TBARS,carnitine, and reduced glutathione levels in human bladder carcinoma

In this study, we investigated tissue levels of reduced glutathione (GSH) and carnitine as well as thiobarbituric acid reactive substances (TBARS, as a marker of lipid peroxidation) levels in bladder carcinoma and control group of patients. The average GSH, carnitine and TBARS levels for tumor group were respectively 7.11 ± 3.3 g/mg protein, 1.81 ± 0.39 nmol/mg protein, and 4.29 ± 3.2 mol/mg protein, versus 14.45 ± 4.11 g/mg protein, 2.14 ± 0.66 nmol/mg protein, and 2.3 ± 0.6 mol/mg protein for normal bladder tissues. Thus, tissue reduced glutathione levels (GSH) were significantly lower in patients as compared with the control group (p < 0.001) whereas average TBARS levels in the tumor group were found to be higher than those in control group. The average tissue carnitine levels in the patient group were found to be lower compared with the control group but the difference was not statistically significant (p > 0.05).     

Uptake and Retention of Selenite and Selenomethionine in cultured K-562 cells

The selenium uptake and retention have been studied in K-562 cells exposed to selenite or selenomethionine. In the uptake experiments the cells were exposed to two doses of selenite (5 or 50 M) or selenomethionine (10 or 50 M). In the retention study the cells were treated for 2 h with the above mentioned doses of the selenocompounds before being observed at different times. The selenium uptake in cells exposed to selenite 5 M began to saturate at 8 h, but increased again between 48 and 96 h. In cells exposed to selenite 50 M the selenium uptake never reached a maximum, however, at 48 and 96 h the cell viability decreased strongly. The two doses of selenite showed different retention patterns, with a relatively small cellular decrease of selenium after treatment with selenite 5 M compared to treatment with 50 M of selenite. The selenium uptake in cells exposed to selenomethionine 10 M or selenomethionine 50 M began to saturate at 24 h and 48 h, respectively. The retention patterns were similar for both selenomethionine doses with a continuous decrease of the selenium concentration during the whole observation period. The results indicated a more controlled uptake and retention pattern of selenomethionine compared to selenite.     

Quinolinic acid is a potent lipid peroxidant in rat brain homogenates

In this study, we describe the lipoperoxidative effect of quinolinic acid (QUIN) in vitro. The formation of thiobarbituric acid reactive products (TBA-RP), an index of lipid peroxidation, was measured in rat brain homogenates after incubation at 37°C for 30 min in the presence of QUIN and some structurally and metabolically related compounds such as Kynurenine, Kynurenic acid, Glutamate, Aspartate and Kainate. Concentrations of QUIN in the range of 20 to 80 M increased lipid peroxidation in a concentration-dependent manner from about 15% to about 50%. Kynurenic acid, a compound metabollically related to QUIN that can block its neurotoxic actions in vivo, also inhibited completely the QUIN-induced TBA-RP formation in our system. Lipid fluorescent material, another index of lipid peroxidation was also found increased by 49% after incubation with 40 M QUIN. It is concluded that lipid peroxidation may be a damaging process involved in the neurotoxicity of QUIN.     

Oxytocin receptor mimetics prepared by molecular imprinting

Oxytocin receptor mimetics were prepared by molecularimprinting using Z-oxytocin as the template. Comparative binding studies with reference polymersshowed that the imprinted polymers recognized bothZ-oxytocin and unprotected oxytocin selectively. Thedissociation constants were 47 M and 102 M,respectively, and the density of binding sites was12 mol/g. The synthetic oxytocin receptors wereeasily prepared, possessed high mechanical andchemical stability, and were reused without loss ofselectivity and capacity after regeneration byextraction.     

