Influence of putrescine, silver nitrate and polyamine inhibitors on the morphogenetic response in untransformed and transformed tissues of Cichorium intybus and their regenerants

The addition of 40 mM putrescine (Put) to Murashige and Skoog's (MS) medium resulted in increased shoot multiplication and shoot growth in untransformed plants relative to transformed plants of Cichorium intybus L. Put at a concentration of 40 mM also resulted in flowering in both systems on the 28th day, with elevated titers of endogenous conjugated Put and spermine (Spm) in both untransformed and transformed plants. The addition of 40 µM AgNO₃ to untransformed axillary buds of C. intybus L. cultured on MS media resulted in increased shoot multiplication (36.9DŽ.63 shoots per culture) and increased shoot growth (7.82ǂ.76 cm) as compared to transformed ones (11.6ǂ.89 shoots per culture; 3.20ǂ.24 cm). Moreover, cultures treated with 40 µM AgNO₃ showed in vitro flowering on the 28th day in both systems, with the endogenous levels of conjugated spermine being higher in untransformed plants than in transformed ones. The morphogenetic response and the endogenous conjugated pool of polyamines were lower following !-DL-difluromethylarginine and !-DL-difluromethylornithine treatments; the addition of put (40 mM) and AgNO₃ (40 µM) restored these to normal levels. Under exogenous put feeding, ethylene production was lower in both the untransformed and transformed cultures. We believe that an interplay between polyamine and ethylene biosynthesis is involved in regulating the morphogenetic response in both transformed and untransformed shoots of C. intybus. The response to AgNO₃ and Put treatment was not altered by the transformation process.     

Induction of multiple shoots by thidiazuron from caryopsis cultures of minor millet (Paspalum scrobiculatum L.) and its effect on the regeneration of embryogenic callus cultures

Caryopsis culture of a minor millet (Paspalum scrobiculatum L. cv. PSC 1) on N₆ medium supplemented with high concentrations of thidiazuron (TDZ, 11.25 µM and 22.5 µM), a phenylurea derivative known to simulate cytokinin action, resulted in the formation of multiple shoots from the base of the seedling. This is the first time that multiple-shoot formation by a seedling cultured on TDZ without a callus interphase has been reported in graminaceous crop plants. The presence of a cytokinin, 6-benzylaminopurine (BAP), at low or high concentrations failed to evoke any morphogenic response. The presence of the auxin 2,4-dichlorophenoxyacetic acid (2,4-D, 4.5 µM) either alone or with BAP (4.5 µM) resulted in the formation of embryogenic callus from the base of the seedlings, which subsequently differentiated into somatic embryos. The combination of TDZ and the auxin (4.5 µM, 2,4-D) in the medium stimulated the differentiation of shoot buds in embryogenic callus cultures. This effect of TDZ, noted for the first time in a monocotyledonous plant, was evident in terms of a significant increase in the frequency of shoot-bud formation in embryogenic callus cultures and occurred only at a high concentration of TDZ (11.25 µM). This requirement for a high concentration of TDZ for the induction of multiple shoots from cultured seedlings or shoot buds in an embryogenic callus culture of a monocot is contrary to its effect at low concentrations in dicotyledonous plants. Complete plantlets, derived either from somatic embryos or shoot buds, could be regenerated on hormone-free basal medium or on basal medium fortified with activated charcoal (0.5%). Following a gradual acclimatization in a culture room, these regenerants survived on transfer to soil and ultimately set seed.     

Germination of synthetic seeds of pineapple (Ananas comosus L. Merr.)

