In vitro culture

The ultimate success of micropropagation on a commercial scale depends on the ability to transfer plants out of culture on a large scale, at low cost and with high survival rates. During field transfer the in vitro grown plantlets are unable to compete with soil microbes and to cope with the environmental conditions. The in vitro culture conditions result in the plantlets with altered morphology, anatomy and physiology. In order to increase growth and reduce mortality in plantlets at the acclimatisation stage, efforts are focused on the control of both physical and chemical environment and biohardening of micropropagated plantlets. This review describes the abiotic and biotic stresses and current developing methods for the acclimatization of microshoots.

Dynamic, multicompartment in vitro gastrointestinal simulators are often used to monitor gut microbial dynamics and activity. These reactors need to harbor a microbial community that is stable upon inoculation, colon region specific, and relevant to in vivo conditions. Together with the reproducibility of the colonization process, these criteria are often overlooked when the modulatory properties from different treatments are compared. We therefore investigated the microbial colonization process in two identical simulators of the human intestinal microbial ecosystem (SHIME), simultaneously inoculated with the same human fecal microbiota with a high-resolution phylogenetic microarray: the human intestinal tract chip (HITChip). Following inoculation of the in vitro colon compartments, microbial community composition reached steady state after 2 weeks, whereas 3 weeks were required to reach functional stability. This dynamic colonization process was reproducible in both SHIME units and resulted in highly diverse microbial communities which were colon region specific, with the proximal regions harboring saccharolytic microbes (e.g., Bacteroides spp. and Eubacterium spp.) and the distal regions harboring mucindegrading microbes (e.g., Akkermansia spp.). Importantly, the shift from an in vivo to an in vitro environment resulted in an increased Bacteroidetes/Firmicutes ratio, whereas Clostridium cluster IX (propionate producers) was enriched compared to clusters IV and XIVa (butyrate producers). This was supported by proportionally higher in vitro propionate concentrations. In conclusion, high-resolution analysis of in vitro-cultured gut microbiota offers new insight on the microbial colonization process and indicates the importance of digestive parameters that may be crucial in the development of new in vitro models.

A reliable and standard method was established for micropropagation of the A70-34 selected genotype of lucerne (Medicago sativa L ., genotype A70-34), aimed at reducing contamination problems, seasonal and phenological influences on regeneration and phytosanitary problems of the mother plants, while maintaining the regenerative potential for somatic embryogenesis of the plant tissues . Mother plants were routinely maintained for several subcultures and somatic embryogenesis was regularly obtained from the subcultured explants . Proliferation, rooting and embryogenetic ability of plants cultured for 30 days was greater than those cultured for 20 days . The regenerative potential of tissues from different organs and of triturated and intact whole plants was also tested . Petioles were confirmed as the best source for embryogenesis as far as efficiency and repeatability were concerned, even though regeneration from other explant types was also achieved . Production of somatic embryos through mechanical trituration of the in vitro cultured plants was obtained ; the regeneration ability of the triturated plants was greater than that observed in the intact plants .

ContentsBovine demi-embryos were produced by micromanipulation of day 6–7 embryos from superovulated cows. Monolayers of bovine oviduct epithelial cells (BOEC) were produced from slaughterhouse material. Half of the demi-embryos were cultured on a confluent layer of bovine oviduct epithelial cells either in Hams F-10 with 10% NCTC-135 and with 10% FCS or in Earles Medium 199 with 10% FCS for 4–24 hours, followed by freezing of the survivors, while the corresponding other halfs were used for sexing. three 16-Cell, 1 morula, 41 compact morulae, 4 early blastocysts, 7 blastocysts and 2 expanded blaszocysts were besected and cultured. An overall survival rate of 83% among the 63 in vitro cultured demi-embryos was seen.Bovine demi-embryos were produced by micromanipulation of day 6–7 embryos from superovulated cows. Monolayers of bovine oviduct epithelial cells (BOEC) were produced from slaughterhouse material. Half of the demi-embryos were cultured on a confluent layer of bovine oviduct epithelial cells either in Hams F-10 with 10% NCTC-135 and with 10% FCS or in Earles Medium 199 with 10% FCS for 4–24 hours, followed by freezing of the survivors, while the corresponding other halfs were used for sexing. three 16-Cell, 1 morula, 41 compact morulae, 4 early blastocysts, 7 blastocysts and 2 expanded blaszocysts were besected and cultured. An overall survival rate of 83% among the 63 in vitro cultured demi-embryos was seen.Inhalt: In vitro Kultivierung von geteilten Rinderembryonen mit bovinen Ovidukt-Epithel-AmmenzellenSechs bis sieben Tage alte Embryonen von superovulierten Rindern wurden mit Hilfe eines Mikromanipulators geteilt. Monolyer von bovinen Eileiterepithelzellen (BOEC) wurden aus Schlachthofmaterial gerüchtet. Eine Hälfte der Embryonen diente der Geschlechtsbestimmung, die andere Hälfte wurde auf konfluierenden Monolayern entweder mit Hams F-10, 10% NCTC-135 und 10′% FCS oder in TCM 199 rnit 10% FCS für 2 bis 4 Stunden kultiviert. Die überlebenden Stadien wurden eingefroren. Drei 16-Zeller, 1 Morula, 41 kompaktierte Morulae, vier frühe und sieben Blastozysten sowie 2 expandierte Blastocysten wurden geteilt und kultiviert. Insgesamt betrug die Oberlebensrate bei 63 in vitro kultivierten Zwillingsembryonen 83%.Sechs bis sieben Tage alte Embryonen von superovulierten Rindern wurden mit Hilfe eines Mikromanipulators geteilt. Monolyer von bovinen Eileiterepithelzellen (BOEC) wurden aus Schlachthofmaterial gerüchtet. Eine Hälfte der Embryonen diente der Geschlechtsbestimmung, die andere Hälfte wurde auf konfluierenden Monolayern entweder mit Hams F-10, 10% NCTC-135 und 10′% FCS oder in TCM 199 rnit 10% FCS für 2 bis 4 Stunden kultiviert. Die überlebenden Stadien wurden eingefroren. Drei 16-Zeller, 1 Morula, 41 kompaktierte Morulae, vier frühe und sieben Blastozysten sowie 2 expandierte Blastocysten wurden geteilt und kultiviert. Insgesamt betrug die Oberlebensrate bei 63 in vitro kultivierten Zwillingsembryonen 83%.

