Growth characteristics of a continuous cell line from the velvetbean caterpillar,Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae)

Summary The cell line UFL-AG-286 was established from the embryos ofAnticarsia gemmatalis (Lepidoptera: Noctuidae). The cell line was characterized by isozyme analysis and from molecular weight determination of the restriction endonuclease bands of the mitochondrial DNA. Cells seeded in 24-cluster wells had a doubling time of 5.9 d when seeded at 2×10⁵ cells ml and 6.7 d when seeded at 3×10⁵ cells/ml. This work was funded by U.S. Department of Agriculture grant 84-CRCR-1-1431. Florida Agricultural Experiment Station Journal Series, no. 8181.     

Enhancement of Sf9 Cells and Baculovirus Production Employing Grace’s Medium Supplemented with Milk Whey Ultrafiltrate

Animal cells can be cultured both in basal media supplemented with fetal bovine serum (FBS) and in serum-free media. In this work, the supplementation of Grace’s medium with a set of nutrients to reduce FBS requirements in Spodoptera frugiperda (Sf9) cell culture was evaluated, aiming the production of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) at a cost lower than those for the production using Sf900 II medium. In Grace’s medium supplemented with glucose, Pluronic F68 (PF68) and yeast extract (YE), the effects of FBS and milk whey ultrafiltrate (MWU) on cell concentration and viability during midexponential and stationary growth phase were evaluated. In spite of the fact that FBS presented higher statistical effects than MWU on all dependent variables in the first cell passage studies, after cell adaptation, AgMNPV polyhedra production was comparable to that in Sf900 II. Batch cultivation in Grace’s medium with 2.7 g l⁻¹ glucose, 8 g l⁻¹ YE and 0.1% (w/v) PF68 supplemented with 1% (w/v) MWU and 3% (v/v) FBS increased viable cell concentration to about 5-fold (4.7×10⁶ cells ml⁻¹) when compared to Grace’s containing 10% (v/v) FBS (9.5×10⁵ cells ml⁻¹). AgMNPV polyhedra (PIBs) production was around 3-fold higher in the MWU supplemented medium (1.6×10⁷ PIBs ml⁻¹) than in Grace’s medium with 10% FBS (0.6×10⁷ PIBs ml⁻¹). This study therefore shows a promising achievement to significantly reduce FBS concentration in Sf9 insect cell media, keeping high productivity in terms of cell concentration and final virus production at a cost almost 50% lower than that observed for Sf900 II medium. C.A. Pereira is recipient of a CNPq fellowship.     

The role of peritrophic membrane in the resistance of Anticarsia gemmatalis larvae (Lepidoptera: Noctuidae) during the infection by its nucleopolyhedrovirus (AgMNPV)

The aim of this study was to analyze morphologically the peritrophic membrane (PM) of Anticarsia gemmatalis larvae resistant (RL) and non-resistant (susceptible) (SL) to the A. gemmatalis multicapsid nucleopolyhedrovirus (AgMNPV), in the presence of viral infection. Also, in this investigation the results between SL and RL were compared to improve the understanding of the resistance mechanisms to the virus. The PM of SL of A. gemmatalis was less efficient as a barrier against the viral infection since it was found to be more fragile than the PM of RL. The lower chitin content as seen from weaker fluorescent staining in SL as well as the abundance of non-solubilized vesicular materials in the ectoperitrophic space, would cause the malformation of this membrane, facilitating the passage of the virus toward the epithelium of the midgut. On the other hand, in RL, the intensity of WGA (wheat germ agglutinin)-conjugated FITC (fluorescein) reaction of the PM was greater than in SL, making this insect more resistant to infection. We can conclude that the effectiveness of the PM in protecting against pathogens is dependent on the integrity of the epithelial cells of the midgut and of the structural preservation of the PM, being directly implicated in the resistance of A. gemmatalis larvae to AgMNPV.     

