Influence of Dimilin on a microsporidian infection in the gypsy moth Lymantria dispar (L.) (Lepidoptera: Lymantriidae)

Bioassay studies were conducted to investigate the influence of Dimilin (diflubenzuron), a chitinsynthetase inhibitor used for insecticidal control of the gypsy moth, Lymantria dispar, on the development and viability of a microsporidian pathogen of L. dispar. Before or after an infection with a Nosema species, L. dispar larvae were fed Dimilin in sublethal dosages. Dimilin fed to L. dispar larvae at 0.65 ng/cm² diet surface resulted in a total larval mortality of 53%. Although the microsporidian infection alone did not cause high mortality rates (9%), mortality increased to 96% when L. dispar larvae were inoculated with both Dimilin and Nosema spores. When Dimilin was fed to the larvae 24 h before or 6 days after inoculation with the microsporidium, the number of mature spores produced was significantly reduced. When Dimilin was fed to the larvae 24 h after microsporidian inoculation, the number of spores produced was not significantly reduced. Spores that were produced in larvae after Dimilin had been ingested with the diet were less infectious than spores produced in control larvae; the experimental infection rate decreased from 94% when spores obtained from control larvae were used, to 48 or 10% when spores obtained from larvae fed Dimilin 24 h or 6 days after Nosema inoculation, respectively, were used. Mature microsporidian spores washed in Dimilin solution prior to oral inoculation, however, were as infectious as spores stored in liquid nitrogen. We have shown that Dimilin interferes with the establishment of the parasite in its host. In addition, when Nosema sp. succeeds in infecting the L. dispar host despite treatment with Dimilin, the microsporidium does not develop optimally and spore production is reduced.     

In vitro induction of haploid,diploid and triploid plantlets by anther culture of Iochroma warscewiczii Regel

Pollen of Iochroma warscewiczii Regel (Solanaceae) produced embryogenic calli or embryos inside anthers cultured on Nitsch & Nitsch medium. Two distinct pathways could be recognized in this process, one involving mainly the vegetative cell, and the second starting with two equal cells in the pollen grains.In all media tested, androgenesis initiation was highest when anthers contained pollen at the first mitosis, or close to it, at inoculation. High sucrose (7%) and calcium (11.3 mM) concentrations were found to be highly desirable for the induction of androgenesis in this species. Addition of benzylaminopurine (0.5 mg l^–1) to the culture medium seems to slightly improve callus or embryo production. When all three factors were present at optimal concentrations as much as 13.9% of inoculated anthers were found to be embryogenic.Plantlet development from pollen embryos required lower sucrose (3%) and a combination of 0.1 mg l^–1 benzylaminopurine and 0.5 mg l^–1 gibberellic acid in the culture medium. Cytological analysis of 55 regenerated plantlets showed that about 49% were haploids, but diploid (ca. 49%) and triploid (ca. 2%) plants were also obtained.     

Growth characteristics and catalpol production in chinese foxglove (Rehmannia glutinosa Liboschitz) hairy roots transformed withAgrobacterium rhizogenes ATCC15834

Hairy root clones ofRehmannia glutinosa were established via transformation withAgrobacterium rhizogenes ATCC15834. To optimize the culturing conditions for both root growth and catalpol production, effects of various combinations of seven basal media, pH, and carbon sources were examined under darkness. The fastest root growth was obtained in an SH medium containing 4% sucrose (pH 5.8); the highest catalpol content (0.54% of dry weight) was achieved in a WPM medium supplemented with 4% sucrose (pH 5.8). Effects of plant growth regulators and chitosan were also investigated. IAA at 2 mg L^-1 significantly increased root lengths and the frequency of lateral roots. Chitosan (50 mg L^-1) and CA₃ (0.5 mg L^-1) induced catalpol production, with contents calculated at 0.7% dry weight and 0.65% dry weight, respectively.     

Effect of carbon and nitrogen sources on growth and solasodine production in batch suspension cultures of Solanum eleagnifolium Cav.

The effect of inoculum size, carbon sources (fructose, glucose, maltose, sucrose), nitrate and ammonia on solasodine production by Solanum eleagnifolium Cav. was studied. The specific growth rate was estimated to be 0.15–0.20 d^-1 with all sugars tested at a concentration of 90 mM. Sucrose (180 mM) produced the highest biomass value (about 2.8 mg DW ml^-1) while the lowest one was produced by maltose. Although solasodine productivity values after 11 days of culture were similar for all sugars tested, the maximum values of productivity (0.9 mg g^-1 d^-1) were achieved after 6 days of culture with sucrose (180 mM). Solasodine productivity of cultures conducted with a large inoculum (20% w/v fresh material) was double that with a small inoculum (10% w/v fresh material).     