. Tufts of multiple shoots were produced from dormant, axillary buds of pineapple in vitro. Tiny shoots (2-5 mm) isolated from the tuft of multiple shoots were encapsulated in 3% sodium alginate prepared using hormone-free Murashige and Skoog's basal medium, Murashige and Skoog's vitamins, 0.56 mM myo-inositol and 0.06 M sucrose. The encapsulated shoots represented synthetic seeds that germinated and formed roots in vitro after subculture onto one of the following media solidified with 0.8% agar: (1) hormone-free Murashige and Skoog's basal medium, Murashige and Skoog's vitamins, 0.56 mM myo-inositol and 0.06 M sucrose (Pin1), (2) Murashige and Skoog's basal medium, Murashige and Skoog's vitamins, 0.56 mM myo-inositol, 0.06 M sucrose, 9.67 µM 1-naphthalene acetic acid, 9.84 µM indole-3-butyric acid and 9.29 µM kinetin (Pin2), and (3) White's basal medium, White's vitamins, 0.56 mM myo-inositol, 0.03 M sucrose, 0.54 µM 1-naphthalene acetic acid and 1.97 µM indole-3-butyric acid (Pin3). Pretreatment of shoots in either liquid Pin3 or Pin4 medium (White's basal medium, White's vitamins, 0.56 mM myo-inositol, 0.03 M sucrose, 10.8 µM 1-naphthalene acetic acid and 39.4 µM indole-3-butryic acid) was required for development into plantlets with roots after culture on either Pin1, Pin2 or Pin3 media. One hundred percent germination of synthetic seeds to plantlets occurred after pretreatment of shoots in liquid Pin4 medium for 12 h followed by culture of synthetic seeds on Pin2 medium. Synthetic seeds stored at 4°C remained viable without sprouting for up to 45 days. Plantlets produced in vitro from synthetic seeds were successfully established in soil. The protocol provides an easy and novel propagation system for pineapple, an otherwise vegetatively propagated fruit crop.     

Cryopreservation of alfalfa (Medicago sativa L.) cells by encapsulation-dehydration

Our objective was to establish a cryopreservation protocol for alfalfa (Medicago sativa L.) cells and study the physiological changes occurring in cells during cryopreservation treatment. Cell cultures of Pioneer cvs. 5262 (fall-dormant, winter-hardy) and 5929 (non-dormant, non-hardy) plants initiated regrowth after cryopreservation by encapsulation-dehydration (ED). Pre-treatment of the encapsulated cells for 4 days in B5 medium containing 0.75 M sucrose and dehydration for 4 h in a laminar flow hood were necessary to achieve maximum cell viability after ED and cryopreservation in liquid N₂ (EDN). Viability (measured as triphenyl tetrazolium chloride reduction) of the cv. 5262 cells after cryopreservation was two- to three-fold greater than that of the cv. 5929 cells. Cold acclimation of the cells (10 days at 2°C) improved viability after cryopreservation. The addition of 7.6 µM ABA to the B5 medium enhanced viability in ED but did not improve cell cryopreservability. Cold-acclimated cells had higher protein concentrations, but neither ABA nor cold acclimation influenced protein composition of cold-acclimated cells determined using SDS-PAGE. Encapsulated cells pre-treated for 4 days in B5 medium containing 0.75 M sucrose showed an increased concentration of cell protein and an altered protein composition. Suspension cultures were re-initiated from both ED and EDN treatments by transferring beads sequentially to B5 media containing 0.75, 0.5, 0.25 M sucrose and then to fresh B5 medium. The ED cells resumed rapid growth after two subcultures, whereas EDN cells needed four or five subcultures to resume rapid growth.     

Cyanobacterial GR6 glutamate-1-semialdehyde aminotransferase: a novel enzyme-based selectable marker for plant transformation

The mutant glutamate-1-semialdehyde aminotransferase (GSA-AT) enzyme encoded by the hemL gene of the gabaculine-resistant cyanobacterium Synechococcus PCC6301 strain GR6 was expressed in tobacco following Agrobacterium-mediated transformation of leaf discs. When targeted to plastids, the GR6 hemL gene product conveyed gabaculine resistance to transgenic plants. Selection using 50 and 100 µM gabaculine was shown to produce two distinct explant phenotypes: 'greens' and 'whites'. T₁ progeny displayed Mendelian segregation ratios, and PCR analysis demonstrated the 'green' phenotype corresponded with the presence of the GR6 hemL gene. Furthermore, 'whites' could be rescued after 9 days growth on solid media containing between 5 µM and 25 µM gabaculine, allowing the potential use of this system for the isolation of gabaculine-sensitive transformants in mutagenesis screening. The use of GR6 hemL as a selectable marker gene provides a novel enzyme-based method for the selection of transgenic regenerants without the need for antibiotic-resistance markers.     