Cell Culture Derived AgMNPV Bioinsecticide: Biological Constraints and Bioprocess Issues

We have studied parameters for optimizing the Spodoptera frugiperda (Sf9) cell culture and viral infection for the production of Anticarsia gemmatalis multiple nucleopolyhedrosis virus (AgMNPV) polyhedra inclusion bodies (PIBs) in shaker-Schott or spinner bottles and bioreactors. We have assayed the k_La of the systems, initial cell seeding, cell culture volume, dissolved oxygen (DO), multiplicity of infection (MOI), nutrients consumption, and metabolites production. The medium surface oxygen transfer was shown to be higher in shaker bottles than in spinner ones, which was in direct correlation to the higher cell density obtained. Best quantitative performances of PIBs production were obtained with a SF900II medium volume/shaker-bottle volume ratio of 15% and MOI of 0.5 to 1 performed at a cell concentration at infection (CCI) of 1 to 2.5×10⁶ cells/ml in a medium containing enough glucose and glutamine. Upon infection, a decrease in the cell multiplication was observed to be dependent on the MOI used, and the μX at the exponential growth phase in infected and non-infected cultures were, respectively, of 0.2832 and 0.3914 (day⁻¹). The glucose consumption and lactate production were higher in the infected cultures (μGlucose and μLactate of, respectively, 0.0248 and 0.0089×10⁻⁸ g/cell×day in infected cultures and 0.0151 and 0.0046×10⁻⁸ g/cell×day in non infected ones). The glutamine consumption did not differ in both cultures (μGlutamine of 0.0034 and 0.0037×10⁻⁸ g/cell×day in, respectively, infected and non infected cultures). When a virus MOI of 0.1 to 1 was used for infection, a higher concentration of PIBs/ml was obtained. This was in direct correlation to a higher cell concentration present in these cultures, where a decrease in cell multiplication due to virus infection is minimized. When a MOI of 1 was used, a more effective decrease in cell multiplication was observed and a lower concentration of PIBs/ml was obtained, but with the best performance of PIBs/cell. Correlations between MOI and CCI indicate that a MOI 0.1 to 1.4 and a CCI of 10⁶ to 2×10⁶ cells/ml led to the best PIBs production performances. The virulence of PIBs produced in cultures infected at low or high MOI showed comparable DL₅₀. Culture and infection in scaling-up conditions, performed in a bioreactor, were shown to provide the cells with a better environment and be capable of potentially improving the shaker-Schott findings. For an accurate qualitative control of PIB virulence, hemolymph from AgMNPV infected Anticarsia gemmatalis was used as starting material for passages in Sf9 cells. These led to a loss of virulence among the PIBs with an increase in the DL₅₀. The loss of virulence was accompanied by a loss in budded virus titer, a decreased number of PIBs produced and an altered DNA restriction pattern, suggesting the generation of defective interference particles (DIPs). Transmission electron microscopy (TEM) studies revealed that after cell passages, PIBs lacking virions were progressively synthesized. The study described here point out the biological constraints and bioprocess issues for the preparation of AgMNPV PIBs for biological control.     

In vitro and in vivo host range of Anticarsia Gemmatalis multiple nuclear polyhedrosis virus

Summary A clone of the wild type (wt) Anticarsia gemmatalis multiple nuclear polyhedrosis virus AgMNPV, derived from a geographical isolate (Hondrina, Brazil) and designated AgMNPV-CL4-3A1, was used to determine the host range of this virus in six established lepidopteran cell lines: Anticarsia gemmatalis (BCIRL-AG-AM1), Helicoverpa zea (BCIRL-HZ-AM1), Heliothis virescens (BCIRL-HV-AM1), Helicoverpa armigera (BCIRL-HA-AM1), Trichoplusia ni (TN-CL1), Bombyx mori (BMN), and a coleopteran cell line Anthonomus grandis (BRL-AG-1). In addition, the in vivo host range of this clone was also assayed in larvae of Helicoverpa zea, Heliothis virescens, Trichoplusia ni, and the homologous species Anticarsia gemmatalis by probit analysis. On the basis of temporal studies of TCID₅₀ values, BCIRL-HV-AM1 cells gave the highest extracellular virus (ECV) titer (9.7×10⁶ TCID₅₀/ml) followed by BCIRL-HA-AM1 cells (8.3×10⁵ TCID₅₀/ml) and BCIRL-AG-AM1 cells (3.2×10⁵ TCID₅₀/ml). In addition, a low ECV titer of 1.37×10³ TCID₅₀/ml was detected from TN-CL1 cells 96 h postinoculation, while BRL-AG-1, BMN, and BCIRL-HZ-AM1 cells were nonpermissive to AgMNPV-CL4-3A1 on the basis of TCID₅₀ results. AgMNPV-CL4-3A1 and the wild type AgMNPV had similar restriction profiles that were different from wild type AcMNPV. The LC₅₀ values were 96.9, 564.6, 733.3, and 1.1×10⁴ occlusion bodies/cm² of diet for A. gemmatalis, Helicoverpa zea, Heliothis virescens, and T. ni, respectively. This article presents the results of research only. Mention of proprietary products in this article does not indicate endorsement or a recommendation for use by USDA-ARS. All programs and services of the USDA are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, marital status or handicap.     