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

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

Agrobacterium-mediated transformation of Phalaenopsis by targeting protocorms at an early stage after germination

A transformation procedure for phalaenopsis orchid established by using immature protocorms for Agrobacterium infection was aimed at the introduction of target genes into individuals with divergent genetic backgrounds. Protocorms obtained after 21 days of culture on liquid New Dogashima medium were inoculated with Agrobacterium strain EHA101(pIG121Hm) harboring both -glucuronidase (GUS) and hygromycin resistance genes. Subculture of the protocorms on acetosyringone-containing medium 2 days before Agrobacterium inoculation gave the highest transformation efficiencies (1.3–1.9%) based on the frequency of hygromycin-resistant plants produced. Surviving protocorms obtained 2 months after Agrobacterium infection on selection medium containing 20 mg l^–1 hygromycin were cut transversely into two pieces before transferring to recovery medium without hygromycin. Protocorm-like bodies (PLBs) proliferated from pieces of protocorms during a 1-month culture on recovery medium followed by transfer to selection medium. Hygromycin-resistant phalaenopsis plants that regenerated after the re-selection culture of PLBs showed histochemical blue staining due to GUS. Transgene integration of the hygromycin-resistant plants was confirmed by Southern blot analysis. A total of 88 transgenic plants, each derived from an independent protocorm, was obtained from ca. 12,500 mature seeds 6 months after infection with Agrobacterium. Due to the convenient protocol for Agrobacterium infection and rapid production of transgenic plants, the present procedure could be utilized to assess expression of transgenes under different genetic backgrounds, and for the molecular breeding of phalaenopsis.     

Production of fructosylxyloside by the new fructosyltransferase from Bacillus macerans EG-6

The new fructosyltransferase (FTase) from Bacillus maceransEG-6 showed a broad acceptor specificity, and resulted in the formation of fructosylxyloside (FX) with d-xylose being the most effective acceptor. The optimal FTase concentration for FX production was 0.6 unit per g sucrose, which gave the highest transfer ratio, 83%, of fructosyl moiety from sucrose to d-xylose. Maximum yield of FX was 114 g l^–1with 200 g sucrose l^–1and 300 g d-xylose l^–1.     

Tropane alkaloid production inDatura stramonium root cultures

Summary Tropane alkaloid production was studied in different root cultures ofDatura stramonium. Cultured roots were obtained with 10⁻⁶ M of indolbutyric acid. Their doubling times were from 6 to 19 days. Hyoscyamine content varied from 0.17 to 0.62% dry weight, and scopolamine content from 0.08 to 0.33% dry weight, depending on the lines. A comparison of the bioproductivity of these compounds in the pot-grown plant roots showed that it was two to three orders lower than cultured roots, and it increased one order of magnitude considering the productivity on the whole plant. Bioproductivity, growth capacity and alkaloid production stability during subsequent transfers (more than 2 yr) are reported. Only one root line (N5) showed excretion of the alkaloids to the culture medium. Characterization of three selected lines (N1, N5, and N9) showed that the highest alkaloid production is reached at the stationary phase of growth, with the exception of line N9.     

In vitro corm production of Gladiolus dalenii and G. tristis

The ability of the summer flowering Gladiolus dalenii Van Geel and the winter flowering G. tristis L. to form corms in vitro was investigated. G. dalenii spontaneously formed corms on a shoot induction medium consisting of the basal medium of Murashige & Skoog (1962) with up to 2.0 mg l^-1 benzyladenine (BA), 3% sucrose and solidified with 2 g l^-1 Gelrite^®. The effect of different BA and sucrose concentrations as well as different temperatures on in vitro corm production of G. tristis was further investigated. The best production of shoots per explant was achieved on a medium containing 0.5 to 1.0 mg l^-1 BA, sucrose concentrations of 6 to 9% and cultured at 15°C. The best corm production was achieved at the same temperature and with the same medium with the exception that BA was omitted from the medium. To test the effect of the osmotic potential on the formation of shoots and corms, sucrose was substituted by mannitol at various concentrations. Sucrose proved to be essential for both shoot and corm production and the use of mannitol had no beneficial effect.     

