Subculture Period Research Articles

GROWTH INHIBITORY EFFECT OF TRICLOSAN ON EQUINE AND BOVINE BABESIA PARASITES

We evaluated the growth inhibitory effect of triclosan, which has recently been reported to inhibit the growth of Plasmodium species and Toxoplasma gondii, on bovine and equine Babesia parasites in in vitro cultures The growth of Babesia bovis and B. bigemina was significantly inhibited in the presence of 100 microg/ml of triclosan, while B. caballi and B. equi were susceptible to as low as 50 microg/ml. Babesia bigemina and B. caballi were completely cleared as early as on the first and second day of the treatment, respectively. These parasites did not exhibit any growth in the subsequent five-day period of subculture without triclosan. Drug-treated parasites appeared pycnotic and atypically shaped, and ultrastructurally showed pronounced vacuolations, leading to complete destruction of parasites. Light microscopy showed that used concentrations of triclosan showed no toxicity against the host cells. The results suggest that triclosan can be used for chemotherapy of babesiosis.

Some features of somatic embryo maturation of algerian fir

Maturation of Abies numidica De Lann. somatic embryos was tested on media with abseisic acid and various maltose and polyethylene glycol-4000 concentrations. The effect of basal medium and subculture period on maturation was also examined. The maturation of somatic embryos was promoted by polyethylene glycol-4000, at 7.5–10%. Three to 6% maltose significantly enhanced the yield of mature embryos. The most effective somatic embryo maturation occurred when embryogenictissue was transferred to maturation medium after 14–21 d cultivation on proliferation medium. The ability for A. numidica cultures to form cotyledonary somatic embryos was assessed over a period of 3 yr. Plantlets germinated on half-strength SH (Schenk and Hildebrandt) medium with charcoal and survived transfer to the soil.

Alkaloid formation in cell suspension cultures of Tabernaemontana elegans after feeding of tryptamine and loganin or secologanin

A cell suspension culture of Tabernaemontana elegans lost its ability to produce alkaloids after a prolonged period of subculture. To determine whether it was still capable of performing the later steps of the alkaloid biosynthetic pathway, the culture was fed with tryptamine and loganin. The precursors and alkaloids were determined in the biomass and in the medium during a growth cycle. In this culture, an increase in the amount of serotonin was found in the biomass after feeding of tryptamine and loganin. Secologanin was detected in small amounts but strictosidine was not. Therefore, a limitation in alkaloid formation in this T. elegans cell line occured in the formation of secologanin from loganin. After feeding of secologanin alone, strictosidine, 10-hydroxy strictosidine, strictosidinic acid and two other indole alkaloids, as yet unidentified, were formed. However, the alkaloids originally produced by this cell line were not found. As the biosynthesis is impaired at several steps, it seems that the loss of productivity is more likely to be to a change on the level of the regulation of the pathway, than due to the loss of the capacity to express an individual biosynthetic gene of the pathway.

The effect of AOA on ethylene and polyamine metabolism during early phases of somatic embryogenesis in Medicago sativa.

Changes in the levels of ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC), 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC) and polyamines were simultaneously investigated during the early phases of alfalfa somatic embryogenesis. These included the period of induction and subculture of callus, and 3- and 7-day suspension cultures for the induction of somatic embryogenesis. The polyamines contained in the embryogenic callus were found to include putrescine (Put), spermidine (Spd) and spermine (Spm), but the level of Spm was much less than that of Put and Spd. There was a dramatic increase in MACC after induction of embryogenesis, and ACC levels were lower in somatic embryos than in embryogenic callus. Induction of embryogenesis for 3 days increased the levels of ACC and polyamines to a maximum level, and these then reduced as the embryogenesis proceeded. The ratios of Put/Spd and ACC/MACC were decreased during the induction. This indicated that both high levels of ACC and polyamines might be a prerequisite for early differentiation during the induction of the embryogenesis. Thus, there appears not to be competition between polyamine biosynthesis and ethylene biosynthesis at least during the induction of somatic embryogenesis, because both the polyamines and ACC were simultaneously increased during the induction period. Conversion of ACC into MACC and the maintenance of a relatively high level of polyamines, especially Spd, appear to be important for further development of the embryos. When aminooxylvinylglycine (AOA) was added at the initiation of the callus subculture, it had no significant effect on the callus growth, the ethylene production and ACC level of the callus. However, AOA increased the numbers of the embryos accompanying an increase in Spd level and S-adenosylmethionine decarboxylase (SAMDC) activity. Thus, the AOA effect could be associated with Spd increase rather than with the effect of ethylene biosynthesis.

