Violacein cytotoxicity and induction of apoptosis in V79 cells

Summary Violacein, a pigment produced by Chromobacterium violaceum, is reported to be a potential drug for the treatment of Chagas' disease. Violacein is also effective against leukemia and lymphoma cells in culture (IC₅₀ 10⁻⁸ M). Changes in the nuclear acid content, 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide reduction and neutral red uptake in these cells were used to evaluate the cytotoxicity of violacein in V79 Chinese hamster (M-8) fibroblasts. Violacein was highly cytotoxic to V79 fibroblasts (IC₅₀ 5–12 μM). Using the TUNEL method and the Feulgen reaction coupled to image analysis, violacein (5 and 10 μM) was found to trigger apoptosis but not necrosis in V79 cells. The morphological changes seen in the nuclei of these cells included chromatin condensation and a decrease in deoxyribonucleic acid content. These results demonstrating that violacein induces apoptosis in V79 cells strengthen its potential as a therapeutic agent.     

<Emphasis Type="Italic">Alternaria</Emphasis> toxins: DNA strand-breaking activity in mammalian cells<Emphasis Type="Italic">in vitro</Emphasis>

Treatment, for 1 h, of cultured Chinese hamster V79 cells, human liver HepG2 cells, and human colon HT-29 cells with theAlternaria toxins alternariol (AOH) and alternariol methyl ether (AME) caused a concentration-dependent induction of DNA strand breaks at concentrations ranging from 5 to 50 micromolar. After treatment for 24 h, DNA strand breaks were observed in HepG2 but not HT-29 cells. Analysis of the 24 h-incubation media of HT-29 cells showed that both toxins were completely conjugated, whereas 75% were still present as unconjugated compounds in the 24 h-media of HepG2 cells. Lysates of both cell types fortified with UDPGA were found to convert both toxins into two glucuronides each, but HT-29 cells exhibited a much high activity than HepG2 cells and gave rise to a different ratio of glucuronides. It is concluded that glucuronidation eliminates the DNA strandbreaking potential of AOH and AME, and that the two glucuronides of eachAlternaria toxin are generated by different UGT isoforms. Presented at the 29^th Mykotoxin-Workshop, Fellbach, Germany, May 14–16, 2007 Financial support: State of Baden-Württemberg (Research Program “Mycotoxins” as part of the Research Initiative “Food and Health”)     

Additive coclastogenicity of sodium selenite and caffeine in CHO cells treated withN-methyl-N′-Nitro-N-Nitrosoguanidine

The clastogenic effect ofN-methyl-N′-nitro-N-nitrosoguanidine (MNNG) in Chinese hamster ovary (CHO) cells and its modulation by Na₂SeO₃ and caffeine were studied by metaphase analysis of chromosome aberrations (CA) as well as by measuring the formation and repair of single-strand (ss) DNA breaks employing hydroxylapatite chromatography. Treatment of CHO cells with MNNG (1.25 or 2.5 × 10-5M) for 3 h caused CA in 11 and 19% of metaphases scored, respectively. Pretreatment of cells with Na₂SeO₃ (1–5 μg/mL) or caffeine (0.2–2.0 mg/mL) for 2 h resulted in a 2–3.5-fold increase of CA frequency. Addition of both modulators during the mutagen exposure tended to cause a slight inhibition of clastogenic activity of MNNG (1.25 × 10⁻⁵ M) or had no effect on CA number when MNNG was used at a concentration of 2.5 × 10−5M. Posttreatment of CHO cells with Na₂SeO₃ for 20 h after MNNG was ineffective in influencing the number of metaphases with CA, whereas, at these conditions, caffeine enhanced up to 6-7-fold the clastogenic activity of MNNG. Addition of both modulators during the whole experiment, 2 h pretreatment included, resulted in a further significant increase of CA frequency up to the total pulverization of chromosomes in all metaphases scored. The coclastogenic effect of caffeine was greater in this case. The enhancement of chromosome-damaging activity of MNNG by selenite and caffeine was better expressed when this carcinogen was applied at the higher concentration used. An additive coclastogenic effect was observed in CHO cells treated simultaneously with Na₂SeO₃ and caffeine plus MNNG. In addition, the treatment of CHO cells with MNNG (5 × 10⁻⁶ M) caused a rapid increase of ssDNA breaks number reaching maximal values after 30–45 min. However, up to 50–60% of MNNG-induced ssDNA breaks were repaired during the first 60–150 min after the mutagen exposure. The 2 h pretreatment of CHO cells with Na₂SeO₃ (2 μg/mL) or the addition of this trace element after MNNG had no effect on formation and repair of MNNG-induced ssDNA breaks. The coclastogenic effect of Na₂SeO₃ in CHO cells treated with MNNG was not directly linked to the induction and disappearance of ssDNA breaks measured by hydroxylapatite chromatography.     

