Ultrastructure of the osphradium of Aplysia californica

Summary The osphradium of Aplysia californica, a sensory organ, is a small yellow-brown epithelial patch located in the mantle cavity immediately anterior to the rostral attachment of the gill. Scanning electron microscopy reveals a round ellipsoid structure of 0.6–1 mm in diameter with a central, occasionally folded, sensory epithelium. The central area is covered with microvilli and surrounded by a densely ciliated epithelium. Transmission electron micrographs show that the columnar supporting cells in the sensory epithelium contain an abundance of apical pigment granules and microvilli. Between the epithelial-supporting cells, the putative sensory elements consist of thin neurites (0.4–1.5 m in diameter) that reach the sea-water side of the osphradium. The neurites contain many neurotubules, mitochondria, vesicles and cilia in their apices. The nerve endings originate from cell bodies up to 40 m below the epithelium or in the osphradial ganglion itself, as revealed by electron microscopy and retrograde labeling with Lucifer yellow. There appear to be two populations of putative sensory cells, a large population of heavily stained cell bodies 4–10 m in diameter and a few scattered cells of large diameter (25–60 m). Following lanthanum impregnation, septate junctions can be seen between all types of cells in the epithelium, 3–5 m below the sea-water surface. This study provides new information for further investigation of osmo- and mechanosensation in Aplysia californica.     

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.     

Tissue culture ofKarwinskia humboldtiana—a plant producing toxins with antitumoural effects

Isolated embryos ofKarwinskia humboldtiana were cultured in vitro. The growth of embryos and development to plantlets on woody plant medium supplemented with indole-3-acetic acid 6.10^-2 mol l^–1, gibberellic acid (GA₃) 3.10^-2 mol l^–1, and 6-benzylaminopurine (BA) 2 mol l^–1 was obtained. Multiplication of shoots and rooting of excised shoots has been achieved. Callus formation on modified Murashige-Skoog medium supplemented with 1-naphthaleneacetic acid 10 mol l^–1, GA₃ 14 mol l^–1, and kinetin 5 mol l^–1 on hypocotyls, or on root cultures on medium supplemented with 2.4-dichlorophenoxyacetic acid 10 mol l^–1 and BA 10 mol l^–1 was induced.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - TEM transmission electron microscopy     

Untersuchungen an der Regio olfactoria des Aals,Anguilla anguilla L.

Zusammenfassung Das Flimmerepithel von Anguilla anguilla besteht aus 4 Zellarten: Flimmerzellen, Stützzellen, Basalzellen und Schleimbecherzellen. Flimmerzellen enthalten im oberen Zelldrittel zahlreiche Mitochondrien und tragen an ihrer Oberfläche bis zu 140 Kinocilien. Die Basalkörper dieser Kinocilien haben lange Wurzelfilamente, von denen ein Teil ins Zellinnere zieht; der andere Teil verläuft parallel zur Oberfläche und verbindet benachbarte Basalapparate. — Ein Übergangsepithel verknüpft das Flimmerepithel mit dem Riechepithel. Im Riechepithel finden sich außer den Zellarten des Flimmerepithels die Rezeptoren. Bei einheitlichem Aufbau des Zellkörpers lassen sich aufgrund rein morphologischer Unterschiede der Vesiculae olfactoriae 3 Rezeptortypen unterscheiden: 1. Cilien-Rezeptor, 2. Mikrovilli-Rezeptor und 3. Pfriem-Rezeptor. — Der Cilien-Rezeptor trägt unterhalb der Vesicula olfactoria in einer Einschnürung 4–8 sensorische Cilien, die alle auf gleicher Höhe entspringen. Zwei gegenüberliegende sensorische Cilien schließen einen konstanten Winkel von 60° ein. — Der Mikrovilli-Rezeptor trägt auf seiner abgerundeten Vesicula olfactoria 30 bis 60 Mikrovilli von 0,1 m Dicke und bis zu 5 m Länge. Der Mikrovillus wird von einem zentralen, 160 Å weiten, Tubulus durchzogen. Unterhalb der Vesicula olfactoria liegen mehrere Centriolen. Die Rezeptornatur dieser Zellen wird durch ein Axon unterstrichen. — Der Pfriem-Rezeptor besitzt eine 0,8 m breite und bis zu 4 m lange Vesicula olfactoria ohne sensorische Cilien und ohne Mikrovilli. Im Lumen der Vesicula olfactoria befinden sich neben Neurotubuli auch Fibrillen von 40–50 Å Durchmesser, die gebündelt auftreten. An der Basis des Köpfchens liegen mehrere Centriolen.