Tight control of growth and cell differentiation in photoautotrophically growing moss (<Emphasis Type="Italic">Physcomitrella</Emphasis><Emphasis Type="Italic">patens</Emphasis>) bioreactor cultures

The use of the moss Physcomitrella patens as a production system for heterologous proteins requires highly standardised culture conditions. For this purpose a semi-continuous photoautotrophic bioreactor culture of Physcomitrella was established. This culture grew stably for 7 weeks in a 5-l bioreactor with a dilution rate of 0.22/day. Enrichment of the air for aeration in a batch bioreactor culture with 2% (v/v) CO₂ resulted in an increase in the specific growth rate to 0.57/day. Changes in the pH of the semi-continuous bioreactor culture medium between pH 4.5 and pH 7.0 influenced protonema differentiation; however it did not negatively affect the growth rate compared to uncontrolled pH. The advantages of Physcomitrella as a system for the production of heterologous proteins in plants are discussed.     

Culture conditions for the production of esterase from Lactobacillus casei CL96

Culture conditions in growth and esterase production by a newly isolated Lactobacillus casei CL96 were investigated using a dextrose-free MRS medium supplemented with different sugars in a 2 l fermentor at different pHs (4.0-9.0) and temperatures (20-50°C). The optimal growth was obtained in basal MRS medium containing 1% (w/v) lactose at pH 7.0 and 30°C. The maximal esterase production was obtained intracellularly during the late logarithmic phase, but during the stationary phase, the esterase activity was released in the culture medium. The enzyme activity was maximal at pH 7.0 and 37°C. Among various substrates (C₂-C₁₆) tested, the highest activity was towards C₆ and C₈. Though the enzyme was produced constitutively, the tributylin induced the enzyme production by 2.5 fold. L. casei CL96 esterase was very active at neutral pH and ambient temperature and might be suitable for biotechnological applications in the dairy industry.     

Growth regulator pretreatment improves somatic embryogenesis from leaves of squash (Cucurbita pepo l.) and melon (Cucumis melo l.)

Leaf explants of squash (Cucurbita pepo L.) and melon (Cucumis melo L.) were pretreated initially with 113.1, 226.2 or 452.4 µM 2,4-dichlorophenoxyacetic acid (2,4-D), 46.5, 93 or 186 µM kinetin or a combination of both at the above concentrations, for 6, 24 or 48 h. After pretreatment, explants were transferred to an agar-solidified medium that was not supplemented with growth regulators or to a species-specific standard induction medium. Control explants from each species were incubated directly on the species-specific standard induction medium. Initial pretreatment of squash explants with 186 µM kinetin and of melon explants with 226.2 µM 2,4-D for 48 h significantly promoted the formation of somatic embryos which developed further to the torpedo-shape stage and germinated. Under these conditions at least four plants can be regenerated per square centimeter of explant surface, thus achieving an increase over non-pretreated cultures of 143% and 130% for squash and melon, respectively.     

Reactor kinetics for submerged aerobic biofilms

Sclerotium rolfsii ATCC 15205 was cultivated in continuous culture (48 l reactor volume) for scleroglucan production. The maximum volumetric productivity (Q_Pvmax) amounted to 7.2 g/ld and was more than twice as high as in comparable batchwise cultivations. In addition, the relation between (a) volumetric productivity (Q_P [g/ld]) and product yield (Y_{Glucan/Glucose} [1]), (b) volumetric productivity and product quality (M_W [g/mol]), and (c) product quality and impeller tip speed (Nd [m/s]) was studied in continuous culture. It was found, that an increase in volumetric productivity led to a decline in product yield and product quality. Furthermore, an impeller tip speed of >0.7 m/s decreased the attainable product quality considerably. Based on these results, the impact of the operational setpoint of the process in terms of oxygen supply and reactor scale on the economics of scleroglucan production was discussed. In contrast to batchwise cultivation, scleroglucan production in continuous culture proved to be not feasible under non-aseptic conditions.     