Antioxidant activity of the flaxseed lignan secoisolariciresinol diglycoside and its mammalian lignan metabolites enterodiol and enterolactone

The antioxidant activities of the flaxseed lignan secoisolariciresinol diglycoside (SDG) and its mammalian lignan metabolites, enterodiol (ED) and enterolactone (EL), were evaluated in both lipid and aqueous in vitro model systems. All three lignans significantly (p 0.05) inhibited the linoleic acid peroxidation at both 10 and 100 M over a 24-48 h of incubation at 40°C. In a deoxyribose assay, which evaluates the non site-specific and site-specific Fenton reactant-induced ·OH scavenging activity, SDG demonstrated the weakest activity compared to ED and EL at both 10 and 100 M; the greatest ·OH scavenging for ED and EL was observed at 100 M in both assays. The incubation of pBR322 plasmid DNA with Fenton reagents together with SDG, ED or EL showed that the inhibition of DNA scissions was concentration dependent. The greatest non site-specific activity of lignans was at 100 M, thus, confirming the results of the deoxyribose test. In contrast, the protective effect of SDG and EL in the site-specific assay was lost and that of ED was minimal. Therefore, the results indicate a structure-activity difference among the three lignans with respect to specific antioxidant efficacy. All three lignans did not exhibit reducing activity compared to ascorbic acid, therefore, did not possess indirect prooxidant activity related to potential changes in redox state of transition metals. The efficacy of SDG and particularly the mammalian lignans ED and EL to act as antioxidants in lipid and aqueous in vitro model systems, at relatively low concentrations (i.e. 100 M), potentially achievable in vivo, is an evidence of a potential anticarcinogenic mechanism of flaxseed lignan SDG and its mammalian metabolites ED and EL.     

Studies on lipid peroxidation in the rat brain

The aim of this study was to set up a simple procedure for assessing lipid peroxidation (L.P.) and testing the activity of antioxidant compounds. L. P. was determined in rat brain homogenates by measuring the endogenous and stimulated accumulation of malonaldehyde (MDA). MDA was assayed by an HPLC method. Homogenates spontaneously formed appreciable amounts of MDA. The addition of increasing concentrations of FeCl₂ resulted in a linear accumulation of MDA, up to 16.6-fold at 50 M. An organic form of iron (Fe-saccharate) was less active on MDA formation (11.4-fold increase at 100 M). The addition of xanthine-xanthine oxidase resulted in only a 2.4-fold increase in MDA formation. Various antioxidant or chelating compounds effectively inhibited L.P., with IC₅₀ between 0.1 M (phenoxazine) and 4–50 M (-tocopherol). Their potencies depended on the iron concentration and time of preincubation with the homogenates. In conclusion, this is a simple and reliable procedure for studying L.P. and inhibiting agents, provided that the experimental conditions are carefully assessed.     

Antioxidant activity of allicin,an active principle in garlic

Garlic has been claimed to be effective against diseases, in the pathophysiology of which oxygen free radicals (OFRs) have been implicated. Effectiveness of garlic could be due to its ability to scavenge OFRs. However, its antioxidant activity is not known. We investigated the ability of allicin (active ingredient of garlic) contained in the commercial preparation Garlicin to scavenge hydroxyl radicals (·OH) using high pressure liquid chromatographic (HPLC) method. ·OH was generated by photolysis of H₂O₂ (1.25–10 moles/ml) with ultraviolet light and was trapped with salicylic acid which is hydroxylated to produce ·OH adduct products 2,3- and 2,5-dihydroxybenzoic acid (DHBA). H₂O₂ produced a concentration-dependent ·OH as estimated by ·OH adduct products 2,3-DHBA and 2,5-DHBA. Allicin equivalent in Garlicin (1.8, 3.6, 7.2, 14.4, 21.6, 28.8 and 36 g) produced concentration-dependent decreases in the formation of 2,3-DHBA and 2,5-DHBA. The inhibition of formation of 2,3-DHBA and 2,5-DHBA with 1.8 g/ml was 32.36% and 43.2% respectively while with 36.0 g/ml the inhibition was approximately 94.0% and 90.0% respectively. The decrease in ·OH adduct products was due to scavenging of ·OH and not by scavenging of formed ·OH adduct products. Allicin prevented the lipid peroxidation of liver homogenate in a concentration-dependent manner. These results suggest that allicin scavenges ·OH and Garlicin has antioxidant activity.     

