Role of vacuolar adenosine triphosphatase in the regulation of cytosolic pH in hepatocytes

The responses of the cytosolic pH of hepatocytes in suspension to agents affecting the activity of vacuolar adenosine triphosphatase (V-ATPase) and Na/H exchange have been studied. Changes of cytosolic pH were determined both with dual-wavelength excitation (500/440 nm) of the fluorescence of 2,7-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein and from the distribution of ¹⁴C-dimethyloxazolidinedione; both methods gave very similar results. Changes of vesicular pH were determined by comparing the fluorescence of fluorescein isothiocyanate-dextran and rhodamine B isothiocyanate-dextran taken up by endocytosis. Nitrate, which inhibits V-ATPase in isolated organelles, induced a concentration-dependent acidification of the cytosol and alkalinization of vesicles, with maximal effects at 25–37.5 mm in each case, indicating that V-ATPase contributes to removal of cytosolic protons. On continued exposure to nitrate, the acidification underwent an amiloride-inhibitable reversal. At the higher concentrations of NO ₃ ^– , both cytosolic acidification and vesicular alkalinization were reduced or absent. Bafilomycin A₁ caused alkalinization of vesicular pH; cytosolic acidification was not observed, possibly because of other ionic exchanges. Recovery of cytosolic pH from an acid load (2 min exposure to 5% CO₂) was sensitive to both 25 mm NO ₃ ^– and to ouabain. The pH dependence of the nitrate effect was tested with media of different pH; the activity was negligible at cytosolic pH 6.2 and rose to a maximum at cytosolic pH 7.3. Treatment of hepatocytes with 0.5–1.0 mm ouabain resulted in an initial alkalinization (0.5–2 min duration) of the cytosol, followed by a spontaneous reversal and, on occasion, further acidification. The alkalinization was blocked by 25 mm NO ₃ ^– , but not by 25 mm gluconate. The results suggest that the cytosolic alkalinization is caused by a stimulation of H⁺ uptake by V-ATPase activity. We conclude that V-ATPases make an important contribution to the regulation of the cytosolic pH of hepatocytes.This work was supported in part by National Institutes of Health B.R.S. Grant 507 RR05417 to Temple University.     

Effect of calcium on the absorption and translocation of heavy metals in excised barley roots: Multi-compartment transport box experiment

Summary The effect of Ca on the absorption and translocation of Mn, Zn and Cd in excised barley roots was studied using a multi-compartment transport box technique. A radioisotope (⁵⁴Mn,⁶⁵Zn or^115mCd)-labelled test solution was supplied to the apexes of excised roots and the distribution pattern in the roots was examined in the absence or presence of Ca. Results obtained were as follows. Addition of Ca to the test solution reduced the absorption of Mn and inhibited drastically its translocation in excised roots. With increasing concentrations of Ca in test solutions, its inhibitory effects on the absorption and translocation of Mn became severe. Similar results were observed for the absorption and translocation of Zn. Ca in the test solution decreased the absorption and inhibited drastically the translocation of Zn; as in the case of Mn, higher concentrations of Ca had severe effects on these functions. It was also evident that the addition of Ca to the test solution reduced the absorption of Cd at all levels of Cd concentration (1, 10, and 100 μM). Cd absorption decreased with increasing concentrations of Ca in the test solution. However, Ca accelerated the translocation of Cd in excised roots supplied with test solutions containing up to 10μM Cd. At 100μM Cd, addition of Ca caused a negligibly small acceleration of Cd translocation. The accelerating effect of Ca on Cd translocation, especially “xylem exudation”, decreased markedly with the addition of 2,4-dinitrophenol, but not with the addition of chloramphenicol or p-chloromercuribenzene sulphonic acid. When barley plants were supplied with only CaSO₄ during the entire growing period, that is, plants were not supplied with nutrient solution on the last day of this period, Ca had no accelerating effect on Cd translocation in excised roots.     