Infection by human hepatitis C virus (HCV) is the principal cause of post-transfusion hepatitis and chronic liver diseases worldwide. A reliable in vitro culture system for the isolation and analysis of this virus is not currently available, and, as a consequence, HCV pathogenesis is poorly understood. We report here the first robust in vitro system for the isolation and propagation of HCV from infected donor blood. This system involves infecting freshly prepared macrophages with HCV and then transmission of macrophage-adapted virus into freshly immortalized B-cells from human fetal cord blood. Using this system, newly isolated HCV have been replicated in vitro in continuous cultures for over 130 weeks. These isolates were also transmitted by cell-free methods into different cell types, including B-cells, T-cells and neuronal precursor cells. These secondarily infected cells also produced in vitro transmissible infectious virus. Replication of HCV-RNA was validated by RT-PCR analysis and by in situ hybridization. Although nucleic acid sequencing of the HCV isolate reported here indicates that the isolate is probably of type 1a, other HCV types have also been isolated using this system. Western blot analysis shows the synthesis of major HCV structural proteins. We present here, for the first time, a method for productively growing HCV in vitro for prolonged periods of time. This method allows studies related to understanding the replication process, viral pathogenesis, and the development of anti-HCV drugs and vaccines.

Unfertilized ovaries isolated from immature female flowers of coconut (Cocos nucifera L.) were tested as a source of explants for callogenesis and somatic embryogenesis. The correct developmental stage of ovary explants and suitable in vitro culture conditions for consistent callus production were identified. The concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) and activated charcoal was found to be critical for callogenesis. When cultured in a medium containing 100 µM 2,4-D and 0.1% activated charcoal, ovary explants gave rise to 41% callusing. Embryogenic calli were sub-cultured into somatic embryogenesis induction medium containing 5 µM abscisic acid, followed by plant regeneration medium (with 5 µM 6-benzylaminopurine). Many of the somatic embryos formed were complete with shoot and root poles and upon Communicated by P. Debergh

Calcium-alginate hydrogel has been widely studied as a material for cell encapsulation for tissue engineering. At present, the effect that cells have on the degradation of alginate hydrogel is largely unknown. We have shown that fibroblasts encapsulated at a density of 7.5 Â 10 5 cells ml À1 in both 2% and 5% w/v alginate remain viable for at least 60 days. Rheological analysis was used to study how the mechanical properties exhibited by alginate hydrogel changed during 28 days in vitro culture. Alginate degradation was shown to occur throughout the study but was greatest within the first 7 days of culture for all samples, which correlated with a sharp release of calcium ions from the construct. Fibroblasts were shown to increase the rate of degradation during the first 7 days when compared with acellular samples in both 2% and 5% w/v gels, but after 28 days both acellular and cell-encapsulating samples retained disc-shaped morphologies and gel-like spectra. The results demonstrate that although at an early stage cells influence the mechanical properties of encapsulating alginate, over a longer period of culture, the hydrogels retain sufficient mechanical integrity to exhibit gel-like properties. This allows sustained immobilization of the cells at the desired location in vivo where they can produce extracellular matrix and growth factors to expedite the healing process.

Isolated somatic embryos from petiole-derived callus cultures of Vitis rupestris Scheele have been employed in experiments on genetic transformation. Co-cultivation of somatic embryos during embryogenesis induction with Agrobacterium tumefaciens strain LBA4404, which contains the plasmid pBI121 carrying the neomycin-phosphotranspherase and the/3-glucuronidase genes, produced transformed cellular lines capable of recurrent somatic embryogenesis. Precocious selection for high levels of kanamycin (100 mgl-~) was an important part of our transformation protocol. Transformed lines still have strong/3-glucuronidase expression as well as stable insertion of the marker genes after 3 years of in-vitro culture, during which they have maintained their capacity to organize secondary embryos and to regenerate transgenic plants with an agreeable efficiency (13%).

