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.     

The electrochemical proton gradient and its influence on citrate uptake in tonoplast vesicles of Hevea brasiliensis

The relationship between the electrochemical proton gradient, _H+ ^– , and citrate transport has been studied in tonoplast vesicles from Hevea brasiliensis (the rubber tree). Vesicles were generated from lyophilized samples of fresh vacuoles obtained from the latex sap. Methylamine was used to measure intravesicular pH and lipophilic ions to determine the electrical potential difference () across the tonoplast. When incubated at pH 7.5 in the absence of ATP, the tonoplast vesicles showed a pH of 0.6 units (interior acid) and a of about-100 mV (interior negative). This potential is thought to be made up of contributions from an H⁺ diffusion potential, diffusion potentials from other cations and a Donnan potential arising from the presence of internal citrate. In the presence of 5 mol m^-3 MgATP the pH was increased to about 1.0 unit and the to about-10 mV. Under these conditions the proton-motive force ( _{p H+} ^– /F) became positive and reached +50 mV. These effects were specific to MgATP (ADP and Mg²⁺ having no significant effect) and were prevented by the protonophore p-trifluoromethoxycarbonylcyanidephenylhydrazone (FCCP). Citrate uptake by the vesicles was markedly stimulated by MgATP; ADP and Mg²⁺ again had no effect. Nigericin greatly increased pH and this was associated with a large increase in citrate accumulation. The results indicate that the vesicle membrane possesses a functional H⁺-translocating ATPase. The _H+ ^– generated by this ATPase can be used to drive citrate uptake into the vesicles. The properties of the tonoplast vesicles are compared with those of the fresh latex vacuoles.Abbreviations _H+ ^– electrochemical proton gradient - electrical potential difference across membrane - p proton-motive force ( _H+ ^– /F) - FCCP p-trifluoromethoxycarbonylcyanidephenylhydrazone - TPMP⁺ triphenylmethylphosphonium ion     

Characterization of catecholamine uptake2 in isolated cardiac myocytes

Extraneuronal catecholamine uptake was investigated in isolated quiescent rat myocardial cells. By administration of (³H-)(–)noradrenaline concentration of 22 nmol/l up to 1000 mol/l the following data were obtained: (1) The K_M of the uptake process amounted to 260 mol/l, the V_max to 4.24 nmol/(10 min × mg Protein) corresponding to 179 nmol/(min × g_WWt)(WWT = Wet Weight). (2) The uptake was largely inhibited by the uptake₂-inhibitors corticosterone (100 mol/l), isoprenaline (IC so = 30.6 mol/l), and O-methylisoprenaline (IC₅₀ = 2.1 pmol/l), but not by the uptake₁-inhibitors cocaine (100 mol/l) and desipramine (10 mol/l). (3) The affinity-values K_M and IC₅₀ closely agreed with those already known, but the V_max-value was higher than those obtained in whole rat hearts by a factor of at least 1.79. This is caused presumably by the voltage dependence of the uptake mechanism and the resulting inhibition of uptake 2 during the periods of depolarisation in beating hearts of other studies.     

Effect of aminooxyacetic acid on the release of preloaded [3H]GABA and radioactive metabolites from slices of developing mouse brain

The release of [³H]-aminobutyric acid (GABA) and its radioactive metabolites from slices of the cerebral cortex, cerebellum, striatum and brain stem of developing and adult mice was studied. The slices were incubated and superfused in the absence and presence of the GABA aminotransferase (GABA-T) inhibitor aminooxyacetic acid (AOAA). Exposure to 100 M AOAA totally inhibited GABA-T and all radioactivity released from slices was in authentic GABA. In studies on developing brain the 10-M concentration was also effective enough, except in cerebellar slices. In the absence of AOAA the major part of radioactivity spontaneously released from slices of adult cerebral cortex and cerebellum was tritiated water and still about one third part in the presence of 10 M AOAA. Potassium stimulation induced only the release of radioactive GABA but not labeled metabolites in both presence and absence of AOAA. AOAA reduced the stimulation-induced release of GABA. It is recommended that the use of GABA-T inhibitors should be discontinued in release experiments. Then labeled GABA must be separated in the effluents from its radioactive breakdown products.     

