In vitro culture of Trichogramma pretiosum on an artificial medium

The composition of an artificial medium and environmental conditions are described for the in vitro rearing of the egg parasite Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae). The medium was composed of defined amounts of protein, carbohydrates, lipid, salts, and vitamins, but also contained up to 40% insect hemolymph. The hemolymph was necessary to induce pupation. T. pretiosum eggs were obtained by dissection of Heliothis virescens (F.) (Lepidoptera: Noctuidae) eggs. In vitro reared T. pretiosum were similar in size to H. virescens reared T. pretiosum, and females were fecund.

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Résumé Les oeufs de Trichogramma pretiosum ont été obtenus par dissection d'oeufs d'Heliothis virescens. T. pretiosum Riley (Hymenoptère, Trichogrammatidae) a été élevé avec succès sur un substrat synthétique. Outre des quantités définies de protéines, glucides, lipides, éléments minéraux et vitamines, la ration contenait aussi jusqu'à 40% d'hémolymphe de Manduca sexta. L'hémolymphe était nécessaire pour induire la nymphose. En plus de la nourriture, les conditions d'environnement sont apparues extrêmement importantes pour élever T. pretiosum dans des conditions satisfaisantes. Le contrôle de l'humidité relative, en particulier, était le facteur le plus important. Les adultes produits au cours de cette étude étaient d'apparence normale; ils se sont accouplés sans problèmes, les femelles étaient fécondes et leur taille ne différait pas de celle d'individus élevés sur H. virescens.

     

In vitro chromosome doubling of nineZantedeschia cultivars

Tetraploid plants have been produced from nineZantedeschia cultivars usingin vitro colchicine treatment. Rapidly-multiplying shoot cultures were treated on a medium containing 0.05% colchicine for 1, 2 or 4 days to induce chromosome doubling. Following the treatment, most shoots were killed but the surviving shoots were multiplied for several subcultures. These shoots were then rootedin vitro and transferred to a greenhouse. Plants were screened 2 months later by measuring stomatal length, and 110 out of 565 plants were selected as putative tetraploids with a stomatal length significantly greater than in diploid control plants. Chromosome counts were carried out on root tips from 44 plants and confirmed that 38 were tetraploids, 2 were chimeras (predominantly tetraploid with a few octoploid cells), and 4 were diploids. Stomatal length has been rechecked in mature tetraploid plants of the cultivar Black Magic, demonstrating that stomatal length is a good indicator of ploidy level inZantedeschia. This study has shown that multiplying colchicine-treated shootsin vitro for several subcultures prior to transfer to soil produced very few chimeras. The stomatal length measurements are non-destructive and allow the rapid screening of a population for tetraploids.     

In vitro assembly of gap junctions

Gap junction structures were assembled in vitro from octyl-β--glucopyranoside-solubilized components of lens fiber cell membranes. Individual pore structures (connexons), short double-membrane structures, and other amorphous material were evident in the solubilized mixture. Following the removal of the detergent by dialysis, these connexons associated to form single- and double-layered, two-dimensional hexagonal arrays (unit cell size a = B = 8.5 nm). The formation of larger arrays was dependent on the lipid-to-protein ratio and the presence of Mg²⁺ ions. Crystallographic analysis of electron micrographs revealed that lens junctional connexons consisted of six subunits surrounding a stain-filled channel. Upon further detergent treatment, in vitro assembled gap junctions were insoluble and formed three-dimensional stacks while other components were solubilized. SDS-PAGE and mass data from scanning transmission electron microscopy strongly suggest that a 38-kDa polypeptide, which is a processed form of the lens specific gap junction protein MP70, is a major component of the arrays. The in vitro assembly of gap junctions opens new avenues for the structural analysis of gap junctions and for the study of the intermolecular interactions of connexons during junctional assembly.     

In vitro organogenesis of Citrus volkameriana and Citrus aurantium

In vitro organogenesis of Citrus volkameriana and C. aurantium was studied considering three explant types: epicotyl segment, internodal segment, and hypocotyl segment with attached cotyledon fragment. The explants were cultured in medium according to Grosser and Gmitter (EME) supplemented with 0, 0.5, 1.0, 1.5, and 2.0 mg dm^{− 3} 6-benzyl-aminopurine (BAP), incubated firstly in darkness for 4 weeks, and then transferred to 16-h photoperiod for 2 weeks. Comparing epicotyl and internodal segments, a higher percentage of responsive explants and a higher number of shoots per explant were obtained with epicotyl segments, regardless of the BAP concentration. For C. volkameriana the highest percentage of responsive epicotyl segments (42 %) was obtained in EME with 1.0 mg dm⁻³ BAP, while for C. aurantium (59 %) in EME with 0.5 mg dm⁻³ BAP. The organogenesis efficiency was the best with the use of the hypocotyl segment with attached cotyledon fragment (77 % for C. volkameriana and to 75 % for C. aurantium). With this explant the morphogenesis occurred only in the hypocotyl region. The in vitro organogenesis was characterized by histological analyses showing that the morphogenic process started in the cambium region near the explant cut end.     

