Relationships between nitrate level,nitrate reductase activity and anaerobic nitrite production inPisum sativum leaf tissue

Anaerobic nitrite production (thein vivo NO₃-R activity) in an incubation medium lacking exogenous nitrate but containing 0.5%n-propanol and 0.1% Triton X-100 showed higher correlation (y - ax ^b) with the level of endogenous nitrate inPisum sativum L. leaves than thein vitro nitrate reductase activity. Thein vivo NO₃-R activity correlated well with thein vitro activity up to the 50 ppm NO₃-N level of endogenous nitrate. The ratioin vivo: in vitro activity slightly decreased with increasing level of endogenous nitrate in leaf tissue.     

Expression ofKlebsiella pneumoniae nif genes inProteus mirabilis

Self-transmissible plasmids carryinghis andnif genes fromKlebsiella pneumoniae have been introduced into threehis mutants ofProteus mirabilis: strains 5006-1, WR19 and WR20. Expression ofhis by the transconjugants was unequivocal, if slightly temperature-sensitive, but none was Nif⁺ when tested for acetylene reduction in anaerobic glucose medium using inocula from rich or glucose-minimal aerobic agar cultures. Succinate or pyruvate in place of glucose, low glucose, lower temperature or elevated Na₂MoO₄ did not allownif expression and no nitrogenase MoFe-protein peptide was detected immunologically after exposure to conditions in which diazotrophic enterobacteria, normal or genetically constructed, derepressnif.One strain,P. mirabilis WR19, carrying thehis nif Km^r plasmid pMF250 was examined in detail. Thenif activator genenifA was introduced on the plasmid pCK1. Such derivatives remained Nif^- when tested, after aerobic growth on rich agar media, with normal or low glucose, with succinate or with elevated Mo. However, pre-conditioning by aerobic growth on glucose-minimal agar led to subsequent anaerobic expression ofnif in glucose medium from pMF250 in WR19 carrying pCK1. NH ₄ ⁺ or proline could serve as N-source in the glucose-minimal agar. Maximum activity was about 5% of that ofK. pneumoniae in our assay conditions. Material cross-reacting with anti-serum to the nitrogenase MoFe protein was formed. Nitrogenase activity was not switched off by NH ₄ ⁺ .P. mirabilis WR19 (pCK1) showed NH ₄ ⁺ -constitutive temperature-sensitive kanamycin resistance (anif-related phenotype of this plasmid) in aerobic glucose minimal medium. Expression ofnif inP. mirabilis WR19 (pCK1, pMF250) was NH ₄ ⁺ -repressible despite the constitutivenifA character of pCK1 and introduction of thentrA ⁺ plasmid pMM17 did not alter this phenotype. However, pCK1 did not give rise to NH ₄ ⁺ -constitutive diazotrophy in the wild-typeK. pneumoniae M5al. A construct of WR19 carrying pMF250 and constitutiventrC plasmid (pMD45) remained Nif^- even after pre-growth on glucose-minimal media.We conclude (a) thatP. mirabilis forms a gene product functionally equivalent to that ofntrA inK. pneumoniae, (b) that it forms no functional equivalent of thentrC product in our growth conditions. The need for pre-conditioning on aerobic glucose media remains perplexing.Non-common abbreviation NFDM Nitrogen-free-Davis-Mingioli medium     

Anaerobic induction of trimethylamine N-oxide reductase and cytochromes by dimethyl sulfoxide inEscherichia coli

Reduction of trimethylamine N-oxide is catalyzed by at least two enzymes inEscherichia coli: trimethylamine N-oxide reductase, which is anaerobically induced by trimethylamine N-oxide, and the constitutive enzyme dimethyl sulfoxide reductase. In this study, an increase in the specific activity of trimethylamine N-oxide reduction was observed in the anaerobic culture with dimethyl sulfoxide, but the specific activity of dimethyl sulfoxide reduction was not changed. The inducible enzyme trimethylamine N-oxide reductase was found in this culture. A marked expression of the structural genetorA for trimethylamine N-oxide reductase was also observed in atorA-lacZ gene fusion strain under anaerobic conditions with either trimethylamine N-oxide or dimethyl sulfoxide.l-Methionine sulfoxide and the N-oxides of adenosine, picolines, and nicotinamide slightly repressed expression of the gene. Membrane-boundb- andc-type cytochromes involved in the trimethylamine N-oxide reduction were also produced in a wild-type strain grown anaerobically with dimethyl sulfoxide. But thec-type cytochrome was not produced in thetorA-lacZ strain grown anaerobically with trimethylamine N-oxide or dimethyl sulfoxide; this suggests that there is a correlation between the expression oftorA and the synthesis of the cytochrome.     

