Antioxidant activity of extracts from Thalictrum minus suspension culture

Low meadow-rue (Thalictrum minus L.) antioxidant complex was studied in cell extracts and culture medium. Its activity was expressed as total polyphenol content in ferulic acid equivalents. In these model systems (cell extracts and culture medium) the inhibition of lipid oxidation and diphenylpicrylhydrazine reduction (EC₅₀ = 12–15 μg/ml) were observed. At the phenolic compound concentration of 8–15 μg/ml, the reducing capacity of cell extracts was equivalent to 1.5 mM ascorbic acid. At the same time, berberine, a major alkaloid synthesized by the culture, manifested a low antioxidant activity. The analysis of phenolic acid composition in low meadow-rue showed that one of the main components of its antioxidant system were caffeic, gallic, chlorogenic, and ferulic acids.     

Antioxidant and antimicrobial actions of the clubmoss <Emphasis Type="Italic">Lycopodium clavatum</Emphasis> L.

Purpose of the present study was to evaluate antioxidant, antibacterial, antifungal, and antiviral activities of the petroleum ether, chloroform, ethyl acetate and methanol extracts as well as the alkaloid fraction of Lycopodium clavatum L. (LC) from Lycopodiaceae growing in Turkey. Antioxidant activity of the LC extracts was evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging method at 0.2 mg/ml using microplate-reader assay. Antiviral assessment of LC extracts was evaluated towards the DNA virus Herpes simplex (HSV) and the RNA virus Parainfluenza (PI-3) using Madin-Darby Bovine Kidney (MDBK) and Vero cell lines. Antibacterial and antifungal activities of the extracts were tested against standard and isolated strains of the following bacteria; Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Acinobacter baumannii, Klebsiella pneumoniae, Staphylococcus aureus, Bacillus subtilis as well as the fungi; Candida albicans and C. parapsilosis. All of the extracts possessed noteworthy activity against ATCC strain of S. aureus (4 μg/ml), while the LC extracts showed reasonable antifungal effect. On the other hand, we found that only the chloroform extract was active against HSV (16–8 μg/ml), while petroleum ether and alkaloid extracts inhibited potently PI-3 (16–4 μg/ml and 32–4 μg/ml, respectively). However, all of the extracts had insignificant antiradical effect on DPPH. In addition, we also analyzed the content of the alkaloid fraction of the plant by capillary gas chromatography-mass spectrometry (GC-MS) and identified lycopodine as the major alkaloid.     

Some inhibitory effects of gentamicin on plant tissue cultures

Summary Tracheary element differentiation in cultured explants of pith parenchyma isolated from heads of romaine lettuce (Lactuca sativa L. var. Romana) was strongly inhibited by concentrations of gentamicin sulfate recommended for tissue culture media (50 to 100 μg/ml). Similar results were obtained with cultured explants of Jerusalem artichoke tuber (Helianthus tuberosus L.). Callus formation was suppressed in the presence of increasing levels of gentamicin sulfate in both tissue systems. Plant tissue culture media employed in studies on cell division and xylem differentiation should be supplemented with this antibiotic in concentrations of 10 μg/ml or less according to these results.     

Cytotoxic activity induced by crude extracts of Ganoderma lucidum (W. Curt.: Fr.) P. Karst. on mouse myeloma cancer cell-line

Ganoderma lucidum powder using hot water and methanol extraction methods indicated a twofold more active cytotoxic activity with IC₅₀ of 44 ± 3.8 μg/ml in the latter method. The representative dose-response curves of the G. lucidum crude extracts on J558 cell-lines revealed that there were great similarities between the curves which reflected rapid killing activities. The percentage viability of the J558 cell exposed to these crude extracts was dose dependent only up to 150 μg/ml. After which, there was no significant reduction when the dose was increased to 200 or 400 μg/ml. The morphological alterations induced by the crude extract were examined under the phase contrast, fluorescent and electron microscopy. When J558 cells were treated with doses higher than 50 μg/ml of the crude extract, obvious morphological changes and apoptosis occurred after 72 h. At 400 μg/ml, most of the cells showed necrosis characterized as small fragments with uniformly stained red nuclei. The apoptotic and necrotic cells increased by 16.5 and 29.1%, respectively whereas the viable cells decreased by as much as 45.6. The mode of cell death via apoptosis was 3.6% higher than necrosis. However, these morphological changes were not observed in the case of 3T3 cells. Results obtained from scanning electron microscopy and transmission electron microscopy further confirmed the occurrence of various apoptotic and necrotic features.     

