Biochemical and pharmacological characterization of cyclic nucleotide phosphodiesterase in airway epithelium

According to their respective elution order, specificity for cAMP and cGMP, their sensitivity to calmodulin, and their modulation by cGMP and rolipram, four cyclic nucleotide phosphodiesterases (PDE) were separated from the cytosol: PDE I (calmodulin-sensitive), PDE II (stimulated by cGMP, PDE IV (cGMP specific-PDE and inhibited by rolipram) and PDE V (cGMP specific). PDE IV (K_m=1.4 M) was competitively inhibited rolipram (K_i=1.2 M) whereas PDE V (K_m=0.83 M) was competitively inhibited by zaprinast in the molar range (K_i=0.12 M). Moreover the microsomal fraction contained three PDE isoforms: PDE II, PDE III (inhibited by cGMP or indolidan) and PDE IV. These results show that cAMP degradation in cytosolic and membrane fractions is modulated by cGMP and selectively inhibited by rolipram and, in addition, by indolidan in membrane fractions. (Mol Cell Biochem140: 171–175, 1994)     

Purine and pyrimidine base and nucleoside concentrations in human cerebrospinal fluid and plasma

Purine and pyrimidine base and nucleoside levels were determined in adult human lumbar (CSF) and plasma by reversed-phase high performance liquid chromatography (HPLC). Guanine, thymine, cytosine and uracil were not detectable (<0.1 M) in human CSF or plasma. Adenine was detectable in plasma (0.3 M) but was not found in CSF (<0.2 M). Hypoxanthine and xanthine levels in CSF were each approximately 2.5 M. Plasma levels of hypoxanthine and xanthine were considerably lower (0.4–0.6 M). Purine and pyrimidine ribouncleosides in human CSF were less than or equal to 0.2 M with the exception of uridine which was present at concentrations of 2–3 M. Although low concentrations of thymidine and deoxyuridine (0.2 M) were present in human plasma, purine and pyrimidine deoxyribonucleosides were less than 0.1 M in human lumbar CSF.     

Callus induction and plant regeneration in Vetiveria zizanioides

Callus induction was obtained from basal parts of Vetiveria zizanioides Stapf. leaves cultured on Murashige and Skoog (MS) medium supplemented with 9.0 M 2,4-dichlorophenoxyacetic acid (2,4-d), 5.7 M indoleacetic acid (IAA) and 4.6 M kinetin. Calli were maintained on MS medium with the addition of 0.9 M 2,4-d and 2.3 M kinetin. Shoot formation was obtained from fast growing 14-day-old callus on the same basal medium supplemented with 0.9 M 2,4-d and 9.3 M kinetin. Embryo-like structures were observed. When transferred to basal medium, shoots readily developed roots. Fully developed regenerated plants were then successfully established in soil.     

Coral growth in high-nutrient,low-pH seawater: a case study of corals cultured at the Waikiki Aquarium,Honolulu, Hawaii

Fifty-seven species of hermatypic corals have been maintained and grown in high-nutrient seawater at the Waikiki Aquarium, Honolulu, Hawaii. In this study we document the chemical conditions of aquarium water in terms of dissolved nutrients and carbon. Aquarium water is characterized by concentrations of inorganic nutrients that are high relative to most natural reef ecosystems: SiO₃ 200 M; PO₄ 0.6 M; NO₃ 5 M; NH₄ 2 M. In contrast, concentrations of organic nutrients are lower than most tropical surface ocean waters: DOP 0.1 M and DON 4 M. The incoming well-water servicing the facility has low pH, crating over-saturation of carbon dioxide. The coral communities in aquaria took up inorganic nutrients and released organic nutrients. Rates of nutrient uptake into aquaria coral communities were similar to nutrient uptake by natural reef communities. Coral growth rates were near the upper rates reported from the field, demonstrating corals can and do flourish in relatively high-nutrient water. The growth of corals does not appear to be inhibited at concentrations of nitrogen up to 5 M. Statements implying that corals can only grow in low nutrient oligotrophic seawater are therefore oversimplifications of processes that govern growth of these organisms. Some basic guidelines are given for maintenance of coral communities in aquaria.     