In vitro production of solasodine from Solanum trilobatum

Sucrose concentration in the culture medium affected chlorophyll content, trichome development and amount of solasodine in regenerated plantlets of Solanum trilobatum. High chlorophyll content and glandular trichomes were observed in the plants grown on Murashige and Skoog basal medium supplemented with 131.85 mM sucrose. The solasodine was quantified using reverse phase high performance liquid chromatography. The plantlets cultivated on this medium yielded 35.97 mg g⁻¹ (d.m.) solasodine whereas the field plants used as control yielded only 2.32 mg g⁻¹ (d.m.) of solasodine.     

In vitro biohydrogenation of four dietary fats

This study was designed to determine in vitro rates of biohydrogenation of dietary unsaturated fatty acids by a mixed population of rumen microbes. The four dietary fats [Alifet High-Energy^® (AHE), Alifet-Repro^® (AR), Megalac^® (MG), and Energy Booster^® (EB)] differ in method of preparation, fatty acid composition, or both of these factors. Dietary fats (20 mg) were incubated with 4 mL strained rumen fluid diluted with 16 mL of medium, 0.8 mL of reducing solution buffer, and 200 mg of a synthetic diet (370 g cellulose, 370 g starch, and 160 g casein per kg DM) at 37 °C. Total contents were collected after 0, 6, 12, 24, or 36 h and change in fatty acid content determined. Disappearance of oleic acid was minimal (0.05–0.20) in AR and MG but moderate (about 0.60) in AHE and EB after 36 h of incubation. Rate of biohydrogenation of linoleic and linolenic acids from AR were similar (0.025 ± 0.009 h⁻¹) and 0.65 of these fatty acids remained intact after 36 h. Rate of biohydrogenation of linoleic acid was four times greater than for oleic acid (0.040 ± 0.013 h⁻¹ versus 0.009 ± 0.002 h⁻¹) in MG. Thus, 0.65 of the linoleic acid but only 0.20 of the oleic acid had disappeared from MG after 36 h. Trans-11 and trans-12 were the predominant trans-isomers in AHE and AR cultures whereas trans-9 and trans-10 were the predominant trans-isomers in EB and MG cultures. None of the dietary fats contained conjugated linoleic acid (CLA) but CLA was present in the incubation inoculum. The amount of CLA decreased with time but this was not affected by source of dietary fat. Most (0.90–0.95) of the long-chain fatty acids eicosapentaenoic (EPA) and docosahexaenoic (DHA) in AR remained after 36 h of incubation. Results demonstrate that biohydrogenation varied among fatty acids and among source of dietary fat and indicate that AR can be used to increase post-ruminal supply of linolenic, EPA and DHA.     

In vitro cultivation of Diplostomum spathaceum and Diplostomum phoxini metacercariae

Using a new egg macerate medium Diplostomum spathaceum metacercariae were cultured to the stage of egg production for the first time, and an improved vitelline and growth response was obtained in Diplostomum phoxini. The importance of physical factors in the cultivation of strigeoid trematodes was demonstrated. Methods involving a continuous circulating system, diphasic media, a double culture tube technique or the chorio-allantoic membrane proved to be of no value in the cultivation of Diplostomum spp. The addition of selected hormones with known growth promoting properties did not have an appreciable effect on D. phoxini cultures. Supplementation of egg macerate cultures with various nutrients had only slight or no beneficial effect. The ability to use stored metacercariae for cultures was demonstrated.     

Chelating agent inhibition of Trypanosoma cruzi epimastigotes In vitro

A number of chelating agents and some of their derivatives are as effective as, or superior to, benznidazole, the compound currently in clinical use, in the suppression of the reproduction of epimastigotes of Trypanosoma cruzi, the protozoa that causes Chagas' disease. All compounds were examined at a culture concentration of 5 μg/mL. The most effective compounds included N, N, N′, N′-tetrakis(2-pyridylmethyl)ethylenediamine, sodium diethylamine-N-carbodithioate, piperidine-N-carbodithioate and several of its analogs, a number of other carbodithioates with two nonpolar groups on the nitrogen, and tetraethylthiuram disulfide, a prodrug of sodium diethylamine-N-carbodithioate and widely used in the treatment of alcoholism. The introduction of additional ionic or nonionic polar groups on the chelating molecule generally results in a loss of tyrpanocidal activity. Common commercially available chelating agents which exhibited no activity included -penicillamine, meso-2,3-dimercaptosuccinic acid, and triethylenetetramine tetrahydrochloride. Dose-response data on the culture indicated that some of these compounds exhibited inhibition of Trypanosoma cruzi epimastigotes at concentrations as low as 0.625 μg/mL. It is proposed that the mechanism of action of these compounds is based on their ability to interface with the essential metal metabolism at intracellular sites of the epimastigote involving iron, copper, or zinc. The results also indicate that a certain degree of hydrophobicity may be necessary for the groups attached to the literal metal-bonding structure if the compounds are to successfully inhibit the epimastigotes of Trypanosoma cruzi. The development of antiprotozoal drugs which are chelating agents specifically designed to selectively disrupt the essential metal metabolism of Trypanosoma cruzi should furnish a new generation of drugs which can be used in the treatment of Chagas' disease.     