An efficient liquid culture system for tea shoot proliferation

The efficiency of thidiazuron in promoting tea shoot proliferation in liquid medium was evaluated. As compared to 6-benzyl adenine which induced hyperhydricity in the proliferated shoots in liquid medium, a progressive increase in the multiplication rate together with healthy shoot growth was achieved when thidiazuron (2.5 to 5.0 μM) was used instead of 6-benzyl adenine. Of the different liquid volumes compared in 250 ml Erlenmyer flasks, 20 ml was the most effective. While an increase in the multiplication rate coupled with normal but healthy shoots was observed under static and agitated conditions at this volume of liquid medium, hyperhydricity was induced in 50 ml liquid medium. Therefore, 20 ml static liquid medium with subculture periods at an interval six to eight weeks seems to be a cost and labour effective process as compared to the existing protocols involving solid media with subculture periods at 4 weeks interval.

ENDOGENOUS BACTERIA IN TISSUE CULTURES OF CONIFERS - APPEARANCE AND ACTION

Investigations were carried out to find the reasons for difficulties and even declines in different conifer micropropagation systems. Especially in embryogenic lines of Norway spruce and hybrid larch the regeneration capacity was dramatically reduced after repeated subculture periods. A slight clouding of nutrient media gave the first clue for the occurrence of endogenous bacteria. Because of their pleiomorphic growth these bacteria were very difficult to determine and only a crude assignment was possible. The addition of a culture filtrate to embryogenic cultures resulted in a reduced survival of calli. The treatment with the antibiotic cefotaxime could enhance the percentage of clean cultures only for some of the embryogenic lines.

One step more towards taxane production through enhanced Taxus propagation.

We have developed a high-yielding procedure for the in vitro propagation of juvenile material of Taxus baccata involving a combination of seed handling and culture on WP culture medium supplemented with sucrose (2%), activated charcoal (0.5%) and BAP (22.19 mM) for 30 days, followed by 40 days on hormone-free medium. Shoot apical ends should be decapitated to obtain propagation rates up to 12- to 18-fold per subculture period (70 days). In this way the high genetic variability of the juvenile material can be used in the most productive way. In addition to producing large numbers of yew plants (difficult to get by traditional methods), this procedure allows the fast screening of individuals for their taxane content. A negative correlation between growth and secondary metabolite content was found for paclitaxel. The positive correlation with 10-deacetyl baccatin III accumulation reflects once more the commercial viability of using 10-deacetyl baccatin III extraction as an alternative to taxane production, but this time opening up the possibility of selecting genotypes with both characteristics: fast growth and high productivity.

Induction of geranylgeranyl diphosphate synthase activity and taxane accumulation in Taxus baccata cell cultures after elicitation by methyl jasmonate

The results presented in this paper describe for the first time the presence of an inducible enzyme in the biosynthetic pathway leading to the anticancer drug paclitaxel. Methyl jasmonate, when administered to Taxus baccata cell suspension cultures, was found to rapidly induce geranylgeranyl diphosphate (GGPP) synthase activity. Experiments showed that after elicitation at day 7 of the subculturing period, the enzyme activity is rapidly induced during the first 6 h and reaches its maximum of 60.7 μkat/kg of protein after 5 days. The enzyme’s induction was followed by a sharp increase in taxane accumulation in the cells, which started 24 h after elicitation and reached its maximum after 5 days. In addition taxine-B was found in the medium of unelicited cultures but not in the medium of elicited cells.