Clozapine and Several Other Antipsychotic/Antidepressant Drugs Preferentially Block the Same ‘Core’ Fraction of GABAA Receptors

Clozapine and several other antipsychotic/antidepressant drugs that fully or partially block GABA_A receptors were tested at concentrations that reversed the inhibitory effect of 1 M GABA on ³⁵S-t-butylbicyclophosphorothionate ([³⁵S]TBPS) binding to rat forebrain membranes only about 20–30%, here designated core fractions. Clozapine at 10 M reverses 1 M GABA 25 ± 4.0% (n = 23) (its core fraction). Fourty three compounds were tested alone, and pairwise together with 10 M Clozapine. The core fractions of some of the compounds yielded significant additive reversals together with 10 M Clozapine, while others did not. A group of 14 compounds of which 7 are clinically effective antipsychotic drugs, including Chlorprothixene, Clomacran, Clopipazan, Fluotracen, Sulforidazine, Thioproperazine, and cis-Thiothixene, were statistically non-additive with 10 M Clozapine, suggesting that all of these drugs selectively block the same core population of GABA_A receptors as Clozapine. These non-additivities also suggest that Clozapine at 10 M fully saturates a subset of GABA_A receptors blocked by 1 M GABA. Therefore, Clozapine probably blocks 2 or more types of GABA_A receptors, but only half of the receptors that are sensitive to 1 M GABA. A second group of 12 compounds of which 6 are clinically active antidepressant/antipsychotic drugs including Amoxapine, Clothiapine, Dibenzepine, Inkasan (Metralindole), Metiapine and Zimelidine were slightly, but significantly, additive with Clozapine suggesting that these compounds block most of Clozapine's core fraction, plus a small additional fraction. A third group consisted of ten compounds that yielded larger (R > 80) and statistically highly significant additivities with Clozapine. Complete additivity was obtained with Bathophenanthroline disulfonate, and Isocarboxazid, suggesting that they block GABA_A receptors other than those blocked by 10 M Clozapine. Seven classical GABA_A receptor blockers, also tested at concentrations yielding 21 to 33% reversal alone, were all significantly additive with 10 M Clozapine, but in no case was the additivity complete. The largest additivity was obtained with Pitrazepine (21%) and the smallest with Tubocurarine (9%). These results provide further support for the notion that selective blockade of the same subset of GABA_A receptors may contribute to the clinical antipsychotic/antidepressant effects of Clozapine. The B_opt values for Clozapine are 50 ± 1.7% and 26 ± 2.6% ( n = 3) in whole rat forebrain and cerebellum, respectively, confirming that clozapine-sensitive GABA_A receptors are unevenly distributed in the brain. The sedative and anxiolytic properties of Clozapine and other antipsychotic drugs may be due to selective blockade of GABergic disinhibition at certain interneurons.     

Nefopam inhibits calcium influx, cGMP formation, and NMDA receptor-dependent neurotoxicity following activation of voltage sensitive calcium channels

Summary. Nefopam hydrochloride is a potent non sedative benzoxazocine analgesic that possesses a profile distinct from that of anti-inflammatory drugs. Previous evidence suggested a central action of nefopam but the detailed mechanism remains unclear. We have investigated the actions of nefopam on voltage sensitive calcium channels and calcium-mediated pathways. We found that nefopam prevented N-methyl-D-aspartate (NMDA)-mediated excitotoxicity following stimulation of L-type voltage sensitive calcium channels by the specific agonist BayK8644. Nefopam protection was concentration-dependent. 47M nefopam provided 50% protection while full neuroprotection was achieved at 100M nefopam. Neuroprotection was associated with a 73% reduction in the BayK8644-induced increase in intracellular calcium concentration. Nefopam also inhibited intracellular cGMP formation following BayK8644 in a concentration-dependent manner, 100M nefopam providing full inhibition of cGMP synthesis and 58M allowing 50% cGMP formation. Nefopam reduced NMDA receptor-mediated cGMP formation resulting from the release of glutamate following activation of channels by BayK8644. Finally, we also showed that nefopam effectively reduced cGMP formation following stimulation of cultures with domoic acid, while not providing neuroprotection against domoic acid. Thus, the novel action of nefopam we report here may be important both for its central analgesic effects and for its potential therapeutic use in neurological and neuropsychiatric disorders involving an excessive glutamate release.     