The present study was undertaken to examine cell cycle characteristics of endangered Goral (CITES Appendix I) adult skin fibroblasts. Seven experiments were performed, each with a one-way completely randomized design involving three replicates. Least significant difference (LSD) was used to determine variation among treatment groups. Experiment I focused on the effects of cycling, serum-starved, and fully confluent stages of Goral cells. In Experiments II and III, the effects of different antioxidants like b-mercaptoethanol (b-ME, 10 mM), cysteine (2 mM), and glutathione (2 mM) were examined after cells were fully confluent without serum starvation for 24 h and 4 h, respectively. In Experiments IV and V, three protease inhibitors, namely 6-dimethylaminopurine (6-DMAP, 2 mM), cycloheximide (7.5 mg/ml) and cytochalasin B (7.5 mg/ml), were used as in Experiment II. In Experiments VI and VII, the effect of different levels of dimethylsulphoxide (DMSO) at 0%, 0.5%, 1.0% and 2.5% were tested by flow cytometry (FACS). In Experiment I, 68.7% of Goral skin fibroblasts reached the G 0 /G 1 stage (2C DNA content) when subjected to the serum-starved medium, which was more than the cycling (64.9%) and fully confluent groups (61.0%) (P > 0.05). Among the chemically treated group, b-ME, cysteine and DMSO showed better results for synchronization of G 0 þ G 1 phases than cycling, serum-starved and fully confluent groups. It can thus be concluded that b-ME, cysteine and DMSO at certain concentrations can synchronize the cell cycle effectively, which could have a positive impact on somatic cell nuclear transfer in the goral. Ó

Partial desiccation treatment was applied to improve plant regeneration response in irradiated in vitro cultures. Embryogenic callus cultures of sugarcane cv. Co-671 were exposed to different doses of gamma radiation (0-80 Gy) and radiation effect was evaluated in terms of post-irradiation callus recovery, growth and regeneration of plants. Proliferative capacity of cultures was inversely correlated with radiation dose as the percentage surviving cultures or white proliferating clumps (WPC) decreased as the radiation dose increased up to 80 Gy. LD50 was found to be around 20-30 Gy and at higher doses, poor regeneration frequency was observed after 4-6 weeks of post-irradiation culture. To stimulate regeneration response, irradiated cultures were subjected to partial desiccation for 6 h and the treatment resulted in enhanced plant regeneration response. The study suggests that partial desiccation treatment can be useful in stimulating regeneration response of irradiated in vitro cultures.

Objective: The damage to cartilage collagen is a central event in the pathogenesis of cartilage aging and osteoarthritis (OA). We have previously developed an in vitro model of cartilage degradation which shows that chondrocyte-dependent lipid peroxidation mediates cartilage collagen degradation. The goal of our study was to investigate the role of vitamin C in this degradation model and to investigate effect of chondrocyte-dependent lipid peroxidation in the oxidation of cartilage collagen. Methods: We studied primary articular chondrocytes. Effect of vitamin C was investigated in the previously described model. Serum-free stimulated and unstimulated chondrocyte-matrix extracts were subjected to SDS-PAGE and immunoblot analysis. Malondialdehyde (MDA)-protein oxidation of cartilage proteins was demonstrated by the reactivity of chondrocyte extracts to a monoclonal antibody, MDA2, which detects MDA-lysine adducts. Results: Vitamin C treatment of chondrocyte cultures resulted in significant enhanced incorporation of 3H-proline label in cell-matrix. Cells treated with vitamin C, as compared to control untreated cells showed decreased spontaneous release of labeled matrix. Vitamin C treated or not treated chondrocytes responded comparably to stimulation with the agonist calcium ionophore A23187. The serum-free in vitro culture of chondrocytes resulted in MDA-protein oxidation. The treatment of chondrocytes with A23187 resulted in the enhancement of MDA-protein oxidation. The immunoblot reactivity pattern of extracts to MDA2 antibody and to polyclonal anti-type II collagen antibody was somewhat similar, which suggests that these two different types of antisera exibit a crossreaction to chondrocyte proteins. Chondrocyte extracts were pretreated both with and without pure collagenase, and then subjected to immunoblot analysis. Only collagenase treated extracts showed a disappearence, or significant reduction, of larger than 60 kDa size MDA2 immunoreactive proteins. This suggests that the proteins that disappeared after the enzyme treatment were collagen proteins and which had also been modified by MDA oxidation. Conclusions: These observations suggest that collagen hydroxylation of matrix by vitamin C does not play a role in this model of chondrocyte-dependent collagen degradation. Also, this study demonstrates that chondrocyte-derived lipid peroxidation product MDA mediates oxidation of cartilage collagens. Oxidative modification of cartilage collagen in vivo could result in alteration of biochemical and biophysical properties of cartilage collagen fibrils, making them prone to degradation, thus initiating the changes observed in aging and OA.

The production of strong healthy plant material ex vitro is essential for an efficient seed potato production programs. Current study shows an applicable method for enhancing performance of potato plantlet subsequent in vitro phase. Using of ventilation and omitting sucrose from the nutrient medium (photoautotrophic culture) resulted in strong plantlets with higher fresh and dry weight performed better during acclimatization. Furthermore, Using of culture media contain macronutrients based on potato leaves macronutrients concentrations produced vigorous plantlets. Varietal differences were recorded between the two tested varieties Cara and Diamant. To obtain healthy start material for seed potato program this study recommend the use of in vitro photoautotrophic culture and modified nutrient media based on potato leaf macronutrient concentrations.

The positive effect of ventilation of the culture container on in vitro shoot proliferation and quality was already proven for different species. Hereafter we report on the evolution of the headspace during in vitro culture of plantain in a Temporary Immersion Bioreactor (TIB) on the one hand, and culture on semi-solid medium on the other hand. The CO2 and C2H4 concentration reached a maximum of 12% and 0.45 μl l−1, respectively in the control treatment on semi-solid medium, compared to 5.7% CO2 and 0.06 μl l−1 C2H4 in TIB. The minimal O2-concentration on semi-solid medium was 15.1%, compared to 19.3% in TIB. The multiplication rate was best in TIB, 6.4 compared to 4.3 in semi-solid conditions, and this was also the case for shoot height (4.3 cm compared to 3.3 cm), and leaf number (2.6 compared to 1.6). Moreover shoots produced on semi-solid medium showed distorted leaves. A typical day-night pattern in CO2 and O2 concentration was observed in TIB, as well as on semi-solid medium; this is illustrative for the photosynthetic capacity of the plant material produced in both systems.