Regeneration of plants from callus and embryos of ‘Royal’ apricot

Immature embryos of apricot (Prunus armeniaca L.) cv. Royal with a PF index of 25–100 were used to regenerate plants in vitro using two methods. In the first case, callus was initiated on MS medium with 4.5 M 2, 4-D plus 0.44 M BA and regeneration of shoots from the callus occurred on MS medium with 4.4 M BA plus 1.0 M 2, 4-D. In the second case, adventitious buds were directly regenerated from the cotyledons on MS medium with 4.4 M BA plus 1.0 M 2, 4-D.Abbreviations BA 6-benzyladenine - IBA dole-3-butyric acid - NAA -naphthylacetic acid - 2, 4-D 2, 4-dichlorophenoxyacetic acid - PF (embryo length/seed length) x 100     

Visualization of the uptake of high-density lipoprotein by rat aortic endothelial cells and smooth muscle cells in vitro

High-density lipoproteins (HDL) were conjugated to Fluorescein 1,1-dioctadecyl 3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI) or colloidal gold for the investigation of ultrastructural aspects of binding and uptake of HDL by cholesterol-loaded cultured endothelial and smooth muscle cells from rat aorta. When cells were incubated for 2h at 4°C, HDL–DiI and HDL–gold conjugates were seen only on the cell surface. When cells were returned to incubation at 37°C for 5min, HDL–DiI appeared in the cytoplasm and colocalized with the fluorescent cholesteryl ester tag BODIPY-FL-C₁₂. HDL–gold conjugates appeared in the plasmalemmal invaginations and plasmalemmal vesicles. After incubation for 15min, most of the HDL–gold conjugates reappeared on the cell surface. After incubation for 30min, only a few conjugates were observed and they localized in lysosomal-like bodies. Quantitative data indicated that when the cholesterol-loaded cells were incubated at 4°C for 2h, the numbers of HDL–gold associated in clusters on the endothelial cell surface was 1.18 clusters/m. When cells were returned to incubation at 37°C for 5min, this value decreased to 0.7, increased again to 1.13 at 15min, and decreased to 0.29 at 30min. The numbers of clusters in the plasmalemmal invaginations were 0.06 clusters/m at 4°C for 2h, increased to 0.34 at 37°C for 5min and decreased gradually to 0.19 and 0.04 at 15 and 30min, respectively. The incidence of clusters in the plasmalemmal vesicles per non-nuclear cytoplasm was 0.01 clusters/m² at 4°C for 2h, increased significantly to 1.08 at 37°C for 5min, and decreased to 0.43 and 0.14 at 15 and 30min, respectively. This work supports that the plasmalemmal invaginations and plasmalemmal vesicles are linked to the HDL uptake in cholesterol-loaded aortic endothelial cells and smooth muscle cells.     

The effect of monoterpenes on astaxanthin production by a mutant ofPhaffia rhodozyma

Summary The total pigment and astaxanthin content ofPhaffia rhodozyma increased with increasing concentrations -pinene up to 500 l -pinene/l. Above this concentration the total pigment and astaxanthin content as well as the biomass production decreased. The addition of 500 l -pinene/l increased the total pigment content from 1652 g/g to 2201 g/g and the astaxanthin content from 1554 g/g to 1883 g/g. A sharp decrease in maximum specific growth rate occurred above 150 l -pinene/l.     

Two new species of Isospora (Apicomplexa: Eimeriidae) from passeriform birds of South America

