited. In this present work, we examined the growth response of U. lactuca exposed to different sources of nitrogen (NH₄⁺; NO₃⁻; and the combination NH₄NO₃) by using photo-scanning technology for monitoring the growth kinetics of U. lactuca. The images revealed differential increases of the surface area of U. lactuca disks with time in response to different N-nutrient enrichments. The results showed a favorable growth response to ammonium as the nitrogen source. The NH₄Cl and NaNO₃ rich media (50 μM of N) accelerated U. lactuca growth to a maximum specific growth rate of 16.4 ± 0.18% day⁻¹ and 9.4 ± 0.72% day⁻¹, respectively. The highest biomass production rate obtained was 22.5 ± 0.24 mg DW m⁻²·day⁻¹. The presence of ammonium apparently discriminated the nitrate uptake by U. lactuca when exposed to NH₄NO₃. Apart from showing the significant differential growth response of U. lactuca to different nitrogen sources, the work exhibits the applicability of a photo-scanning approach for acquiring precise quantitative growth data for U. lactuca as exemplified by assessment of the growth response to two different N-sources.     

Hydroxylamine oxidation and subsequent nitrous oxide production by the heterotrophic ammonia oxidizer Alcaligenes faecalis

Nitrous oxide (N₂O), a greenhouse gas, is emitted during autotrophic and heterotrophic ammonia oxidation. This emission may result from either coupling to aerobic denitrification, or it may be formed in the oxidation of hydroxylamine (NH₂OH) to nitrite (NO₂ ⁻). Therefore, the N₂O production during NH₂OH oxidation was studied with Alcaligenes faecalis strain TUD. Continuous cultures of A. faecalis showed increased N₂O production when supplemented with increasing NH₂OH concentrations. ¹⁵N-labeling experiments showed that this N₂O production was not due to aerobic denitrification of NO₂ ⁻. Addition of ¹⁵N-labeled NH₂OH indicated that N₂O was a direct by-product of NH₂OH oxidation, which was subsequently reduced to N₂. These observations are sustained by the fact that NO₂ ⁻ production was low (0.23 mM maximum) and did not increase significantly with increasing NH₂OH concentration in the feed. The NH₂OH-oxidizing capacity increased with increasing NH₂OH concentrations. The apparent V _max and K _m were 31 nmol min⁻¹ mg dry weight⁻¹ and 1.5 mM respectively. The culture did not increase its growth yield and was not able to use NH₂OH as the sole N source. A non-haem hydroxylamine oxidoreductase was partially purified from A. faecalis strain TUD. The enzyme could only use K₃Fe(CN)₆ as an electron acceptor and reacted with antibodies raised against the hydroxylamine oxidoreductase of Thiosphaera pantotropha. Received: 1 September 1998 / Received revision: 5 November 1998 / Accepted: 7 November 1998     

Effects of nutritional factors on the formation of Anthraquinones in callus cultures of Rheum ribes

Rheum ribes L. (Polygonaceae) is the source of one of the most important crude drugs in Asiatic regions .The medicinal character of rhubarb is due to its anthraquinone content . Different parts of sterile seedlings were cultured on MS medium to study the generation of callus. The explants were cultured with different ranges of plant growth regulators and the best range of plant growth regulators for generation of callus was IBA (1 mg l^–1) and BA (1 mg l^–1). The content of anthraquinones were determined by HPLC . The concentration of sucrose, vitamins and Myo-inositol and ratio of NO₃ to NH₄ in the medium was changed and growth rate and content of 2 anthraquinones was determined . The growth rate of callus declined with increased rate of secondary metabolites production. Myo-inositol 100 mg l^–1 in the medium increased anthraquinone content and in medium that had NO₃/NH₄:1/1 the maximum content of anthraquinone was obtained.     