Cotyledons from seedlings of various ages of Aegle marmelos were cultured on Murashige and Skoog (MS) medium supplemented with different combinations of phytohormones. The optimum seedling age was 10 days for shoot induction response and benzyladenine (BA) was superior to either kinetin, isopentenyladenine or zeatin. The optimum cytokinin (BA) concentration for bud induction was 2 mg 1-i. The addition of indole-3acetic acid (IAA; 0.2 mg 1-1 ) improved shoot regeneration efficiency. The proximal part of the cotyledon had the highest regeneration potential. Adventitious shoots were elongated on MS medium containing 0.5 mg l-~ kinetin and 0.1 mg l-1 gibberellic acid. Approximately 25% of regenerated shoots were induced to differentiate roots on half-strength MS medium with 0.5 mg 1-t indole-3-butyric acid. The rooted plantlets were successfully transplanted to soil.

ABSTRACT: Cassava is one of the most important food crops in Africa. Meristem culture is an effective method of eliminating viruses and other systemic diseases spread through the vegetative propagation of stems. However, in semi-arid conditions, survival of ex vitro plants in the field is often disappointing. When an increasing range of light regimes in vitro was provided, the fresh and dry masses more than doubled their values between 29 and 369 mmol s−1 m−2 PPFDs. Increases in numbers of senescent leaves and stem thickness were also recorded with increasing PPFD. However, PPFD above 101 mmol s−1 m−2 resulted in 30–70% reduction in plant survival, with the thin plants with the smallest fresh and dry masses being the ones with highest survival rates. High light and temperature levels in the greenhouse were also found to be critical for plant survival. It was also shown that transpirational loss from detached leaves and epicuticular wax deposits were not good indicators for predicting survival of ex vitro cassava plantlets during acclimatisation.

Examination by scanning electron microscopy showed abaxial stomata on in vitro cultured apple (Malus pumila Mill.) leaves. With leaf ontogeny, most of these stomata appeared to lose their regulatory ability while developing wide vestibules of up to 20 #m in diameter. It is proposed that these deformed stomata may be a possible cause for the excessive transpirational water loss and consequent dehydration associated with transferring plants regenerated in vitro from culture.

We obtained callus, cell suspension and hairy root cultures of Tropaeolum majus and we demonstrated their ability to produce glucotropaeolin and myrosinase. In hairy roots glucotropaeolin content and myrosinase activity were higher in comparison with callus, cell suspension and leaves of intact plants. In hairy root cultures the highest glucotropaeolin contents were detected on the 9th day of culture. In

The present work evaluates a newly developed silated hydroxypropylmethylcellulose (Si-HPMC)-based hydrogel as a scaffold for 3D culture of osteogenic cells. The pH variation at room temperature catalyzes the reticulation and self-hardening of the viscous polymer solution into a gelatine state. We designed reticulation time, final consistency and pH in order to obtain an easy handling matrice, suitable for in vitro culture and in vivo injection. Three human osteogenic cell lines and normal human osteogenic (HOST) cells were cultured in 3D inside this Si-HPMC hydrogel. We show here that osteosarcoma cells proliferate as clonogenic spheroids and that HOST colonies survive for at least 3 weeks. Mineralization assay and gene expression analysis of osteoblastic markers and cytokines, indicate that all the cells cultured in 3D into this hydrogel, exhibited a more mature differentiation status than cells cultured in monolayer on plastic. This study demonstrates that this Si-HPMC hydrogel is well suited to support osteoblastic survival, proliferation and differentiation when used as a new scaffold for 3D culture and represents also a potential basis for an innovative bone repair material. r

Soria-Soto, M., Molina-Fernández, D., Hidalgo-Hidalgo, V., Adroher, F.J., Benítez, R., Malagón, D., 2013. In vitro cultivation of Anisakis simplex, causal agent of the human anisakiasis, in: III Jornadas Internacionales y V Nacionales de Ciencias de la Salud, Facultad de Ciencias de la Salud, Universidad de Granada (Eds.), "Nuevos Retos en Investigación, Docencia y Clínica en Ciencias de la Salud". Granada, pp. 223–227.
Anisakis simplex is a parasite ascaridoid nematode, main etiological agent of the parasitic illness known as human anisakiasis, which affects the gastrointestinal tract. The natural final hosts are cetaceans. The intermediate/paratenic hosts are crustaceans, fishes, and cephalopods, which harbor the third larval stage (L3) of this nematode. When the
human, who acts as an accidental host, consumes these raw, marinated or undercooked hosts harboring L3 of Anisakis, has a high risk of acquiring human anisakiasis.