Transport and utilization of methionine sulfoxide in the rabbit

Methionine sulfoxide is transported into purified intestinal and renal brush border membrane vesicles from rabbit by an Na⁺-dependent mechanism and is accumulated inside the vesicles against the concentration gradient. Both in intestine and kidney, the rate of transport is enhanced with increasing concentrations of Na⁺ in the external medium. Increasing the Na⁺ gradient reduces the apparent K_t for methionine sulfoxide without causing any change in V_max. With an outward K⁺ gradient (vesicle > medium), valinomycin stimulates the Na⁺-gradient-dependent transport of methionine sulfoxide in the kidney, showing the electrogenicity of the transport process. A number of amino acids inhibit methionine sulfoxide transport in both the intestine and kidney. An enzymatic activity capable of reducing methionine sulfoxide to methionine is present in the intestinal mucosa, renal cortex and liver. The activity is highest in renal cortex and lowest in intestine. The methionine sulfoxide-reducing activity is stimulated by NADH, NADPH, glutathione and dithiothreitol and the potency of the stimulation is in the order: dithiothreitol > NADPH > glutathione > NADH.     

Rudimentary mutants ofDrosophila melanogaster

Summary The X-linkedrudimentary (r) mutants ofDrosophila melanogaster are pyrimidine auxotrophs and require exogenous pyrimidines (Nørby, 1970; Falk, 1976). We have established a set ofrudimentary cell lines that are derived from embryos, homozygous for eitherr ¹ orr ³⁶. The enzymatic activities of the pyrimidine synthesizing enzymes were measured in the mutant lines. We have further investigated the nutritional requirements of the mutant cells in vitro by using a pyrimidine free culture medium.Ther ¹ cell lines were found to express 3–7%dihydroorotase (DHOase) activity as compared to a wildtype cell line. Reducedaspartate transcarbamylase (ATCase) activity was measured in somer ¹ cell lines whereas wildtypecarbamylphosphate synthetase (CPSase) activity is expressed in allr ¹ cell lines. Ther ³⁶ cell line expresses wildtype activity ofDHOase andCPSase. ATCase activity was found to be reduced to 10% of the wildtype activity.The mutant cell lines do not proliferate in pyrimidine free minimal medium and cell proliferation is obtained by the addition of crude RNA. Proliferation of ther ¹ cells is restored by the supplementation of the minimal medium withdihydroorotate whereas proliferation of ther ³⁶ cells is restored by supplementation with eitherdihydroorotate orcarbamylaspartate.The results demonstrate that therudimentary phenotypesr ¹ andr ³⁶ are expressed at the cellular level and that the two mutant cell types behave as cellular pyrimidine auxotrophs in vitro.     

Characterization of a potassium carrier in rabbit reticulocyte cell membrane

Summary In this study we present evidence that high ouabain-resistant Rb influx, carried out by the rabbit reticulocyte membrane, is composed of carrier-mediated Rb influx and passive diffusion across the cell membrane. To meet this end, an assay was developed by which the two ouabain-resistant Rb influxes could be measured separately.Whereas theK _m for Rb of the carrier (12.5mm) did not change by increasing the pH, theVm was markedly reduced. At the optimal pH (6.0–6.5) theVm was 6–8 mmol h^–1 liter^–1 and fell to zero at pH 8.0. This may indicate a possible role of H⁺ ions in this transport mechanism.The carrier is inhibited by furosemide and ethacrynic acid, similarly to pump II in the erythrocyte and kidney. In addition, its activity is dependent upon the ionic content of the medium. The K(Rb) carrier appeared not to be involved in an active transport since depletion of ATP had no effect on the carrier activity. The carrier activity was also measured in rabbit erythrocytes and was found to be 10 times lower than that of rabbit reticulocytes. TheK _m for Rb, optimal pH, and high sensitivity to furosemide and ethacrynic acid of the erythrocyte and the reticulocyte carrier are similar.Our study suggests that maturation of reticulocytes to erythrocytes is accompanied by a loss or inactivation of most of a K (or Rb) carrier very active in the reticulocyte cell.     