Role of cytokinin in enhanced productivity of maize supplied with NH4 + and NO3 −

Supplying both N forms (NH₄ ⁺+NO₃ ⁻) to the maize (Zea mays L.) plant can optimize productivity by enhancing reproductive development. However, the physiological factors responsible for this enhancement have not been elucidated, and may include the supply of cytokinin, a growth-regulating substance. Therefore, field and gravel hydroponic studies were conducted to examine the effect of N form (NH₄ ⁺+NO₃ ⁻ versus predominantly NO₃ ⁻) and exogenous cytokinin treatment (six foliar applications of 22 μM 6-benzylaminopurine (BAP) during vegetative growth versus untreated) on productivity and yield of maize. For untreated plants, NH₄ ⁺+NO₃ ⁻ nutrition increased grain yield by 11% and whole shoot N content by 6% compared with predominantly NO₃ ⁻. Cytokinin application to NO₃ ⁻-grown field plants increased grain yield to that of NH₄ ⁺+NO₃ ⁻-grown plants, which was the result of enhanced dry matter partitioning to the grain and decreased kernel abortion. Likewise, hydroponically grown maize supplied with NH₄ ⁺+NO₃ ⁻ doubled anthesis earshoot weight, and enhanced the partitioning of dry matter to the shoot. NH₄ ⁺+NO₃ ⁻ nutrition also increased earshoot N content by 200%, and whole shoot N accumulation by 25%. During vegetative growth, NH₄ ⁺+NO₃ ⁻ plants had higher concentrations of endogenous cytokinins zeatin and zeatin riboside in root tips than NO₃ ⁻-grown plants. Based on these data, we suggest that the enhanced earshoot and grain production of plants supplied with NH₄ ⁺+NO₃ ⁻ may be partly associated with an increased endogenous cytokinin supply.     

Effects of increased CO2 and N on CH4 efflux from a boreal mire: a growth chamber experiment

Increases in the supply of atmospheric CO₂ and N are expected to alter the carbon cycle, including CH₄ emissions, in boreal peatlands. These effects were studied in a glasshouse experiment with peat monoliths cored from an oligotrophic pine fen. The cores with living plants were kept in 720 ppm_v and 360 ppm_v CO₂ atmospheres for about 6 months under imitated natural temperature cycle. Fertilisation with NH₄NO₃ (3 g m⁻² for 25 weeks) was applied to 18 of the 36 monoliths. The rate of CH₄ flux was non-linearly dependent on the number of Eriophorum vaginatum shoots growing in the monoliths, probably due to the gas transport properties of the aerenchyma. The average CH₄ efflux rate, standardised by the number of shoots, was increased by a maximum of 10–20% in response to the raised CO₂ level. In the raised-NH₄NO₃ treatment, the increase in CH₄ release was lower. The effect of combined CO₂+NH₄NO₃ on CH₄ release was negligible and even lower than in the single treatments. Both potential CH₄ production and oxidation rates at 5, 15 and 25°C were higher near the surface than at the bottom of the core. As expected, the rates clearly depended on the incubation temperature, but the different treatments did not cause any consistent differences in either CH₄ production or oxidation. The determination of potential CH₄ production and oxidation in the laboratory is evidently too crude a method of differentiating substrate-induced differences in CH₄ production and oxidation in vivo. These results indicate that an increase in atmospheric CO₂ or N supply alone, at least in the short term, slightly enhances CH₄ effluxes from boreal peatlands; but together their effect may even be restrictive. Received: 18 June 1998 / Accepted: 25 January 1999     

In vitro shoot growth of Brugmansia × candida Pers

The objective of this study was to improve the growth of in vitro shoot cultures of Brugmansia × candida ‘Creamsickle’. Several mineral nutrient experiments were conducted to determine the effect of NH₄⁺, NO₃⁻, K⁺, FeSO₄/EDTA, ZnSO₄, MnSO₄, and CuSO₄ on quality, leaf width and length, size and weight of shoot mass, and shoot number. The experiment to determine the levels of NH₄⁺, NO₃⁻, and K⁺, was conducted as a 2-component NH₄⁺: K⁺ mixture crossed by [NO₃⁻] and resulted in an experimental design free of ion confounding and capable of separating the effects of proportion and concentration. The results of the NH₄⁺-K⁺-NO₃⁻ experiment revealed a region in the design space where growth was significantly improved; the region generally had lower total nitrogen and lower NH₄⁺:K⁺ ratios than MS medium. The experiments to determine the appropriate levels of Fe, Zn, Mn, and Cu were conducted at six log levels ranging from 0 to 1 mM. Of the four metal salts tested, MnSO₄ had the least effect on in vitro shoot growth and its concentration was reduced from 0.1 mM (MS level) to 0.001 mM. CuSO₄ had large effects on in vitro shoot growth and was increased from 0.0001 mM to 0.001 mM. A 2-level factorial of NH₄⁺-K⁺-NO₃⁻, FeSO₄/EDTA, and ZnSO₄ was conducted and several formulations identified for their improvements of quality and growth. In addition to the changes to MnSO₄ and CuSO₄, these formulations were characterized by lower levels of NH₄⁺, K⁺, NO₃⁻ and Zn, and higher levels of FeSO₄/EDTA. Overall, several nutrient formulations were identified as superior to MS medium for growth of in vitro shoot cultures of B. ‘Creamsickle’.     