Summary Two new species of Isospora (Apicomplexa: Eimeriidae) are described from the faeces of passeriform birds of South America. I. cyanocoracis n. sp. is described from Cyanocorax chrysops (Passeriformes: Corvidae) and I. paroariae n. sp. from Paroaria coronata (Passeriformes: Emberizidae). I. cyanocoracis oocysts are spherical or subspherical, 28.7×26.8 m (25.0–30.5×24.5–29.0), with bi-layered wall about 2.0 m thick. Micropyle and oocyst residuum are absent; large polar granule present. Sporocysts are ovoid, 19.3×11.4 m (17.0–21.0×10.5–12.2), with smooth, single-layered wall about 0.8 m thick. Stieda and substiedal bodies and sporocyst residuum are present. Sporozoites 12.2×4.2 m (10.5–15.0×3.5–4.5), possess spherical anterior and posterior refractile bodies. I. paroariae oocysts are spherical or subspherical, 22.3×21.4 m (19.5–25.5×18.5–24.0), and have bi-layered wall about 1.8 m thick. Micropyle, polar granule, and oocyst residuum are absent. Sporocysts ovoid, 15.2×10.0 m (14.0–16.5×8.0–11.5), possess smooth, single-layered wall about 0.7 m thick. Stieda and substiedal bodies and sporocyst residuum are present. Sporozoites elongate, 11.3×3.4 m (10.0–13.5×3.2–4.0), have single, large, posterior refractile body. ac]19840712     

Isolation and identification of plant growth inhibitors as candidate(s) for allelopathic substance(s), from aqueous leachate from mesquite (Prosopis juliflora (Sw.) DC.) leaves

As candidate(s) for allelopathic substance(s), two plant growthinhibitors were isolated from aqueous leachate of leaves of mesquite, whichshowa strong allelopathy, and which were identified as syringin and(–)-lariciresinol by their spectral analyses. Syringin inhibited root andshoot growth of lettuce seedlings at concentrations greater than 0.8M, and root and shoot growth of barnyard grass seedlings atconcentrations greater than 2.7 and 26.9 M, respectively. Onthe other hand, (–)-lariciresinol inhibited root and shoot growth oflettuce seedlings at concentrations greater than 2.8 and 0.8M,and root and shoot growth of barnyard grass seedling at concentrations greaterthan 0.8 and 2.8 M, respectively. The contents of syringin and(–)-lariciresinol in the rhizosphere soil of mesquite were 0.34 and 0.38g/g soil, respectively. These results indicate that syringinand (–)-lariciresinol are allelopathic substance(s), and may play rolesinthe allelopathy of mesquite.     

Somatic embryogenesis in Centella asiatica L. an important medicinal and neutraceutical plant of India

Leaf segments excised from Centella asiatica, a medicinal and neutraceutical plant, produced abundant somaticembryoswhen cultured onMS mediumwith 9.29 Mkinetin in combination with 2.26 M2,4-D. Granular, white,shiny clusters of callus developed after 1 week of culture, and then formed heart and cotyledonary stage embryoson the same medium after 4 weeks. Somatic embryos matured and germinated in the presence of MS mediumcontaining 2.32 M kinetin with (2.89M) GA3. Plantlets were successfully transferred to pots containing amixture of soil and vermiculite (1:1).     

Problems in estimating growth rates of marine phytoplankton from short-term14C assays

Growth rate estimates () of phytoplankton populations that were sampled from nitrogen-limited continuous cultures and then incubated for short durations in batch culture with added¹⁴C-HCO₃ ^– were significantly different than steady-state growth rates () for 3 of 5 marine phytoplankton species. Two diatoms,Thalassiosira weissflogii andChaetoceros simplex, displayed virtually identical growth rates (=) over a wide range of, whereas for a third diatom,Phaeodactylum tricornutum, was overestimated by an average of 40% compared to. In contrast, was underestimated by the¹⁴C technique for the two remaining species: up to 40% at a steady-state of 1.0 day^–1 for the chlorophyteDunaliella tertiolecta and up to 100% at of 1.4 day^–1 for the haptophytePavlova lutheri. For the latter two species the divergence between and appeared to increase with increasing steady-state. A simple model of labeled and total carbon flow between the aqueous phase and cellular biomass was constructed to demonstrate that respiration was negligible when=, but was significant when>. In the cases in which<, a rapid physiological alteration presumably took place once the steady state was disturbed and cells were placed in the incubation chambers, which perhaps was related to the nutritional state of the cultures at the time of sampling. Questions thus are raised regarding our ability to measure accurately primary productivity from shipboard experiments with confined samples of phytoplankton from nutrient-impoverished waters that probably are less hardy than the laboratory cultures used in these studies.     

