Cloning and expression of a cDNA encoding mouse kidney -amino acid oxidase

A cDNA encoding -amino acid oxidase (DAO;EC 1.4.3.3) has been isolated from a BALB/c mouse kidney cDNA library by hybridization with the cDNA for the porcine enzyme. Analysis of the nucleotide (nt) sequence of the clone revealed that it has a 1647-nt sequence with a 5′-terminal untranslated region of 68 nt that encodes 345 amino acids (aa), and a 3′-terminal untranslated region of 544 nt that contains the polyadenylation signal sequence ATTAAA. The deduced aa sequence showed 77 and 78% aa identity with the porcine and human enzymes, respectively. Two catalytically important aa residues, Tyr²²⁸ and His³⁰⁷, of the porcine enzyme, were both conserved in these three species. RNA blot hybridization analysis indicated that a DAO mRNA, of 2 kb, exists in mouse kidney and brain, but not liver. Synthesis of a functional mouse enzyme in Escherichia coli was achieved through the use of a vector constructed to insert the coding sequence of the mouse DAO cDNA downstream from the tac promoter of plasmid pKK223-3, which was designed so as to contain the lac repressor gene inducible by isopropyl-β- -thiogalactopyranoside. Immunoblot analysis confirmed the synthesis and induction of the mouse DAO protein, and the molecular size of the recombinant mouse DAO was found to be identical to that of the mouse kidney enzyme. Moreover, the maximum activity of the mouse recombinant DAO was estimated to be comparable with that of the porcine DAO synthesized in E. coli cells.     

Induction of hepatic heme oxygenase activity by bromobenzene

Hepatic heme oxygenase, an enzyme which converts heme to carbon monoxide and bile pigment in vitro, is inducible by heme but also by large “toxic” doses of such nonheme substances as hormones, endotoxin, and heavy metal ions. When we gave rats a single hepatotoxic dose of allyl alcohol, ethionine, acetaminophen, furosemide, or endotoxin, hepatic heme oxygenase activity rose modestly (two- to fivefold) after 20 h. In contrast, administration of bromobenzene (5 mmol/kg) induced heme oxygenase in the liver an average of 15-fold after 20 h but was without effect on the enzyme in the kidney or spleen. The change in heme oxygenase was accompanied by a loss in cytochrome P-450 concentration and, in rats labeled with 5-δ-amino[¹⁴C]levulinic acid, an increased rate of degradation of hepatic [¹⁴C]heme to ¹⁴CO. Induction of heme oxygenase by bromobenzene was blocked by cycloheximide, an inhibitor of protein synthesis, but not by actinomycin D, an inhibitor of RNA synthesis. This suggests that bromobenzene stimulates de novo enzyme synthesis at the step of translation. Subtoxic doses of bromobenzene (less than 1 mmol/kg) gave proportionately greater induction of heme oxygenase. Furthermore, induction of the enzyme remained unaffected when bromobenzene hepatotoxicity was blocked by pretreatment of rats with SKF-525A, 3-methylcholanthrene, or cysteine (which supplements liver sulfhydryl content), or when hepatotoxicity was enhanced by pretreatment with phenobarbital or with diethylmaleate (which depletes hepatic glutathione). These data suggest that with induction of heme oxygenase by bromobenzene, neither liver cell necrosis nor alteration in hepatic sulfhydryl metabolism is indispensible. The latter characteristic differs from induction of the enzyme by metal ions in which depletion of sulfhydryl-containing constituents has been thought to be essential. We conclude that bromobenzene is a novel inducer of heme oxygenase activity in the liver, differing from other nonheme substances in potency and specificity for the liver, and in utilizing mechanism(s) which require neither production of hepatotoxicity, depletion of hepatic glutathione, nor sensitivity to actinomycin D.     

Renal handling of Ca2+ in diabetes

Ca²⁺ transport in kidney has gained considerable attention in the recent past. Our laboratory has been involved in understanding the regulatory mechanisms underlying Ca²⁺ transport in the kidney across the renal basolateral membrane. We have shown that ANP, a cardiac hormone, mediates its biological functions by acting on its receptors in the kidney basolateral membrane. Furthermore, it has been established that ANP receptors are coupled with Ca²⁺ ATPase, the enzyme that participates in the vectorial translocation of Ca²⁺ from the tubular lumen to the plasma. It is possible that a defect in the ANP-receptor-effector system in diabetes (under certain conditions such as hypertension) may be associated with abnormal Ca²⁺ homeostasis and the development of nephropathy. Accordingly, future studies are needed to establish this hypothesis.     