The effect of oxygen tension on rat hepatocytes in short-term culture

Summary Cell viability, cytochrome P-450 content, cell respiration, and lipid peroxidation were all investigated as a function of oxygen tension in adult rat hepatocytes in short-term culture (less than 9 h). The various oxygen tensions used in this study were obtained by equilibrating culture medium with air, air + nitrogen, or air + oxygen. Cell viability, as assessed by trypan blue exclusion, was significantly greater at all time points tested when hepatocytes were cultured in Ham's F12 medium containing 132 μM O₂, as compared to medium equilibrated with air (220 μM O₂) or air + oxygen (298 μM O₂). Cells cultured in 220 μM O₂ (air) also exhibited a gradual loss of cytochrome P-450, so that by 9 h of incubation less than 60% of the active material remained. This loss of P-450 was minimized when cells were cultured in 163 μM O₂ and abolished when cells were cultured in 132 μM O₂. The 132 μM O₂ exposure conditions also maintained cell respiration at the 1 h incubation values, whereas there was a continuous loss in cell respiration over time when the cells were cultured in either 220 μM O₂ (air) or 298 μM O₂ (air:O₂). These cytotoxicity findings may be related to oxidative cell damage inasmuch as it was additionally demonstrated that lipid peroxidation (as measured by malondieldehyde equivalents) was consistantly lower in hepatocytes cultured in air:N₂ as compared to air or air:O₂. These results suggest that hepatocyte culture in low oxygen tension improves not only cell viability but also maintains other functional characteristics of the cell. This work was supported by a Biomedical Research Support Grant S-S07-RR 05448 awarded to the University of Minnesota School of Public Health by the Biomedical Research Grant Program, Division of Research and Resources, National Institutes of Health, Bethesda, MD.     

Protective mechanism of sodium molybdate against the acute toxicity of cadmium in rats. II. Prevention of cytoplasmic acidification

In order to clarify the protective mechanism of sodium molybdate against the acute toxicity of cadmium chloride in rat, the effect of in vivo sodium molybdate pretreatment on the cytotoxic action of cadmium in isolated hepatocytes was studied. The cytosolic pH of hepatocytes isolated from untreated rats immediately decreased with incubation in either neutral Hank's balanced salt solution (HBS), pH 7.4, containing 5 µM cadmium chloride minimum or acidic HBS (pH 7.1, 6.8, 6.5, and 6.2). The presence of 5 µM cadmium in HBS adjusted to pH 7.1 aggravated cytosalic acidification induced by the acidic medium alone. Cell viability of hepatocytes incubated in HBS at pH 6.2 was significantly reduced as compared to that of control cells in HBS at pH 7.4, but the presence of cadmium in the acidic HBS had no aggravating action against such a toxic action of the acidic medium although cellular uptake of the metal in the medium increased, as compared to that in HBS at pH 7.4. Molybdenum pretreatment alleviated cytoplasmic acidification induced by the treatment with HBS at pH 7.4 or 7.1 containing cadmium or by extracellular acid load wothout cadmium. This pretreatment also prevented the loss of cell viability induced by the treatment with HBS at pH 6.2 but could not attenuate that when cadmium was present in the medium.These facts suggest that molybdenum pretreatment alleviated the acute toxicity of cadmium in rat by preventing cytoplasmic acidification caused by the harmful metal.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Eucalyptus phylacis</Emphasis> L. Johnson and K. Hill., A critically endangered relict from Western Australia

Summary In vitro methods were applied to the only remaining plant of the Meelup Mallee (Eucalyptus phylacis), a critically endangered species from the southwest of Western Australia. Shoot explants were initiated into culture using a 1/2 MS [Murashige and Skoog basal medium (BM) for all experiments] liquid medium supplemented with 1% (w/v) activated charcoal, which was replenished twice daily, followed by transfer of explants to agar medium supplemented with 0.5 μM zeatin. Explants were cultured under low intensity lighting (PPFD of 5–10 μmol m⁻²s⁻¹) to minimize blackening of tissues, and some explants were induced to produce nodular green calluses in response to BM supplemented with 5 μM thidiazuron. Nodular green calluses were induced to form adventitious shoots following transfer to medium supplemented with 0.5 μM zeatin and 1 μM gibberellic acid, A₄ isomer (GA₄). Development of shoots was completed on 1 μM zeatin + 0.1 μM 6-benzylaminopurine (BA) in vented culture tubes. Regenerated shoots were sequentially cultured on medium containing 0.5 μM zeatin + 0.2 μM indoleacetic acid (IAA) followed by either 0.5 μM zeatin + 1μM GA₄ for shoot elongation or 1 μM zeatin + 0.5 μM IAA to optimize shoot growth. Rooted microshoots were produced after 4 weeks on 5 μM indolebutyric acid (IBA) and survived acclimatization and transfer to potting mixture.     