Genetic transformation and regeneration of rubber tree (Hevea brasiliensis Muell. Arg) transgenic plants with a constitutive version of an anti-oxidative stress superoxide dismutase gene

Agrobacterium tumefaciens-mediated genetic transformation and the regeneration of transgenic plants was achieved in Hevea brasiliensis. Immature anther-derived calli were used to develop transgenic plants. These calli were co-cultured with A. tumefaciens harboring a plasmid vector containing the H. brasiliensis superoxide dismutase gene (HbSOD) under the control of the CaMV 35S promoter. The -glucuronidase gene (uidA) was used for screening and the neomycin phosphotransferase gene (nptII) was used for selection of the transformed calli. Factors such as co-cultivation time, co-cultivation media and kanamycin concentration were assessed to establish optimal conditions for the selection of transformed callus lines. Transformed calli surviving on medium containing 300 mg l^-1 kanamycin showed a strong GUS-positive reaction. Somatic embryos were then regenerated from these transgenic calli on MS2 medium containing 2.0 mg l^-1 spermine and 0.1 mg l^-1 abscisic acid. Mature embryos were germinated and developed into plantlets on MS4 medium supplemented with 0.2 mg l^-1 gibberellic acid, 0.2 mg l^-1 kinetin (KIN) and 0.1 mg l^-1 indole-3-acetic acid. A transformation frequency of 4% was achieved. The morphology of the transgenic plants was similar to that of untransformed plants. Histochemical GUS assay revealed the expression of the uidA gene in embryos as well as leaves of transgenic plants. The presence of the uidA, nptII and HbSOD genes in the Hevea genome was confirmed by polymerase chain reaction amplification and genomic Southern blot hybridization analyses.Communicated by L. Peña     

Cryopreservation of the edible alkalophilic cyanobacterium Arthrospira platensis

Efficient cryopreservation conditions for the edible alkalophilic cyanobacterium Arthrospira (Spirulina) platensis were investigated using a model strain A. platensis NIES-39. As a result, it was found that more than 60% of cells were viable upon thawing, when they had been frozen at a cooling rate of approximately ?1 °C min^?1 in the presence of 10% (v/v) dimethyl sulfoxide. Further examination with other Arthrospira strains showed that many of them had strain-dependent optimal conditions for cryopreservation. For example, the best freezing conditions for A. platensis SAG 21.99 were snap-freezing in liquid nitrogen in the presence of 5% (v/v) dimethyl sulfoxide, while they were slow cooling at approximately ?1 °C min^?1 in the presence of 10% (v/v) methanol for A. platensis NIES-46, NIES-2308 and UTEX 1926. The variety of successful cryopreservation conditions presented in this study is useful when attempting to cryopreserve various Arthrospira strains.     

Baclofen-induced,calcium-dependent stimulation of in vivo release ofd-[3H]aspartate from rat hippocampus monitored by intracerebral microdialysis

The release ofd-[³H]aspartate (used as a tracer for endogenous glutamate and aspartate) was studied at high K⁺ (100 mM) and under ischemia in rats implanted with 0.3 mm diameter dialysis tubing through the hippocampus. The effect on thed-[³H]aspartate release of the two -aminobutyric acid (GABA) agonists 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol (THIP) and (±)--(p-chlorophenyl)GABA (baclofen), which specifically activate GABA_A and GABA_B receptors, respectively, was studied. Initial experiments employing HPLC analysis showed a coincident increase in the amounts of glutamate, aspartate and the amount of radioactivity following introduction of K⁺ (100 mM) or a period of ischemia suggesting that thed-[³H]aspartate labels the transmitter pools of the two amino acids under the present experimental conditions. The presence of 10 mM baclofen or 10 mM THIP in the perfusion medium did not inhibit ischemia inducedd-[³H]aspartate release. On the contrary, 10 mM baclofen alone (but not 0.1 or 1 mM) in the perfusion medium induced release ofd-[³H]aspartate in a calcium dependent manner, whereas 10 mM THIP had no significant releasing effect.Special issue dedicated to Dr. Elling Kvamme     

Characteristics of 3-O-methylfluorescein phosphate hydrolysis by the (Na+ + K+)-ATPase