Hormonal regulation of ornithine aminotransferase biosynthesis in rat liver and kidney.

The relative rates of ornithine aminotransferase (OAT) synthesis in vivo were studied by pulse-labeling rats with [4,5-³H]leucine, isolating the mitochondrial enzyme protein by immunoprecipitation with a monospecific antibody, dissociating the immunoprecipitates on sodium dodecyl sulfate-acrylamide gels, and determining the radioactivity in OAT. After 4 days of treatment with triiodothyronine (T₃), both the enzyme activity level and the relative synthetic rate of OAT in rat kidney were elevated over twofold. The level of hepatic OAT activity was unaffected by this treatment. Thyroidectomy caused a 50% drop in the basal level of OAT activity and synthesis in kidney but not in liver. Although the basal levels of activity and synthesis of both renal and hepatic OAT were unaffected by adrenalectomy, the glucagon induction of the enzyme in liver was enhanced by about one-third and the T₃ induction in kidney was suppressed 50% by this operation. After 4 days of treatment with estrogen, both the enzyme activity level and the relative synthetic rate of OAT in male rat kidney were elevated nearly 10-fold. Hepatic OAT activity and synthesis were unaffected by this regimen. Thyroidectomy almost completely abolished the estrogen induction of OAT in kidney. OAT induction by estrogen could be restored by treating thyroidectomized rats with T₃. Simultaneous administration of T₃ plus estrogen to intact rats produced a multiple effect, resulting in a striking 20-fold induction of renal OAT. Although administration of either T₃ or estrogen causes an increase in the synthesis of immunoprecipitable OAT protein in rat kidney, each of these hormones may induce OAT by a different mechanism.     

Purification,kinetic studies,and homology model of Escherichia coli fructose-1,6-bisphosphatase

Previous kinetic characterization of Escherichia coli fructose 1,6-bisphosphatase (FBPase) was performed on enzyme with an estimated purity of only 50%. Contradictory kinetic properties of the partially purified E. coli FBPase have been reported in regard to AMP cooperativity and inactivation by fructose-2,6-bisphosphate. In this investigation, a new purification for E. coli FBPase has been devised yielding enzyme with purity levels as high as 98%. This highly purified E. coli FBPase was characterized and the data compared to that for the pig kidney enzyme. Also, a homology model was created based upon the known three-dimensional structure of the pig kidney enzyme. The k_cat of the E. coli FBPase was 14.6 s⁻¹ as compared to 21 s⁻¹ for the pig kidney enzyme, while the K_m of the E. coli enzyme was approximately 10-fold higher than that of the pig kidney enzyme. The concentration of Mg²⁺ required to bring E. coli FBPase to half maximal activity was estimated to be 0.62 mM Mg²⁺, which is twice that required for the pig kidney enzyme. Unlike the pig kidney enzyme, the Mg²⁺ activation of the E. coli FBPase is not cooperative. AMP inhibition of mammalian FBPases is cooperative with a Hill coefficient of 2; however, the E. coli FBPase displays no cooperativity. Although cooperativity is not observed, the E. coli and pig kidney enzymes show similar AMP affinity. The quaternary structure of the E. coli enzyme is tetrameric, although higher molecular mass aggregates were also observed. The homology model of the E. coli enzyme indicated slight variations in the ligand-binding pockets compared to the pig kidney enzyme. The homology model of the E. coli enzyme also identified significant changes in the interfaces between the subunits, indicating possible changes in the path of communication of the allosteric signal.     

Induction of L-lysine-2-oxoglutarate reductase by glucagon and glucocorticoid in developing and adult rats in vivo and in vitro studies

L-Lysine-2-oxoglutarate reductase (EC 1.5.1.8, NADP⁺) in the liver of adult rats increased 4–5-times when the animals were treated with alloxan. In diabetic rats injection of insulin or adrenalectomy prevented the increase in enzyme activity. The activity of the similar enzyme in kidney was not changed by these treatments. The enzyme activity in primary cultured adult rat hepatocytes was also induced by addition of dexamethasone and glucagon together, and glucagon could be replaced by dibutyryl cyclic AMP. Insulin inhibited the induction. The hormonal induction was also inhibited by actinomycin D and by cycloheximide. During development of rats, fetal liver showed very low activity, but the activity appeared on day 1 after birth and then increased rapidly, reaching the adult level by day 5. The activity of the kidney enzyme increased more slowly and reached the adult level 1 month after birth. Intra-uterine injection of glucagon caused precocious induction of the liver enzyme in fetuses. These results indicate that the activity of L-lysine-2-oxoglutarate reductase in the adult liver and in part in neonatal liver also, is controlled by both glucagon and glucocorticoid.     