Evaluation of radioprotective activities of Rhodiola imbricata Edgew – A high altitude plant

The present study reports the radioprotective properties of a hydro-alcoholic rhizome extract of Rhodiola imbricata (code named REC-7004), a plant native to the high-altitude Himalayas. The radioprotective effect, along with its relevant superoxide ion scavenging, metal chelation, antioxidant, anti-lipid peroxidation and anti-hemolytic activities was evaluated under both in vitro and in vivo conditions. Chemical analysis showed the presence of high content of polyphenolics (0.971 ± 0.01 mg% of quercetin). Absorption spectra analysis revealed constituents that absorb in the range of 220–290 nm, while high-performance liquid chromatography (HPLC) analysis confirmed the presence of four major peaks with retention times of 4.780, 5.767, 6.397 and 7.577 min. REC-7004 was found to lower lipid oxidation significantly (p < 0.05) at concentrations viz., 8 and 80 g/ml respectively as compared to reduced glutathione, although the optimally protective dose was 80 g/ml, which showed 59.5% inhibition of induction of linoleic acid degradation within first 24 h. The metal chelation activity of REC-7004 was found to increase concomitantly from 1 to 50 g/ml. REC-7004 (10–50 g/ml) exhibited significant metal chelation activity (p < 0.05), as compared to control, and maximum percentage inhibition (30%) of formation of iron-2,2-bi-pyridyl complex was observed at 50 g/ml, which correlated well with quercetin (34.9%), taken as standard. The reducing power of REC-7004 increased in a dose-dependent manner. The absorption unit value of REC-7004 was significantly lower (0.0183± 0.0033) as compared to butylated hydroxy toluene, a standard antioxidant (0.230± 0.091), confirming its high reducing ability. Superoxide ion scavenging ability of REC-7004 exhibited a dose-dependent increase (1–100 g/ml) and was significantly higher (p < 0.05) than that of quercetin at lower concentrations (1–10 g/ml), while at 100 g/ml, both quercetin and REC-7004 scavenged over 90% superoxide anions. MTT assay in U87 cell line revealed an increase in percent survival of cells at doses between 25 and 125 g/ml in case of drug + radiation group. In vivo evaluation of radio-protective efficacy in mice revealed that intraperitoneal administration of REC-7004 (maximally effective dose: 400 mg/kg b.w.) 30 min prior to lethal (10 Gy) total-body -irradiation rendered 83.3% survival. The ability of REC-7004 to inhibit lipid peroxidation induced by iron/ascorbate, radiation (250 Gy) and their combination [i.e., iron/ascorbate and radiation (250 Gy)], was also investigated and was found to decrease in a dose-dependent manner (0.05–2 mg/ml). The maximum percent inhibition of formation of MDA-TBA complex at 2 mg/ml in case of iron/ascorbate, radiation (250 Gy) and both i.e., iron/ascorbate with radiation (250 Gy) was 53.78, 63.07, and 51.76% respectively and were found to be comparable to that of quercetin. REC-7004 (1 g/ml) also exhibited significant anti-hemolytic capacity by preventing radiation-induced membrane degeneration of human erythrocytes. In conclusion, Rhodiola renders in vitro and in vivo radioprotection via multifarious mechanisms that act in a synergistic manner.     