With 3-O-methylfluorescein phosphate (3-OMFP) as substrate for the phosphatase reaction catalyzed by the (Na⁺ + K⁺)-ATPase, a number of properties of that reaction differ from those with the common substratep-nitrophenyl phosphate (NPP): theK _m is 2 orders of magnitude less and the V_max is two times greater, and dimethyl sulfoxide (Me₂SO) inhibits rather than stimulates. In addition, reducing the incubation pH decreases both theK _m and V_max for K⁺-activated 3-OMFP hydrolysis as well as theK _0.5 for K⁺ activation. However, reducing the incubation pH increases inhibition by P_i and the V_max for 3-OMFP hydrolysis in the absence of K⁺. When choline chloride is varied reciprocally with NaCl to maintain the ionic strength constant, NaCl inhibits K⁺-activated 3-OMFP hydrolysis modestly with 10 mM KCl, but stimulates (in the range 5–30 mM NaCl) with suboptimal (0.35 mM) KCl. In the absence of K⁺, however, NaCl stimulates increasingly over the range 5–100 mM when the ionic strength is held constant. These observations are interpreted in terms of (a) differential effects of the ligands on enzyme conformations; (b) alternative reaction pathways in the absence of Na⁺, with a faster, phosphorylating pathway more readily available to 3-OMFP than to NPP; and (c) a (Na⁺ + K⁺)-phosphatase pathway, most apparent at suboptimal K⁺ concentrations, that is also more readily available to 3-OMFP.Abbreviations Et₃N triethyl amine - FITC fluorescein isothiocyanate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonate - MES 2-(N-morpholino)ethanesulfonate - Me₂SO dimethyl sulfoxide - NPP p-nitrophenyl phosphate - 3-OMFP 3-O-methylfluorescein phosphate - TNP-ATP 2, (or 3)-O-(2,4,6-trinitrophenyl)-ATP     

Tryptic digestion of the (Na + K)-ATPase is both sensitive to and modifies K+ interactions with the enzyme

Tryptic digestion of the (Na + K)-ATPase in the presence of choline chloride or NaCl (Na-type) and in the presence of KCl (K-type) produced distinct patterns of peptide fragments and losses of catalytic activity. TheK _0.5 for K⁺ to shift digestion from the Na-type, and its sensitivity to dimethyl sulfoxide and Triton X-100, were consistent with K⁺ acting at sites on the cytoplasmic face of the enzyme through which the K-phosphatase reaction also is activated. Reagents favoring the E₁ conformational states, oligomycin, Triton, and ATP, shifted the pattern toward the Na-type, whereas those favoring E₂ states, dimethyl sulfoxide, MgCl₂, and MnCl₂, shifted the pattern toward the K-type. Na-type digestion caused a greater loss of K-phosphatase than (Na + K)-ATPase activity, and the residual K-phosphatase activity was more sensitive to inhibition by Triton and ATP but stimulated more by dimethyl sulfoxide and inhibited less by P_i and MnCl₂; all these effects are consistent with such digestion shifting equilibria toward E₁ enzyme states. Accordingly, theK _0.5 for K⁺ to activate the (Na + K)-ATPase was increased. However, theK _0.5 for the K-phosphatase was unchanged; this observation requires revision of previous formulations, and bears on additional aspects of enzyme activity as well.     

Relationship between the F0F1-ATPase and the K+-transport system within the membrane of anaerobically grownEscherichia coli. N,N′-dicyclohexylcarbodiimide-sensitive ATPase activity in mutants with defects in K+-transport

A considerable (2-fold) stimulation of the DCCD-sensitive ATPase activity by K⁺ or Rb⁺, but not by Na⁺, over the range of zero to 100mM was shown in the isolated membranes ofE. coli grown anaerobically in the presence of glucose. This effect was observed only in parent and in thetrkG, but not in thetrkA, trkE, ortrkH mutants. ThetrkG or thetrkH mutant with anunc deletion had a residual ATPase activity not sensitive to DCCD. A stimulation of the DCCD-sensitive ATPase activity by K⁺ was absent in the membranes from bacteria grown anaerobically in the presence of sodium nitrate. Growth of thetrkG, but not of othertrk mutants, in the medium with moderate K⁺ activity did not depend on K⁺ concentration. Under upshock, K⁺ accumulation was essentially higher in thetrkG mutant than in the othertrk mutant. The K⁺-stimulated DCCD-sensitive ATPase activity in the membranes isolated from anaerobically grownE. coli has been shown to depend absolutely on both the F₀F₁ and theTrk system and can be explained by a direct interaction between these transport systems within the membrane of anaerobically grown bacteria with the formation of a single supercomplex functioning as a H⁺-K⁺ pump. ThetrkG gene is most probably not functional in anaerobically grown bacteria.This study was performed at the Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, Illinois 60637.     