Multiple shoot regeneration and tissue culture studies on Bacopa monnieri (L.) Pennell

Adventitious shoot buds were induced from leaf and stem explants of Bacopa monnieri on Murashige and Skoog medium supplemented with benzyladenine or kinetin. The source of the explants as well as different gelling agents in the medium were found to influence shoot induction and eventual shoot growth. The best response was obtained in leaf explants taken from shoot cultures grown in medium supplemented with 2 μM benzyladenine and gelled with 0.2% gelrite. A transverse section of the leaf explant incubated in this medium showed several shoot primordia emerging from the leaf surface. This system exhibited a potential for repeated harvesting of the shoots from the original leaf explant as the latter continued to expand and regenerate new shoots, upon repeated periodical subculturing onto fresh medium. However, the callusing response of the plant was very low. Qualitative TLC studies of the regenerated shoots revealed a phytochemical profile similar to that of the field grown-plants.

The role of rejuvenated and adult forms of English oak (Quercus robur) in in vitro cultures

In vitro plant material of clones (Q. robur) NL 100 A (adult) and NL 100 R (rejuvenated) received from Germany (A. Meier-Dinkel, 1995) were used in these experiments. WPM medium was used for the multiplication phase. Plantlets were subcultured monthly. Differences in quality and colour of the adult and rejuvenated cultures induced us to follow and compare the changes of mineral- and chlorophyll content and dry weight during the propagation phase. Mineral and chlorophyll content as well as dry weight were measured weekly on three samples during the subculture period.
 In the case of propagation rates we stated, they were similar around the year, but both clones had a high peak in April. Examining the cation-content, we detected that, the plantlets had a highest quantity of several elements during the 2nd and 3rd week of subculture. The iron content was the highest in the 1st week and after that it decreased continuously. It is supposed, that the content of iron is not enough in the media. The chlorophyll content of the rejuvenated clone was higher than that of the adult one.
 In the rooting experiments it was stated that, after one-week cold treatment the rooting ability was the best.

Evaluation of microprojectile-mediated DNA delivery and reporter genes for genetic transformation of the Mediterranean cypress (Cupressus sempervirens L.).

Embryonal-suspensor tissue (EST) of Mediterranean cypress (Cupressus sempervirens L.) was tested for microprojectile-DNA delivery (by the PDS-1000/He device) for different subculture periods (9, 15, and 21 days) using the plasmid vectors pRT99GUS [containing the β-glucuronidase (GUS) and neomycin phosphotransferase (NPT II) genes, and the CaMV 35S promoter], pBI426 (with a GUS::NPT II fusion gene under the control of a duplicated 35S RNA promoter), and pCGUδ0 (containing the GUS gene with the ubiquitin intron, under the control of the sunflower ubiquitin promoter). The relative strengths of the promoters as determined by GUS assays were sunflower ubiquitin>35S-35S-AMVE>35S. The highest expression level was observed when 15-day-subcultured EST was bombarded with the pCGUδ0 gene construct, which also showed high activity of the chloramphenicol acetyltransferase and NPT II genes. Green fluorescent areas were observed on EST when bombarded with the p35S-GFP plasmid, carrying the gene for the green fluorescent protein from the bioluminescent jellyfish Aequorea victoria.

Growth characteristics and transformability of soybean embryogenic cultures.

Embryogenic cultures of soybean [Glycine max (L.) Merr. cv. Jack and Asgrow A2872] were established in liquid Finer and Nagasawa medium, maintained by transfer to fresh medium at biweekly intervals, and subjected to microprojectile bombardment over time. Cultures were not amenable to transformation until they were at least 6 months old. Over time, different cell lines of the same genotype acquired very different culture phenotypes. Histological analysis of cell lines differing in transformation ability showed that the most transformable cultures had cytoplasmic-rich cells in the outermost layers of the tissue. In contrast, the outer layers of less transformable cultures contained cells with prominent vacuoles. Although fresh weight accumulation of the cultures was curvilinear during the 2-week subculture period, a burst of mitotic activity was evident shortly after transfer to fresh medium. This activity usually lasted from the 2nd to the 6th day following subculture, and peaked on the 4th day. Tissues at or near this stage always produced more transient expression of a reporter gene than did bombardments at other times. In addition, the cell lines most amenable to transformation also exhibited the highest mitotic index. Thus any treatment to increase the mitotic index, especially when the cell lines are less than 6 months old, may facilitate the transformation of cell lines from which efficient recovery of transgenic plants is still possible.

Flow cytometric analyses in embryogenic and non-embryogenic callus lines of Cyclamen persicum Mill.: relation between ploidy level and competence for somatic embryogenesis.