An efficient direct induction of protocorm-like bodies from leaf subepidermal cells of Doritaenopsis hybrid using thin-section culture

High-frequency protocorm-like body (PLB) formation directly from thin leaf sections of Doritaenopsis hybrid was achieved in order to develop a mass-scale propagation system. Concentrated efforts were made to study the effects of different cytokinins on in vitro PLB induction from thin leaf sections. Among the cytokinins tested, thidiazuron (TDZ) was found to be a more effective inducer of PLBs than benzyladenine and zeatin. A modified Murashige and Skoog medium supplemented with 9.0 µM TDZ was found to be the optimum concentration for PLB development from thin leaf sections of Doritaenopsis hybrid. Of the two different explant types used in the present experiment, the highest percentage of PLB formation (72.3%) and highest number of PLBs (18) per explant were observed on thin leaf sections (1 mm thick), while only 20% (4.3 per explant) of comparatively large leaf segments (5 mm thick) were able to produce PLBs under the same culture conditions. Light microscopy observations indicated that the initial cell divisions for PLB formation occurred on the region near the cut surface and that an intact epidermal layer appeared to play an important role in PLB formation. Proembryo initiation occurred from several cells just beneath the intact epidermal cell, and globular PLBs were clearly visible after 3 weeks of culture and subsequently developed into mature PLBs.     

Microbial community composition and function beneath temperate trees exposed to elevated atmospheric carbon dioxide and ozone

We hypothesized that changes in plant growth resulting from atmospheric CO₂ and O₃ enrichment would alter the flow of C through soil food webs and that this effect would vary with tree species. To test this idea, we traced the course of C through the soil microbial community using soils from the free-air CO₂ and O₃ enrichment site in Rhinelander, Wisconsin. We added either ¹³C-labeled cellobiose or ¹³C-labeled N-acetylglucosamine to soils collected beneath ecologically distinct temperate trees exposed for 3 years to factorial CO₂ (ambient and 200 µl l^-1 above ambient) and O₃ (ambient and 20 µl l^-1 above ambient) treatments. For both labeled substrates, recovery of ¹³C in microbial respiration increased beneath plants grown under elevated CO₂ by 29% compared to ambient; elevated O₃ eliminated this effect. Production of ¹³C-CO₂ from soils beneath aspen (Populus tremuloides Michx.) and aspen-birch (Betula papyrifera Marsh.) was greater than that beneath aspen-maple (Acer saccharum Marsh.). Phospholipid fatty acid analyses (¹³C-PLFAs) indicated that the microbial community beneath plants exposed to elevated CO₂ metabolized more ¹³C-cellobiose, compared to the microbial community beneath plants exposed to the ambient condition. Recovery of ¹³C in PLFAs was an order of magnitude greater for N-acetylglucosamine-amended soil compared to cellobiose-amended soil, indicating that substrate type influenced microbial metabolism and soil C cycling. We found that elevated CO₂ increased fungal activity and microbial metabolism of cellobiose, and that microbial processes under early-successional aspen and birch species were more strongly affected by CO₂ and O₃ enrichment than those under late-successional maple.     

Methyl laurate and 6-benzyladenine promote the germination of somatic embryos of a hybrid rose

Globular stage somatic embryos were induced in callus cultures of Rosa Heritage 2 Alister Stella Gray on medium containing 13.5 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and developed to the cotyledonary stage on medium containing 9 µM 2,4-D. Cotyledonary-stage embryos were transferred to germination media with or without 1.5 µM 6-benzyladenine (BA) and with or without 44 µM methyl laurate (Mela). BA and Mela both promoted the development of shoots and roots and increased the frequency of bipolar germinations. An average of 56.5% (SEdž.1%) embryos on medium containing both BA and Mela underwent bipolar germinations compared with less than 20% in treatments where either or both were excluded. The effectiveness of BA and Mela was reduced if Mela was included in the development medium or if the concentration of salts and vitamins in the germination media was sub-optimal. There was evidence that growth at one pole of the somatic embryo promoted development at the other.     

Chromosome number stability and mitotic activity of cryopreserved Hypericum perforatum L. meristems

Meristems of in vitro-grown Hypericum perforatum L. plants were precultured for 3, 10, or 14 days in the presence of 0.5 M mannitol, or 0.076 µM or 0.76 µM abscisic acid, in RM basal liquid culture medium supplemented with 0.5 mg/l 6-benzylaminopurine and subsequently subjected to cryopreservation by the slow freezing method. The survival rate - determined as the percentage of meristems capable of differentiating plantlets - varied between 10% and 48%. Chromosome number stability of the cryopreserved meristems was determined by chromosome counting. The mitotic index of the control did not significantly differ from that of the treated samples.     