Five clones of date palm (Phoenix dactylifera L.) corresponding to three reputed Moroccan cultivars (BFG, JHL and BSK) and two selected seedling genotypes (S16 and S35), derived from in vitro culture, were acclimatized and planted in Errachidia soil (South Morocco) in 1989. After 10 years of culture, clones were evaluated on the presence/absence of the two principal morphological characters (offshoots, inflorescences). Then, their leaf extracts were subjected to electrophoresis on polyacrylamide gels in order to determine their isoenzyme polymorphism. Inflorescence formation is relatively more frequent in the clones S16 and S35 than in clones JHL and BSK. Clone BFG showed an intermediate situation. High isoenzyme variations were found for the oxidoreductases (peroxidases (POX) and polyphenoloxidases (PPO)), the transferase (glutamate oxaloacetate transaminase (GOT)) and the hydrolases (esterases (EST) and endopeptidases (ENP)) enzymes. The factorial component analysis exhibited a negative correlation between the two principal morphological characters (presence of inflorescences and offshoots formation). Date palm clones typified respectively by high inflorescences and high offshoots formed two separated groups showing each one some isoenzyme characteristics. All morphological and enzyme variations were discussed in respect to the genotype effect on the micropropagation process.

Mass proliferation from the lower half bulb scales and adventitious bulb regeneration from in vitro cultured leaf explants of Madonna Lilly (Lilium candidum L.) was achieved using various concentrations of BAP-IBA. Regenerated plantlets were transferred to greenhouse for adaptation where they flowered after two years.

The main hard tissues of teeth are composed of dentin and enamel, synthesized by the mesenchymederived odontoblasts and the epithelial-derived ameloblasts, respectively. Odontoblasts are highly differentiated post-mitotic cells secreting the organic matrix of dentin throughout the life of the animal. Pathological conditions such as carious lesions and dental injuries are often lethal to the odontoblasts, which are then replaced by other pulp cells. These cells are able to differentiate into odontoblast-like cells and produce a reparative dentin. In this study we reproduced this physiological event in an in vitro culture system using pulps of human third molars. Pulp cells cultured in presence of ␤-glycerophosphate formed mineralization nodules, which grew all over the culture period. The immunohistochemical study revealed that, as odontoblasts, pulp cells contributing to the nodule formation express type I collagen, osteonectin, and nestin. By the exception of nestin, these proteins are also detected in the nodules. The composition of the nodules was also analyzed by Fourier transform infrared microspectroscopy. The spectra obtained showed that both the organic and the mineral composition of the nodules have the characteristics of the human dentin and differ from those of enamel and bone. Taken together, these results show that both the molecular and the mineral characteristics of the human dentin matrix are respected in the in vitro culture conditions.

Plants were regenerated from root explants of Arabidopsis halleri (L.) O'Kane and Al-Shehbaz via a three-step procedure: callus induction, induction of somatic embryos and shoot development. Callus was induced from root segments, leaflets and petiole segments after incubation for 2 weeks in Murashige and Skoog medium (MS) supplemented with 0.5 mg/l)1 (2.26 lM) 2,4-D D (2,4-dichlorophenoxyacetic acid) and 0.05 mg/l)1 (0.23 lM) kinetin. Only calli developed from root segments continued to grow when transferred to a regeneration medium containing 2.0 mg/l)1 (9.8 lM) 6-cc -(dimethylallylamino)-purine (2ip) and 0.05 mg/l)1 (2.68 lM) a-naphthalenacetic acid (NAA) and eventually 40% of them developed embryogenic structures. On the same medium 38% of these calli regenerated shoots. Rooting was achieved for 50% of the shoots subcultured in MS medium without hormones. The regeneration ability of callus derived from root cuttings, observed in this study, makes this technique useful for genetic transformation experiments and in vitro culture studies.

A recently developed, new theoretical absorption model for passive diffusion through biological membranes describing the dependency of membrane permeability on lipophilicity and molecular size, predicts different sigmoid-hyperbolic permeability-lipophilicity relationships for different molecular weight ranges. This model has been tested with experimental in vitro cultured epithelial cell (Caco-2) permeability data for structurally diverse drugs differing in lipophilicity, ionization state and molecular size. These data were pooled with literature values. Using this simple physicochemical approach, the permeability of a compound through Caco-2 cells by passive diffusion can be predicted from the compounds' distribution coefficient in 1-octanol / water (log D ) and its molecular weight (MW). Deviations oct from this expected behaviour may point to the involvement of biological components in the transport process, which may require further investigations.

The influence of the plant growth regulators 2,4-D, GA 3 , BA and kinetin, and the polyamines putrescine, spermidine and spermine were tested on axenic in vitro cultures of carposporelings of Grateloupia doryphora. The auxin 2,4-D (10 3 M) and the polyamine spermine (10 6 M and 10 3 M) induced a callus (disorganised cell mass that arose from the organised tissue of the carposporeling, as demonstrated by microscopic monitoring of the tissue). Putrescine and spermidine (10 3 M) transformed the carposporelings into cell masses that produced shoots. BA (10 3 M) and kinetin (10 6 M and 10 3 M) were inhibitory. In 10 1 M glycerol-containing culture medium, which is known to induce the formation of morphogenic cell masses, the addition of GA 3 M) resulted in the inhibition of the morphogenesis (i.e. shoot emission) in the cell mass. The kinetin at 10 6 M inhibited morphogenesis, whilst at 10 3 M inhibited even the formation of the cell masses. The combination of glycerol (10 1 M) and the auxin 2,4-D (10 6 and 10 3 M) or the polyamines putrescine, spermidine and spermine (10 6 and 10 3 M) resulted in a bigger size of the cell masses that led to a higher amount of shoots per cell mass than in glycerol alone.