Optimization of culture conditions for determining hepatobiliary disposition of taurocholate in sandwich-cultured rat hepatocytes

Summary This study was undertaken to examine the influence of time and volume of collagen overlay, type of media, and media additives on taurocholate (TC) accumulation and biliary excretion in hepatocytes cultured in a collagen-sandwich configuration. Hepatocytes were isolated from male Wistar rats by in situ perfusion with collagenase, seeded onto collagencoated 60-mm dishes, overlaid with gelled collagen, and cultured for 4 d. Experiments to examine the influence of time and volume of collagen overlay were conducted in Dulbecco's modified Eagle's medium (DMEM)+1.0μM dexamethasone (DEX)+5% fetal bovine serum (FBS). Hepatocytes were overlaid at 0 h with 0.1 or 0.2 ml collagen, or at 24 h with 0.1 or 0.2 ml collagen. The influence of media type and additives was examined in hepatocytes overlaid at 0 h with 0.2 ml collagen and incubated in DMEM+0.1μM DEX, DMEM+0.1μM DEX+5% FBS, Williams' medium E+0.1μM DEX+1% ITS^Θ+, DMEM +1.0μM DEX, DMEM+1.0 μM DEX+5% FBS, or modified Chee's medium (MCM)+0.1 μM DEX+1% ITS^Г+. [³H] TC accumulation by hepatocytes in Hank's balanced salt solution (HBSS) and Ca²⁺-free HBSS was measured, and the biliary-excretion index (BEI: percentage of accumulated TC localized in the canalicular compartment) was calculated. Light microscopy and carboxydichlorofluorescein fluorescence were employed to examine the cellular and canalicular morphologies. The volume of collagen used for both the substratum and the overlay did not affect TC accumulation or biliary excretion. The BEI tended to be higher in cells overlaid at 24 h (BEI=0.649 [0.1 ml collagen]; BEI=0.659 [0.2 ml collagen]) compared with those overlaid at 0 h after seeding (BEI=0.538 [0.1 ml collagen]; BEI=0.517 [0.2 ml collagen]), although the differences were not statistically significant. Hepatocytes cultured in MCM produced consistently the lowest BEI of TC (BEI=0.396). Differing DEX concentration (0.1 μM versus 1.0 μM) with or without 5% FBS did not appear to have a significant effect on the BEI of TC.     

Effects of plant growth regulators on high frequency shoot multiplication and callus regeneration of an important Indian medicinal plant, nirgundi (Vitex negundo L.)

Summary A rapid shoot multiplication protocol was established for an important medicinal plant, Vitex negundo L., belonging to the family Verbenaceae, using Murashige and Skoog medium, achieved by shoot multiplication as well as callus regeneration. Shoot multiplication was induced by different concentrations of 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ), Benzyladenine and 6-furfuryl amino purine separately along with 10% (v/v) coconut water. Green organogenetic callus was obtained by the combined effect of 0.5–2.15 μM TDZ and 1.7 μM indole-3-acetic acid (IAA) along with 1% polyvinylpyrrolidone (PVP), and produced the maximum number of shoots when subcultured onto medium containing 2.7 μM TDZ alone. Elongation of in vitro shoots was observed in MS medium containing 2.4 μM gibberellic acid and rooting was induced by the combined effect of 1.71 μM IAA and 1.62 μM α-naphthalene acetic acid.     

Plant regeneration via organogenesis from adventitious bud explants of a medicinal herb species, <Emphasis Type="Italic">Polygonatum cyrtonema</Emphasis>

Summary Explants derived from adventitious buds, rhizomes, stems, and leaves of a medicinal plant, Polygonatum cyrtonema, were studied for plantlet regeneration, and only adventitious bud explants were able to be regenerated into plantlets. Regeneration was also accompanied by the formation of rhizome-like tissue, the medicinal portion of the plant. The optimum hormone combination for plantlet regenertion was 4.44 μM benzyladenine plus 2.26 μM 2,4-dichlorophenoxyacetic acid, at which new adventitious buds were obtained from 96.6% of the adventitious bud explants, with an average of 5.2 buds per explant. The best medium for root induction was half-strength Murashige and Skoog medium with 4.57 μM α-naphthaleneacetic acid, as 92% of regenerated buds rooted. Regenerated plantlets were successfully transferred to a greenhouse with 86% survival. Histological observation indicated that new adventitious buds originated from the superficial meristematic cell cluster of the granular callus induced from adventitious bud explants via organogenesis.     