Effects of the sporulation conditions on the lovastatin production by Aspergillus terreus

The production of biomass and lovastatin by spore-initiated submerged fermentations of Aspergillus terreus ATCC 20542 was shown to depend on the age of the spores used for inoculation. Cultures started from older spores produced significantly higher titers of lovastatin. For example, the lovastatin titer increased by 52% when the spore age at inoculation rose from 9 to 16 days. The lovastatin titer for a spore age of 16 days was 186.5±20.1 mg L⁻¹. The time to sporulation on surface cultures was sensitive to the light exposure history of the fungus and the spore inoculation concentration levels. A light exposure level of 140 μE m⁻² s⁻¹ and a spore concentration of 1,320 spore cm⁻² produced the greatest extent of sporulation within about 50 h of inoculation. Sporulation was slowed in the dark and with diluted inoculants. A rigorous analysis of the data of statistically designed experiments showed the above observations to be highly reproducible.     

Pinoresinol and matairesinol accumulation in a Forsythia × intermedia cell suspension culture

A newly established Forsythia × intermedia cell suspension culture was shown to accumulate (+)- and (–)-pinoresinol as well as matairesinol. The influence of the sucrose content of the culture medium and of the cultivation time on pinoresinol and matairesinol accumulation was evaluated. The highest pinoresinol yield was achieved from cells grown in medium containing 6% sucrose for 12 ± 2 days with levels of 0.6–0.8 mg g^–1 dry weight and an average enantiomeric composition of 75 ± 5% (+)-pinoresinol. The highest matairesinol amount was reached in the same medium at the 14th ± 2 culture day with levels of 1.0–2.7 mg g^–1 dry weight. To our knowledge, this is the first report on pinoresinol accumulation in Forsythia × intermedia plants or cell suspension cultures.     

Intermittent maltose feeding enhances paclitaxel production in suspension culture of Taxus chinensis cells

High paclitaxel production (26 mg l^–1) was achieved through the intermittent feeding of 3, 1, and 2% (w/v) sucrose at days 0, 7, and 21, respectively, to a suspension culture of Taxus chinensis cells. Intermittent feedings of 1 and 2% (w/v) maltose at days 7 and 21, respectively, to the culture increased the paclitaxel production up to 67 mg l^–1.     

Production of poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-3-hydroxyvalerate) P(3HB-<Emphasis Type="Italic">co</Emphasis>-3HV) with a broad range of 3HV content at high yields by <Emphasis Type="Italic">Burkholderia sacchari</Emphasis> IPT 189

The biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from sucrose and propionic acid by Burkholderia sacchari IPT 189 was studied using a two-stage bioreactor process. In the first stage, this bacterium was cultivated in a balanced culture medium until sucrose exhaustion. In the second stage, a solution containing sucrose and propionic acid as carbon source was fed to the bioreactor at various sucrose/propionic acid (s/p) ratios at a constant specific flow rate. Copolymers with 3HV content ranging from 40 down to 6.5 (mol%) were obtained with 3HV yield from propionic acid (Y _3HV/prop) increasing from 1.10 to 1.34 g g⁻¹. Copolymer productivity of 1 g l⁻¹ h⁻¹ was obtained with polymer biomass content rising up to 60% by increasing a specific flow rate at a constant s/p ratio. Increasing values of 3HV content were obtained by varying the s/p ratios. A simulation of production costs considering Y _3HV/prop obtained in the present work indicated that a reduction of up to 73% can be reached, approximating US$ 1.00 per kg which is closer to the value to produce P3HB from sucrose (US$ 0.75 per kg).     

Enhancement of phenylethanoid glycosides biosynthesis in cell cultures of <Emphasis Type="Italic">Cistanche deserticola</Emphasis> by osmotic stress