In vitro cloning of Azadirachta indica from root explants

In vitro cultures of Azadirachta indica A. Juss. were raised by first culturing the root segments on modified Murashige and Skoog (MS) medium supplemented with 8.88 μM 6-benzylaminopurine (BAP), 9.84 μM N⁶-(2-isopentenyl) adenine (2iP), 5.71 μM indole-3-acetic acid (IAA), 81.43 μM adenine hemisulphate and 2.27 μM putrescine for 2 d followed by their transfer to the same medium except containing one-tenth of the initially used concentrations of BAP, 2iP and IAA. The regenerated shoots sustained proliferation in the basal medium supplemented with 1.11 μM BAP, 1.43 μM IAA and 135.72 μM adenine hemisulphate. The isolated shoots were rooted to produce plantlets in the presence of 2.46 μM indole-3-butyric acid (IBA). The plantlets showed uniform luxuriant growth under field conditions. True-to-type nature of the field-grown root-regenerated plants was ascertained by random amplified polymorphic DNA (RAPD) analysis.     

In vitro rearing ofExorista larvarum on tissue culture-based diets

Exorista (=Tachina) larvarum (L.) (Diptera, Tachinidae), a polyphagous parasitoid that attacksLymantria dispar L. andHyphantria cunea (Drury), was rearedin vitro from egg to adult on four tissue culture media-based diets (TMM-FH, SCHNEIDER'S, EX-CELL 400, and SF-900). The kind of tissue culture media in the diets did not influence the adult yield (34 to 55%) and puparium weight (26–27 mg). Adult yield and the puparium weight ofE. larvarum developed on TNM-FH and SCHNEIDER'S-based diets containing different amounts ofGalleria mellonella pupal extract (PE) (0, 1.25, 2.5 and 5%), were lower on diets without PE. In diets without PE development times from oviposition to adult emergence, were shorter on TNM-FH (19 days) than on SCHNEIDER'S-based diet (25–26 days). The adults that developed on artificial diets were able to parasitize the factitious hostG. mellonella and produce viable progeny. The results demonstrate thatE. larvarum is the most promising parasitoid ever studied forin vitro mass production.     

In vitro cytostatic and immunomodulatory properties of the medicinal mushroom Lentinula edodes

Lentinula edodes, known as “shiitake” is one of the widely used medicinal mushrooms in the Orient. Antitumour activity of extracts of this mushroom has been widely demonstrated in animals and humans. However, this activity was shown to be host mediated and not by direct cytotoxic activity to cancer cells. This study demonstrates cytotoxic and cell growth inhibitory (cytostatic) effect of aqueous extracts of the mushroom on MCF-7 human breast adenocarcinoma cell line using an MTT cytotoxicity assay. Such effect was demonstrated with fruit body and mycelial extracts, the difference being that there was no significant suppression on normal cells with the latter. Furthermore mycelial extracts did not induce any cytostatic effect in both cancer and normal cell lines based on a DNA synthesis assay. The significant suppression of the proliferation of cancer cells was reflected by the comparatively low IC₅₀ values and the simultaneous higher respective values on normal fibroblast cells. The immunostimulatory activity of both fruit body and mycelial extracts was tested by the lymphocyte transformation test (LTT), which is based on the capacity of active immunomodulators to augment the proliferative response of rat thymocytes to T mitogens in vitro. Both fruit body and mycelial preparations were able to enhance the proliferation of rat thymocytes directly and act as co-stimulators in the presence of the T-mitogen PHA. Interestingly both extracts, similarly to zymosan showed SI_comit/SI_mit ratios of about 2, indicating adjuvant properties. Overall L. edodes aqueous extracts have demonstrated direct inhibition of the proliferation of human breast cancer cells in vitro and immunostimulatory properties in terms of mitogenic and co-mitogenic activity in vitro.     