The success of various in vitro micrografting techniques, establishment of the rootstock, size of the microscion, and the effects of culture medium on the grafted seedling development for almond cultivars "Ferragnes" and "Ferraduel" were studied. In vitro germinated wild almond seedlings developed from seeds were used as rootstocks. Shoot culture initiation was successfully achieved from the above almond cultivars by culturing mature shoot tips from forced nodal buds, about 3-5 mm, on 0.7 mg/L BA and 0.01 mg/L NAA containing a MS medium. The regenerated adventitious shoots from in vitro cultures were maintained and proliferated by sub-culturing on a fresh medium every three to 4 weeks. Regenerated shoot tips, which were micrografted onto in vitro seedlings, resulted in the restoration of shoot proliferation. The results indicated that the most successful method for the grafting of tested almond cultivars was slit micrografting. High levels of micrograft take were achieved with all ranges of scions (4-15 mm) obtained from the regenerated shoot tips. Slow growth and lack of axillary shoot development on the micrografts were noticeable when the micrografts were cultured on hormone-free germination medium. In vitro micrografted plantlets were successfully acclimatized and no problems were encountered with the establishment of micrografted plants in vivo. The developed technique has demonstrated a high potential for application in the micropropagation of almond cvs. "Ferragnes" and "Ferraduel" and thereby, represents a feasible method for the renewal of almond orchards in Turkey and elsewhere in the world.

In vitro culture establishment, shoot proliferation and ex vitro rooting responses of Mongolian cherry (Prunus fruticosa L.), and Nanking cherry (Prunus tomentosa L.), were examined using various combinations of growth regulators. Dormant buds, taken during winter months, were used as explants. In both species, Murashige and Skoog Minimal Organic (MSMO) solid medium supplemented with 0.49 lM indole-3-butyric acid (IBA) and either 4.44 or 8.88 lM 6-benzylaminopurine (BA), was the best for culture initiation, and with 8.88-15.16 lM BA for shoot proliferation. Good rooting responses were also obtained with shoots produced on media containing 0.91 lM thidiazuron (TDZ). Auxin treatments were required for ex vitro rooting of approximately 20 mm long shoots in peat/perlite (1:1 v/v) mixture, at 25°C, under mist. The best rooting (79%) was obtained with IBA/NAA (naphthaleneacetic acid) (9.80/2.69 lM) combination. A commercial rooting powder, Rootone F, containing IBA/NAA (0.057/0.067%), was also effective (73%). The ex vitro rooted plantlets did not require any additional acclimatization prior to transplanting to the regular greenhouse conditions.

The present study was conducted at ICAR-Central Potato Research Institute, Regional Station Gwalior during 2007-08 to 2016-17 for 10 years on micro-propagted material multiplied at the station to identify suitable tissue culture/ hi-tech based seed potato production systems using micro-plants, micro-tubers and mini-tubers and performance of seven popular cultivars viz Kufri Lauvkar, Kufri Chandramukhi, Kufri Surya, Kufri Bahar, Kufri Chipsona-1, Kufri Chipsona-3 and Kufri Sindhuri planted during 10 years under these three hi-tech systems for potato breeder seed production. Variation in performance among different varieties for establishment/emergence, mean tuber weight and seed multiplication rate (both by number and weight), percent

Brachypodium distachyon is a novel model system for structural and functional genomics studies of temperate grasses because of its biological and genetic attributes. Recently, the genome sequence of the community standard line Bd21 has been released and the availability of an efficient transformation system is critical for the discovery and validation of the function of Brachypodium genes. Here, we provide an improved procedure for the facile and efficient Agrobacterium-mediated transformation of line Bd21. The protocol relies on the transformation of compact embryogenic calli derived from immature embryos using visual and chemical screening of transformed tissues and plants. The combination of green fluorescent protein expression and hygromycin resistance enables early identification of transformation events and drastically reduces the quantity of tissue to be handled throughout the selection process. Approximately eight independent fully developed transgenic Bd21 plants can be produced from each immature embryo, enabling the generation of thousands of T-DNA lines. The process-from wild-type seeds to transgenic T 1 seeds-takes B8 months to complete.

Opisthorchiasis is the major public health problems in Laos PDR and Thailand. The disease becomes chronic and persists for many years, leading to hepatobiliary disease and cholangiocarcinoma. Less severe manifestations include cholangitis, chronic cholecystitis and cholelithiasis. A significant degree of humoral and cell mediated immune responses to the parasite can be detected both in patients and animal models. The patients IgG levels appear to correlate with gall bladder size and dysfunction and correlated significantly with opisthorchis egg count and decrease after treatment. However, the possible significance of these immune responses to protective immunity is presently unknown. The development of immunodiagnostic method for Opisthorchis viverrini detection has been attempted. The components with molecular weight >116, 89, 78 and 20 kDa appear to be specifically associated with the somatic extract of adult fluke. The 89 kDa protein is the most prominent component found in the in vitro culture fluid of adult worms and the metacercarial extract that can be a candidate with significant immunodiagnostic potential. Highly specific and sensitive monoclonal antibodies for O. viverrini antigens were prepared to detect parasite antigens in stool and antibody in serum. Information regarding the molecular approaches of O. viverrini is very limited. The genome of O. viverrini has neither CpG nor A methylated as found in other parasites. The total length O. viverrini ribosomal DNA is approximately 13 kb. and the presence of a highly repeated DNA specific for the parasite was demonstrated. A O. viverrini specific DNA probe was constructed and PCR based detection with high specificity for amplification of the repeated sequences is performed to detect the presence of eggs' DNA in stool samples in comparison with classical methods. (S. Wongratanacheewin).