Classical biological control in an ephemeral crop habitat with Anticarsia gemmatalis nucleopolyhedrovirus

Anticarsia gemmatalis nucleopolyhedrovirus(AgNPV) was released as a single spray in soybean at two sites in Louisiana, near Crowley and Baton Rouge, after which viral prevalence and population density were monitored for 3--4 years. Each site had a plot with no treatment (control) and two virus-treated plots, one planted with soybean yearly for 3-4 years, and the other planted with soybean for 3-4 years except for rotation to a different crop in year 2. In year 1, the single spray ofAgNPV resulted in viral prevalence rates ranging from 25--100% A. gemmatalis mortality over the entire growing season. By the end of this season, viral accumulation in soil averaged 4.1 ×10⁴ occlusion bodies (OB)/g at Crowley and 7.4× 10³ OB/g at Baton Rouge, which had a sandier soil than Crowley. At Crowley, prevalence of AgNPVreached 49% insect mortality in the unrotated plot in year 2, and 31% in the rotated plot and 38% in the unrotated plot in year 3, in spite of moderate to low population densities of A. gemmatalis. At Baton Rouge, AgNPV prevalence decreased to peak prevalence rates of 25% insect mortality in year 2,4% in year 3, and 11% in year 4, even though A. gemmatalis population densities were moderate in years 2 and 3. Viral concentration in soil decreased to 2.7 × 10² OB/g (rotated plot) and1.4 × 10⁴ OB/g (unrotated plot) by the end of year 3 at Crowley and to 10 OB/g (rotated plot) and31 OB/g (unrotated plot) by the end of year 4 at Baton Rouge. In forward stepwise multiple regressions, the concentration of OB in soil was significantly (p = 0.0001) and positively correlated with AgNPV prevalence, but correlations of the latter parameter with host population density and rainfall were not significant (p > 0.05). Prevalence rates of the fungal entomopathogen Nomuraea rileyi were significantly (p < 0.01) and negatively correlated with AgNPV prevalence at Crowley but not in the combined data set. Prevalence of N. rileyi was negatively correlated (p = 0.0001) with precipitation and positively correlated (p = 0.0001) with A.gemmatalis population density. The research demonstrated that AgNPV can be introduced and established for long-term suppression of A.gemmatalis in an ephemeral crop habitat, but certain site-related conditions, perhaps relating to soil, are necessary for its continued success. This revised version was published online in July 2006 with corrections to the Cover Date.     

Inactivation of the ecdysteroid UDP-glucosyltransferase (egt) gene of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) improves its virulence towards its insect host

Some baculovirus have been genetically modified for the inactivation of their ecdysteroid glucosyltransferase (egt) gene, and these viruses were shown to kill infected larvae more rapidly when compared to wild-type virus infections. We have previously identified, cloned, and sequenced the egt gene of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV). Here we present data regarding the construction of an egt minus (egt−) AgMNPV and its virulence towards its insect host. We have inserted an hsp70-lacZ (3.7 kb) gene cassette into the egt gene open reading frame (ORF) and purified a recombinant AgMNPV (vAgEGTΔ-lacZ). Bioassays with third-instar A. gemmatalis larvae showed that viral occlusion body (OB) production were consistently lower from infections with vAgEGTΔ-lacZ compared to the wild-type virus. A mean of 20.4×10⁸ OBs/g/larva and 40.7×10⁸ OBs/g/larva was produced from vAgEGTΔ-lacZ and AgMNPV infections, respectively. The mean lethal concentration which killed 50% of insects in a treatment group (LC₅₀) for the 10th day after virus treatment (DAT) was 3.9-fold higher for the wild-type virus compared to vAgEGTΔ-lacZ. The recombinant virus killed A. gemmatalis larvae significantly faster (ca. 1–2.8 days), than the wild-type AgMNPV. Therefore, the vAgEGTΔ-lacZ was more efficacious for the control of A. gemmatalis larvae (in bioassays) compared to wild-type AgMNPV.     