In vitro plant regeneration of spinach from mature seed-derived callus

Summary A system for the regeneration of spinach (Spinacia oleracea L.) from mature dry seed explants has been established. The response of two commercial spinach cultivars, ‘Grandstand’ and ‘Baker’, was examined. Callus proliferation was most prominent on MS medium supplemented with 9.3 μM of 6-furfurylaminopurine (kinetin) and 3.39 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Adventitious shoot formation was observed within 8 wk after callus was transferred onto regeneration medium. Shoot regeneration was best from callus induced on 9.3 μM kinetin and 4.56 μM 2,4-D. The regeneration medium contained 9.3 μM kinetin, 0.045 μM 2,4-D, and 2.89 μM gibberellic acid (GA₃). Shoots were rooted on hormone-free medium, and plants grown in a greenhouse showed normal phenotype. This system is beneficial in rapid propagation of spinach plants, particularly when only a limited number of seeds are available.     

Effects of Metal Combinations on the Production of Phytochelatins and Glutathione by the Marine Diatom Phaeodactylum tricornutum

Copper, Cd and Zn can be found at elevated concentrations in contaminated estuarine and coastal waters and have potential toxic effects on phytoplankton species. In this study, the effects of these metals on the intracellular production of the polypeptides phytochelatin and glutathione by the marine diatom Phaeodactylum tricornutum were examined in laboratory cultures. Single additions of Cu and Cd (0.4 μM Cu² and 0.45 μM Cd²⁺) to the culture medium induced the production of short-chained phytochelatins ((γ-Glu-Cys)_n-Gly where n = 2–5), whereas a single addition of Zn (2.2 μM Zn²⁺) did not stimulate phytochelatin production. Combination of Zn with Cu resulted in a similar phytochelatin production compared with a single Cu addition. The simultaneous exposure to Zn and Cd led to an antagonistic effect on phytochelatin production, which was probably caused by metal competition for cellular binding sites. Glutathione concentrations were affected only upon exposure to Cd (85% increase) or the combination of Cd with Zn (65% decrease), relative to the control experiment. Ratios of phytochelatins to glutathione indicated a pronounced metal stress in response to exposures to Cu or Cd combined with Zn. This study indicates that variabilities in phytochelatin and glutathione production in the field can be explained in part by metal competition for cellular binding sites.     

Effect of zinc and cadmium on physiological and production characteristics in <Emphasis Type="Italic">Matricaria recutita</Emphasis>

Effects of zinc (12–180 μM) alone and in mixtures with 12 μM Cd on metal accumulation, dry masses of roots and shoots, root respiration rate, variable to maximum fluorescence ratio (F_V/F_M), and content of photosynthetic pigments were studied in hydroponically cultivated chamomile (Matricaria recutita) plants. The content of Zn in roots and shoots increased with the increasing external Zn concentration and its accumulation in the roots was higher than that in the shoots. While at lower Zn concentrations (12 and 60 μM) the presence of 12 μM Cd decreased Zn accumulation in the roots, treatment with 120 and 180 μM Zn together with 12 μM Cd caused enhancement of Zn content in the root. Presence of Zn (12–120 μM) decreased Cd accumulation in roots. On the other hand, Cd content in the shoots of plants treated with Zn + Cd exceeded that in the plants treated only with 12 μM Cd. Only higher Zn concentrations (120 and 180 μM) and Zn + Cd mixtures negatively influenced dry mass, chlorophyll (Chl) and carotenoid content, F_V/F_M and root respiration rate. Chl b was reduced to a higher extent than Chl a.     