Ranunculus asiaticus is an important ornamental species mainly cultivated in the countries surrounding the Mediterranean sea. So far the multiplication of this plant has been mainly carried out by seed and rhizome division; however, these systems present many drawbacks. Tissue culture is an attractive alternative for accelerated propagation of selected and indexed genotypes. In this paper we review the work carried out on in vitro culture of R. asiaticus and present a flow chart for its commercial production, using axillary budding and embryogenesis. Although the price of micropropagated plants is higher compared to traditional material (seedlings and rhizomes from seed populations), it should be considered that tissue cultured plants have a better rhizome yield per plant; moreover, the tissue culture approach allows to offer clonal material of selected lines.

Canine visceral leishmaniasis (CVL) is a zoonosis and a chronic systemic disease of the dog caused by a protozoan by the species Leishmania infantum in the Old World and Leishmania chagasi in the New World. Several methods are currently employed for the diagnosis of CVL including microscopic detection of the parasite in bone marrow and lymph node aspirates, demonstration of specific antibodies anti-Leishmania in sera from infected animals, and isolation of the parasite by in vitro culture or by inoculation of laboratory animals. However, a definitive diagnosis is based on the actual detection of the parasite, which is conventionally achieved by examining Giemsastained smears or histopathological sections stained with hematoxylin and eosin. These methods have a low sensitivity, and therefore, they are often inconclusive. This is particularly true in canine organs that have a low level of parasitism such as kidneys, lungs, central nervous system, and testis, or, in some cases, the skin. The technique for immunohistochemical detection of leishmanial amastigotes in canine tissues has been reported previously and has proved to be undoubtedly efficient for the diagnosis. In this paper, we describe a straightforward and inexpensive immunohistochemical approach for Leishmania detection in formalin-fixed paraffin-embedded canine tissues. Amastigote forms of Leishmania were easily observed within macrophages in several organs from naturally infected dogs using the streptavidin-biotin immunohistochemical method with canine hyperimmune serum as the primary antibody. In addition, the secondary antibody used was not specific to canine immunoglobulin, characterizing a cross-immune 0022-1759/$ -see front matter D reaction. Our results indicate that this technique could be a useful tool for epidemiological, clinical, and histopathological studies. D

Fertilized embryo sacs of Zea mays were isolated and cultured in vitro. Each explant contained one zygote and 2-4 endosperm nuclei which formed, respectively, embryo and cellular endosperm during the culture. In our double-layer/two-phase culture system, NBM medium (M61 et al. 1993) supplemented with 0.1-1.0 mg'1-1 zeatin and 12 % sucrose showed the best results. On this medium, embryos were isolated from 37-54 % of two-week-old explants. They were similar to maize embryos developing in vivo. We have shown that development of stage-2 embryos (according to Abbe and Stein 1954) with two leaf primordia and normally differentiated provascular tissue is possible from the maize zygote in an in vitro culture system. Some embryos with enlarged and deformed scutellum or whole apical parts were also found. Up to 62 % of the embryos germinating on a simple medium regenerated into mature and fertile plants; i.e. 23 % of explants yielded plants. This improved culture method results in better embryo differentiation and 14-fold increase of regeneration frequency than previous protocol.

An early postfertilization ovule culture technique in Alstroemeria was described in which young ovules were cultured in vitro two days after pollination. This resulted in the direct production of seedlings in a normally incongruous cross combination. A histological study of the in vitro cultured ovules revealed the development of the embryo inside the ovule in the absence of normal endosperm. The in vivo grown ovules showed a total breakdown of the developing seed within four weeks after pollination due to a postfertilization barrier. This ovule culture technique was applied in a diallel cross of seven Alstroemeria species, five from Chile and two from Brazil. The selfings and the reciprocal crosses between the Brazilian species A. inodora and A. brasiliensis were congruous and gave seed set. The cross A. inodora × A. pelegrina gave viable seeds. In all other 39 combinations no viable seeds were harvested. In those cross combinations both pre-and postfertilization barriers were found. After early ovule culture hybrid plants were obtained in 27 of the 39 incongruous species combinations of the diallel.

Casein phosphopeptides (CPPs) obtained by enzymatic hydrolysis in vitro of caseins, have been shown to enhance calcium solubility and to increase the calcification of embryonic rat bones in their diaphyseal area. Little is known about the direct effects of CPPs on cultured osteoblastic cells. Calcium in the microenvironment surrounding bone cells is not only important for the mineralization of the extracellular matrix, but it is believed to provide preosteblasts with a signal that modulates their proliferation and differentiation. The aim of the present study was to investigate the direct effects of four selected casein phosphopeptides on osteoblastic cell (MC3T3-E1 cells) viability and differentiation. The selected peptides have been obtained by chemical synthesis and differed in the number of phosphorylated sites and in the amino acid spacing out two phosphorylated sites, in order to further characterize the relationship between structure and function. The results obtained in this work demonstrated that CPPs may directly affect osteoblast-like cell growth, calcium uptake and ultimately calcium deposition in the extracellular matrix. The effects exerted by distinct CPPs on osteogenesis in vitro can be either stimulatory or inhibitory. Differential short amino acid sequences in their molecules, like the -SpEE-and the -SpTSpEE-motifs, are likely the molecular determinants for their biological activities on osteoblastic cells. Moreover, two genetic variants of CPPs showing one amino acid change in their sequence may profoundly differ in their biological activities. Finally, our data may also suggest important clues about the role of intrinsic phosphorylated peptides derived from endogenous phosphorylated proteins in bone metabolism, apart from extrinsic CPPs.