A lyophilized aqueous extract of<Emphasis Type="Italic"> Maytenus ilicifolia</Emphasis> leaves inhibits histamine-mediated acid secretion in isolated frog gastric mucosa

Lyophilized aqueous extract of Maytenus ilicifolia leaves (LAEMIL) is commonly used in Brazilian folk medicine in the treatment of dyspepsia as well as gastric ulcers. We have investigated the effect and the possible mechanism of action of the LAEMIL on acid secretion in isolated frog gastric mucosa incubated in an Ussing chamber. It was observed that LAEMIL (7–28 mg%) as well as cimetidine (125–4,000 M), a well-known histamine H₂ receptor antagonist, decreased basal acid secretion in a concentration-dependent manner. Similarly to cimetidine (190 M), LAEMIL (21 mg%) also inhibited gastric acid secretion induced by increasing concentrations of histamine (50–800 M). The EC₅₀ values for histamine alone and histamine in the presence of LAEMIL or cimetidine were 94.6 M (71.1–125.9 M), 244.9 M (209.4–286.4 M) and 142.2 M (23.6–855.0 M), respectively. LAEMIL, histamine and cimetidine were effective on acid secretion only when added to the serosal surface of the mucosa. Furthermore, simultaneous addition of LAEMIL and cimetidine at concentrations, per se, ineffective, caused a 16% reduction in the basal acid secretion [from 8.3±0.3 to 6.9±0.2 Eq g^–1 (15 min)^–1, n=4]. Although effects such as inhibition of histamine biosynthesis and/or histamine release can not be ruled out, our data suggest that LAEMIL, like cimetidine, reduces acid secretion in the isolated frog gastric mucosa by antagonising histamine H₂ receptors.Abbreviations LAEMIL Lyophilized aqueous extract of Maytenus ilicifolia leaves - Hist Histamine - Cim Cimetidine     

In vitro somatic embryogenesis and plant regeneration in Acacia arabica

In vitro somatic embryogenesis and subsequent plant regeneration was achieved in callus cultures derived from immature zygotic embryos of Acacia arabica on semi-solid Murashige and Skoog (MS) basal salts and vitamins supplemented with 8.88 MBA, 6.78 M2,4-D and 30 g l^–1 (w/v) sucrose. Somatic embryos proliferated rapidly by secondary somatic embryogenesis after transfer to MS medium supplemented with 6.66 M BA, 6.78 M 2,4-D. The maximum number of somatic embryos per callus was 72.6 after 8 weeks of culture on medium containing 6.66 M BA and 6.78 M 2,4-D. The isolated somatic embryos germinated on half-strength basal MS salts and vitamins supplemented with 0.04 M BA, 0.94 M ABA and 2% (w/v) sucrose. The embryo-derived plantlets were acclimatized in the greenhouse and subsequently showed normal growth.     