Production,cross reactivity,and epitope analysis of monoclonal antibodies against rat kidney gamma glutamyltransferase

Eighteen IgGl monoclonal antibodies (blabs) have been produced against gamma-glutamyl transferase (GGT) from rat kidney. They were specific to the light subunit of the enzyme with affinity constants ranging from 0.3 to 7.5 10⁸ M^–1, while they did not react with GGT from other sources i.e. human and pig kidney, rat and guinea pig liver, suggesting species and organ specificity. Two of the blabs (N° 11 and 21) lost their immunoreactivities towards rat kidney GGT in the presence of N-acetyl-neuraminic acid, while immunoreactivities of the other blabs were unchanged. Furthermore, Mabs No 11 and 21 did not react with desialylated rat kidney GGT. These findings suggest that N-acetyl-neuraminic acid is involved in the epitopes recognized by these two Mabs.Abbreviations ELISA enzyme linked immunosorbent assay - GGT gamma-glutamyltransferase - Mab monoclonal antibody - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Biochemical and morphological characterization of primary kidney cell cultures from beige mutant mice

Summary Primary kidney cultures from adult beige-J (bg ^J/ bg ^J) mice were selected for epithelial cell growth using D-valine medium. After 2 weeks of attachment and proliferation in vitro, the cells form a confluent or nearly confluent monolayer that retains several phenotypic characteristics of the beige-J mutant. These include large, multilamellar inclusion bodies that are apparently dysmorphic lysosomes, and higher concentrations of neutral glycosphingolipids and dolichols than control cells. -Glucuronidase activity, used as a lysosomal enzyme marker, is not elevated in beige-J-cultured kidney cells compared with controls, as it is in the intact kidney. The high levels of -glucuronidase activity in both control and mutant cells may mask expression of this difference in vitro. The action of the beige-J mutation in kidney cells is thought to be due to a block in exocytosis that results in the accumulation of abnormal lysosomes and their components. The maintenance of the beige phenotype in vitro indicates that the mutation is not suppressed in primary kidney cell cultures. The expression of the beige phenotype in vitro should be useful for studies concerning the primary lesion of this mutation.     

Glucocorticoid induction of tyrosine aminotransferase in kidney cortex

It has recently been reported that the glucocorticoid receptors present in kidney occur as two distinct forms which are segregated in the cortex and the medulla. We were interested in determining if glucocorticoid induction of the enzyme tyrosine aminotransferase (L-tyrosine: 2 oxoglutarate aminotransferase, E.C.2.6.1.5) also differed in these two areas of the kidney. Administration of the synthetic glycocorticoid, dexamethasone, resulted in a 2-fold induction of tyrosine aminotransferase in kidney cortex of adrenalectomized rats and no induction of the enzyme in kidney medulla. Examination of this response in rat brain revealed no induction of the enzyme by dexamethasone in this tissue.     

TheN haplotype of the murineβ-glucuronidase gene is altered in both its systemic regulation and its response to androgen induction

A new haplotype of the -glucuronidase gene complex, [Gus]^N, has been characterized following its transfer from the PAC/Cr strain to the standard strain C57BL/6J. TheN haplotype contains a novel structural gene allele which encodes an allozyme differing from all previously characterized allozymes in both size and charge. Altered systemic regulation is exhibited by the [Gus]^N haplotype. Multiple tissues contain levels of GUS protein that are 60±15% those found in the standardB haplotype. The regulatory mechanism for reduction is complex, involving tissue-specific changes in both enzyme synthesis and enzyme turnover. The changes in GUS protein synthesis do not result from changes in GUS mRNA levels. Instead, the amount of mature enzyme formed per mRNA molecule, or translational yield, is altered. These regulatory changes parallel those seen in other systemic regulatory variants of GUS which are also altered in translational yield. A commonality of mechanism among systemic regulatory variants of this gene is suggested. TheN haplotype is also exceptional in the nature of its response to androgenic induction in kidney proximal tubule epithelial cells. The time course for GUS induction consists of a lag period followed by a progressive increase in mRNA, rate of enzyme synthesis, and enzyme activity. For the [Gus]^N haplotype the lag is of an exceptionally short duration and the plateau is of a greater magnitude than for any haplotype previously described.This work was supported by United States Health Service Research Grant GM 31656.     