The production of reactive oxygen species (ROS) in plants is a common event in metabolic and physiological processes as well as in the response to biotic and abiotic stress. In this paper we will report that root tissue from axenically grown tomato cultivars and Lycopersicon wild species can be used for the determination of superoxide production. Superoxide generation was evaluated following the treatment of root tissues with two general elicitors of the defence response: laminarin and calcium ionophore A23187. Results demonstrated that elicitor reactivity in terms of superoxide generation of the tomato cultivars and the wild species used was different. This suggested varying levels of competence for non-specific active defence. The proposed technique merges the advantages of in vitro cultures and of whole tissues and also demonstrates that root tissue is a suitable material for evaluating free radical release.

Naturally acquired immunity to Plasmodium falciparum is related to immune system that changes during normal development and ageing. The effects of repeated infections during the early life on the maturation of the immune system are still unknown. Elucidation of these effects is of considerable interest given that malaria originates high mortality, especially during the first years of life.

Callus cultures were initiated from leaf bases of turmeric on Murashige and Skoog's basal medium (MS) supplemented with dicamba, picloram (2 mg l −1 ) or 1-naphthaleneacetic acid (NAA) (5 mg l −1 ) in combination with benzyladenine (BA) (0.5 mg l −1 ). On transfer of callus cultures to medium supplemented with benzyladenine (BA) (5 mg l −1 ) in combination with triiodebenzoic acid (TIBA) or 2,4-dichlorophenoxyacetic acid (2,4-D) (0.1 mg l −1 ), green shoot primordia were seen to differentiate from the surface of the callus. On transfer of regenerating cultures to half MS media supplemented with Kn, shoot primordia developed into well developed shoots. When shoots were transferred to medium devoid of phytohormones, complete rooted plants were obtained. Ninety percent of the plants survived to maturity on transfer to soil. Random Amplified Polymorphic DNA (RAPD) analysis of eight regenerated plants using 14 primers when separated on non-denaturing polyacrylamide gels showed 38 novel bands. About 51 bands present in the control were absent in the regenerants. The result indicates that variation at DNA level has occurred during in vitro culture.

The present study was conducted at ICAR-Central Potato Research Institute, Regional Station Gwalior during 2007-08 to 2016-17 for 10 years on micro-propagted material multiplied at the station to identify suitable tissue culture/ hi-tech based seed potato production systems using micro-plants, micro-tubers and mini-tubers and performance of seven popular cultivars viz Kufri Lauvkar, Kufri Chandramukhi, Kufri Surya, Kufri Bahar, Kufri Chipsona-1, Kufri Chipsona-3 and Kufri Sindhuri planted during 10 years under these three hi-tech systems for potato breeder seed production. Variation in performance among different varieties for establishment/emergence, mean tuber weight and seed multiplication rate (both by number and weight), percent

We report here the successful establishment of callus, cell and root cultures from explants of in-vitrogrown plantlets of the soapwort Saponaria officinalis L. The production of saporin in the different tissue systems was evaluated by determining the capability of crude extracts to inactivate protein synthesis and by Western blotting analysis. Protein synthesis inhibition varied in callus and derived cell suspensions and in cultured roots, the latter, in particular, showing the lowest specific activity. The ribosome-inactivating principle from root cultures was purified to homogeneity by cation exchange chromatography. Key words Ribosome-inactivating protein • Roots • Saponaria officinalis • Saporin Abbreviations BAP 6-Benzylaminopurine • 2,4-D 2,4-Dichlorophenoxyacetic acid • IAA Indole-3-acetic acid • KIN Kinetin • MS Murashige and Skoog medium • NAA α-Naphthaleneacetic acid • PBS Phosphate buffer saline • RIP Ribosome-inactivating protein

The present study was conducted at ICAR-Central Potato Research Institute, Regional Station Gwalior during 2007-08 to 2016-17 for 10 years on micro-propagted material multiplied at the station to identify suitable tissue culture/ hi-tech based seed potato production systems using micro-plants, micro-tubers and mini-tubers and performance of seven popular cultivars viz Kufri Lauvkar, Kufri Chandramukhi, Kufri Surya, Kufri Bahar, Kufri Chipsona-1, Kufri Chipsona-3 and Kufri Sindhuri planted during 10 years under these three hi-tech systems for potato breeder seed production. Variation in performance among different varieties for establishment/emergence, mean tuber weight and seed multiplication rate (both by number and weight), percent <3 g tuber (both by number and weight) and tuber yield (m 2) were recorded during the study. Kufri Sindhuri, Kufri Bahar and Kufri Surya under micro-plant based, Kufri Bahar and Kufri Surya and Kufri Chipsona-1 under micro-tuber and mini-tuber based multiplication system performed better than other varieties under G-0 conditions for yield attributing parameters. Seed multiplication rate by number was higher in micro-plant system of seed production (5.50) followed by micro-tuber systems (5.31) and mini-tuber (4.43) based system of seed production. Mean tuber weight was higher for mini-tuber system of seed production (22.46 g) followed by micro-plant (15.59 g) and micro-tuber systems (14.62) and showed higher tuber yield i.e 130 tuber/m 2 and 3.10 Kg/m 2 in mini-tuber based system followed by111 tuber/m 2 and 1.74 kg/m 2 in micro-plant based system and by 86 tuber/m 2 and 1.16 kg/m 2 in micro-tuber based seed potato production system. Hence performance of Kufri Sindhuri, Kufri Bahar and Kufri Surya under micro-plant based, Kufri Bahar and Kufri Surya and Kufri Chipsona-1 under micro-tuber and mini-tuber based seed multiplication system was better in 10 years of the study.