The detection and characterization of bacteria-sized protists in “Protist-free” filtrates and their potential impact on experimental marine ecology

Nuclepore filters of 0.6–1.0m pore size have been used to prepare protist-free water for a number of studies in microbial ecology. This procedure has been called into question by a recent study claiming that a significant portion of bacterial loss in filtrates could be due to uncharacterized predators passing through 0.6m filters. We were unable to directly observe protists in 0.6m filtrates using phase contrast, epifluorescence, or transmission electron microscopy. Using the culture techniques of rice grain enrichment and most probable number, however, we were able to observe and quantify several species of bacterivorous nanoflagellates that developed not only in 0.6m, but also in 0.4m seawater filtrates. The ability of predacious nanoflagellates to squeeze through bacteria-sized pores questions studies of bacterial production and chemical cycling that have assumed protist-free filtrates.     

The contribution of size-fractionated plankton to biomass and primary production of phytoplankton in saline–alkaline ponds

Primary productivity, biomass and chlorophyll-a of size fractionated phytoplankton (<0.22 m, <3 m, <8 m, <10 m, <40 m, <64 m, <112 m and <200 m) were estimated in 6 ponds and 5 experimental enclosures. The results showed that the planktonic algae less than 10 m are important in the biomass and production of phytoplankton in saline–alkaline ponds. The production of size fractionated phytoplankton corresponding to <112 m, <10 m and <3 m in saline–alkaline ponds were 10.5 ± 6.6 , 8.6 ± 5.4 and 0.33 ± 0.1 mgC l^–1 d^–1, respectively. Mean community respiration rate was 1.80 ± 0.73, 1.69 ± 0.90 and 1.38 ± 1.12 mgC l^–1 d^–1, respectively. The average production of phytoplankton corresponding to micro- (10–112 m), nano- (3–10 m) and pico- (<3 m) were 1.61, 8.30 and 0.33 mgC l^–1 d^–1, respectively. The ratio of those to the total phytoplankton production was 15%, 79% and 3%, respectively. The mean respiration rate of the different size groups was 0.11, 0.31 and 1.38 mgC l^–1 d^–1; the ratio of those to total respiration of phytoplankton was 6%, 17% and 77%, respectively. The production of size-fractionated phytoplankton corresponding to <200 m, <10 m and <3 m in enclosures was 2.19 ± 1.63, 2.08 ± 1.75 and 0.22 ± 0.08 mgC l^–1 d^-1, respectively. Mean community respiration rates were 1.25 ± 1.55, 1.17 ± 1.42 and 0.47 ± 0.32 mgC l^–1 d^–1, respectively. The average production of phytoplankton corresponding to micro- (10–200 m), nano- (3–10 m) and pico- (<3 m) plankton was 0.11, 1.86 and 0.22 mgC l^–1 d^–1, respectively. The ratio of those to the total production of phytoplankton was 5%, 85% and 10%, respectively. The mean respiration rate of different size groups were 0.08, 0.72 and 0.46 mgC l^–1 d^–1, the ratio of those to total respiration of phytoplankton was 6%, 57% and 37%, respectively. The concentrations of chlorophyll-a of the phytoplankton in the corresponding size of micro- (10–112 m), nano- (3–10 m) and pico- (<3 m) plankton in the experimental ponds were 19.3, 98.2 and 11. 9 g l^–1, respectively. The ratio of those to the total chlorophyll-a was 15%, 76% and 9%, respectively. The concentrations of chlorophyll-a of phytoplankton micro- (10–200 m), nano- (3–10 m) and pico- (<3 m) plankton in enclosures were 1.7, 34.3 and 3.0 g l^–1, respectively. The ratio of those to the total chlorophyll-a was 4%, 88% and 8%, respectively.     