Effect of plant growth regulators on regeneration of plantlets from bud cultures ofCymbopogon nardus L. and the detection of essential oils from thein vitro plantlets

Cymbopogon nardus L. could be propagated via tissue culture using axillary buds as explants. The aseptic bud explants obtained using double sterilization methods produced stunted abnormal multiple shoots when they were cultured on Murashige and Skoog medium (MS) supplemented with 1.0 mg L^-1 or 2.0 mg L^-1 benzyladenine (BA). Stunted shoots that cultured on MS + 1.0 mg L^-1 BA + 1.0 mg L^-1 N⁶-isopentenyl-adenine (2iP) could induce elongation of shoots from about 60% of the stunted shoots. Normal multiple shoots could be induced at the highest (19.7 shoots per bud) from the bud explants within six weeks when cultured on proliferation medium consisted of MS supplemented with 0.3 mg L^-1 BA and 0.1 mg L^-1 indole-3-butyric acid (IBA). The separated individual shoot produced roots when transferred to basic MS solid medium. The essential oils that were contained in the mature plants namely citronellal, geraniol and citronellol were also found in thein vitro C. nardus plantlets. Citronellal was the main essential oil component in the matured plants while geraniol was the main component in thein vitro plantlets.     

TheChlamydomonas plus and minus agglutinin: difference in sensitivity to dithiothreitol

The effect of disulfide-reducing agent dithiothreitol (DTT) on the plus and minus agglutinins ofChlamydomonas reinhardtii gametes was studied. Live gametes of mating-type plus (mt ⁺) lost their flagellar agglutinability by DTT treatment without any loss of cell motility and concurrently released into the medium agglutinin in an inactive form. DTT treated cells also lost completely their cell body-agglutinin. By contrast, the mating-type minus (mt ^-) gametes neither lost their agglutinability nor released agglutinin into the medium by DTT even at very high concentrations. In vitro experiments showed that plus agglutinin in solution is just as sensitive as that in vivo to DTT, whereas minus agglutinin is totally insensitive, and the sulfhydryl-oxidizing agent diamide restores the plus agglutinin activity immediately and completely. Isolated flagella from themt ⁺ gametes were also inactivated by DTT, but they retained the inactivated agglutinin on the surfaces. The results indicate that plus agglutinin, but not minus agglutinin, possesses disulfide bonds which are essential for the recognition/adhesion activity.Abbreviations mt ^+/- mating-type plus or minus - DTT dithiothreitol     

Enhanced oxygen transfer rates in fermentation using soybean oil-in-water dispersions

Summary The effect of soybean oil on the volumetric oxygen transfer coefficient during the cultivation ofAerobacter aerogenes cells is presented. For our aeration-agitation conditions (0.278 vvm and 500 rpm), it has been demonstrated that the use 19% (v/v) of soybean oil enabled a 1.85-fold increase of thek _l a coefficient (calculated on a per liter aqueous phase basis). For smaller volumetric oil fractions,k _L a increased linearly with the oil loading. Because of the oxygen-vector properties of soybean oil, this oil is able to significantly increase thek _L a of a bioreactor.Nomenclature C^*, C saturation and actual dissolved oxygen concentrations respectively (g/m³) - K_La volumetric oxygen transfer coefficient (h^–1) - K_La_initial k _La measured before the oil addition (h^–1) - M_O2 molar mass of oxygen (dalton) - N oxygen transfer rate (g/m³. h) - P_O2. P_N2 partial pressures ofO ₂ andN ₂ in the gas (atm) - P_H2OT partial pressure of water in air at the temperatureT (atm) - P_T total pressure (atm) - Q₀ volumetric flow rate of outlet air before seeding (m³/h) - Sp spreading coefficient (dynes/cm) - T absolute temperature of outlet gas (K) - V_i volume of the liquidi in the fermentor (m³) - V_M molar volume at 273 K and 1 atm (m³/mole) - _ij interfacial tension betweeni andj componants (dynes/cm) - _v volumetric fraction of the oil (v/v) - G gas - O oil - W water - i inlet - o outlet     

Die Wirkung eines Komplexbildners und von Metallionen auf die Auspr?gung dessnaky- undMonstra-Charakters beiParamaecium aurelia

Summary This paper presents new facts on the problem of the inherited defects of cell separation inParamecium aurelia (Mo-strain andsnaky, cf.Maly 1958, 1960).The complexing agent EDTA, when added to the culture medium, causes a drastic increase in monster formation in thesnaky-strain, a much smaller increase in theMo-strain and no effect in the normal strain. This effect can be compensated by the addition of different metal ions and evidence is presented that the addition of iron or cobalt to the medium normalises thesnaky- andMo-strains: the separation of the cells runs normally. It is suggested that the aldolase in thesnaky-strain and in the defective particles of theMo-strain differs from that in the normal strain.     