Yield and nitrogen uptake of rapessed (Brassica campestris L.) with ammonium and nitrate

Water culture, growth chamber, greenhouse and field experiments were conducted to compare the effect of NH₄−N and NO₃−N on yield and N uptake of rapeseed (Brassica campestris L.). In water culture, the yields of 28-day old rapeseed plants grown at 14 μg N ml⁻¹ were double with NO₃ compared to NH₄, but N uptake was little affected. There was no such effect when concentration was reduced to 3.5 or 7 μg N ml⁻¹. The yield and N uptake of 26-day old rapeseed grown on six soils (pH 4.6 to 6.5) in pots in a growth chamber were much greater with NO₃ than with NH₄, although N concentration was more in the NH₄- than the NO₃-grown plants. In a greenhouse experiment with rapeseed grown on 12 potted soils, the N uptake of applied N was greater with NO₃ than with NH₄ on all soils. Averages were 63% with NH₄ and 78% with NO₃. However, NH₄-fixation capacities of the soils were only weakly correlated with yield from the two sources of N (r=0.48) and the relation was similar with N uptake. In contrast to the behavior of water culture, growth chamber and greenhouse experiments, the 33 field experiments did not show consistent difference in seed yield with NH₄ and NO₃ applied at time of seeding. In nine field experiments where band application was used for Ca(NO₃)₂, (NH₄)₂ SO₄, NH₄ NO₃, yield tended to be greatest for (NH₄)₂SO₄. However, in 19 experiments on acid soils with and without lime, yields in most cases were similar with (NH₄)₂SO₄ and NH₄ NO₃. Nitrification inhibitors were added to spring banded NH₄-based fertilizers in five experiments, but the yields were not influenced. Scientific Paper No. 558, Lacombe Research Station, Agriculture Canada.     

Optimization of growth regulators and silver nitrate for micropropagation of <Emphasis Type="Italic">Dianthus caryophyllus</Emphasis> L. with the aid of a response surface experimental design

This paper reports on the optimum concentrations of naphthalene acetic acid (NAA) and 6-benzyladenine (BA) to stimulate callus growth and NAA; kinetin and silver nitrate (AgNO₃) for callus redifferentiation in Dianthus caryophyllus L. Meristems were excised and placed in MS medium with 30 g l⁻¹ sucrose and 9.0 μM 2,4-d. Callus clusters were transferred to MS medium containing NAA (0, 1.7, 3.3, and 5.0 μM) and BA (0, 1.7, 3.3, and 5.0 μM) for proliferation and to MS medium with 30 g l⁻¹ sucrose, 2.5 g l⁻¹ phytagel, kinetin (0, 33, and 66 μM); NAA (0, 7.95, and 15.9 μM) and AgNO₃ (0, 23.54 and 47.08 μM) for shoot and root induction. Treatments were applied according to a Box–Behnken design. After callus growth and redifferentiation, plants were incubated in the greenhouse at 18 ± 2°C for 4 wk and at 20–26°C for 4 wk. Finally, plants were changed to near-commercial greenhouse conditions with different day (30–35°C) and night (16–24°C) temperatures. Results showed better callus growth at higher NAA concentrations. A maximum callus weight was found with 5.0 μM NAA but without BA. A maximum of 78% calluses with shoots was obtained with 15.9 μM NAA, 47.08 μM AgNO₃, and 0.74 μM kinetin and 58% with roots with 15.7 μM NAA and 47.08 μM AgNO₃, but without kinetin. The shoots obtained showed little hyperhydricity. Vigorous plants were obtained after gradual acclimatization with an 80% survival rate under nursery conditions.     