The effect of osmotic stress on cell growth and phenylethanoid glycosides (PeGs) biosynthesis was investigated in cell suspension cultures of Cistanche deserticola Y. C. Ma, a desert medicinal plant grown in west region of China. Various initial sucrose concentrations significantly affected cell growth and PeGs biosynthesis in the suspension cultures, and the highest dry weight and PeGs accumulation reached 15.9 g l⁻¹-DW and 20.7 mg g⁻¹-DW respectively at the initial osmotic stress of 300 mOsm kg⁻¹ where the sucrose concentration was 175.3 mM. Stoichiometric analysis with different combinations of sucrose and non-metabolic sugar (mannitol) or non-sugar osmotic agents (PEG and NaCl) revealed that osmotic stress itself was an important factor for enhancing PeGs biosynthesis in cell suspension cultures of C. deserticola. The maximum PeGs contents of 26.9 and 23.8 mg g⁻¹-DW were obtained after 21 days at the combinations of 87.6 mM sucrose with 164.7 mM mannitol (303 mOsm kg⁻¹) or 20 mM PEG respectively, which was higher than that of C. deserticola cell cultures grown under an initial sucrose concentration of 175.3 mM after 30 days. The stimulated PeGs accumulation in the cell suspension cultures was correlated to the increase of phenylalanine ammonium lyase (PAL) activity induced by osmotic stress.     

Light-susceptibility of camptothecin production from<Emphasis Type="Italic">in vitro</Emphasis> cultures of<Emphasis Type="Italic">Camptotheca acuminata</Emphasis> Decne

Production of camptothecin (CPT) from callus cultures ofCamptotheca acuminata Decne was affected by light and culture conditions. Among the culture media tested, modified B5 medium containing 3% (w/v) sucrose, 2 mg/L 2,4-D, 2 times of MS medium vitamins, 500 mg/L casein hydrolysate, 250 mg/L myo-inositol, 0.05% (w/v) activated charcoal, and 0.15% (w/v) gelite was used for callus induction. The highest cell growth and CPT production were obtained in dark and green light condition, respectively. Photoperiod has no effect on cell growth and CPT production. Both cell growth and CPT production were also influenced by combination ratio of red and blue light. Cell growth and CPT production were the highest in the ratio of red and blue light 90∶10.     

Effects of Octadecanoylsucrose Derivatives on the Production of Inulinase by Apergillus niger SL-09

Summary The effect of the addition of octadecanoylsucrose esters to the growth medium on the production of inulinase by Aspergillus niger SL-09 was studied in batch culture using shake flasks. The activities of inulinase in vitro and in vivo formed by Aspergillus niger SL-09 was enhanced dramatically by the addition of sucrose ester S-770 to the medium, and it was confirmed that sucrose ester acted as a very efficient inducer for inulinase production. As a result, with the addition of 6 g sucrose ester l⁻¹ at the beginning of the culture, the enzyme activities were enhanced near 7-fold higher than that obtained in the basal medium.     

Effects of inoculum concentration,temperature, and carbon sources on tannase production during solid state fermentation of cashew apple bagasse

In this study, the optimization of tannase production by solid state fermentation was investigated using cashew apple bagasse (CAB), an inexpensive residue produced by the cashew apple agroindustry, as a substrate. To accomplish this, CAB was enriched with 2.5% (w/w) tannic acid and 2.5% (w/w) ammonium sulphate and then moistened with water (60 mL/100 g of dry CAB). The influence of inoculum concentration (10⁴ to 10⁷ spores/g), temperature (20, 25, 30, and 35°C) and several additional carbon sources (glucose, starch, sucrose, maltose, analytical grade glycerol, and glycerol produced during biodiesel production) on enzyme production by Aspergillus oryzae was then evaluated. Supplementation with maltose and glycerol inhibited tannase synthesis, which resulted in lower enzyme activity. Starch and sucrose supplementation increased enzyme production, but decreased the enzyme productivity. The maximum tannase activity (4.63 units/g of dry substrate) was obtained at 30°C, using 10⁷ spores/g and 1.0% (w/v) sucrose as an additional carbon source.     

Direct somatic embryogenesis and synthetic seed production from<Emphasis Type="Italic"> Paulownia elongata</Emphasis>