Phosphorylation Sites Within α4 Subunits of α4β2 Neuronal Nicotinic Receptors: A Comparison of Substrate Specificities for cAMP-Dependent Protein Kinase (PKA) and Protein Kinase C (PKC)

The present study determined whether putative phosphorylation sites within the M3/M4 cytoplasmic domain of the human 4 subunit of 42 neuronal nicotinic receptors are substrates for cAMP-dependent protein kinase (PKA) or protein kinase C (PKC). Five peptides corresponding to predicted phosphorylation sequences were synthesized, and phosphorylation was compared with standard peptide substrates for each kinase, that is, Kemptide for PKA and glycogen synthase (GS) 1-8 for PKC. VRCRSRSI had the highest affinity for PKA, with a Km of 44.5 M; Kemptide had a Km of 7.7 M. LMKRPSVVK and KARSLSVQH were also phosphorylated by PKA, but had lower affinities of 593 M and 2896 M, respectively. LMKRPSVVK had the highest affinity for PKC with a Km of 182 M; GS 1–8 had a Km of 2.1 M. VRCRSRSI had a comparative affinity for PKC with a Km of 327 M. PCKCTCKK was not phosphorylated by PKA, but was a substrate for PKC with a Km of 1392 M, whereas PGPSCKSP was not phosphorylated by either kinase. Based on these findings, results suggest that Ser-362 and Ser-486 on the human 4 subunit may be phosphorylated by either PKA or PKC, Ser-467 is a putative PKA site, and Thr-532 represents a likely PKC substrate; Ser-421 does not appear to be phosphorylated by either kinase.     

The effect of avermectins on feeding,salivary fluid secretion,and fecundity in some ixodid ticks

We tested the effects of the potent acaricides, avermectin B₁a (AVM) and 22,23-dihydroavermectin B₁ (ivermectin; IVM) when injected directly into partially fed and fully engorged female ticks. When injected into small ticks (Amblyomma hebraeum Koch), neither drug (up to 100 g/kg b.w.) inhibited subsequent engorgement nor affected oviposition latency, weight of total egg mass laid nor viability of laid eggs. At higher concentrations (1000 and 5000 g/kg b.w.), AVM and IVM were markedly toxic. When injected into engorged ticks, both drugs increased oviposition latency, and reduced fecundity at about 75–100 g/kg b.w. Vitellogenesis, as assessed by a spectrophotometric assay of the ovaries, was not inhibited. Also at 50–100 g/kg b.w., AVM and IVM caused paralysis of the abdominal dorso-ventral muscles and the leg muscles. Both drugs, at 7 days post-injection, proved detrimental to salivary gland function in both small and large ticks, but had little effect on salivary gland weight. At concentrations which did not inhibit oviposition (20–50 g/kg b.w.) many of the eggs dried out even though they were kept at high RH. We then demonstrated inAmblyomma americanum, Dermacentor andersoni andD. albipictus that removal of egg wax (by extraction with hexane) induced a marked increase in water permeability. IVM neither increased water permeability ofD. andersoni eggs nor diminished the amount of egg wax deposited on the surface of the eggs, when injected posteriorly through the alloscutum. However, injection of IVM, dimethylsulphoxide (vehicle for IVM) or distilled water through the articulation between the capitulum and scutum (anterior injection), did markedly reduce the wax coating and increased egg permeability. We suggest that anterior injection damages Gené's organ and thus causes the latter effects.     

Al-induced inhibition of root elongation in corn,Zea mays L. is overcome by Si addition

The effect of silicic acid on Al-induced inhibition of root elongation was investigated in corn roots (Zea mays L. cv. golden cross bantam) in 100 t M CaCl2 solution at pH 4.3. Twenty t M Al inhibited root elongation (20 h) about 70%, however, inhibition was alleviated by addition of silicic acid. The alleviative effect increased with higher silicic acid concentrations. The concentration of Al3+, the toxic species, in solution was decreased to about 15, 10, and 5 t M, respectively, from the initial concentration of 20 t M by addition of silicic acid at 500, 1000, and 2000 t M Si. Under the same concentration of Al3+, Al-induced inhibition of root elongation showed the same extent regardless of the addition of silicic acid or not by comparing 5 t M Al treatment with 20 t M Al + 2000 t M Si treatment, and 10 t M Al treatment with 20 t M Al +1000 t M Si treatment. Viability of cells on the root tip surface was decreased by Al addition. Cell viability was not improved by addition of silicic acid under the same concentration of Al3+. All these facts suggest that the alleviative effect of silicic acid on Al toxicity resulted from decreasing toxic Al3+ concentration by forming Al-Si complexes rather than from other physiological effects of silicic acid in corn roots.     