培养条件对三七愈伤组织生长和皂苷积累的影响

以MS为基础培养基,改变激素配比、氮源和光照等因素,以分光光度法和HPLC法分析三七愈伤组织培养过程中皂苷含量的变化。结果表明:培养条件对三七愈伤组织中皂苷积累有一定影响,激素配比对愈伤组织中皂苷含量的影响最大,在0.5 mg·L-12,4-D+1.0 mg·L-16-BA组合下,培养物中总皂苷含量最多,达到4.72%±0.29%;在总氮量为60 mmol·L-1条件下,45 mmol·L-1KNO3+7.5 mmol·L-1NH4NO3(NO3-/NH4+=7∶1)时,愈伤组织皂苷含量最多,达到4.71%±0.17%;分别在1 000 lx和500 lx光强下每天光照12 h的愈伤组织,皂苷含量均低于黑暗培养的愈伤组织,三者皂苷含量分别为1.94%±0.31%、2.38%±0.12%和3.57%±0.27%,光照引起愈伤组织表面变绿及细胞分化,可能是抑制愈伤组织中皂苷合成与积累的主要原因;HPLC检测发现,三七愈伤组织和根中均含有Rg1、Re、Rb1及Rd四种皂苷,但栽培三七根含有R1皂苷,而三七愈伤组织中未检测到R1,其原因需要进一步研究。该研究结果为未来愈伤组织培养成为部分代替人工栽培生产三七天然产物的潜在途径提供了研究基础。     

Distribution of nitrogen-15 tracers applied to the canopy of a mature spruce-hemlock stand,Howland, Maine,USA

In N-limited ecosystems, fertilization by N deposition may enhance plant growth and thus impact C sequestration. In many N deposition–C sequestration experiments, N is added directly to the soil, bypassing canopy processes and potentially favoring N immobilization by the soil. To understand the impact of enhanced N deposition on a low fertility unmanaged forest and better emulate natural N deposition processes, we added 18 kg N ha⁻¹ year⁻¹ as dissolved NH₄NO₃ directly to the canopy of 21 ha of spruce-hemlock forest. In two 0.3-ha subplots, the added N was isotopically labeled as ¹⁵NH₄ ⁺ or ¹⁵NO₃ ⁻ (1% final enrichment). Among ecosystem pools, we recovered 38 and 67% of the ¹⁵N added as ¹⁵NH₄ ⁺ and ¹⁵NO₃ ⁻, respectively. Of ¹⁵N recoverable in plant biomass, only 3–6% was recovered in live foliage and bole wood. Tree twigs, branches, and bark constituted the most important plant sinks for both NO₃ ⁻ and NH₄ ⁺, together accounting for 25–50% of ¹⁵N recovery for these ions, respectively. Forest floor and soil ¹⁵N retention was small compared to previous studies; the litter layer and well-humified O horizon were important sinks for NH₄ ⁺ (9%) and NO₃ ⁻ (7%). Retention by canopy elements (surfaces of branches and boles) provided a substantial sink for N that may have been through physico-chemical processes rather than by N assimilation as indicated by poor recoveries in wood tissues. Canopy retention of precipitation-borne N added in this particular manner may thus not become plant-available N for several years. Despite a large canopy N retention potential in this forest, C sequestration into new wood growth as a result of the N addition was only ~16 g C m⁻² year⁻¹ or about 10% above the current net annual C sequestration for this site.     