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Studies of the regio olfactoria in the eel, Anguilla anguilla I. Fine structure of the olfactory epithelium

Summary The ciliary and olfactory epithelia of the olfactory folds in Anguilla anguilla were studied with the electron microscope. The ciliary epithelium is composed of ciliary cells, supporting cells, basal cells, and mucous cells. The ciliary cells contain numerous mitochondria in their apical portion and bear up to 140 cilia. The ciliary basal bodies have rootlets, some of which project towards the central part of the cell, and others parallel to the cell surface thereby connecting neighbouring basal bodies. A transitional epithelium is located between the ciliary and olfactory epithelia. The olfactory epithelium is composed of the same 4 cell types of the ciliary epithelium and besides contains three morphologically different receptor cell types: ciliary receptor cells, microvillous receptor cells, and receptors with a single rodshaped free appendage. The ciliary receptors have 4 to 8 sensory cilia which project from below the vesicula olfactoria, each forming a constant angle of about 30° with the vertical cell axis. The vesicula olfactoria of the microvillous receptors bears from 30 to 60 microvilli, each of 0.1 m diameter and up to 5 m length. Each microvillus of this receptor type contains a central tubulus of 160 Å diameter. Few centrioles are located closely to the vesicula olfactoria. The third receptor type, which has neither cilia nor microvilli, is characterised by a single rod-shaped appendage of 0.8 m diameter which projects up to 4 m above the epithelial surface. This appendage contains neurotubules and fibril bundles; some centrioles lie close to the base of the appendage.

     

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     

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.     

Phenylacetic acid-induced somatic embryogenesis in cultured hypocotyl explants of geranium (Pelargonium x hortorum Bailey)

Summary The effect of a non-indole compound, phenylacetic acid (PAA), on the induction of somatic embryogenesis in tissue cultures of geranium (Pelargonium x hortorum Bailey cv. Scarlet Orbit Improved) was investigated. Hypocotyl explants derived from young, dark-grown seedlings were cultured on Murashige and Skoog (1962) medium (MS) supplemented with PAA or IAA (0.01–120 M) alone or in combination with BAP (8 M). Somatic embryogenesis was induced by both PAA and IAA at 0.01–20 M with 8 M BAP, however, the optima differed considerably for the two compounds. Maximal activity of IAA for somatic embryogenesis was found at 0.1–2.5 M, whereas PAA gave best results at 10 and 20 M under identical culture conditions. Higher concentrations (30–120 M) of IAA or PAA in the medium induced callusing in the explants, but the callus was neither embryogenic nor morphogenic.Abbreviations BAP N⁶-benzylaminopurine - IAA indole-3-acetic acid - MS Murashige and Skoog (1962) - PAA phenylacetic acid     

Copper but not iron limitation increases astaxanthin production by Phaffia rhodozyma in a chemically defined medium

The influence of copper (0–32 M) and iron (0–108 M) on growth and astaxanthin production by Phaffia rhodozyma was studied. Copper below 3.2 M increased the astaxanthin content of the cells (from 220 to 287 g g^–1) but at the expense of a slightly decreased growth (from 11.3 to 10.2 mg ml^–1). In contrast, iron below 1 M decreased both the growth and astaxanthin content of the cells. Using copper limitation instead of toxic respiratory inhibitors to improve astaxanthin production has obvious advantages from the product quality, environmental and process operation points of view.     

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.     

Adventitious shoot formation and plant regeneration from tissues of tomatillo (Physalis ixocarpa Brot.)

Cultured hypocotyl explants of tomatillo (Physalis ixocarpa Brot.), were evaluated with regard to their morphogenic responses to combinations of benzyladenine (BA, 0–5 M) with either naphthaleneacetic acid (NAA, 0–50 M) or 2,4-dichlorophenoxyacetic acid (2,4-D, 0–50 M). The induction of shoots or roots was dependent on the cytokinin/auxin combination.Hypocotyl explants failed to form shoots when they were grown on media containing either a cytokinin or an auxin alone. The highest frequency of shoot formation was observed on media containing 12.5–25 M BA and 5 M NAA. Likewise the highest frequency of root formation was observed on media supplemented with 1 M BA and 1 M NAA. Complete plants were regenerated and transferred to soil, where they reached maturity.     

Direct somatic embryogenesis and plantlet regeneration from cotyledonary leaves of safflower

Somatic embryos were induced directly on adaxial surface of cotyledonary leaves within 8–10 days of culture on Murashige and Skoog medium containing 5.37 to 10.74 M 1 — napthaleneacetic acid and 2.22 M benzyl adenine. Germinated embryos with shoot axes developed into complete plants after transfer onto half stength Murashige and Skoog medium containing 1.07 M 1 — napthaleneacetic acid. Histological studies suggested direct origin of somatic embryos with broad-base attachment.Abbreviations BA benzyl adenine - MS Murashige and Skoog - NAA 1-napthaleneacetic acid - RH relative humidity     