The size and form of DNA molecules from microsporocytes of Lilium longiflorum at different stages of meiosis

DNA molecules from lysates of microsporocytes at different stages of meiosis of Lilium longiflorum were examined in the electron microscope. In all of the preparations, only linear (non-circular) molecules were found. They had a mean length of between 59 and 75 , and a range of lengths which varied from 1 to 115 , to 1 to 210 . From the data obtained, there was no indication of a change in molecular form or size of DNA molecules associated with the process of meiosis.     

Effects of GABA antagonists,SR 95531 and bicuculline,on GABAA receptor-regulated chloride flux in rat cortical synaptoneurosomes

Synaptoneurosomes isolated from cerebral cortices of male Sprague-Dawley rats were used for studying GABA_A receptor-regulated chloride influx. The in vitro effects of GABA antagonists, SR 95531 (a pyridazinyl GABA derivative) and bicuculline, on pentobarbital-stimulated, muscimol-stimulated or flunitrazepam-enhanced, muscimol-stimulated chloride uptake were studied. The chloride uptake was determined at 30°C, for 5 sec. Pentobarbital and muscimol produced a maximal stimulation of chloride uptake in cortical synaptoneurosomes at 500 M and 50M, respectively. SR 95531 as well as bicuculline had no effect on the basal uptake of chloride. Whereas, SR 95531 (0.3–30 M) and bicuculline (0.1–100 M), when added 5 min before muscimol (50 M), produced a significant concentration-dependent inhibition of muscimol (50 M)-stimulated chloride uptake (IC₅₀ s of 0.89±0.11 M and 13.45±2.10M, respectively). In studies of the inhibitory effects of SR 95531 and bicuculline on pentobarbital (500 M)-stimulated chloride uptake, the IC₅₀ s were 0.81±0.12 M and 3.86±1.14 M, respectively. SR 95531 exhibited a more potent inhibitory effect than bicuculline on flunitrazepam-enhanced, muscimol-stimulated chloride uptake. The results revealed that SR 95531 has a more potent antagonistic effect than bicuculline on GABA_A-regulated chloride flux.     

Arsenic concentrations in water and fish from Chautauqua Lake,New York

Synopsis Arsenic persists in Chautauqua Lake, New York waters 13 years after cessation of herbicide (sodium arsenite) application and continues to cycle within the lake. Arsenic concentrations in lake water ranged from 22.4–114.81 g l^–1, = 49.0 ag l^–1. Well water samples generally contained less than 10 g l^–1 arsenic. Arsenic concentrations in lake water exceeded U.S. Public Health Service recommended maximum concentrations (10 g l^–1) and many samples exceeded the maximum permissible limit (50 g l^–1). Fish accumulated arsenic from water but did not magnify it. Fish to water arsenic ratios ranged from 0.4–41.6. Black crappie (Pomoxis nigromaculatus) contained the highest arsenic concentrations (0.14–2.04 g g^–1 ), X = 0.7 g g^–1) while perch (Perca flavescens), muskellunge (Esox masquinongy) and largemouth bass (Micropterus salmoides) contained the lowest concentrations (0.02–0.13 g g^–1). Arsenic concentrations in fish do not appear to pose a health hazard for human consumers.     