Isolation of anthglutin, an inhibitor of gamma-glutamyl transpeptidase from Penicillum oxalicum.

Anthglutin, a new inhibitor of γ-glutamyl transpeptidase, has been isolated from the cultured medium of Penicillium oxalicum and its structure established as l-γ-l-glutamyl-2-(2-carboxyphenyl)hydrazine. The isolation of anthglutin was achieved by ion-exchange chromatography. Anthglutin inhibited γ-glutamyl transpeptidase specifically and the kinetic analysis of the inhibition showed that anthglutin inhibited the enzyme competitively with regard to the glutamyl donor, γ-glutamyl-p-nitroanilide, and noncompetitively with regard to the glutamyl acceptor, glycylglycine. K₁ values were 5.7 μm for the hog kidney enzyme, 18.3 μm for the human kidney enzyme, 13.6 μm for the human liver soluble enzyme, and 10.2 μm for the bound enzyme. After oral administration of [¹⁴C]methionine and anthglutin to rats, no effect of anthglutin was observed on the absorption of methionine in the intestine.     

Large scale isolation,purification, and partial characterization of the intermediate filament-specific,Ca2+-activated proteinase from porcine kidney and Ehrlich ascites tumor cells: A comparative study

The Ca²⁺-activated, neutral thiol proteinase specific for intermediate filament subunit proteins was isolated at large scale from the postribosomal supernatant of a low-ionic-strength extract of porcine kidney and Ehrlich ascites tumor (EAT) cells, respectively. The purification procedure encompassed DEAE-Sephacel ion exchange chromatography of the material precipitating between 23 and 55% (NH₄)₂SO₄ saturation, followed by hyroxylapatite chromatography and activated thiol Sepharose 4B covalent chromatography. On the average, 25 mg of 62% pure enzyme was obtained from 500 g frozen kidney and 55 mg of 51% pure enzyme from 500 g EAT cells within a week. Both enzyme preparations were free of Ca²⁺-independent proteolytic activities and indistinguishable with respect to their physicochemical and functional properties; their catalytic properties were indistinguishable from those of enzyme purified to homogeneity on arginine methylester Sepharose 4B. Because of this identity, porcine kidney proves to be an inexpensive source for the Ca²⁺-activated proteinase which had previously been isolated and purified at small scale from EAT cells (W. J. Nelson and P. Traub, (1983) J. Biol. Chem.257, 5544–5553). Despite a 38% protein contamination, the partially purified enzyme from porcine kidney is useful for the in vitro study of structure-function relationships of intermediate filaments and their subunit proteins. During affinity chromatography of the partially purified proteinase from EAT cells on arginine methylester Sepharose 4B, a 100-kDa protein was purified which has a high affinity for arginine residues. It also occurs in porcine kidney, although at a considerably lower concentration. Its cellular localization and function remain to be determined.     

Inhibition of phytoalexin biosynthesis in elicitor-treated tobacco cell-suspension cultures by calcium/calmodulin antagonists

A role for calcium/calcium-binding proteins in a mechanism of signaling elicitor-inducible phytoalexin biosynthesis was investigated. Two classes of calcium/calmodulin antagonists, phenothiazines and naphthalenesulfonamides, inhibited sesquiterpene phytoalexin accumulation in tobacco (Nicotiana tabacum) cell-suspension cultures when added 1 h before elicitor. The antagonists also inhibited the induction of sesquiterpene cyclase enzyme activity, a key regulatory enzyme for sesquiterpene biosynthesis. The antagonists suppressed the induction of sesquiterpene cyclase only if added before or simultaneously with elicitor. Additionally, the antagonists inhibited (a) accumulation of the cyclase protein as measured in immunoblots; (b) the in vivo synthesis rate of the cyclase protein, measured as the incorporation of [³⁵S]methionine into immunoprecipitable cyclase protein; and (c) the cyclase mRNA translational activity, measured as the incorporation of [³⁵S]methionine into immunoprecipitable cyclase protein synthesized by in vitro translation of RNA isolated from antagonist-treated, elicitor-induced cells. In contrast, elicitor-inducible phenylalanine ammonia lyase enzyme activity, the level of the enzyme protein, the in vivo synthesis rate, and the mRNA translational activity were not affected by any of the antagonist treatments. Uptake and incorporation of [³⁵S]methionine into total cellular proteins and total in vitro translation products were also not indiscriminately altered by the antagonist treatments. The current results suggest that calcium and/or calmodulin-like proteins may be elements of a signal transduction pathway mediating elicitor-induced accumulation of phytoalexins in tobacco.     