Micropropagation of parsley through axillary shoot proliferation

A micropropagation protocol of parsley,Petroselinum crispum (Mill.) Nyman (curled type) has been developed. Surface-sterilized axillary buds cultured on Murashige and Skoog medium supplemented with benzyladenine, kinetin, or thidiazuron developed axillary shoots (rosettes). Kinetin resulted in only a low proliferation rate. The concentrations of thidiazuron or benzyladenine that were optimal for shoot proliferation, resulted in shoots with a low capability to root. During the rooting treatment, these shoots showed wilting signs. Rooting was increased significantly by using a two-week inductive stage with 2.5 M naphthaleneacetic acid directly followed by acclimatization. Two proliferation media (5 M benzyladenine and 0.5 M naphthaleneacetic acid or 5 M kinetin and 2.5 M naphthaleneacetic acid) resulted in moderate proliferation but produced shoots that were easy-to-root. These media have been tested by repeated axillary proliferation on the same medium. The medium with 5 M benzyladenine and 0.5 M naphthaleneacetic acid was optimal.Abbreviations BA 6-Benzyladenine - IBA Indole-3-butyric acid - MS Murashige & Skoog - NAA -Naphthaleneacetic acid     

Stepwise decrease of 2,4-D and addition of BA in subculture medium stimulated shoot regeneration and somatic embryogenesis in buffalograss

Plant regeneration of buffalograss `Texoka' was achieved through both somatic embryogenesis and organogenesis by culturing immature male inflorescences collected from field-grown plants. Three passages of subculture for calluses derived from male `Texoka' on medium containing 2.25, 4.5, or 9 M 2,4-D combined with either 0.44 M or 1.32 M BA led to shoot formation via organogenesis. Higher concentrations of 2,4-D (4.5 or 9 M) resulted in higher percentages of embryogenic callus while 2,4-D at 2.25 M generated shoot-producing callus but with a lower percentage of embryogenic callus. Transfer of calluses from medium containing 4.5 M 2,4-D and 0.44 M BA to the somatic embryo initiation medium containing 0.9 M 2,4-D gelled with either 7 g 1^–1 agar or 3 g 1^–1 Gelrite led to the formation of somatic embryos. Somatic embryo initiation medium gelled with 3 g 1^–1 Gelrite led to significantly higher frequency of somatic embryo formation than in medium gelled with 7 g 1^–1 agar. Callus of a female genotype `315' generated under similar treatments did not produce shoots or somatic embryos.     

Somatic embryogenesis in cultured mature zygotic embryos of Abies balsamea

Embryo suspensor masses (ESMs) were induced by culture of isolated mature zygotic embryos of balsam fir [Abies balsamea (Mill.)] on media containing 10 M cytokinin [6-(--dimethylallylamino)purine (2iP), 6-benzyladenine (BA), or thidiazuron (TDZ)]. Once induced, ESMs proliferated on media containing 2iP, BA or TDZ (10 M) or on 4.5 M BA in combination with 10 M naphthyl-1-acetic acid. When ESMs were transferred to media containing 5–80 M abscisic acid, cotyledonary-stage embryos were formed. Embryos were readily germinated on medium lacking growth regulators.Abbreviations ABA abscisic acid - BA 6-benzyladenine - ESM embryo-suspensor mass - 2iP 6-(--dimethylallylamino)purine - NAA naphthyl-1-acetic acid - TDZ N-phenyl-N-1,2,3-thiadiazol-5-yl urea (thidiazuron)     

In vitro plant regeneration via somatic embryogenesis from root culture of some rhizomatous irises

A method for plant regeneration of Iris via somatic embryogenesis is described. Root and leaf pieces from in vitro-grown plants of several genotypes of rhizomatous Iris sp. were cultured in vitro. Callus induction occurred only on root cultures incubated under low light intensity (35 mol m^-2 s^-1) on two induction media containing 2,4-D (4.5 or 22.5 M), NAA (5.4 M) and kinetin (0.5 M). Somatic embryos developed after transfer of callus onto four regeneration media containing 9 or 22 M BA, or 5 M kinetin and 2 M TIBA or 9 M BA and 4 M TIBA. Plantlets could be obtained from these somatic embryos. Genotypic differences were found both in callus induction and somatic embryo formation, with I. pseudacorus responding better than I. versicolor or I. setosa. Cytological analysis performed on root tips of 80 regenerated plants revealed that two of the I. pseudacorus regenerants were tetraploid.Abbreviations 2,4-D dichlorophenoxy acetic acid - NAA naphthaleneacetic acid - BA 6-benzyladenine - TIBA 2,3,5-triiodobenzoic acid - IBA indolebutyric acid     

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.     