Aluminum-induced secretion of both citrate and malate in rye

Aluminum (Al)-resistant mechanisms responsible for Al-induced secretion of organic acids are poorly understood. In this study, we characterized the Al-induced secretion of both citrate and malate from rye (Secale cereale L. cv. King). Secretion of organic acids increased with increasing concentration (10, 30 and 50 M) and duration of Al treatments. Neither phosphorous (P) deficiency up to 15 days nor addition of 50M lanthanum, 50 M lead, 10 M cadmium, or 200 M manganese caused secretion of organic acids, suggesting that this secretion was a specific response to Al stress. Aluminum activated citrate synthase, the main enzyme for the synthesis of citrate, but its activation occurred only in the root tip. The elongation of roots of an Al-sensitive cultivar of wheat (Tritium aestivum L. cv. Scout 66) was not inhibited by 50 M Al in the presence of externally applied 50 M citrate or 400 M malate. The secretion of citrate and malate from intact rye roots exposed to 50 M Al corresponded to 31.3 ± 1.7 M and 11.5 ± 2.5 M, respectively, in the rhizosphere based on an assumption of a 2 mm thick unstirred layer around root tips. This result indicated that Al-resistance in rye was achieved by the Al-induced synthesis of citrate in root apices followed by Al-induced specific secretion of citrate from root tips.     

Artemisia annua L.: dedifferentiated and differentiated cultures

Dedifferentiated and differentiated tissue cultures ofArtemisia annua L. for artemisinin production were carried out. The calluses were initiated on MS medium supplemented with sucrose (30 g l^-1), myoinositol (100 mg l^-1) and RT vitamins. The auxins used were naphtaleneacetic acid (NAA), indoleacetic acid (IAA), indolebutyric acid (IBA) and 2,4-dichlorophenoxyacetic acid (2,4-d). These were added to the basal medium either singly or in combination. The best results were obtained with 2.4-d (4.5 M : 0.02 d^-1) and NAA (5.4 M : 0.06 d^-1). Cell suspensions were established on the same media without agar. Suspension cultures showed different morphological characteristics according to the plant growth regulator supplied. Organized cultures were initiated from callus obtained on 2,4-d (4.5 M) and from bud cultures. Medium containing 6-benzylaminepurine (BA) (8.9 M)+NAA (0.54 M); Zeatin (45.62 M)+NAA (5.37 M) or BA (8.9 M) stimulated both organogenesis in callus (frequency of induction =50%) and semi-organized tissue in shoot buds. BA (13.32 M)+NAA (1.08 M) or BA (13.32 M) only stimulated multiple shoot cultures (frequency of induction =80%). Regarding artemisinin content, while the values obtained were 1.13 and 0.78 mg gDW^-1 in primary callus, artemisinin was not detected in cell suspension and only traces of it were found in multiple shoot cultures.     

Plant catechols prevent lipid peroxidation in human plasma and erythrocytes

The antioxidant activity of several plant catechol derivatives was tested in buffer, plasma, and human erythrocytes. In buffer, chlorogenic acid (CGA), caffeic acid (CA), and dihydrocaffeic acid (DCA) reduced ferric iron equally well in the ferric reducing antioxidant power (FRAP) assay. Low concentrations of the polyphenols enhanced the ability of plasma to reduce ferric iron by about 10%. In plasma, lipid hydroperoxide and F₂-isoprostane formation induced by a water-soluble free radical initiator were reduced by CGA at concentrations as low as 20 M. During incubation at 37°C, human erythrocytes took up DCA, but not CGA, and intracellular DCA enhanced the ability of erythrocytes to reduce extracellular ferricyanide. When intact erythrocytes were exposed to oxidant stress generated by liposomes containing small amounts of lipid hydroperoxides, extracellular CGA at a concentration of 5 M decreased both lipid peroxidation in the liposomes, and spared -tocopherol in erythrocyte membranes. These results suggest that the catechol structure of these compounds convey the antioxidant effect in plasma and in erythrocytes.     