On the role of K+ on succinic dehydrogenase activity

The effect of potassium ions on succinic dehydrogenase activity of mitochondria was studied. The results showed that in these organelles K⁺ induces inhibition of the respiratory control; moreover, in submitochondrial particles potassium inhibits the rate of oxidation of succinate. The results showed also that K⁺ does not changes theK _m for succinate but diminishes theV _max. In addition, the data provide evidence that mitochondria oxidizing glutamatemalate in a sucrose medium show a higher activity of succinate dehydrogenase than mitrochondria incubated in KCl.     

Antagonism of cadmium cytotoxicity by differentiation inducers

Studies on the antagonism of toxicity can provide information about toxic mechanisms and suggest chemotherapeutic strategies. A rapid cell growth assay that measures the effects of test agents on the accumulation of cell protein (Shopsis and Eng,Toxicol. Lett. 1985;26:1) has been applied to studies of the antagonism of the cytotoxicity of cadmium. Exposure of Balb/c mouse 3T3 cells to 15 mol/L Cd²⁺ for 24 h or 7 mol/L Cd²⁺ for 48 h caused a 50% decrease in total cell protein. Zn²⁺ and selenite ion, antagonists of Cd toxicityin vivo, antagonized Cd²⁺ cytotoxicity when added in micromolar concentrations at the initiation of exposure to Cd²⁺. A diverse group of chemicals that can induce differentiationin vitro in cultured erythroleukemia and other cells were also found to antagonize the cytotoxic effects of Cd²⁺ to 3T3 cells. Dimethyl sulfoxide (DMSO), hexamethylene bisacetamide,N,N-dimethyl formamide,N-methyl formamide, dimethyl acetamide, hypoxanthine, hemin, ouabain, and sodium butyrate, when added to cultures simultaneously with Cd²⁺, each antagonized Cd²⁺ toxicity. These agents were used at concentrations equal to or lower than the concentrations at which they induce cellular differentiation. Other cytotoxicity assays and morphological studies confirmed these observations. DMSO added as much as 6 h after the initiation of a 24-h exposure to Cd²⁺ still protected cells; conversely, pretreatment of cultures with butyrate or DMSO for 24 h followed by their removal did not confer protection against subsequent Cd²⁺ challenge. Ethanol and methanol (noninducers of differentiation) did not antagonize Cd²⁺ cytoxicity, and differentiation-inducing agents did not protect the cells from Zn²⁺-or Hg²⁺-induced cytotoxicity. DMSO treatment does not induce an increase in the concentrations of metallothionein or glutathione in these cells.Abbreviations AZA 5-azacytidine - BSA bovine serum albumin - DMEM Dulbecco's modified Eagle's medium - DMSO dimethyl sulfoxide - DTNB 5,5-dithiobis(2-nitrobenzoic acid) - EDTA ethylenediaminetetraacetic acid - FCS fetal calf serum - GSH reduced glutathione - GSSG oxidized glutathione - HMBA hexamethylene bisacetamide - MELC murine erythroleukemia cells - MT metallothionein - MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide - SEM standard error of the mean - Tris tris(hydroxymethyl)aminomethane     

Efficient transgenic plant regeneration throughAgrobacterium-mediated transformation of Chickpea (Cicer arietinum L.)

Three genotypes of chickpea ICCV-1, ICCV-6 and a Desi (local) variety were tested for plant regeneration through multiple shoot production. The embryo axis was removed from mature seeds, the root meristem and the shoot apex were discarded. These explants were cultured on medium containing MS macro salts, 4X MS micro salts, I35 vitamins, 3.0 mg/1 BAP, 0.004 mg/1 NAA, 3% (w/v) sucrose and incubated at 26⁰C. The explants were transformed withAgrobacterium tumefaciens strain LBA4404 with binary vector pBI121 containing theuidA andnptIl genes. Multiple shoots were repeatedly selected with kanamycin. The selected kanamycin resistant shoots were rooted on MS medium supplemented with 0.05 mg/1 113A. The presumptive transformants histochemically stained positive for GUS. Additionally, nptll assay confirmed the expression ofnptII in kanamycin resistant plants. Transgenic plants were transferred to soil and grown in the green house.Abbreviations BAP 6-benzylamino purine - 2,4-D 2,4dichlorophenoxy acetic acid - IAA Indole acetic acid - IBA Indole butaric acid - NAA Naphthalene acetic acid