Modulation of cellular defense processes in human lymphocytes in vitro by a 1,4-dihydropyridine derivative

The aim of this pilot study was to assess whether a compound of the β-carbonyl-1,4-dihydropyridine series (AV-153 or sodium 3,5-bis-ethoxycarbonyl-2,6-dimethyl-1,4-dihydropyridine-4-carboxylate), which has high efficiency in stimulating DNA repair, can simultaneously modulate apoptosis in human cells. Peripheral blood lymphocytes of healthy donors were used in this study. DNA strand-break rejoining was assessed with the alkaline comet assay after a 3-h incubation of lymphocytes in the presence of a wide range of concentrations of AV-153 (10⁻¹⁰–10⁻⁵ M). Apoptotic and micronucleated (MN) cells were scored in phytohaemagglutinin-stimulated lymphocytes after a 72-h incubation with AV-153, using the standard cytokinesis-blocked micronucleus test. The study revealed dual effects of AV-153 on cellular defense systems against endogenously generated DNA damage: the compound per se simultaneously reduces DNA strand breaks and stimulates apoptosis, with a maximal efficiency of 76% and 42%, respectively; in contrast, after genotoxic stress (2 Gy of gamma-radiation) AV-153 reduces DNA strand breaks, the number of MN cells and apoptotic cells in a similar dose-dependent manner. A maximal efficiency of 67% was found for reduction of DNA strand breaks, while for MN cells and apoptotic cells the efficiencies were, respectively, 47% and 44%. While limited in number, these preliminary studies show the direct correlation between the efficiency of AV-153 in reduction of radiation-induced DNA breaks and MN cells on one side, and in reduction of apoptosis on the other. It suggests that the major target of the compound's action on genotoxic stress is DNA repair, followed by reduction of the number of damaged cells entering apoptosis.     

Genotoxicity of chloroquine in rat liver cells: Protective role of free radical scavengers

The genotoxic effect of chloroquine (CQ), a 4-aminoquinoline antimalarial drug was investigated in rat liver cells using the alkaline comet assay. Chloroquine (0–1000 μmol/L) significantly increased DNA strand breaks of rat liver cells dose-dependently. Rat liver cells exposed to CQ (100–500 μmol/L) and treated with endonuclease III and formamidopyrimidine-DNA glycosylase, the bacterial DNA repair enzymes that recognize oxidized pyrimidine and purine, respectively, showed greater DNA damage than those not treated with the enzymes, providing evidence that CQ induced oxidation of purines and pyrimidines. Treatment of cells with 5 mmol/L N-acetylcysteine, an intracellular reactive oxygen species (ROS) scavenger, and 100 μmol/L and 250 μmol/L deferoxamine, an established iron chelator, significantly decreased the CQ-induced strand breaks and base oxidation, respectively. Similarly, the formation of DNA strand breaks and oxidized bases was prevented by vitamin C (10 μmol/L) (a water-soluble antioxidant), quercetin (50 μmol/L) (an antioxidant flavonoid), and kolaviron (30 μmol/L and 90 μmol/L) (an antioxidant and a liver hepatoprotective phytochemical). The results indicate that the genotoxicity of CQ in rat liver cells might involve ROS and that free radical scavengers may elicit protective effects in these cells.     

Callus formation and plant regeneration from tubercles of Coryphantha elephantidens (Lem.) Lem.