Regeneration of Acacia mangium through somatic embryogenesis

 Somatic embryogenesis and whole plant regeneration were achieved in callus cultures derived from immature zygotic embryos of Acacia mangium. Embryogenic callus was induced on MS medium containing combinations of TDZ (1–2 mg/l), IAA (0.25–2 mg/l) and a mixture of amino acids. Globular embryos developed on embryogenic callus cultured on the induction medium. Nearly 42% of embryogenic cultures with globular embryos produced torpedo- and cotyledonary-stage embryos by a two-step maturation phase. The first stage occurred on 1/2-strength MS basal medium containing 30 g/l sucrose and 5 mg/l GA₃ followed by the second stage on 1/2-strength MS basal medium containing 50 g/l sucrose. Of the cotyledonary-stage somatic embryos, 11% germinated into seedlings that could be successfully transferred to pots. Light- and scanning electron microscopy showed that the somatic embryos originated from single cells of the embryogenic callus. Further, a single cell layer could be detected beneath the developing somatic embryos that appeared to be a demarcation layer isolating the somatic proembryonic structure from the rest of the maternal callus. A suspensor-like structure connected the globular embryos to the demarcation layer. This is the first successful report of plant regeneration through somatic embryogenesis for this economically important tropical forest species. Received: 20 January 2000 / Revision received: 28 September 2000 / Accepted: 29 September     

A macronutrient optimization platform for micropropagation and acclimatization: using turmeric (<Emphasis Type="Italic">Curcuma longa</Emphasis> L.) as a model plant

Tissue culture medium is often overlooked as a factor in plant biotechnology. Most work uses Murashige and Skoog (MS; Physiol Plant in 15:473–497, 1962) inorganic medium formulation, which is not likely optimal for many of the plant systems where it is used. This current study of macronutrient factors simultaneously altered media volume and amount of tissue (plants per vessel), sucrose, nitrogen (as NO₃⁻ and NH₄⁺ ions), and K⁺ in a d-optimal design space with only 55 experimental units (including five true replicates). Meso- and micro-nutrient concentrations were lowered (5% of MS) to determine which elements were most critical to plantlet quality. Plantlet quality was quantified by multiplication in the laboratory and survival and growth in the greenhouse. Plantlets grown at the lowest plant density, the lowest macronutrient concentration (20 mM), and equi-molar proportions of NH₄⁺/K⁺ resulted in the best multiplication ratio and 100% greenhouse survival. Multiplication ratio in vitro and survival in the greenhouse were well correlated with one another. Laboratory dry mass, media use, sucrose use, and the uptake of the macronutrients NO₃⁻, NH₄⁺, and K⁺ were not well correlated with plantlet quality. Plantlets with the greatest uptake of P, Ca, Mg, and Mn had the best multiplication in the laboratory and on subsequent transfer, acclimatized and grew fastest in the greenhouse. Phosphorus was shown to be most depleted in media. This work demonstrates a platform to simultaneously optimize several nutritive components of tissue culture media to produce plantlets that perform well in both laboratory and greenhouse environments. Plant quality was related with factors outside the macronutrient design, and this platform indicated where to expand the experimental space. Fixed, flat-screen presentations revealed less of the response surface than interactive profiles driven by the reader.     

Adventitious root suspension cultures of <Emphasis Type="BoldItalic">Hypericum perforatum</Emphasis>: effect of nitrogen source on production of biomass and secondary metabolites

The present study investigated the effect of nitrogen source (NH₄⁺; NO₃⁻) at different concentrations on the accumulation of biomass and secondary metabolites in adventitious root cultures of Hypericum perforatum L. Cultures were initiated in shake flasks by using half-strength Murashige and Skoog (MS) medium with B5 vitamins, 1.0 mg l⁻¹ indole-3-butyric acid, 0.1 mg l⁻¹ kinetin, 3% (w/v) sucrose, and different ratios of ammonium and nitrate (0:30, 5:25, 10:20, 15:15, 20:10, 25:5, and 30:0 mM, using NH₄Cl and KNO₃). The cultures were maintained in darkness. The medium supplemented with 5:25 (mM) NH₄⁺/NO₃⁻ resulted in the optimum accumulation of biomass and total phenols and flavonoids. The antioxidant potential of a methanolic extract, measured as the 1, 1-diphenyl-2-picrylhydrazyl and 2, 2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activities, of H. perforatum adventitious roots showed that antioxidant activity was high from root extracts that were grown on higher concentrations of NO₃⁻ nitrogen (15, 20, and 25 mM). Further, assessment of hydrogen peroxide (H₂O₂) and malondialdehyde content of the root extracts revealed that cultures supplemented with higher levels of NO₃⁻ nitrogen (15–30 mM) were under oxidative stress, which boosted the levels of secondary metabolites in the adventitious roots. These results suggest that optimal adventitious root biomass could be achieved with the supplementation of cultures with 5:25 ratios of MS nitrogen sources.     