Changes in lipid peroxidation, the redox system and ATPase activities in plasma membranes of rice seedling roots caused by lanthanum chloride

Highly purified plasma membranes were isolated by aqueous two-phase partitioning from rice (Oryza sativa) seedling roots. The effects of lanthanum chloride (LaCl₃) on the activities of lipid peroxidation, the redox system and H⁺-ATPase, Ca²⁺-ATPase of plasma membranes were studied. The lipid peroxidation of plasma membranes could be depressed by certain low concentrations of LaCl₃ and enhanced by high concentrations of LaCl₃, while the lipid peroxidation was also dependent on the plasma membrane protein and incubation time. The relative activity of O₂ uptake of plasma membranes was inhibited by all tested LaCl₃ concentrations. In contrast, the reduction rate of Fe(CN)₆ ^3– by plasma membranes was stimulated below 40 M of LaCl₃, but was reduced above 60 M of LaCl₃. The relative activities of both H⁺-ATPase and Ca²⁺-ATPase increased constantly from control to LaCl₃ of concentration 60 M where the activities of both enzymes were the maximum, but decreased remarkably at 80 M LaCl₃ concentrations various LaCl₃ were added to culture solutions. In the other measurement case in which various LaCl₃ concentrations were added directly to reaction medium and the plasma membrane vesicles only came from the control cultured rice seedling roots, the response of H⁺-ATPase activity to La³⁺ was similar to the response in culture solution. However, the La³⁺ concentration was only 20 M when the activity of H⁺-ATPase was the maximum. In contrast to the case of LaCl₃ addition to culture solution, Ca²⁺-ATPase activity was inhibited by all concentrations of La³⁺ which were added directly to the reaction medium. The above results revealed that REEs inhibited electron transfer from NADH to oxygen in plant plasma membranes, depressed the production of active oxygen radicals, and reduced the formation of lipid peroxides through plasma membrane lipid peroxidation. REEs ions also enhanced the H⁺ extrusion by both standard redox system and H⁺-ATPase in plasma membranes at certain concentrations. A possible role for the plant cell wall in REEs effects on plasma membranes was also suggested.     

Methylobacterium rhodesianum MB 126 possesses two acetoacetyl-CoA reductases

Two constitutive acetoacetyl-CoA (AcAc-CoA) reductases were purified from Methylobacterium rhodesianum MB 126, an NADPH-linked d(-)--hydroxybutyryl-CoA forming reductase (enzyme A) and an NADH-and NADPH-linked l(+)--hydroxybutyryl-CoA forming reductase (enzyme B). Enzyme A and B give apparent K _m values of 15 M and 30 M for AcAc-CoA, 18 M for NADPH and 30 M for NADH, respectively. They are inhibited by AcAc-CoA at concentrations higher than 25 M and 50 M, respectively. The contribution of the two reductases to poly--hydroxybutyrate synthesis is discussed.     

Karyometry of nuclei in liver cells

Summary In untreated adult male albino rats nuclear volume and the percentage of binucleate cells were determined in the first layer of hepatocytes adjacent to hepatic venous branches of varying diameters (<40 m, 40 m–80 m, 80 m–120m, 120 m–160 m, >160 m), and in the third and fourth layer of hepatocytes in the remainder of the perivenous parenchyma. In the first layer of hepatocytes adjacent to the vascular structures means of nuclear volume are significantly lower and percentage of binucleate cells significantly higher than in the cells of the remainder of the perivenous parenchyma. Within each area measured distribution curves of nuclear volume classes were homogeneous but showed heterogeneity in comparison with each other. The morphometric data presented in this study strongly support the opinion of the heterogeneity of liver cells in the perivenous zone, as previously postulated on the basis of histochemical investigations.Supported by the Deutsche Forschungsgemeinschaft (Hi 318/2-1)     

Endogenous phosphorylation of rat brain synaptosomal plasma membranes in vitro: Some methodological aspects

The time course of endogenous phosphorylation in vitro of total or separted synaptic plasma membrane proteins (SPM) has been correlated with that of hydrolysis of the phosphate donor (ATP) in the incubation medium. The ATP/SPM ratio in the medium was varied. In a low-ratio medium (7.5 M ATP; 2.2 g SPM/l) a complete hydrolysis of ATP occurred almost instantaneously as was measured by the release of free phosphate in and the disappearance of ATP from the medium. As a consequence, only a very short peak of phosphorylation, followed by dephosphorylation was observed. However, when higher ATP/SPM ratios were used (200 M ATP; 0.4 g SPM/l and 500 M ATP; 0.4 g SPM/l), the incorporation of phosphate into SPM proteins was linear for 20 sec, and the maximum level of phosphate incorporation was increased. Similar results were obtained after separation of³²P-labeled phosphoproteins by slab gel electrophoresis. However, analysis of the autoradiographs obtained fromone SPM preparation under different ATP/SPM ratios revealed dependence of phosphorylation of individual protein bands on the conditions used.     