Summary Callus cultures were established from pith tissue of Coryphantha elephantidens (Lem.) Lem. on Murashige and Skoog (MS) basal medium supplemented with 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.3 μM kinetin. Highest shoot regeneration frequency was observed on a medium containing 6.9 μM kinetin and 2.3 μM 2,4-D under 30 μE m⁻² s⁻¹ light intensity with a 16-h photoperiod. Calluses retained organogenic potential throughout several passages of subculture (18 mo.). Shoots were rooted on MS medium without plant growth regulators. All (100%) plantlets transplanted to soil survived acclimatization. Regenerated plants showed good overall growth and were morphologically similar to the mother plants.     

Stable transformation of a recalcitrant kentucky bluegrass (Poa pratensis L.) cultivar using mature seed-derived highly regenerative tissues

Summary An efficient method to produce highly regenerative tissues from seeds of a previously recalcitrant cultivar of Kentucky bluegrass (Poa pratensis L. ev. Kenblue) was established under dim-light conditions (10–30 μE m⁻²s⁻¹, 16-h light) using media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D; 4.5 or 9.0 μM), 6-benzylaminopurine (BA; 0.44 or 2.2 μM), and a high level of cupric sulfate (5.0 μM). The tissues were co-transformed with three plasmids containing the genes for hygromycin phosphotransferase (hpt), β-glucuronidase (uidA; gus), and a synthetic green fluorescent protein gene [sgfp(S65T)]. From 463 individual explants bombarded, 10 independent transgenic events (2.2%) were obtained after a 3–4-month selection period for hygromycin resistance using 30–100 mg l⁻¹ hygromycin B; of the 10 independent events, seven (70%) were regenerable. Stable integration of the transgene(s) in transgenic plants was confirmed by polymerase chain reaction and DNA blot hybridization analyses. Coexpression frequency of all three genes was 20%; for two transgenes, either hpt/uidA or hpt/sgfp(S65T), coexpression frequency was 30–40%.     

Tissue culture and plant regeneration of blue grama grass, Bouteloua gracilis (H.B.K.) Lag. Ex Steud

Summary As a first step towards applying biotechnology to blue grama, Bouteloua gracilis (H. B. K.) Lag. ex Steud., we have developed a regenerable tissue culture system for this grass. Shoot apices were isolated from 3-d-old seedlings and cultured in 15 different growth regulator formulations combining 2,4-dichlorophenoxyacetic acid (2,4-D), Picloram (4-amino-3, 5,6-trichloropicolinic acid), N⁶-benzyladenine (BA) or adenine (6-aminopurine). The highest induction of organogenic callus was obtained with formulations containing 1 mg l⁻¹ (4.52 μM) 2,4-D plus 0.5 mg l⁻¹ (2.22 μM) BA. and 2 mg l⁻¹ (8.88 μM) BA plus 1 mg l⁻¹ (4.14 μM) Picloram with or without 40 mg l⁻¹ (296.08 μM) adenine. Lower frequencies of induction were obtained for embryogenic as compared to organogenic callus. The most efficient treatments for induction of embryogenic callus contained 2 mg l⁻¹ (9.05 μM) 2,4-D combined with 0.25 (1.11 μM) or 0.50 mg l⁻¹ (2.22 μM) BA, or 1 mg l⁻¹ (4.52 μM) 2,4-D with 0.50 mg l⁻¹ (2.22 μM) BA. Regeneration was achieved in hormonefree Murashige anmd Skoog (MS) medium, half-strength MS medium or MS medium plus 1 mg l⁻¹ (1.44 μM) gibberellic acid. The number of plantlets regenerated per 500 mg callus fresh weight on MS medium ranged from 9 for 2 mg l⁻¹ (9.05 μM) 2,4-D to 62.2 for induction medium containing 2 mg l⁻¹ (8,28 μM) Picloram, 1 mg l⁻¹ (4.44 μM) BA and 40 mg l⁻¹ (296.08 μM) adenine. Regnerated plants grown in soil under greenhouse conditions reached maturity and produced seeds.     