Somatic embryogenesis in callus cultures of Rosa hybrida L. cv. Landora

Callus was initiated from immature leaf and stem segments of rose (Rosa hybrida cv. Landora) and subcultured every four weeks on a basal medium of half-strength Murashige & Skoog (1962) salts plus 30 g l^-1 sucrose (1/2 MS) and supplemented with 2.2 M BA, 5.4 M NAA and 2.2–9.0 M 2,4-D. Embryogenic callus and subsequently somatic embryos were obtained from 8-week-old callus culture on 1/2 MS+2.2 M BA+0.05 M NAA+0.3 M GA₃+200–800 mg l^-1 L-proline. Long-term cultures were established and maintained for up to 16 months by repeated subculture of embryogenic callus on L-proline deficient medium. About 12% of cotyledonary stage embryos taken from cultures cold-stored at 8±1°C for 4 days germinated on 1/2 MS+2.2 M BA+0.3 M GA₃+24.7 M adenine sulphate.Abbreviations BA benzyladenine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid     

Rapid clonal propagation of Dioscorea zingiberensis

A protocol was developed for rapid in vitro propagation of Dioscorea zingiberensis Wright using stems as explants. MS medium with the macroelements at half strength and supplemented with 20.0 g l^–1 sucrose and 8.0 g l^–1 agar was used as basal medium. Lateral buds on nodal cuttings grew into shoots within 20 days after culture on basal medium supplemented with 4.4 M 6-benzylaminopurine (BAP) and 1.1 M -naphthalene acetic acid (NAA). The shoots were cut into segments and cultured on medium with 8.9 M BA and 5.4 M NAA for 30 days for callus formation. The callus was cut into pieces and cultured on medium containing 22.2 M BAP and 1.1 M NAA, on which 87.5% of the callus pieces regenerated multiple shoots within 50 days. The shoots were rooted on medium containing 4.9 M indole-3-butyric acid (IBA) for 20 days. Adventitious buds and shoots could be repeatedly formed after the calli were cut into pieces and cultured on the medium containing 8.9 M BAP plus 1.1 M NAA. More than 85% of the regenerated plantlets survived and grew vigorously 1 month after they were transplanted in vermiculite and each plant formed 2–4 microtubers 3 months of transplanting.     

Modulation of specific [3H]phenytoin binding by benzodiazepines

We have shown that diazepam (ED₅₀ 2.4 M), flunitrazepam (ED₅₀ 10.2 M) and Ro5-4864 (ED₅₀ 5 M) are able to enhance both total and specific [³H]phenytoin binding. Picrotoxin (IC₅₀ 1.43 M) and chloride, either NaCl or KCl (IC₅₀ 42.4 M) inhibit both the increase in total and specific binding of [³H]phenytoin, Ro 15-1788 does not. The optimum time for this enhancement was 3–4 hours. While the ED₅₀'s for the benzodiazepines are high their order of potency suggests that an involvement of both the peripheral type benzodiazepine receptor and the GABA-chloride ionophore complex is likely. Clonazepam (IC₅₀ 23 M), oxazepam (IC₅₀ 12 M) chlordiazepoxide (IC₅₀ 35 M) and Ro8682-10, a convulsant benzodiazepine (IC₅₀ 16 M) all inhibit both total and specific [³H]phenytoin binding. These effects were not blocked by chloride ions, picrotoxin or Ro 15-1788, and reached equilibrium within 45 minutes. This order of potency also parallels that for the peripheral benzodiazepine receptor in rat brain. These data suggest the presence of a micromolar benzodiazepine receptor site which may play a role in the control of CNS excitability. Nitrazepam, medazepam, bromazepam and the tetralobenzodiazepines U38335, U42794, U43434, and U37834 had no effect on total or specific [³H]phenytoin binding nor on the actions of the other benzodiazepines described in concentrations up to 50 M.     