Induction of L-lysine-2-oxoglutarate reductase by glucagon and glucocorticoid in developing and adult rats in vivo and in vitro studies

L-Lysine-2-oxoglutarate reductase (EC 1.5.1.8, NADP⁺) in the liver of adult rats increased 4–5-times when the animals were treated with alloxan. In diabetic rats injection of insulin or adrenalectomy prevented the increase in enzyme activity. The activity of the similar enzyme in kidney was not changed by these treatments. The enzyme activity in primary cultured adult rat hepatocytes was also induced by addition of dexamethasone and glucagon together, and glucagon could be replaced by dibutyryl cyclic AMP. Insulin inhibited the induction. The hormonal induction was also inhibited by actinomycin D and by cycloheximide. During development of rats, fetal liver showed very low activity, but the activity appeared on day 1 after birth and then increased rapidly, reaching the adult level by day 5. The activity of the kidney enzyme increased more slowly and reached the adult level 1 month after birth. Intra-uterine injection of glucagon caused precocious induction of the liver enzyme in fetuses. These results indicate that the activity of L-lysine-2-oxoglutarate reductase in the adult liver and in part in neonatal liver also, is controlled by both glucagon and glucocorticoid.     

A simple and sensitive assay for 9-hydroxyprostaglandin dehydrogenase

The tritium recovery assay of 9-hydroxyprostaglandin dehydrogenase [Pace-Asciak, C. (1975) J. Biol. Chem.250, 2789] has been modified to ensure its applicability to both crude and purified enzyme preparations. The stereospecificity of NAD⁺-dependent 9-hydroxyprostaglandin dehydrogenase with respect to NAD⁺ was determined first and found to be A-side specific. Based on the stereospecificity of the enzyme, a simple and sensitive assay method for 9-hydroxyprostaglandin dehydrogenase has been developed. The assay is able to detect picomole quantities of substrate conversion. When 15-keto-13,14-dihydro-[9β-³H]PGF_2α is employed as substrate, the tritium label of the tritiated prostaglandin is effected to transfer to lactate stereospecifically by coupling 9-hydroxyprostaglandin dehydrogenase with a saturating level of lactate dehydrogenase. The amount of prostaglandin oxidized is quantitated by the radioactivity of the labeled lactate produced, which is separated from labeled prostaglandin by charcoal precipitation. Simultaneous assays with the current tritium-release and thin-layer chromatography methods indicated excellent correlation. Using this method we have found that rat kidney possesses the highest enzyme activity among those tissues examined. Rat kidney enzyme activity is linear for the first 10 min it is studied and is nonlinear with increasing amounts of crude enzyme extract, indicating the possible presence of endogenous inhibitor(s). The apparent K_m for 15-keto-13,14-dihydro-PGF_2α is 0.66 μm. The enzyme is activated by imipramine, inhibited by indomethacin, but not affected by furosemide and ethacrynic acid. These results confirm previous findings reported in the literature.     

Phosphorylated intermediate of the ouabain-insensitive,Na-stimulated ATPase in rat kidney cortex and rainbow trout gills

Several tissues from different animals, including the rat kidney and the freshwater rainbow trout gills, show an ouabain-insensitive, furosemide-sensitive, Na⁺-stimulated ATPase activity, which has been associated with the active control of the cell volume. This Na-ATPase is Mg²⁺ dependent and it is inhibited by vanadate, which can be taken as an indication that this enzyme is a P-type ATPase. The P-type ATPases are known to form a phosphorylated intermediate during their catalytic cycle, where the phosphate binds an aspartyl residue at the enzyme's substrate site. In the current study, we partially characterized the phosphorylated intermediate of the ouabain-insensitive Na-ATPase of rat kidney cortex homogenates and that of gill microsomes from freshwater rainbow trout. While the kidney cortex homogenates, under our assay conditions, show both Na- and Na,K-ATPase activities, the gill microsomes, when assayed at pH 5.2, only show Na-ATPase activity. Both preparations showed a Mg²⁺-dependent, Na⁺-stimulated phosphorylated intermediate, which is enhanced by furosemide. Incubation of the phosphorylated enzyme with 0.6 N hydroxylamine (NH₂OH) showed that it is acid-stable and sensitive to hydroxylamine, either when phosphorylated in the presence or absence of furosemide. Addition of ADP to the incubation medium drives the reaction cycle of the enzyme backward, diminishing its phosphorylation. Na⁺ seems to stimulate both the phosphorylation and the dephosphorylation of the enzyme, at least for the Na-ATPase from gill microsomes. In a E1–E2 reaction cycle of the Na-ATPase, furosemide seems to be blocking the transition step from Na·E1∼P to Na·E2-P.     