Application of alkaline unwinding assay for detection of mutagen-induced DNA strand breaks

The frequency of single-strand breaks in parental DNA and gaps in nascent DNA in various cells exposed to methyl methanesulfonate (MMS) or methylnitrosourea (MNU) was investigated by alkaline unwinding assay using two types of alkaline lysis conditions, 22°C lysis versus 0°C lysis. The DNA damage induced by MMS and MNU is considered to be characteristic of lesions produced in DNA by alkylating agents. The aim of our research project was to adjust this method to be able to detect the greatest number of DNA lesions induced by alkylating agents in parental DNA of different mammalian cells. In our experiments we used human cell lines EUE, GM637 and XP12, Chinese hamster V79 cells, and Syrian hamster embryo cells. The higher level of strand interruptions was detected under conditions of lysis of cells at 22°C. Probably the level of strand interruptions found after the lysis of cells at 22°C correlates with the increased number of disrupted alkali-labile sites of DNA. It is remarkable that the different lysis conditions did not influence the number of gaps detected in nascent DNA of alkylated cells. Comparing induction of breaks and gaps in radiolabelled strands of parental and daughter DNA under different lysis conditions, we succeeded in defining the optimum conditions for detection of alkali-labile sites of parental DNA.     

Dose-related influence of sodium selenite on apoptosis in human thyroid follicles in vitro induced by iodine, EGF, TGF-β, and H2O2

Apoptosis of thyroid follicular cells is induced by high doses of iodide, epidermal growth factor (EGF), transforming growth factor-β (TGF-β), as well as H₂O₂ and might be attenuated by antioxidants. Therefore, we examined the apoptotic index induced by these substances in selenium-treated vs untreated human thyroid follicular cells. Reconstituted human thyroid follicles were incubated with sodium selenite (10 or 100 nM) for 72 h; controls received none. The follicles were then distributed to 24-well plates and incubated with potassium iodide (5, 10, or 20 nM), EGF (5 ng/mL), TGF-β (5 ng/mL), or H₂O₂ (100 μM). Apoptosis was determined by a mitochondrial potential assay and the number of apoptotic cells counted by two independent, experienced technicians and the glutathione peroxidase (GPx) activity was determined. A significant increase of apoptic cells was obtained in control thyroid follicles treated with iodine (5, 10, or 20 μM), thyroid-stimulating hormone (TSH) 1, or 10 mU/mL in combination with 5 and 10 μM iodine, EGF (5 ng/mL) and TGF-β (5 ng/mL), or H₂O₂ (100 μM) (p<0.001). In contrast, in thyroid follicles preincubated with 10 or 100 nM sodium selenite, the apoptototic index was identical to the basal rate. In H₂O₂-treated follicles, the apoptotic index was still significantly elevated but 50% lower compared to control cells. The GPx activity increased from 1.4±0.2 to 2.25±0.4 mU/μg DNA with 10 nM selenite and 2.6+0.4 mU/μg DNA with 100 nM selenite. Sodium selenite might increase the antioxidative potential in human thyroid follicles in vitro and therefore diminish the apoptosis induced by TGF-β, EGF, iodide, and even H₂O₂     

Plant regeneration through somatic embryogenesis and rapd analysis of regenerated plants in Tylophora indica (Burm. F. Merrill.)

Summary A procedure for the regeneration of complete plantlets of Tylophora indica from cultured leaf callus via somatic embryogenesis is described. Callus induction from leaf explants was on Murashige and Skoog (MS) medium with different concentrations of 2,4-dichlorophenoxyacetic acid (2.4-D; 0.03–3 mg l⁻¹; 0.0–13.56 μM) and kinetin (Kn; 0.01 mg l⁻¹; 0.05 μM). The best response for callus induction was obtained on MS medium containing 2 mg l⁻¹ (9.04 μM) 2.4-D and 0.01 mg l⁻¹ (0.05 μM) Kn. After two subeultures on the same medium the embryogenic callus was transferred to MS medium with different concentrations of the cytokinin, 6-benzyladenine (0.5–3 mg l⁻¹; 2.22–13.32 μM) and 2-isopentenyladenine (2ip; 0.53 mg l⁻¹; 2.46–14.76 μM) along with 0.01 mg l⁻¹ (0.05 μM) indole-3-butyric acid (IBA) for somatic embryo development and maturation. MS medium with 2 mg l⁻¹ (9.84 μM) 2ip produced the maximum number of mature somatic embryos. The mature embryos were bipolar and on transfer to MS basal medium produced complete plantlets. After hardening the regenerants were planted in the Gudalur forests of Western Ghats. Total DNA was extracted from 14 regenerants and the mother plant. Random amplified polymorphic, DNA (RAPD) analysis was carried out using 20 arbitrary oligonucleotides. The amplification products were monomorphic among all the plants revealing the genetic homogeneity and true-to-type nature of the regenerants.     