Cryopreservation of in vitro-grown shoot tips of 'Troyer' citrange [Poncirus trifoliata (L.) Raf. × Citrus sinensis (L.) Osbeck.] by encapsulation-dehydration

. In vitro-grown shoot tips excised from preconditioned stock shoots of 'Troyer' citrange were successfully cryopreserved by encapsulation-dehydration. Optimal survival of cryopreserved shoot tips was achieved when encapsulated shoot tips were dehydrated to 17.1% water content. The sucrose concentration in the preconditioning medium significantly influenced the growth and dry matter percentage of the stock shoots as well as subsequent survival of the cryopreserved shoot tips. Maximal growth of stock shoots was obtained in sucrose concentrations in the range of 0.15 M to 0.29 M, while the dry matter percentage increased as sucrose concentration increased up to 0.44 M. The survival of cryopreserved shoot tips increased from 40% to approximately 80% as the sucrose concentration for stock shoots increased from 0.09 M to 0.22 M or 0.29 M. The benzyladenine concentration in the post-culture medium significantly affected the survival and regrowth of the cryopreserved shoot tips. Survival of the shoot tips was lowest when they were post-cultured on benzyladenine-free medium. However, high benzyladenine concentrations (3-4 µM) induced callus formation. Optimal recovery was obtained in post-culture medium containing 2 µM benzyladenine and 0.05 µM !-naphthalene acetic acid. The extraction of shoot tips from alginate beads greatly improved the regrowth of cryopreserved shoot tips.     

Primary production, light and vertical mixing in Potter Cove, a shallow bay in the maritime Antarctic

Phytoplankton photosynthesis was measured during spring-summer 1991-1992 in the inner and outer part of the shallow Potter Cove, King George Island. Strong winds characterise this area. Wind-induced turbulent mixing was quantified by means of the root-mean square expected vertical displacement depth of cells in the water column, Z_t. The light attenuation coefficient was used as a measure of the influence of the large amount of terrigenous particles usually present in the water column; 1% light penetration ranged between 30 and 9 m, and between 30 and 15 m for the inner and outer cove, respectively. Obvious differences between photosynthetic capacity [P*_max; averages 2.6 and 0.6 µg C (µg chlorophyll-a)^-1 h^-1] and photosynthetic efficiency {!*; 0.073 and 0.0018 µg C (µg chlorophyll-a)^-1 h^-1 [(µmol m^-2 s^-1)^-1]} values were obtained for both sites during low mixing conditions (Z_t from 10 to 20 m), while no differences were found for high mixing situations (Z_t>20 m). This suggests different photoacclimation of phytoplankton responses, induced by modifications of the light field, which in turn are controlled by physical forcing. Our results suggest that although in experimental work P*_max can be high, wind-induced mixing and low irradiance will prevent profuse phytoplankton development in the area.     

Comparative study of the fermentation of D-glucose/D-xylose mixtures with Pachysolen tannophilus and Candida shehatae

We have performed a comparative analysis of the fermentation of the solutions of the mixtures of D-glucose and D-xylose with the yeasts Pachysolen tannophilus (ATCC 32691) and Candida shehatae (ATCC 34887), with the aim of producing bioethanol. All the experiments were performed in a batch bioreactor, with a constant aeration level, temperature of 30v°C, and a culture medium with an initial pH of 4.5. For both yeasts, the comparison was established on the basis of the following parameters: maximum specific growth rate, biomass productivity, specific rate of substrate consumption (q_s) and of ethanol production (q_E), and overall ethanol and xylitol yields. For the calculation of the specific rates of substrate consumption and ethanol production, differential and integral methods were applied to the kinetic data. From the experimental results, it is deduced that both Candida and Pachysolen sequentially consume the two substrates, first D-glucose and then D-xylose. In both yeasts, the specific substrate-consumption rate diminished over each culture. The values q_s and q_E proved higher in Candida, although the higher ethanol yield was of the same order for both yeasts, close to 0.4 kg kg^у.     