Effects of Methyl jasmonate with indole-3-acetic acid and 6-benzylaminopurine on the secondary metabolism of cultured <Emphasis Type="Italic">Onosma paniculatum</Emphasis> cells

Summary Methyl jasmonate (MeJA) interacted significantly with both indole-3-acetic acid (IAA) and 6-benzylaminopurine (BA) to influence cell growth of cultured Onosma paniculatum cells. Cell growth decreased with increasing concentrations of MeJA from 0.004–4.45 μM with or without IAA and BA. The same concentrations of MeJA (0–4.45 μM) increased the cell growth with IAA and BA, when administered to the cultured cells in M₉ medium. This was found to enhance the production of shikonin. The optimum time for MeJA addition for enhanced shikonin formation was 4 d after cell inoculation in M₉ medium. Furthermore, shikonin formation was affected significantly by both MeJA/IAA and MeJA/BA combinations. Shikonin content was enhanced by increasing MeJA concentrations with IAA concentrations in the range of 0–28 μM and with BA concentrations in the range of 0–44.38 μM in MeJA/BA experiments, respectively. The optimal combination of MeJA and IAA was 4.45 μM and 0.28 μM, while MeJA and BA concentrations of 4.45 μM and 2.22 μM were optimal for shikonin formation. The result also showed that MeJA increased phenylalanine ammonia-lyase (PAL) and p-hydroxybenzoic acid-geranyltransferase (PHB-geranyltransferase) activites during the course of shikonin formation, but decreased the activity of PHB-O-glucosyltransferase within 9 d after inoculation. These results suggest that enhanced shikonin formation in cultured Onosma paniculatum cells induced by MeJA involves regulation of the key enzyme activities.     

Metallothionein induction in freshly isolated rat hepatocytes

The control of metallothionein (MT) synthesis was investigated in freshly prepared rat hepatocytes in experiments of short-term duration. Viability and metabolic function were maintained in incubations of 6-h duration. MT synthesis was measurable in hepatocytes from fed rats at Zn concentrations down to 1 μM. Zn and dexamethasone induced concentration-dependent increases in the synthesis of MT with maximal increases above the 5-h control of 3.2- and 2.5-fold, respectively. Zn induction of MT was first measurable at 2 h and was inhibited by actinomycin C. Although initial (0 h) MT concentrations in hepatocytes from fasted rats were double those from fed rats, after 6-h incubation in the presence of 50 μM Zn, the fasted rat hepatocytes showed only half the MT concentrations of the fed rat hepatocytes. Glucagon and interleukin-6 (IL-6) were less effective inducers and increased MT synthesis by 28 and 17%, respectively. IL-6 (100 U/mL) was found to have an additive effect on MT synthesis above that of Zn alone (1–50 μM) or Zn plus dexamethasone (1 μM). A supernatant from LPS-stimulated macrophages increased MT synthesis by 40%. The basal MT synthesis was not increased by either tumor necrosis factor-α (TNF-α) or interleukin-1 (IL-1). All incubations were carried out in the presence of RPMI 1640 medium with Hepes (20 mM), bicarbonate (24 mM), and fatty acid-free albumin (FAFA; 0.5% w/v). MT synthesis was also seen using Krebs bicarbonate buffer with glucose (10 mM), Hepes (20 mM), and FAFA (0.5% w/v), and although the level of MT synthesis was less than in RPMI, the increases in concentrations of MT at 5 h were 225, 139, 36 and 20% for Zn, dexamethasone, glucagon, and control, respectively. It is concluded that MT synthesis occurs in freshly prepared hepatocytes and that these cells are responsive to some of the established inducers of MT. This system enables the study of MT synthesis in individual rats in various metabolic and pathological states.     

Somatic embryogenesis and plant regeneration from petioles of Parthenocissus tricuspidata planch

Summary A protocol of somatic embryogenesis and plant regeneration from petiole segments of Parthenocissus tricuspidata Planch. has been developed. Embryogenic tissue was induced on B5 (Gamborg) basal medium supplemented with 2.25–9.0 μM 2,4-dichlorophenoxyacetic acid, 500 mg l⁻¹ casein hydrolysate (CH), and 0.1 gl⁻¹ activated charcoal. Somatic embryos were induced on B5 medium containing various concentrations of benzyladenine (BA) (4.44, 6.66, and 8.88 μM) and α-naphthaleneacetic acid (NAA) (0, 0.54, and 1.61 μM) plus 500 mg l⁻¹ CH. Ninety percent of normal somatic embryos were converted into plantlets directly on Murashige and Skoog (MS) medium free of plant growth regulators. Shoots could be induced from abnormal somatic embryos on MS medium containing 4.44 μM BA, 0.05 μM NAA, and 500 mg l⁻¹ CH. Genotypic differences were found in the process of somatic embryogenesis and plant regeneration. Histological analysis confirmed the process of somatic embryogenesis. Regenerated plantlets with well-developed roots were successfully acclimatized in greenhouse and all plants showed normal morphological characteristics.     