We have developed a reproducible system for efficient direct somatic embryogenesis from leaf and internodal explants of Paulownia elongata. The somatic embryos obtained were subsequently encapsulated as single embryos to produce synthetic seeds. Several plant growth regulators [6-benzylaminopurine, indole-3-acetic acid, -naphthaleneacetic acid, kinetin and thidiazuron (TDZ)] alone or in combination were tested for their capacity to induce somatic embryogenesis. The highest induction frequencies of somatic embryos were obtained on Murashige and Skoog (MS) medium supplemented with 3% sucrose, 0.6% Phytagel, 500 mg l^-1 casein hydrolysate and 10 mg l^-1 TDZ (medium MS10). Somatic embryos were induced from leaf (69.8%) and internode (58.5%) explants on MS10 medium after 7 days. Subsequent withdrawal of TDZ from the induction medium resulted in the maturation and growth of the embryos into plantlets on MS basal media. The maturation frequency of somatic embryos from leaf and internodal explants was 50.8% and 45.8%, respectively. Subculturing of mature embryos led to their germination on the same medium with a germination frequency of 50.1% and 29.8% from leaf and internode explants, respectively. Somatic embryos obtained directly on leaf explants were used for encapsulation in liquid MS medium containing different concentrations of sodium alginate with a 30-min exposure to 50 mM CaCl₂. A 3% sodium alginate concentration provided a uniform encapsulation of the embryos with survival and germination frequencies of 73.7% and 53.3%, respectively. Storage at 4°C for 30 days or 60 days significantly reduced the survival and complete germination frequencies of both encapsulated and non-encapsulated embryos relative to those of non-stored somatic embryos. However, the survival and germination rates of encapsulated embryos increased following storage at 4°C. After 30 days or 60 days of storage, the survival rates of encapsulated embryos were 67.8% and 53.5% and the germination frequencies were 43.2% and 32.4%, respectively. These systems could be useful for the rapid clonal propagation and dissemination of synthetic seed material of Paulownia elongata.Abbreviations BAP 6-Benzylaminopurine - IAA Indole-3-acetic acid - NAA -Naphthaleneacetic acid - TDZ ThidiazuronCommunicated by H. Lörz     

Controlling the sucrose concentration increases Coenzyme Q10 production in fed-batch culture of <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

The production yield of Coenzyme Q₁₀ (CoQ₁₀) from the sucrose consumed by Agrobacterium tumefaciens KCCM 10413 decreased, and high levels of exopolysaccharide (EPS) accumulated after switching from batch culture to fed-batch culture. Therefore, we examined the effect of sucrose concentration on the fermentation profile by A. tumefaciens. In the continuous fed-batch culture with the sucrose concentration maintained constantly at 10, 20, 30, and 40 g l⁻¹, the dry cell weight (DCW), specific CoQ₁₀ content, CoQ₁₀ production, and the production yield of CoQ₁₀ from the sucrose consumed increased, whereas EPS production decreased as maintained sucrose concentration decreased. The pH-stat fed-batch culture system was adapted for CoQ₁₀ production to minimize the concentration of the carbon source and osmotic stress from sucrose. Using the pH-stat fed-batch culture system, the DCW, specific CoQ₁₀ content, CoQ₁₀ production, and the product yield of CoQ₁₀ from the sucrose consumed increased by 22.6, 13.7, 39.3, and 39.3%, respectively, whereas EPS production decreased by 30.7% compared to those of fed-batch culture in the previous report (Ha SJ, Kim SY, Seo JH, Oh DK, Lee JK, Appl Microbiol Biotechnol, 74:974–980, 2007). The pH-stat fed-batch culture system was scaled up to a pilot scale (300 l), and the CoQ₁₀ production results obtained (626.5 mg l⁻¹ of CoQ₁₀ and 9.25 mg g DCW⁻¹ of specific CoQ₁₀ content) were similar to those obtained at the laboratory scale. Thus, an efficient and highly competitive process for microbial CoQ₁₀ production is available.     

Callus Growth and Proline Accumulation in Response to Sorbitol and Sucrose-Induced Osmotic Stress in Rice

This study investigated the influence of osmotic stress, induced by sorbitol and sucrose combinations, on growth and proline accumulation in callus cultures of rice (Oryza sativa L.). Dehusked mature seeds, cv. Hassawi, were induced to callus on MS medium supplemented with 4.52 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.32 µM 6-furfurylaminopurine (kinetin). The medium also contained 29.2, 58.4, 87.6, and 116.8 mM sucrose combined with 0, 54.9, 109.8, and 164.7 mM sorbitol. Callus formation was observed in about 35 % of the cultured seeds irrespective of the sugar treatment. An increase in callus mass was observed as sucrose concentration increased reaching a maximum growth at 87.6 mM. Callus growth was enhanced in response to 54.9 mM sorbitol but at higher concentration it was inhibitory. Best callus growth was obtained on a medium containing 54.9 mM sorbitol combined with 87.6 mM sucrose. Increasing osmotic stress, as a consequence of increasing sucrose and sorbitol concentrations, induced proline accumulation and the highest concentration of proline, 5.8 µmol g^–1(f.m.), was obtained on 164.7 mM sorbitol combined with 116.8 mM sucrose.