In vitro chiral nematic ordering of chitin crystallites

Microfibrillar fragments of purified crab and shrimp chitin were prepared by hydrolysis in 3 HCl at its boiling point (104°C). After removal of the acid by centrifugal washing and dialysis, an ultrasound treatment converts the residual product to a colloidal suspension stabilized by NH₃⁺ charges. When dewatered to a critical concentration, spontaneous formation of a two-phase equilibrium system occurs. The upper phase (lower concentration) is isotropic and the lower phase is anisotropic. The latter displays chiral nematic order and dries to a solid film which mimics the helicoid organization characteristic of the chitin microfibrils in the cuticle of arthropods.     

In vitro responses of grape rootstocks to NaCl

An investigation was undertaken to adjudge the in vitro salt tolerance and biochemical changes due to NaCl on four grape rootstocks (Dogridge, SO4, H-144 and 3309C). The in vitro two-node micro-cuttings from the established cultures were sub-cultured on rooting medium comprising Murashige and Skoog (MS) basal medium supplemented with 0.5 μM indolebutyric acid (IBA), 200 mg dm⁻³ activated charcoal and different NaCl concentrations ranging from 0 to 125 mM. The surviving and proliferated cultures were further sub-cultured four times on respective media. Dogridge and H-144 tolerated 125 and 100 mM NaCl, respectively, while SO4 and 3309C survived only up to 75 mM NaCl. Contents of proteins, proline, K⁺ and Na⁺ in tissue increased in all genotypes due to NaCl supplementation, while contents of chlorophyll and total soluble sugars declined. Higher K⁺/Na⁺ ratio was registered in Dogridge and H-144 than in SO4 and 3309C. The relative NaCl tolerance for different grape rootstocks under study could be ranked as Dogridge > H-144 > SO4 and 3309C.     

In vitro regeneration of an endangered medicinal plant Picrorhiza scrophulariiflora

A reproducible in vitro regeneration system for Nepalese kutki (Picrorhiza scrophulariiflora Pennell) was developed from in vitro leaf derived callus. Induction of more than seven shoot buds per explant was achieved on Woody plant medium (WPM) supplemented with 0.53 μM α-napthaleneacetic acid (NAA) and 0.23 μM kinetin (KIN). The shoots were elongated on WPM supplemented with 0.44 μM 6-benzylaminopurine (BAP) and rooted on WPM supplemented with 5.3 μM NAA within 2 weeks. The random amplified polymorphic DNA (RAPD) analysis indicated genetic uniformity of the micropropagated plants with its donor plants.     

In vitro regeneration of Solanum nigrum with enhanced solasodine production

High frequency of shoot formation was achieved from Solanum nigrum L. leaves on Murashige and Skoog (MS) medium without any callusing stage. Shoot forming ability was more pronounced on leaves positioned dorsally. For shoot induction, 2.0 mg dm⁻³ benzylaminopurine and 1.5 mg dm⁻³ kinetin were observed to be the most effective plant growth regulators (PGRs). The present paper also describes first successful induction of in vitro flowering in S. nigrum. The leaf derived shoots were excised and treated with various root promoting PGRs and 0.25 mg dm⁻³ indole-3-butyric acid produced maximum number of roots (15.2 per plant). Plants were later transplanted in field with 100 % survival. Solasodine content was higher in in vitro raised shoots and leaf derived callus, compared to ex vitro grown shoots.     

In vitro propagation of plant virus using different forms of plant tissue culture and modes of culture operation

Plant virus accumulation was investigated in vitro using three different forms of plant tissue culture. Suspended cells, hairy roots and shooty teratomas of Nicotiana benthamiana were infected with tobacco mosaic virus (TMV) using the same initial virus:biomass ratio. Viral infection did not affect tissue growth or morphology in any of the three culture systems. Average maximum virus concentrations in hairy roots and shooty teratomas were similar and about an order of magnitude higher than in suspended cells. Hairy roots were considered the preferred host because of their morphological stability in liquid medium and relative ease of culture. The average maximum virus concentration in the hairy roots was 0.82 ± 0.14 mg g⁻¹ dry weight; viral coat protein represented a maximum of approximately 6% of total soluble protein in the biomass. Virus accumulation in hairy roots was investigated further using different modes of semi-continuous culture operation aimed at prolonging the root growth phase and providing nutrient supplementation; however, virus concentrations in the roots were not enhanced compared with simple batch culture. The relative infectivity of virus in the biomass declined by 80–90% during all the cultures tested, irrespective of the form of plant tissue used or mode of culture operation. Hairy root cultures inoculated with a transgenic TMV-based vector in batch culture accumulated green fluorescent protein (GFP); however, maximum GFP concentrations in the biomass were relatively low at 39 μg g⁻¹ dry weight, probably due to genetic instability of the vector. This work highlights the advantages of using hairy roots for in vitro propagation of TMV compared with shooty teratomas and suspended plant cells, and demonstrates that batch root culture is more effective than semi-continuous operations for accumulation of high virus concentrations in the biomass.