Ontogeny of somatic embryos in cucumber (<Emphasis Type="Italic">cucumis sativus</Emphasis> L.)

Summary Suspension culture of cucumber (Cucumis sativus L.) has been an inefficient method for production of somatic embryos owing to problems with embryo maturation and conversion. Embryogenic callus of cv. Green Long was induced on semisolid Murashige and Skoog (MS) medium containing 6.8 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.2 μM 6-benzylaminopurine (BA). A large number of globular somatic embryos were obtained on transfer of the callus to MS liquid medium supplemented with 87.6 mM sucrose, 1.1 μM 2,4-D, and improved by the addition of 342.4 μM l-glutamine. MS medium supplemented with 87.6 mM sucrose was more effective in somatic embryo production than other sugars. Subsequent development led to the formation of heart-and torpedo-shaped embryos. Maturation of somatic embryos occurred on plant growth regulator-free MS semi-solid medium containing 175.2 mM sucrose and 0.5 gl⁻¹ activated charcoal. Conversion of embryos into plants was achieved on half-strength MS semi-solid medium containing 87.6 mM sucrose and 1.4 μM gibberellic acid (GA₃) in a 16h photoperiod. Twenty-seven percent of embryos were converted into normal plants.     

AMP-activated protein kinase plays a role in initiating metabolic rate suppression in goldfish hepatocytes

In the present study, we test the hypothesis that AMP-activated protein kinase (AMPK) initiates metabolic rate suppression in isolated goldfish hepatocytes. To accomplish this, we attempted to pharmacologically activate AMPK in goldfish hepatocytes with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and the thienopyridone, A769662, to examine the effects of AMPK activation on eukaryotic elongation factor-2 (eEF2), protein synthesis, and cellular oxygen consumption rate ( [(M)\dot]_{\textO 2} \dot{M}_{{{\text{O}}_{ 2} }} ). Goldfish hepatocytes treated with 1 mM AICAR under normoxic conditions (>200 μM O₂) showed a modest but significant 1.1-fold increase in AMPK phosphorylation, a 7.5-fold increase in AMPK activity, a 1.4-fold increase in eEF2 phosphorylation, and a 24% decrease in [(M)\dot]_{\textO 2} \dot{M}_{{{\text{O}}_{ 2} }} . At physiologically relevant [O₂] (<40 μM O₂), the addition of 1 mM AICAR resulted in only a 13% decrease in cellular [(M)\dot]_{\textO 2} \dot{M}_{{{\text{O}}_{ 2} }} with no change in sensitivity to [O₂] as assessed by estimates of cellular P₅₀ and P₉₀ values. The addition of compound C, a general protein kinase inhibitor, after AICAR incubation did not reverse the effects of AICAR on [(M)\dot]_{\textO 2} \dot{M}_{{{\text{O}}_{ 2} }} in normoxia. Treatment of hepatocytes with ≤200 μM A769662 did not affect AMPK activity, AMPK phosphorylation, eEF2 phosphorylation, or cellular [(M)\dot]_{\textO 2} \dot{M}_{{{\text{O}}_{ 2} }} . These data suggest that A769662 is not an activator of AMPK in goldfish hepatocytes. Although our study provides support for the hypothesis that AMPK plays a role in initiating metabolic rate suppression in goldfish hepatocytes, this support must be viewed cautiously because of the known off-target effects of the pharmacological agents used.     

In vitro propagation of caper (Capparis spinosa L.)

Summary A twenty fold multiplication per twenty days of caper was achieved by culturing nodal shoot segments in the presence of BAP (4 μM) plus IAA (0.3 μM) and GA₃ (0.3 μM). The use of a modified MS medium facilitated this response. Plantlet regeneration was induced on single shoots taken from proliferating clusters subcultured for 20 days on a reduced BAP (2 μM) without auxin and gibberellin Higher rooting responses (70%) were obtained after a 20-day incubation period in darkness on solid half-strength MS1 medium plus IAA (30 μM), followed by a subsequent 20 day culture period on half-strength MSI basal medium. Proliferation was mainly due to axillary shoot-bud development as revealed by histological studies. The extensive meristematic activities observed indicated the enormous morphological potential of this species.     