Epsilon-alkyllysinase. New assay method, purification, and biological significance

?-Alkyllysinase (EC 1.5.3.4.) has been solubilized and purified approximately 15-fold from rat kidney. Flavin adenine dinucleotide stimulated the partially purified enzyme preparation. The enzyme produces an equimolar amount of l-lysine and formaldehyde from ?-N-monomethyl-l-lysine while consuming half a mole of oxygen. Based on the determination of radioactive formaldehyde from ?-N-mono[¹⁴C]methyl-lysine a new, highly sensitive assay method has been developed. All available evidence indicates that ?-alkyllysinase is identical to histone demethylase which has been previously reported to be rich in rat kidney mitochondria. The ?-alkyllysinase activity in the liver and kidney of a young rat are relatively low, and reaches the adult level during or before puberty. On the other hand, protein methylase III, which methylates histones using S-adenosyl-l-methionine as methyl donor, is high in the young rat liver and kidney, and decreases to the adult level in a pattern opposing that of ?-alkyllysinase. This opposing pattern of change of protein methylase III and ?-alkyllysinase activity is also found in fast-growing Novikoff hepatoma: While ?-alkyllysinase activity is practically nil in the hepatoma, protein methylase III is very high.     

The relationship between rooting of poplar shoots and ATPase activity of their crude microsomal vesicles

Poplar shoots raised in vitro were induced to root by a 7 h passage on an auxin (1-naphthaleneacetic acid) medium. The percentage of rooting was reduced from ± 97% to ± 47% when vanadate (200 µM) was included in the auxin medium. Introduction of vanadate in the medium without auxin after the 7 h induction on auxin medium, did not inhibit rooting but affected only the development of the roots produced. The Mg²⁺-dependent ATPase activity of the microsomal vesicles of poplar shoots was increased after 7 h induction on rooting medium and corresponded to an increase in the Vmax of the enzyme. Results from experiments using some inhibitors of the polyamine metabolism suggested that this pathway was not involved in the increase of this activity. The auxin had no effect on the in vitro ATPase activity at any concentration tested except at about 2 mM where it was inhibitory, probably due to a change in the conformation of the enzyme. The transient increase of indole-3-acetic acid during rooting induction could be responsible for the increase in the level of the enzyme. The inhibition of root formation and growth by vanadate indicates strongly that the ATPase activity may be necessary for the induction and expression of rooting.     

In vitro hormone-regulated growth and floral induction of <Emphasis Type="Italic">Cuscuta reflexa</Emphasis>: a parasitic angiosperm

The role of different growth regulators in callus induction, shoot regeneration, floral induction and chlorophyll content of the obligatory parasitic plant Cuscuta reflexa has been studied. Callus development was excellent from the nodal part of the shoot explants in modified Murashige and Skoog (MMS) media supplemented with 2 mg L⁻¹ benzyl adenine (MMS1c). Supplementation of 2 mg L⁻¹ naphthalene acetic acid (NAA) along with MMS1c (MMS2c) was responsible for estimable shoot induction and development in callus. 2,4-Dichloro acetic acid (2,4-D) played a crucial role in the floral induction of C. reflexa in vitro. MMS supplemented with 2 mg L⁻¹ NAA and 2 mg L⁻¹ 2,4-D (MMS3b) supported floral induction after shooting in vitro. MMS supplemented with 3 mg L⁻¹ 2,4-D (MMS4a) rapidly induced flower directly from the stem explants without showing any elongation of shoot. MMS1c along with MMS3b (MMS5a) showed callus proliferation followed by shoot elongation and floral induction. In vitro MMS5a grown plants show a sharp increase in the chlorophyll contents. Cytokinin treatment further increases the chlorophyll level of the plant.