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.     

Stimulation of ethylene production by catecholamines and phenylethylamine in potato cell suspension cultures

The catecholamines (50 M dopamine, 50 M norepinephrine and 100 M epinephrine) and phenylethylamine (200 M) were found to stimulate ethylene production in potato suspension cultures. When 100 M amino-oxyacetic acid was added together with epinephrine, ethylene release returned to control levels. The endogenous 1-aminocyclopropane-1-carboxylic acid levels were increased in parallel with the release of ethylene, suggesting that the observed effect probably occurs via regulation of aCC synthase. Our results suggest that there is a link between these naturally occurring monoamines and ethylene in plants.Abbreviations AOA amino-oxyacetic acid - ACC 1-aminocyclopropane-1-carboxylic acid - DA dopamine - NE norepinephrine - E epinephrine - CA catecholamines - PEA phenylethylamine     

Clonal propagation of Callistemon by tissue culture techniques

Callus development in Callistemon viminalis was readily achieved when axillary buds derived from nodal tissue were placed in a medium containing macro- and micro-nutrients, sucrose (0.06 M), inositol (300 M), nicotinic acid (20 M), pyridoxine hydrochloride (3 M), thiamine hydrochloride (2 M), riboflavin (10 M), cytokinins (5 M) and auxins (0.1 M). The presence of benzylaminopurine (5 M) and p-chlorophenoxyacetic acid (0.1 M) promoted the most vigorous callus development and sprout formation. Rooting of nodal material was rare but occurred readily following the transference of sprouts developed on callus to a basal medium containing sucrose and salts. Root initiation was stimulated, however, by the presence of auxins. Chlorophenoxyacetic acid while stimulating root initiation repressed root growth. Indole butyric acid stimulated both root initiation and shoot growth at concentrations of 0.005 to 0.1 M. The treatment of choice for rooting and shoot growth was the addition of indole butyric acid at a concentration of 0.01 M.     

Direct somatic embryogenesis through thin cell layer culture in Panax ginseng

Direct somatic embryos were differentiated on cotyledon transverse Thin Cell Layers (tTCLs) of Panax ginseng after 9 weeks in the Murashige and Skoog basal (MS) medium containing 2,4-d (5M). When MS medium containing 2,4-d (5M) was used for seedling pretreatment and for tTCLs culture, somatic embryos were observed 2 weeks earlier, i.e. after 7 weeks of culture. On the tTCLs from seedlings pretreated with 2,4-d (5M) combined with benzyladenine and zeatin at 0.1 M (BZ), somatic embryos were observed after 6 weeks of culture and the percentage of embryogenesis was higher (62%) than when 2,4-d was used alone for pretreatment (40%). Similar results were also obtained from pretreatment with combinations of 2,4-d (5M) and thidiazuron (TDZ) (0.01, 0.1M). When a combination of 2,4-d (5M) and BZ (0.1M) was used both for seedling pretreatment and for tTCLs culture, both somatic embryos and shoots were observed after only 3 weeks. As the concentration of BZ increased, the percentage of somatic embryogenesis decreased but the percentage of organogenesis increased. Similar responses were obtained with a combination of 2,4-d (5M) and TDZ (0.01M). On the medium containing both NAA (0.3M) and BZ (1M), globular- and heart- stage embryos developed after 4 weeks of culture into cotyledonary-staged embryos which remained dormant after a short elongation of the embryo axis. The importance of seedling pretreatment by growth substances in enhancing somatic embryogenesis is reported.Abbreviations BA 6-benzyladenine - BZ combination of BA and zeatin - 2,4-d 2,4-dichlorophenoxyacetic acid - MS medium Murashige and Skoog basal medium - NAA a-naphthaleneacetic acid - TDZ thidiazuron - tTCLs transverse thin cell layers - TCL longitudinal thin cell layer