Development of callus and suspension cultures of potato resistant to NaCl and mannitol and their response to stress

Callus and suspension cultures adapted to various concentrations of NaCl or mannitol were developed from the cultivated potato Solanum tuberosum cv. Desire. Growth of the calli was less inhibited by mannitol than by iso-osmotic concentrations of NaCl. Reduction of growth by both NaCl and mannitol was considerably lower in osmotically adapted calli than in non-adapted ones. Salt-adapted suspension cultures that grew in the medium to which they had been originally adapted had a shorter lag in growth as well as a shorter time required to achieve the maximum growth, as compared with non-adapted cells. Suspension cultures adapted to NaCl concentrations higher than 150 mM were obtained only after preadaptation to osmotic stress. Adaptation of these cells was found to be stable. Accumulation of Na⁺ was lower and level of K⁺ was more stable in osmotically adapted than in non-adapted calli, when both were exposed to salt. Potassium level in NaCl-adapted calli exposed to saline medium was lower than that in non-adapted calli in standard medium. The maximum of Cl^– and Na⁺ accumulation was reached at higher external salt concentration in salt-adapted than in non-adapted suspension cultures. In both callus and suspension cultures, Cl^– accumulated more than Na⁺. Potassium level decreased more in non-adapted than in NaCl-adapted suspension cultures. The decrease of osmotic potential in osmotically adapted calli exposed to mannitol and in salt-adapted calli and suspension cultures exposed to salt was correlated to the increase of the external concentration. Such a correlation was not found in osmotically adapted calli exposed to salt. Non-electrolytes were found to be the main contributors to the decrease is osmotic potential in both callus and suspension cultures.     

l-Phenylalanine ammonia-lyase in suspension culture cells of spruce (Picea abies)

The activity of l-phenylalanine ammonia-lyase (PAL) (EC 4.3.1.5) was determined in seedlings, callus cells, cell suspension cultures and in young needles of spruce (Picea abies) (L.) (Karst). PAL activity increased up to 10 fold in response to transferring suspension cultured cells into new cultivation medium. PAL was also induced about 10 fold when callus cells were transferrd into liquid medium. The increase was transient and it required the presence of a carbohydrate.In cell suspension cultures, grown in the dark (white cells), but not in light-grown cultures (green cells), PAL activity was induced up to 30 fold by UV-light.With a cell wall preparation of Rhizosphaera kalkhoffii, a forest pathogenic fungus, used as elicitor, the activity of PAL could be induced more than 10 fold. The degree of induction depended on the elicitor concentration. Induction was prevented by cycloheximide but not by actinomycin D.     

Induction of phenoloxidase in cell suspension cultures of Mucuna pruriens L.

The effects of limitating nitrogen-containing compounds in the medium and of adding the amino-acid analogues p-fluorophenylalanine and ethionine on both phenoloxidase activity and the accumulation of L-3,4-dihydroxyphenylalanine (L-DOPA) are reported for cell suspension cultures of Mucuna pruriens. Nitrogen limitation of the cultures, or the addition of p-fluorophenylalanine or ethionine to the culture medium resulted in an increased phenoloxidase activity. There appeared to be an inverse relationship between phenoloxidase activity and the acccumulation of L-tyrosine into L-DOPA by alginate-entrapped cells occurred at a higher rate when phenoloxidase activity was increased.Abbreviations pFPA p-fluorophenylalanine - L-DOPA L-3,4-dihydroxyphenylalanine     

Infectivity of Anticarsia gemmatalis nucleopolyhedrovirus to different insect cell lines: Morphology, viral production, and protein synthesis

The Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) is currently used as an efficient biological pesticide for the control of the velvetbean caterpillar (A. gemmatalis), an important pest of soybean in Brazil. Until now, production of the virus has been achieved mainly by infection of larvae on local soybean farms. Studies for the development of in vitro systems and the optimization of mass production in insects reared on artificial diets is now important to help to meet the actual demand for the bioinsecticide. We therefore, investigated the infectivity of AgMNPV in cell culture, which might contribute to the selection of suitable cell lines that may be used for in vitro production of this virus. The cytopathic effects induced by the virus, the production of viral particles and the synthesis of viral polypeptides were examined and compared in the cell lines from A. gemmatalis (UFL-AG-286), Trichoplusia ni (BTI-Tn-5B1-4 and TN-368), Spodoptera frugiperda (IPLB-SF-21AE and Sf9), Lymantria dispar (IPLB-LD-652Y), and Bombyx mori (BM-5). Whereas, Tn-5B1-4 and AG-286 cells produced large numbers of occlusion bodies, no polyhedra were visualized in either Ld-652Y or BM-5 cells, although extensive cell lysis was observed in BM-5. Analysis of the kinetics of viral protein synthesis by SDS–PAGE after pulse labeling with [³⁵S]methionine, showed similar protein patterns in most of the cell lines tested. Exceptions were the LD-652Y and BM-5 cells, in which viral polypeptides, including polyhedrin, were not synthesized. In parallel, measurement of viral titers (budded virus) by the endpoint dilution method showed that Tn-5B1-4, AG-286, and SF-21AE cells were highly productive. Their TCID₅₀ values, at 48 h p.i., were about 10⁷ IU/ml. In addition to the lower formation of polyhedra, the viral titers determined in Sf9 and TN-368 cells were about 5 to10-fold lower. As expected, the viral titers obtained in LD-652Y and BM-5 cells were similar to basal levels.     

The influence of some inhibitors on the formation of caffeic acid in cultures ofPerilla cell suspensions

A cell suspension culture, prepared fromPerilla frutescens var.crispa callus induced by Murashige and Skoog (1962) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 ml/l) and kinetin (0.1 mg/l), contained caffeic acid derivatives as the phenolic components. Fresh and dry weights of the cells increased exponentially for about 11 days after transfer to a fresh medium. The contents of caffeic acid and protein also reached a maximum on the 11th day, but α-amino nitrogen phenylalanine and tyrosine continued to increase in amount until the 20th to 23rd day. Caffeic acid formation in the cells was increased by lowering the concentration of 2,4-D. The administration ofl-2-aminooxy-3-phenylpropionic acid (l-AOPP), 2-aminooxyacetic acid (AOA) andN-(phosphonomethyl)glycine (glyphosate) to the cells inhibited caffeic acid formation to a large extent. An 80% inhibition of caffeic acid formation was caused by 10⁻⁴Ml-AOPP whereas phenylalanine and tyrosine contents of the cells became 7.5 and 2.3 times higher at thisl-AOPP concentration than those in the control. An 85% inhibition of caffeic acid formation was achieved at 10⁻³M glyphosate concentration, while 10⁻³M AOA inhibited caffeic acid formation by 95% and also growth rate by 80%. The influence of inhibitors on caffeic acid formation is discussed in relation to the level of α-amino nitrogen, particularly aromatic amino acids, in the cell suspension cultures.     

Production of eleutherosides in in vitro regenerated embryos and plantlets of <Emphasis Type="BoldItalic">Eleutherococcus chiisanensis</Emphasis>

High frequency somatic embryogenesis of Eleutheorcoccus chiisanensis was achieved through suspension culture of embryogenic cells in hormone-free Murashige and Skoog liquid medium supplemented with 30 g sucrose l⁻¹. Cotyledonary somatic embryos were germinated and converted into plantlets using 20 μM gibberellic acid which were then grown in a 10 l airlift bioreactor. HPLC analysis revealed the accumulation of eleutheroside B, E and E1 in the embryos and plantlets. Thus mass production of embryos and plantlets of E. chiisanensis can be achieved in liquid cultures and the biomass produced may become an alternative source of eleutherosides.     

Somatic embryogenesis in suspension and suspension-derived callus cultures ofDactylis glomerata

Summary Suspension cultures were initiated from somatic embryos and embryogenic callus ofDactylis glomerata L. in SH-30 liquid medium [Schenk andHildebrandt (1972) containing 30 M 3,6-dichloro-o-anisic acid (dicamba)] with or without 1.5 gl^–1 casein hydrolysate. Established suspension cultures maintained in SH-30 without casein hydrolysate proliferated when cell masses underwent cell division and enlargement. These cultures contained numerous root primordia and increased in volume when the cell masses continued to grow and fragment. Embryos developed only when cell masses were plated on solidified SH-30 medium. Cultures maintained in SH-30 liquid medium with casein hydrolysate also proliferated by the growth and fragmentation of cell masses. However, these cell masses contained numerous developing embryos and possessed few or no root primordia. Embryos were either attached to cell masses by a suspensor-like structure or were free and became fully developed in the liquid medium. Newly formed embryos became callused and produced embryogenic cell masses. Embryos germinated either in liquid or on solid SH medium without dicamba. The resulting plantlets possessed green shoots and well developed roots. Plants from suspension and suspension-derived callus cultures have been established in soil and grown to maturity.     