Cadmium-induced hepatotoxicity in cultured rat hepatocytes as evaluated by morphometric analysis

Summary Freshly isolated hepatocytes from neonatal rats were cultured for approximately 24 h; incubated for 5, 30, or 60 min in solutions containing 0, 50, 100, or 200 μM cadmium; embedded in plastic; and sectioned for optical microscopy. The exeent of cadmium-induced hepatotoxicity was evaluated by double-blind morphometric analysis (a geometricostatistical processing of two-dimensional data for the collection of threedimensional information) whereby hepatocytes were classified on the basis of the severity of morphologic damage at the optical level. Both time and concentration effects were studied. Cultures exposed to 200 μM cadmium, for various intervals of time from 5 to 60 min, showed statistically significant reductions in the relative volume percent of normal hepatocytes, elevations (then reductions) in the relative volume percent of slightly damaged hepatocytes, increases in the relative volume percent of moderately damaged cells, and increases in the relative volume percent of severely damaged liver cells. As the concentration of cadmium was increased from 50 to 200 μM cadmium (during both 30 and 60-min exposures), significant trends were observed in cellular distribution patterns based on relative volume percent. Morphologically normal cells decreased, both slightly damaged and moderately damaged cells increased, and severely damaged cells remained unchanged. These results indicated that morphometric analysis at the optical level provided quantitative estimates for the evaluation of time- and concentration-effects of cadmium on cultured hepatocytes. This work was supported by a grant from the Johns Hopkins University Center for Alternatives to Animal Testing.     

adventitious shoot regeneration from petiole explants of Heracleum candicans wall

Summary A method for adventitious shoot induction from petiole explants of Heracleum candicans is reported. Shoot buds were induced on Murashige and Skoog (MS) medium with 4.4μM 6-benzylaminopurine (BA) and 1.1 μM 2,4-dichlorophen-oxyacetic acid (2,4-D). A wound response in the presence of BA and 2,4-D at the time of culture was necessary for inducing shoot buds. The shoot bud regeneration was significantly influenced by size, type and orientation of explants on the culture medium. These shoot buds developed into 4–5 cm shoots upon transfer to a medium containing 1.1μM BA and 0.5 μM α-naphthaleneacetic acid (NAA). The regenerated shoots formed rooted plantlets on MS medium supplemented with 4.9 μM indole-3-butyric acid (IBA). About 15 plants were established in the field for further evaluation.     

Somatic embryogenesis from stigmas and styles of grapevine

Summary An in vitro protocol has been developed for callus indiction, somatic embryogenesis, and plant regeneration from stigma-style culture of grapevine. Four different grapevine cultivars (Vitis vinifera L.: cvs. ‘Bombino Nero’, ‘Greco di Tufo’, ‘Merlot’, and ‘Sangiovese’) were tested. Exlants were cultured on Nitsch and Nitsch medium (NN) supplemented with various combinations of 6-benzylaminopurine (BA: 4.5 and 9.0 μM) and β-naphthoxyacetic acid (NOA; 5.0 and 9.9 μM). Sucrose (88 mM) was used as the carbon source. Somatic embryogenesis was induced within 3–7 mo. after culture initiation. Even though explants of different origin (unfertilized ovules and anthers) regenerated somatic embryos, the higher embryogenic potential was observed in stigma and style explants, with the exception of ‘Merlot’, which regenerated somatic embryos only from unfertilized ovules. The percentages of stigma-style explants producing somatic embryos was 7% in ‘Bombino Nero’ (cultured on NN medium supplemented 9.0 μM BA and 9.9 μM NOA). 14% in ‘Greco di Tufo’ (4.5 μM BA and 9.9 μM NOA), and 8% in ‘Sangiovese’ (9.0 μM BA and 9.9 μM NOA). The presence of growth regulators (BA and NOA) in the medium was essential for induction of somatic embryogenesis. Plants were regenerated on hormone-free NN medium containing 88 mM sucrose.