Plant regeneration by somatic embryogenesis and organogenesis in commercial pineapple (<Emphasis Type="Italic">Ananas comosus</Emphasis> L.)

Summary Axillary and terminal buds from suckers of Ananas comosus cv. Phuket were established on Murashige and Tucker-based (MT) medium with 2.0 mgl⁻¹ (9.8 μM) indolebutyric acid, 2.0 mgl⁻¹ (10.74 μM) naphthaleneacetic acid, and 2.0 mgl⁻¹ (9.29 μM) kinetin, followed by multiplication on Murashige and Skoog-based (MS) medium containing 2.0 mgl⁻¹ (8.87 μM) benzyladenine (BA) to provide a continuous supply of axenic shoots. Leaves, excised from such cultured shoots, produced adventitious shoots from their bases when these explants were cultured on MS medium containing 0.5 mgl⁻¹ (2.26 μM) 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.0 mgl⁻¹ (8.87 μM) BA. Embryogenic callus was produced when leaf explants were cultured on MS medium with 3.0 mgl⁻¹ (12.42 μM) 4-amino-3,5,6-trichloropicolinic acid (picloram). Somatic embryos developed into shoots following transfer of embryogenic tissues to MS medium with 1.0 mgl⁻¹ (4.44 μM) BA. Cell suspensions, initiated by transfer of embryogenic callus to liquid MS medium with 1.0 mgl⁻¹ (4.14 μM) picloram or 1.0 mgl⁻¹ (4.52 μM) 2,4-D, also regenerated shoots by somatic embryogenesis, on transfer of cells to semisolid MS medium with 1.0 mgl⁻¹ (4.44 μM) BA. All regenerated shoots rooted on growth regulator-free MS medium, prior to ex vitro acclimation and transfer to the glasshouse. These studies provide a baseline for propagation, conservation, and genetic manipulation of elite pineapple germplasms.     

DNA damage in mouse lymphocytes exposed to curcumin and copper

Dietary polyphenolics, such as curcumin, have shown antioxidant and anti-inflammatory effects. Some antioxidants cause DNA strand breaks in excess of transition metal ions, such as copper. The aim of this study was to evaluate thein vitro effect of curcumin in the presence of increasing concentrations of copper to induce DNA damage in murine leukocytes by the comet assay. Balb-C mouse lymphocytes were exposed to 50 μM curcumin and various concentrations of copper (10 μM, 100 μM and 200 μM). Cellular DNA damage was detected by means of the alkaline comet assay. Our results show that 50 μM curcumin in the presence of 100–200 μM copper induced DNA damage in murine lymphocytes. Curcumin did not inhibit the oxidative DNA damage caused by 50 μM H₂O₂ in mouse lymphocytes. Moreover, 50 μM curcumin alone was capable of inducing DNA strand breaks under the tested conditions. The increased DNA damage by 50 μM curcumin was observed in the presence of various concentrations of copper, as detected by the alkaline comet assay.     

Alteration of cadmium-induced mutational spectrum by catalase depletion in Chinese hamster ovary-K1 cells

Previously, we have demonstrated that cadmium acetate significantly induces hprt mutation frequency in Chinese hamster ovary (CHO)-K1 and that 3-amino-1,2,4-triazole (3AT), a catalase inhibitor, potentiates the mutagenicity of cadmium [Chem. Res. Toxicol. 9 (1996) 1360–1367]. In this study, we investigate the role of intracellular peroxide in the molecular nature of mutations induced by cadmium. Using 2′,7′-dichlorofluorescin diacetate and fluorescence spectrophotometry, we have shown that cadmium dose-dependently increased the amounts of intracellular peroxide and the levels were significantly enhanced by 3AT. Furthermore, we have characterized and compared the hprt mutation spectra in 6-thioguanine-resistant mutants derived from CHO-K1 cells exposed to 4 μM of cadmium acetate for 4 h in the absence and presence of 3AT. The mutation frequency induced by cadmium and cadmium plus 3AT was 11- and 16-fold higher than that observed in untreated populations (2.2×10⁻⁶), respectively. A total of 40 and 51 independent hprt mutants were isolated from cadmium and cadmium plus 3AT treatments for mRNA-polymerase chain reaction (PCR), genomic DNA-PCR and DNA sequencing analyses. 3AT co-administration significantly enhanced the frequency of deletions induced by cadmium. Cadmium induced more transversions than transitions. In contrast, 3AT co-administration increased the frequency of GC→AT transitions and decreased the frequencies of TA→AT and TA→GC transversions. Together, the results suggest that intracellular catalase is important to prevent the formation of oxidative DNA damage as well as deletions and GC→AT transitions upon cadmium exposure.     