Effects of aluminum and cadmium toxicity on growth and antioxidant enzyme activities of two barley genotypes with different Al resistance

A hydroponic experiment was carried out to study genotypic differences in effect of Al and Cd on growth and antioxidant enzyme activities by using 2 two-row winter barley genotypes (Hordeum vulgare L.) with different Al resistance, the relatively resistant Gebeina and the sensitive Shang 70–119. The seedling growth, presented as shoot height, root length and dry weight of root and shoot, and tillers per plant were inhibited by all stress treatments, including low pH, 100 M Al (pH 4.0) and 1.0 M Cd+100 M Al (pH 4.0), while 1.0 M Cd showed a slight stimulation of growth. The inhibition was more severe in 1.0 M Cd +100 M Al (pH 4.0) than in 100 M Al (pH 4.0), indicating that the effect of Cd and Al is synergistic. Al-sensitive genotype Shang 70–119 was more inhibited than Al-resistant genotype Gebeina. Proline concentration in leaves was significantly increased when plants were exposed to all stress treatments, being more pronounced in Shang 70–119 than in Gebeina. A highly significant increase in malonaldehyde (MDA) concentration, and a stimulation of superoxide dismutase (SOD) and peroxidase (POD) activities were recorded in the plants subjected to low pH, 100 M Al (pH 4.0) and 1.0 M Cd +100 M Al(pH 4.0) treatments, and the extent of the increase varied greatly depending on concentration and time of exposure. Shang 70–119 had a higher MDA concentration, and less increase in SOD activity when first exposed than Gebeina had.     

Qualitative and quantitative freeze-fracture studies on olfactory and nasal respiratory epithelial surfaces of frog,ox, rat,and dog

Summary The densities and diameters of intramembranous particles in olfactory and nasal respiratory structures of frog, ox, rat and dog have been compared using the freeze-fracture technique. Dendritic endings and the various segments of the cilia of the olfactory receptor cells of a given species have identical particle densities (700–1,800 particles/m² in P-and 100–600 in E-faces). Densities in P-faces of respiratory cilia are about 1/3 of those in the olfactory cilia. E-face particle densities of these respiratory cilia are often higher than P-face densities. Microvillus P-face densities range from 700–2,000 (respiratory cell microvilli) to 1,800–3,400 particles/m² (olfactory supporting and Bowman's gland microvilli). Microvillus E-faces show no conspicuous mutual differences. Literature comparisons showed that odour concentrations at threshold are considerably lower (10⁵–10¹⁰ times) than the concentrations of olfactory receptor ending intramembranous particles (5 M–30 M) expressed in the same units.Relative differences in particle distributions of the various cell structures studied are usually species-independent. Absolute values vary considerably with the species. Relative P-face particle densities of the supporting cell microvilli tend to correlate with those of dendritic ending structures. Particle diameters are usually similar for corresponding structures and fracture faces in the four species. Apical structures of supporting and Bowman's gland cells in rat and dog show rod-shaped particle aggregates in their P-and pits in their E-faces. Neither sex-dependency nor an influence related to physiological treatments on the particle distributions could be demonstrated.     

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     

Establishment and multiplication of colchi-autotetraploids of Rauvolfia serpentina L. Benth. ex Kurz. through tissue culture

A tissue culture procedure was developed for the establishment and propagation of a colchi-autotetraploid of Rauvolfia serpentina for possible commercial exploitation. Multiplication of autotetraploid shoots was obtained either through axillary bud elongation on Murashige and Skoog [1] medium (MS) containing 2.65 M (0.5 mgl^–1) -naphthaleneacetic acid and 0.33 M (0.05 mgl^–1) kinetin, or via multiple shoot formation on MS medium supplemented with 4.44 M (1.0 mgl^–1) 6-benzylaminopurine and 0.53 M (0.1 mgl^–1) -naphthaleneacetic acid. Rooting could be induced by transferring the shoots to MS medium containing 7.95 M (1.5 mgl^–1) -naphthaleneacetic acid alone. The plantlets, thus formed, were tetraploid in nature by cytological observations of the root tips. They exhibited 80–90% success in establishment under glass house and field conditions.     

Influence of inorganic microelements on the production of camptothecin with suspension cultures of <Emphasis Type="Italic">Camptotheca acuminata</Emphasis>

The effects of eight microelements (I^–, BO₃ ^3–, MoO₄ ^2–, Co²⁺, Cu²⁺, Mn²⁺, Fe²⁺, Zn²⁺) on the biosynthesis of camptothecin and the growth of suspension cultures of Camptotheca acuminata were studied. The increase of I^– to 25 M l^–1, Cu²⁺ to 1 M l^–1, Co²⁺ to 2 M l^–1 and MoO₄ ^2– to 10 M l^–1 in Murashige and Skoog (MS) medium resulted in 1.66, 2.84, 2.53 and 2.04 times higher of camptothecin yield than that in standard MS medium respectively. Combined treatment of I^– (25 M l^–1), Cu²⁺ (1 M l^–1), Co²⁺ (2 M l^–1) and MoO₄ ^2– (10 M l^–1) lead to improve cell dry weight, camptothecin content, and camptothecin yield to 30.56 g l^–1, 0.0299%, and 9.15 mg l^–1, respectively, which were 20.2, 208.9 and 273.8% increment respectively when compared with those of control.     