Experimental behavior of a whole cell immobilized dextransucrase biocatalyst in batch and packed bed reactors

Dextransucrases from Leuconostoc mesenteroides have been used to produce a diversity of controlled structure oligosaccharides with potential industrial applications. This is the case of !(1̄) branched glucooligosaccharides produced by L. mesenteroides NRRL B-1299 dextransucrase. In order to establish an industrial scale process with the immobilized enzyme, a biocatalyst was produced by whole cell entrapment in alginate beads. The main physical and physicochemical properties of the biocatalyst were determined and the hydrodynamic behavior in a packed bed reactor studied. It was possible to produce spherical beads of 0.2 cm diameter containing the insoluble part of L. mesenteroides culture (cells and insoluble polymer) with an activity of 4 IU/g. Immobilization yield reached 93% with an effectiveness factor of 0.995 for particles of d_p < 0.2 cm. Due to the complexity of dextransucrase mechanism and kinetics, data obtained from initial rate measurements failed to describe the results obtained from the batch and continuous reactors. Therefore, apparent K_M and V_max data were used for the reactor modeling. It was found that under the conditions studied, the reaction rate was controlled by external mass transfer limitations.     

Optimization of in vitro micropropagation and regeneration for Populus × interamericana and Populus × euramericana hybrids (P. deltoides, P. trichocarpa, and P. nigra)

A rapid and efficient micropropagation method has been established for six European poplar cultivars of economic interest - four Populus 2 interamericana and two Populus 2 euramericana. Using a three-step procedure, we were able to regenerate plantlets from callus and acclimate them within 4 months. In the first step, callogenesis was induced when explants were cultured for 25 days on culture medium supplemented with 10 µM !-naphthaleneacetic acid and 5 µM N⁶(2-isopentenyl)adenine. Bud regeneration followed by shoot elongation was then obtained from callus tissue by combining the cytokinin-like compound thidiazuron with the surfactant Pluronic F-68 at concentrations adjusted for each cultivar. The usefulness of this procedure in the area of genetic engineering is discussed.     

Effect of gaseous composition on cell growth and secondary metabolite production in suspension culture of Stizolobium hassjoo cells

The gaseous composition is an important factor affecting the performance of plant cell cultures. Gaseous metabolites, especially O₂, CO₂ and C₂H₄, play important roles in cell physiology. Forced aeration in bioreactors usually results in poor cell growth and secondary metabolite production. In this work, the effects of gaseous metabolites on cell growth, secondary metabolite formation as well as PPO activity were investigated with respect to Stizolobium hassjoo cell culture producing l-DOPA (3,4-dihydroxyphenylalanine). A device allowing the control of the partial pressures of gaseous metabolites in shake flasks was designed. In addition, a recirculating gas system with a P_O2 controller was designed for a bioreactor. This device could maintain constant P_O2 and P_CO2 in the bioreactor headspace. The results showed that the highest l-DOPA content was attained at P_O2=0.30 atm. Higher P_O2 values retarded cell growth and increased the pH of the culture broth. High P_O2 also enhanced the formation of ethylene and inhibited l-DOPA formation. Carbon dioxide concentrations lower than 5% enhanced cell growth and l-DOPA formation. Cell growth was retarded by 0.3 ppm of ethylene in 2~5 carbon dioxide. Oxygen concentration and D.O. in the broth could be controlled at constant levels in the recirculating culture system. Enrichment of P_O2 up to 0.3 atm during the later stage of cultivation facilitated l-DOPA formation. The interaction among the gaseous metabolites and their influences on cell metabolism and l-DOPA formation were elucidated. This information will facilitate the rational operation of plant cell culture systems producing secondary metabolites.     

Modelling of aerobic growth of Saccharomyces cerevisiae in a pH-auxostat

A model for growth and overflow metabolism of Saccharomyces cerevisiae was applied to simulate continuous cultivations in a pH-auxostat. The concentrations of glucose, biomass and ethanol are controlled by the flow ratio r between fresh medium and titrant solution, both of which are pH-regulated. A critical value of r could be derived, below which the culture becomes substrate depleted, resulting in a stop-flow condition with retained biomass but without growth. At r-values slightly above the critical value the pH-auxostat is substrate limited and unstable. Further increase of the r value results in a stable continuous culture growing at w_max. Thus, the pH-auxostat complements the chemostat in the growth range at or close to w_max. Even at w_max conditions, the ethanol concentration can be controlled at a low level.