DNA double-strand breaks in mammalian cells exposed to Á-rays and very heavy ions

The spatial distribution of DNA double-strand breaks (DSB) was assessed after treatment of mammalian cells (V79) with densely ionizing radiation. Cells were exposed to beams of heavy charged particles (calcium ions: 6.9 MeV/u, 2.1⋅10³ keV/μm; uranium ions: 9.0 MeV/u, 1.4⋅10⁴ keV/μm) at the linear accelerator UNILAC of GSI, Darmstadt. DNA was isolated in agarose plugs and subjected to pulsed-field gel electrophoresis under conditions that separated DNA fragments of size 50 kbp to 5 Mbp. The measured fragment distributions were compared to those obtained after γ-irradiation and were analyzed by means of a convolution and a deconvolution technique. In contrast to the finding for γ-radiation, the distributions produced by heavy ions do not correspond to the random breakage model. Their marked overdispersion and the observed excess of short fragments reflect spatial clustering of DSB that extends over large regions of the DNA, up to several mega base pairs (Mbp). At fluences of 0.75 and 1.5/μm², calcium ions produce nearly the same shape of fragment spectrum, merely with a difference in the amount of DNA entering the gel; this suggests that the DNA is fragmented by individual calcium ions. At a fluence of 0.8/μm² uranium ions produce a profile that is shifted to smaller fragment sizes in comparison to the profile obtained at a fluence of 0.4/μm²; this suggests cumulative action of two separate ions in the formation of fragments. These observations are not consistent with the expectation that the uranium ions, with their much larger LET, should be more likely to produce single particle action than the calcium ions. However, a consideration of the greater lateral extension of the tracks of the faster uranium ions explains the observed differences; it suggests that the DNA is closely coiled so that even DNA locations several Mbp apart are usually not separated by less than 0.1 or 0.2 μm. Received: 27 January 1998 / Accepted in revised form: 15 April 1998     

Changes in the DNA lesions induced by alkylating agents during the post-treatment washing and redrying of barley seeds

During the 24 hours’ washing of¹⁴C MNU and¹⁴C MMS-treated seeds the amount of DNA 7-methylguanine as well as the total alkylation of DNA, RNA and proteins in cells of the embryo was enhanced. The drying of shortly washed¹⁴C MNU-treated seeds to 30% and 15% water content led to, a similar increase of alkylation. In accordance with the changes in the biological damage, the amount of single strand breaks and/or alkali labile sites in DNA dropped down during a 19 h washing period of MNU-treated seeds but changed only slightly in MMS-treated and 19 hours washed seeds. The drying of 0·5 or 18 h resp. 19 h washed seeds treated either with MNU or MMS to 15 per cent water content increased the amount of single strand breaks and/or alkali labile sites in DNA.     

Photoperiodically independent flowering of Pharbitis nil plants regenerated from flower buds

Summary Flower buds and anthers of the short-day plant Pharbitis nil were treated either with thermic shock (7 or 35°C) or osmotic/trophic shock (12% sucrose) for 24 h. Explants were transferred either to Murashige and Skoog medium (MS) with addition of 6-benzylaminopurine (BA; 4.4μM) and 6% sucrose or to the same growth medium containing 22 μM BA and 3% sucrose. Both media were supplemented with α-naphthaleneacetic acid (NAA; 0.55 μM). Osmotic/trophic shock stimulated the occurrence of shoots on flower buds grown on medium containing 22 μM BA. Thermic shock (7 and 35°C) inhibited this process on both types of explants. Regenerated plantlets were transferred to MS medium supplemented with 6% sucrose, gibberellic acid (GA₃; 1.44μM), NAA (0.55 μM) and Ca²⁺ (0.66 mgl⁻¹). After 3–4 wk they were able to produce flowers without photoperiodic induction.     