Direct Organogenesis from Mature Leaf and Petiole Explants of Eryngium Foetidum L.

Eryngium foetidum L. plants were regenerated from mature leaf and petiole explants through direct organogenesis without intervening callus phase. From leaf explants, adventitious multiple shoots raised on Murashige and Skoog (MS) medium supplemented with 4.43 M benzylaminopurine (BAP) and 0.57 M indole-3-acetic acid (IAA), whereas in petiole explants shoot regeneration occurred at 8.86 M BAP and 0.57 M IAAA. 80% of the leaf explants and 44% of petiole explants produced shoots after four weeks of culture. The regenerated plants were rooted on MS medium supplemented with 2.46 M indole-3-butyric acid and 2.88 M gibberellic acid. The plants were successfully established in the soil and showed 70.9% survival in the field.     

In vitro propagation of Asparagus maritimus – A rare Mediterranean salt-resistant species

Asparagus maritimus L. Miller is a rare species growing of the Mediterranean region and is morphologically similar to A. officinalis. In order to establish an efficient in vitro propagation protocol, explants were excised from spear segments and cultured on Murashige and Skoog (1962) medium containing 3% sucrose and various concentrations of growth regulators. The best shoot initiation (3–4 per explant) was achieved on a medium containing 0.88 M N⁶-benzyladenine (BA), 0.93 M kinetin, 1.07 M -naphthaleneacetic acid (NAA) and 3.90 M ancymidol. Shoot initiation could also be achieved without ancymidol but the shoots were thinner and longer. A very high shoot multiplication rate was achieved on media supplemented with 3% sucrose, 1.07 M NAA, 0.93 M kinetin, 0.44 M BA and various concentrations of ancymidol. The lowest concentration of ancymidol (0.39 M) significantly promoted the highest shoot multiplication rate (11.9 shoots/crown). For root formation, media were supplemented with 6% sucrose, 1.07 M NAA and various concentrations of ancymidol. Rooting frequency increased with higher ancymidol concentration up to 5.07 M (82.0% rooting). The number of ex vitro shoots formed was strongly correlated (r=0.66) with the length of roots formed in vitro, which was the highest at a 1.95 M ancymidol.     

Plant development in long-term embryogenic callus lines of Cucurbita pepo

Embryogenic callus derived from pumpkin hypocotyl segments was induced and maintained for 15 years on MS medium supplemented with the auxins IBA (4.9 M), 2, 4-D (4.5 M) or IAA (5.7 M). On induction media continued embryo maturation and development of adult plants typically failed. Therefore, small embryogenic clumps and individually isolated embryos were subcultured two to four times on one of the conversion media: MS supplemented with 1.5% sucrose and (a) no hormone, (b) 2.9 M IAA, (c) 5.7 M IAA, (d) 11.4 M IAA, (e) 12 M IEt, (f) 3.8 M ABA or (g) 2% activated charcoal. The cell line and the kind of auxin used in the induction and maintenance medium, both had a marked influence on the development of plantlets. The best result was achieved with a line that has been induced and maintained for 15 years on MS with IBA. In the IBA line, out of 100 embryos, 77 developed into plantlets on MS medium supplemented with 11.4 M IAA.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid - BA 6-benzylaminopurine - ABA abscisic acid - 2, 4-D 2, 4-di-chlorophenoxyacetic acid - IEt indole-3-etha-nol - MS Murashige and Skoog (1962) medium