Increased taxane accumulation in callus cultures of Taxus cuspidata and Taxus × media by some elicitors and precursors

In Taxus cuspidata callus, vanadyl sulfate (10 mg l^–1) induced a high (146 g g^–1 dry wt) production of 10-deacetylbaccatin III in comparison to 7 g g^–1 dry wt of the control. The content of paclitaxel in this species increased from 16 g g^–1 to 74 g g^–1 dry wt when 20 mg phenylalanine l^–1 was used. In T. media, p-aminobenzoic acid induced the highest content of 10-deacetylbaccatin III (481 g g^–1 dry wt) versus 181 g g^–1 in the control. Paclitaxel increased from 89 to 139 g g^–1 dry wt after adding chitosan (20 mg l^–1) to the cultures.     

In vitro clonal propagation of Capsicum spp.

The effect of various concentrations of benzyladenine (BA 4.4–177.5 M) or kinetin (4.7–185.9 M) on shoot proliferation from shoot-tip explants was investigated in C. praetermissum Heiser & Smith and C. annuum L. Maximum number of shoots were obtained on Murashige & Skoog's medium with 66.6 M BA or 92.9 M kinetin in C. praetermissum, and 88.8 M BA or 116.2 M kinetin in C. annuum after 4 weeks of culture. Combining 1 M 2, 3, 5-triiodobenzoic acid (TIBA) with low levels of BA or kinetin significantly increased shoot number as compared to using either cytokinin alone. Rooting of regenerated shoots was achieved on MS medium containing 5.7 M indoleacetic acid. Best rooting (80–100%) was observed in shoots from TIBA plus BA or kinetin media while only 40–50% of shoots from the BA or kinetin treatments were rootable. Plantlets obtained from TIBA plus BA or kinetin were normal diploids while those from BA or kinetin alone revealed distinct chromosomal aberrations in their root tip squashes. Regenerants from TIBA plus BA or kinetin media were successfully established in the soil (86% survival rate), where they flowered and showed normal meiotic behaviour with 100% pollen viability.Abbreviations BA benzyladenine - IAA indole-3-acetic acid - MS Murashige & Skoog's medium - TIBA 2, 3, 5-triiodobenzoic acid     

Rapid clonal propagation of guava through in vitro shoot proliferation on nodal explants of mature trees

In vitro clonal propagation of guava Banaras local was achieved by culturing nodal explants of mature trees on Murashige and Skoog (MS) revised medium supplemented with 4.5 M 6-benzyladanine (BA) alone or in combination with either 0.6 M indole-3-acetic acid (IAA), 0.5 M indole-3-butyric acid (IBA) or 0.3 M gibberellic acid (GA₃). Multiple shoots were induced to form by enhancement of axillary branching and BA (4.5 M) without any auxin and gibberellin was found to give best shoot multiplication rate. In this medium 3–6 shoots developed on explants collected from field-grown plants and 5–10 shoots developed on explants taken from in vitro proliferated shoots within 12 wk of culture. A prior transfer of shoot clumps to a medium containing a lower concentration of BA (0.5 M) before harvesting of cuttings for rooting allowed rapid extension growth and increased the number of usable shoots per culture. Adventitious rooting occurred after subculturing excised shoots on a medium containing 1/2 strength MS salts, 1.5% sucrose, 1 M each of IBA and -naphtha-leneacetic acid (NAA), and 1 gl^-1 activated charcoal. Regenerated plantlets were successfully established on soil.     

Factors influencing shoot regeneration from cotyledonary explants ofCucumis melo

Cotyledonary explants of 4-day-oldCucumis melo cv. Hale's Best Jumbo in vitro seedlings showed maximum initiation of shoot buds when cultured onto a revised Murashige & Skoog medium supplemented with 5 M indole-3-acetic acid and 5 M benzylaminopurine and cultured at 25–29°C under low light intensity (5–30 mol m^-2 s^-1). Subculture of the shoot buds onto the same medium without auxin and supplemented with 3 M benzylaminopurine caused the development of shoots from 30% of the buds. The presence of abscisic acid significantly increased the number of explants producing shoot buds. Bud initiation was affected by genotype, seedling age, light intensity, and temperature. Addition of gibberellic acid, thidiazuron or silver nitrate to regeneration medium did not improve either bud initiation or shoot regeneration.