Plant regeneration from mesophyll protoplasts of a medicinal plant, Phellodendron amurense rupr.

Summary A regeneration system from protoplast to plantlet for a medicinal plant species, Phellodendron amurense Rupr., has been developed. Leaves of micropropagated shoots or plantlets were selected as plant materials for protoplast isolation. The yield and viability of leaf protoplasts were greatly influenced by enzyme combination, treatment time and osmoticum. The highest viability (86%) with a yield of 7.1×10⁵ protoplasts per gram fresh weight was obtained with a 6-h digestion in 1% Cellulase Onozuka R-10 plus 1% Driselase-20. Sustained cell division and colony formation from the protoplasts were best supported at a plating density of 4×10⁵−6×10⁵ protoplasts per milliliter using a 0.2% gellan gum-solidified or liquid MS (Murashige and Skoog, 1962) medium containing 0.6M mannitol, 2.0μM 6-benzylaminopurine (BA) with 4.0 μM α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), or 2,4-dichlorophenoxyacetic acid (2,4-D). The protoplast-derived colonies formed green compact calluses when transferred to a solidified MS medium containing 2.0 μM BA with 4.0μM NAA of IBA. Shoot regeneration from protoplast-derived calluses was induced on MS medium supplemented with 2.0 μM BA and 1.0μM NAA or 2.5μM IBA. Shoot multiplication and elongation occurred on MS medium containing 1.0μM BA. In vitro-grown shoots were rooted on MS medium with either 0.5–4.0μM IBA or NAA. Regenerants were transferred to the Kanuma soil and successfully established under greenhouse conditions.     

The effects of low concentrations of aluminium on the growth and uptake of nitrate-N by white clover

Summary The effects of aluminium (Al³⁺) at concentrations of 0, 25, 50 and 100 μM on the growth of white clover, dependent upon N supplied as NO ₃ ⁻ , were examined in flowing solution culture. Plants were established with a normal nutrient supply for 7 weeks and then grown with carefully controlled pH (at 4.5) and P concentrations, and with 0, 25, 50 or 100 μM Al³⁺ for a further three weeks. There were rapid visual effects (i.e. symptoms of P deficiency and reduction in root extension) and the dry weights of shoots and roots were reduced at 50 and 100 μM. Less than 10% of Al absorbed from solution was transported to the shoots. The uptake of P, and its transport between roots and shoots, were reduced in plants grown with Al. The uptake of NO ₃ ⁻ stopped immediately after the introduction of 50 or 100 μM Al, and was significantly reduced at 25 μM after three weeks. During a second phase of the experiment, plants previously grown at 0, 25, 50 and 100 μM Al, were grown for a further 2 weeks either with NO ₃ ⁻ (with and without 50 μM Al³⁺) or without NO ₃ ⁻ but with inoculation by Rhizobia (and with or without 50 μM Al³⁺). The effects of the previous treatments with Al on N uptake were small during the second phase, but uptake by all plants was restricted when Al was present. Inoculation did not result in nodulation in the second phase when Al³⁺ was present in the solution, but Al already in the plant from the first phase did not prevent nodulation in the absence of Al during the second phase.     

Improved method of regeneration of black gram (Vigna mungo L.) through liquid culture

Summary A very rapid and efficient regeneration method of Vigna mungo L. has been established using liquid culture. A highly regenerable explant, viz., young multiple shoots obtained by germinating the seeds in 2 mgl⁻¹ (8.9μM) N⁶-benzyladenine-supplemented Murashige and Skoog (MS) medium, was used as a source of tissue to initiate the liquid culture. The liquid medium consisted of half-strength B5 or MS salts supplemented with MS organics, α-naphthaleneacetic acid (0.1 mgl⁻¹, 0.54μM) and N⁶-benzyladenine (0.5mgl⁻¹, 2.2μM). Transferring the growing tissues to fresh medium every third day resulted in ca. 142% increase in the number of shoot buds produced after 24d. Shoot buds elongated on one-third-strength MS (MS_1/3) semisolid medium and plantlets were obtained by transferring the shoots onto MS_1/3 semisolid medium supplemented with indolebutyric acid (1 mgl⁻¹, 4.9 μM).