The spermatozoon of Oikopleura dioica Fol (Larvacea,Tunicata)

Summary The spermatozoon of Oikopleura dioica is about 30 m long, with a spherical head, about 1 m wide, a 3 m long and 1 m wide midpiece, and a 25 m long tail with a tapered end piece. The head contains a nucleus with the chromatin volume limited to about 0.1 m³. A small acrosome is found in an anterior inpocketing, and a flagellar basal body in a posterior inpocketing of the nucleus. The midpiece contains a single mitochondrion with the flagellar axoneme embedded in a groove along its medial surface. The flagellar axoneme has the typical 9 + 2 substructure, and the basal body the typical 9+0 substructure. A second centriole and special anchoring fibres are absent.     

An abscisic acid analog inhibits abscisic acid-induced freezing tolerance and protein accumulation, but not abscisic acid-induced sucrose uptake in a bromegrass (Bromus inermis Leyss) cell culture

The application of abscisic acid (ABA), either as a racemic mixture or as optically resolved isomers, increases freezing tolerance in a bromegrass (Bromus inermis Leyss) cell culture and induces the accumulation of several heat-stable proteins. Two stereoisomers of an ABA analog, 23 dihydroacetylenic abscisyl alcohol (DHA), were used to study the role of ABA-induced processes in the acquisition of freezing tolerance in these cells. Freezing tolerance was unchanged in the presence of (–) DHA (LT₅₀ -9°C), and no increase in heat-stable protein accumulation was detected; however, the (+) enantiomer increased the freezing tolerance (LT₅₀ -13°C) and induced the accumulation of these polypeptides. All three forms of ABA increased freezing tolerance in the bromegrass cells, although (–) ABA was less effective than either (+) or (±) ABA when added at equal concentrations. Cells pretreated with 20 or 50 M (–) DHA displayed lower levels of freezing tolerance following the addition of 2.5, 7.5 or 25 M (±) ABA. Full freezing tolerance could be restored by increasing the concentration of (±) ABA to > 25 M. Pretreatment of cells with (–) DHA (20 or 50 M) had no effect on freezing tolerance when 25 M (+) ABA was added. The induction of freezing tolerance by 25 M (–) ABA was completely inhibited by the presence of 20 M (–) DHA. The accumulation of ABA-responsive heat-stable proteins was inhibited by pretreatment with 20 M (–) DHA in cells treated with 2.5 or 7.5M (+) ABA, and in cells treated with 25 M (–) ABA. The accumulation of these polypeptides was restored when (±) or (+) ABA was added at a concentration of 25 M. The analysis of proteins which cross-reacted with a dehydrin antibody revealed a similar inhibitory pattern as seen with the other ABA-responsive proteins. The effects of the various isomers of ABA and DHA on cell osmolarity and sucrose uptake was also investigated. In both cases, (±) and (+) ABA had pronounced effects on the parameters measured, whereas (–) ABA treated cells gave substantially different results. In both sucrose uptake and cell osmolarity, DHA had no significant effect on the results obtained following (±) or (+) ABA treatment. Maximum freezing tolerance was only observed in cells when both heat-stable protein accumulation and sucrose uptake were observed.Abbreviations ABA abscisic acid - DHA 2,3 dihydroacetylenicabscisyl alcohols - DMSO dimethyl sulfoxide - LT₅₀ temperature at which 50% of cells are killed The authors would like to acknowledge the technical assistance of Angela Bollman, Bruce Ewan and Angela Shaw. This work was supported by grants from the Natural Science and Engineering Research Council of Canada to L.V.G. and N.H.L., and a grant from the University of Saskatchewan to R.W.W.     

Plant regeneration from embryo-derived callus of oil palm – the effect of exogenous polyamines

Regeneration in oil palm was achieved through somatic embryogenesis/organogenesis from embryo-derived callus. Callus was induced from mature embryos of the cross 281 (D)×18 (P) on modified MS medium supplemented with 2,4-D (113.12 M) and 2-iP (14.76 M). The embryogenic calluses obtained were transferred to Blaydes medium supplemented with 2,4-D (0.045 M) and one of the following growth regulators: TDZ (4.54 M), zeatin riboside (2.85 M), putrescine (1 mM) and spermine (100 M). Secondary somatic embryogenesis was found to occur in media supplemented with polyamines. The efficiency of formation of somatic embryos, secondary somatic embryos and shoot meristemoids were significantly higher in putrescine containing medium. Histological studies were also undertaken.