Isolation and characterization of nitrite-reductase-deficient mutants of Chlorella sorokiniana (strain 211-8k)

 A method is presented to isolate mutants of Chlorella sorokiniana with defects in NO₃ ⁻ metabolism. Three nitrite-reductase (NIR; E.C.1.7.7.1)-deficient mutants were obtained from 500 pinpoint-colony-forming clones. The final screening was performed using NO₃ ⁻, NO₂ ⁻ or NH⁺ ₄ as N-source. The mutants isolated absorb NO₃ ⁻ with rates close to those measured for the wild type and they excrete NO₂ ⁻ into the medium. The ratio between NO₃ ⁻ uptake and NO₂ ⁻ excretion was 1:1. The sensitivity of NO₃ ⁻ uptake to NH⁺ ₄ was reduced in the mutant strains as it was in the N-starved wild type of Chlorella. Nitrate reductase (NR; EC 1.6.6.1) expression and NR activity were slightly reduced compared to the wild type due to feedback regulation in the mutant strains. No NIR protein was found in the three mutants. However, NIR activity was obtained (50% of the wild-type) for one mutant strain. The NIR-deficient mutants and the already available NR-deficient mutants will be promising tools for investigations of the nitrate assimilation pathway on the molecular level and for studies searching for signaling of C and N metabolism by inorganic N-compounds. Received: 8 October 1999 / Accepted: 25 January 2000     

Effects of macro elements and nitrogen source on biomass accumulation and bacoside A production from adventitious shoot cultures of <Emphasis Type="Italic">Bacopa monnieri</Emphasis> (L.)

The present work deals with optimization of adventitious shoot culture of Bacopa monnieri for the production of biomass and bacoside A and has investigated the effects of macro elements (NH₄NO_3, KNO_3, CaCl_2, MgSO₄ and KH₂PO₄) and nitrogen source [NH₄ ⁺/NO₃ ⁻] of Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium (MS) on accumulation of biomass and bacoside A content. Optimum number of adventitious shoots (99.33 shoots explant⁻¹), fresh weight (1.841 g) and dry weight (0.150 g) were obtained in the medium with 2.0× strength of NH₄NO₃. The highest production of bacoside A content was also recorded in the medium of 2.0× NH₄NO₃, which produced 17.935 mg g⁻¹ DW. The number of adventitious shoot biomass and bacoside A content were optimum when the NO₃ ⁻ concentration was higher than that of NH₄ ⁺. Maximum number of shoots (70.00 shoots explant⁻¹), biomass (fresh weight 1.137 g and dry weight 0.080 g) and also bacoside A content (27.106 mg g⁻¹ DW) were obtained at NH₄ ⁺/NO₃ ⁻ ratio of 14.38/37.60 mM. Overall, MS medium supplemented with 2.0× NH₄NO₃ is recommended for most efficient bacoside A production.     

Plant regeneration in Arachis pintoi (Leguminosae) through leaf culture

Plant regeneration in Arachis pintoi was obtained via two developmental pathways: organogenesis and somatic embryogenesis. Organogenic callus cultures were initiated from pieces of leaf on MS medium supplemented with NAA or 2,4-D in combination with BA, KIN or 2iP. The most suitable combination for plant regeneration through organogenesis was an initial medium composed of 10 mg/l NAA+1 mg/l BA followed by transfer of the callus to a shoot induction medium (MS+1 mg/l BA). Rooting of regenerated shoots was readily achieved by culture on MS+0.01 mg/l NAA. Embryogenic callus cultures were initiated from pieces of leaf on MS medium supplemented with PICL in combination with KIN, ZEA, BA or 2iP, and the most suitable combinations were 20 mg/l PICL+1 mg/l BA or 2iP. When pieces of embryogenic callus were subcultured on MS+1 mg/l BA, somatic embryos were differentiated and developed further into well-developed plants in MS+1 g/l AC followed by MS medium devoid of plant growth regulators. Received: 29 April 1999 / Revision received: 24 November 1999 / Accepted: 18 December 1999