New cell lines derived from the black cutworm, Agrotis ipsilon, that support replication of the A. ipsilon multiple nucleopolyhedrovirus and several group I nucleopolyhedroviruses

New cell lines were recently developed from the embryos of the black cutworm, Agrotis ipsilon (Lepidoptera: Noctuidae). A primary culture was initiated from 4-day-old A. ipsilon eggs in ExCell420 medium supplemented with 5% fetal bovine serum. This initial culture produced sufficient cell growth to allow subcultivation and eventually led to the establishment of eight distinct strains. Two of these strains (AiE1611T and AiEd6T) were selected for further characterization. Extracts of these strains were compared to an extract from A. ipsilon eggs by isozyme analysis and shown to be from the same species. Both strains were susceptible to infection by the A. ipsilon multiple nucleopolyhedrovirus (AgipMNPV), as well as to lepidopteran group I NPVs from A. californica, Anagrapha falcifera, Anticarsia gemmatalis, Galleria mellonella, Helicoverpa armigera, Plutella xylostella, and Rachiplusia ou, with large numbers of occlusion bodies produced in most of the inoculated cells. The cell lines did not support the replication of group II NPVs from Helicoverpa zea, Lymantria dispar, and Spodoptera exigua. Both cell lines produced confluent monolayers in plaque assays and supported the formation of plaques upon infection with AgipMNPV and Autographa californica (Ac)MNPV. Twenty AgipMNPV plaques were picked from either AiE1611T or AiEd6T monolayers, and the plaque isolates were serially passaged three times through A. ipsilon cells. Only one isolate from AiE1611T cells exhibited genotypic variation in the form of an altered restriction fragment profile. Our results suggest these new lines can be useful in the study of AgipMNPV and A. ipsilon cellular and molecular biology.     

Establishment of in vitro plants, cell and tissue cultures from Oldenlandia affinis for the production of cyclic peptides

In vitro cultured plants from Oldenlandia affinis were established from seeds and grown on a hormone-free medium. In vitro plants produced the cyclic peptide kalata B1 in concentrations of 0.67 mg g⁻¹ dry weight after growth of 30 days. This was approximately 50% of the concentration analysed in green house plants (shoot tips), where different concentrations have been determined in leaves (1.82 mg g⁻¹), shoot tips (1.36 mg g⁻¹), stems (0.36 mg g⁻¹), and in flowers (0.16 mg g⁻¹). Callus and cell suspension cultures could be initiated from aseptic root, stem and leaf explants of O. affinis seedlings and plants. Different light intensities were shown to affect culture growth as well as chlorophyll synthesis. The friable callus was then used for the establishment of a cell suspension culture. Fresh and dry weight measurements showed that growth was optimal on MS medium supplemented with 0.4 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-d). Leaf suspensions cultured on this medium showed a 4-fold increase of biomass by the first week of incubation. No quantifiable amounts of kalata B1 were produced under these conditions. Morphological differentiation seems to be essential for cyclic peptide production. Therefore, several undifferentiated as well as organised cell lines of O. affinis have been developed. These cell lines will constitute a worthwhile starting point for the optimisation of kalata B1 synthesis in liquid media to the objective of producing cyclic peptides under controlled and defined conditions in bioreactors.     

Enhancing the productivity of hybrid yellow-poplar and hybrid sweetgum embryogenic cultures