Embryogenic and non-embryogenic callus lines derived from the same diploid Cyclamen persicum genotype (`Purple Flamed') were analyzed by flow cytometry and compared to the initial plant material. The DNA content of the diploid plant in the greenhouse was 1.12 pg DNA/2C as estimated in relation to the internal standards tomato nuclei and chicken erythrocytes. In both callus lines the majority of cells contained the same amount of DNA as the initial plant, indicating that no polyploidization has taken place after 5 years of culture on medium containing 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.8 mg/l 6-(γ-γ-dimethylallylamino)purine(zip). Thus, our data suggest that in Cyclamen callus lines there was no strict correlation between the ploidy level and the ability to produce somatic embryos. Furthermore, following the proportion of cells in the three phases of the cell cycle (G0/G1, S, G2/M) during one subculture period of 4 weeks revealed high division activity within the first 2 weeks for both callus lines cultured on the 2,4-D-containing medium. However, when transferred to hormone-free medium, the division activity of the embryogenic cell line decreased markedly, corresponding to the differentiation of somatic embryos. In contrast, for the non-embryogenic callus an increase in cells in the G2/M phase was observed.

Embryogenesis in vitro of several Citrus species and cultivars

SummaryA number of experiments were performed to obtain somatic embryos and embryogenie cell lines from several species and cultivars of citrus by in vitro ovule culture. The source of ovules (pre-anthesis and post-anthesis), incubation conditions (light and darkness) and several modifications of the culture medium were evaluated. All the species and cultivars assayed produced somatic embryos and “pseudobulbi”, but production of friable embryogenie callus was species dependent. The small proliferation of callus and globular bodies recovered from the primary cultures were suitable for recovering embryogenie callus lines by periodical subculturing into fresh medium. After several monthly subcultures discarding embryos and “pseudobulbi”, cell lines virtually devoid of organized structures were obtained from three cultivars of sweet orange, sour orange and Cleopatra mandarin. In all these species, embryogenesis was restored spontaneously and/or by substitution of sucrose by maltose or lactose in the culture medium. The embryos germinated and produced phenotypically normal plants.

Growth Estimation of in vitro Cultured Callus and Plant Regeneration from Leaf Disk and Petiole Callus of Musk Strawberry (Fragaria moschata Duch.) Subcultured for 18 Months.

Callus was induced from leaves and petioles of in vitro cultred Fragaria moschata shoots. The highest callus formation rate was obtained on MS supplemented with NAA 1.0 mg·1-1 and BA 0.025 mg·1-1. These calli were transferred to eight MS media supplemented with different combinations of NAA and BA. After 18 months with periodic subcultures, callus growth was estimated by their area during a 8-week culture period. The highest growth rate was observed on medium supplemented with NAA 0.1 mg·1-1 and BA 0.25 1 mg·1-1 and the lowest on the one supplemented with NAA 0.5 mg·1-1 and BA 0.025 mg·1-1. Callus weight was measured to determine the fidelity of the callus area calculation method as a non-destructive way of estimation the grwoth rate. High correlation was observed between callus area and callus weight (r2=0.974 to 0.996). Afler 18 months in culture, calli were transferred to different regeneration-inducing media enriched with TDZ, BA, ZEA, IBA and NAA. After nearby four months on these media, the calli regenerated shoots which when transferred to a root-inducing medium, they formed normal roots.

Asymmetric Somatic Cell Hybrids between Alfalfa and Birdsfoot Trefoil.

Production of intergeneric somatic hybrids between two sexually incompatible leguminous species, alfalfa (Medicago sativa L.) and birdsfoot trefoil(Lotus corniculatus L.) was carried out. Donor protoplasts of alfalfa were given lethal doses of X-irradiation, and recipient protoplasts of birdsfoot trefoil were inactivated with iodoacetamide. Donor and recipient protoplasts were fused with PEG. Fusion products initiated cell division and colony formation in the culture medium and resulted in callus formation. By using isozyme analysis, it was confirmed that 17 out of 155 calli were somatic cell hybrids. Sorne of the hybrid calli regenerated shoots. By using isozyme analysis it was clarified that the chromosomes or chromosome segments were randomly eliminated from both parents at an early stage of hybrid callus culture. However, after a long period of subculture the isozyme pattern of the recipient type was seen in all of the regenerated shoots which were analyzed. This indicates that the genomes in the calli rapidly converged on the recipient type as subcultures progressed. Southern blot analyses of the chloroplast genomes indicated that either genome of both parents sorted out in all calli investigated.