Summary High-frequency embryogenesis systems were established for hybrid yellow-poplar (Liriodendron tulipifera×L. chinense) and hybrid sweetgum (Liquidambar styraciflua×L. formosana) by modifying a medium originally developed for embryogenic yellow-poplar cultures. Embryogenic cultures of both hybrids, consisting of proembryogenic masses (PEMs), were initiated from immature hybrid seeds on an induction-maintenance medium (IMM) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D), benzyladenine (BA), and casein hydrolyzate (CH). For hybrid yellow-poplar, as many as 2100 germinable somatic embryos per 4000 cells or cell clumps were produced when PEMs were grown in liquid IMM lacking CH, at a pH that varied with genotype (3.5 or 5.6), followed by size fractionation and plating on semisolid embryo development medium (DM; IMM lacking 2,4-D and BA) without CH, but supplemented with 4.0 mgl⁻¹ (15 μM) abscisic acid. For hybrid sweetgum, up to 1650 germinable somatic embryos per 4000 cells or cell clumps were produced when PEMs were grown in liquid IMM without CH, but with 550 mgl⁻¹ l-glutamine, 510 mg l⁻¹ asparagine, and 170 mg l⁻¹ arginine at pH 5.6. Somatic embryos developed from cell clumps on DM without any plant growth regulators or other supplements. Hundreds of somatic embryos of both hybrids were germinated on DM without CH, transferred to potting mix, and hardened off in a humidifying chamber for transfer to the greenhouse.     

Production of yeastolates for uniform stable isotope labelling in eukaryotic cell culture

Preparation of stable isotope-labelled yeastolates opens up ways to establish more cost-effective stable isotope labelling of biomolecules in insect and mammalian cell lines and hence to employ higher eukaryotic cell lines for stable isotope labelling of complex recombinant proteins. Therefore, we evaluated several common yeast strains of the Saccharomycetoideae family as a source of high-quality, non-toxic yeastolates with the major aim to find a primary amino acid source for insect and mammalian cell culture that would allow cost-effective uniform stable isotope labelling (¹³C, ¹⁵N). Strains of the facultative methylotrophic yeasts Pichia pastoris and Hansenula polymorpha (Pichia angusta) as well as a strain of the baker’s yeast Saccharomyces cerevisiae were compared as a source of yeastolate with respect to processing, recovery and ability to sustain growth of insect and mammalian cell lines. The best growth-supporting yeastolates were prepared via autolysis from yeast obtained from fed-batch cultures that were terminated at the end of the logarithmic growth phase. Yeastolates obtained from H. polymorpha performed well as a component of insect cell cultures, while yeastolates from S. cerevisiae and H. polymorpha both yielded good results in mammalian cell cultures. Growth of yeasts in Heine’s medium without lactic acid allows relatively low concentrations of ¹³C and ¹⁵N sources, and this medium can be reused several times with supplementation of the ¹³C source only.     

Isolation and characterization of free glycans of the oligomannoside type from the extracellular medium of a plant cell suspension

The oligosaccharides Man₅GlcNAc and Man₃(Xyl)GlcNAc(Fuc)GlcNAc presumed to originate fromN-glycosyl proteins have been purified from an extracellular medium (concentration: 2–5 mg/l of 14 day cultures) of white campion (Silene alba) suspension culture. Their primary structures have been determined by¹H-400-MHz NMR spectroscopy and FAB-MS spectrometry. They are probably the result of an autophagic process including protein catabolism due to sucrose starvation. Additional identification of digalactosylglycerol (galactolipid breakdown) argues for this hypothesis.Abbreviations Fuc l-fucose - Man d-mannose - Xyl d-xylose - GlcNAc N-acetyl-d-glucosamine - Gal d-galactose - Glc d-glucose - FAB-MS fast atom bombardment mass spectrometry - NMR nuclear magnetic resonance     

Structural and ultrastructural studies of Anticarsia gemmatalis midgut cells infected with the baculovirus A. gemmatalis nucleopolyhedrovirus

Anticarsia gemmatalis is a lepidopteran insect susceptible to A. gemmatalis nucleopolyhedrovirus (AgNPV), which is being used in a large scale, in Brazil, as a biological control agent against this serious soybean pest. Baculovirus usually infects its insect host through the midgut epithelium. In the midgut, it replicates in the nuclei of epithelial cells, producing progeny virus and establishing systemic infection. The AgNPV infection of A. gemmatalis midgut was studied using light and electron microscopy. It was observed that AgNPV enters the midgut mainly through columnar cells. Although the virus was not found in the nuclei of columnar cells until late on infection, it is believed that these cells are the primary sites of infection and replication. This fact can be explained by the continuous regeneration of the midgut epithelium. Besides, the infection may be occurring in isolated cells, making it more difficult to be visualized by electron microscopy. At 48 h post infection, hemocytes and tracheoblasts are infected and polyhedra are formed later in these cells, which are the secondary sites of infection.