Production and cryoconservation of embryogenic cultures of mandarin and mandarin hybrids

Assays were performed to obtain embryogenic callus lines from nine mandarin and mandarin hybrid cultivars by in vitro culture of ovules collected from immature fruits six weeks after anthesis. All cultivars produced embryos and loose friable callus. The small proliferations of nucellar callus from cultured ovules were suitable to recover embryogenic callus lines by periodical subculturing to fresh medium. The embryogenic callus cultures were subjected to cryoprotection with 10% (v/v) (DMSO), freezing by slow cooling, storage in liquid nitrogen and thawing by fast warming. Whole plants from these cryopreserved cultures were recovered through embryogenesis.

Sugar Use in Relation to Shoot Induction by Sorbitol and Cytokinin in Apple

Microshoots of `McIntosh' apple (Malus domestica Borkh.) were grown on Murashige-Skoog (MS) nutrient medium supplemented with either sucrose or sorbitol or with sucrose and an elevated level of cytokinin. Shoot growth was recorded and concentrations of fructose, glucose, sorbitol, and sucrose were analyzed in nutrient media and shoots during a 6-week subculture period. Axillary branching was stimulated by high cytokinin and sorbitol media, with increased biomass production and carbohydrate use on the high-BA medium only. The sucrose in the nutrient medium was hydrolyzed to fructose and glucose, which were equally taken up by shoots. Sorbitol was taken up somewhat less effectively. The elevated level of BA decreased sucrose hydrolysis in the nutrient medium. There were high concentrations of sorbitol in shoots grown on the sorbitol medium, and sorbitol also accumulated at the end of the culture period in shoots grown on sucrose. The amount of sucrose was low, and glucose was more abundant than fructose in microshoots. The starch content of leaves was not affected by treatments or sampling time. Chemical names used: N6-benzyladenine (BA).

Micropropagation of Karwinskia parvifolia and the transfer of plants to ex vitro conditions

Zygotic embryos of Karwinskia parvifolia, isolated from seeds obtained from different regions of Mexico, were cultured on Woody Plant Medium (WPM) supplemented with 0.06 µM indole-3-acetic acid, 0.03 µM gibberellic acid, and 2 µM 6-benzylaminopurine. The growth of embryos and multiplication of shoots from stem segments were achieved. Rooting of excised shoots could be initiated on basal WPM medium with prolonged subculture period to 2 months, or on WPM medium supplemented with 10 µM 1-naphthaleneacetic acid. Multiplication capacity of shoots and rooting of K. parvifolia differed in dependence on the origin of explant material. The shoot multiplication was much lower than that of Karwinskia humboldtiana. The rooting depended on the origin of K. parvifolia seeds. The regenerated plants were successfully transferred to glasshouse.

In planta 2,3,5 truodobenzoic acid treatment promotes high frequency and routine in vitro regeneration of sugarbeet (Beta vulgaris L.) plants

The effect ofin planta treatments with auxin inhibitors such as 2,3,5 triiodobenzoic acid (TIBA) on regeneration of plantsin vitro is not known. Here, we show the beneficial effect of preconditioning sugarbeet plants in the greenhouse with TIBA (3 mg/1) for efficientin vitro plant regeneration via a callus phase from cultured leaf explants. Without this treatment, no shoot developed on the control leaf-calluses. Several hundred plants were routinely regenerated using this protocol. More importantly, the number of shoots per explantcallus increased drastically over the subsequent subculture period. The most favorable media for callus induction contained a combination of an auxin and a cytokinin (0.1 mg/1 2,4-dichlorophenoxyacetic acid and 1 mg/1 N-6 benzylaminopurine) or a cytokinin alone (2.2 mg/1 thidiazuron). However, only the callus derived from leaves of TIBA-treated genotypes and induced on thidiazuron-medium produced numerous shoots. Histological studies showed the formation of meristematic zones only in the organogenic callus developed on thidiazuron-coutaining medium. The analysis of peroxidase activity showed that the activity was higher for the TIBA-treated plants than for the untreated control plants.