Relative resistance to methothexate by proliferating normal rabbit epidermal cells in vitro

Summary Primary cell cultures of normal rabbit epidermal cells (keratinocytes) were established without the use of enzymatic techniques. Six experiments were carried out on cells from six different rabbits. When these cells were exposed to methotrexate (MTX) for 24 h at 1 μg/ml, proliferation, as measured by cells entering mitosis, was significantly inhibited (P<0.05) in only one experiment. When the dose of MTX was elevated to 100 μg/ml, only two experiments showed significant inhibition of mitosis. This minimal inhibition of mitosis by MTX was contrasted by the dramatic inhibitory effect of this antimetabolite on DNA synthesis. At 1 μg/ml MTX for 24 h, DNA synthesis, as measured by [³H]deoxyuridine uptake, was inhibited >95%. We can conclude that under certain conditions, the rabbit keratinocyte may represent a normal cell type that is inherently resistant to the antiproliferative effects of methotrexate. The research was supported by National Cancer Institute Grant CA 11536.     

Isolation and identification of an anticancer drug,taxol from <Emphasis Type="Italic">Phyllosticta tabernaemontanae</Emphasis>, a leaf spot fungus of an angiosperm, <Emphasis Type="Italic">Wrightia tinctoria</Emphasis>

Phyllosticta tabernaemontanae, a leaf spot fungus isolated from the diseased leaves of Wrightia tinctoria, showed the production of taxol, an anticancer drug, on modified liquid medium (MID) and potato dextrose broth (PDB) medium in culture for the first time. The presence of taxol was confirmed by spectroscopic and chromatographic methods of analysis. The amount of taxol produced by this fungus was quantified using high performance liquid chromatography (HPLC). The maximum amount of taxol production was recorded in the fungus grown on MID medium (461 μg/L) followed by PDB medium (150 μg/L). The production rate was increased to 9.2 × 10³ fold than that found in the culture broth of earlier reported fungus, Taxomyces andreanae. The results designate that P. tabernaemontanae is an excellent candidate for taxol production. The fungal taxol extracted also showed a strong cytotoxic activity in the in vitro culture of tested human cancer cells by apoptotic assay.     

Minimum inhibitory concentration of drugs againstMycobacterium leprae as determined by anin vitro assay

The observations that liveMycobacterium leprae after entry into cultured peritoneal macrophages from mice, reduced the EA rosetting macrophages, have been exploited to determine the minimum inhibitory concentration of diamino diphenyl sulphone and rifampicin. Diamino diphenyl sulphone showed a minimum inhibitory concentration of 0.028 μg/ml and rifampicin 0.11μg/ml when given externally. However, there was accumulation of diamino diphenyl sulphone inside the macrophages. At an external concentration of 0.028 μg/ml the concentration inside the macrophage was 0.5μg/ml. The minimum inhibitory concentration for diamino diphenyl sulphone in this assay system is higher by several folds and that for rifampicin is slightly lower, than what is reported earlier with mice foot pad experiments. The minimum inhibitory concentration reported in this assay system is quite close to what is observed forin vitro inhibition ofMycobacterium lufu with both the drugs     

Endophytic fungi with anti-microbial,anti-cancer and anti-malarial activities isolated from Thai medicinal plants

A total of 81 Thai medicinal plant species collected from forests in four geographical regions of Thailand were examined for the presence of endophytic fungi with biological activity. Of 582 pure isolates obtained, 360 morphologically distinct fungi were selected for cultivation on malt Czapek broth and yeast extract sucrose broth, from which extracts were tested for biological activity. Extracts of 92 isolates could inhibit Mycobacterium tuberculosis (MIC 0.0625–200 μg ml⁻¹) when tested by the microplate Alamar blue assay, while extracts of six inhibited Plasmodium falciparum (IC₅₀ of 1.2–9.1 μg ml⁻¹) as determined by the [³H]hypoxanthine incorporation method. Strong anti-viral activity against Herpes simplex virus type 1 was observed in 40 isolates (IC₅₀ of 0.28–50 μg ml⁻¹). The sulphorhodamine B assay for activity against cancer cell lines revealed that 60 were active against human oral epidermoid carcinoma cells (EC₅₀ 0.42–20 μg ml⁻¹) and 48 against breast cancer cells (EC₅₀ 0.18–20 μg ml⁻¹). Bioactivity profile was affected by the type of culture medium. Given the high incidence of bioactive extracts and the fact that most of the isolated fungi could not be identified due to lack of spore formation, the results suggested that Thai medicinal plants can provide a wide variety of endophytes that might be a potential source of novel bioactive compounds. This revised version was published online in August 2006 with corrections to the Cover Date.     

In vitro antibacterial activity of laboratory grown culture of Spirulina platensis

The hexane, ethyl acetate, dichloromethane, methanol extracts and spent media (extracellular substances) were tested in vitro for their antibacterial activity for which one Gram-positive bacterium (Staphylococcus aureus) and four Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, and Klebsiella pneumoniae) were used as test organisms. The methanol extract showed more potent activity than other organic extracts, spent medium of the culture exhibited little activity against E. coli only. No inhibitory effect was found against Klebsiella pneumoniae.The broth microdilution assay gave minimum inhibitory concentrations (MIC) values ranging from 1 to 512 μg/ml. The MIC of methanol extract against S. aureus and E. coli were 128 μg/ml and 256 μg/ml, respectively.     

Duct,exocrine, and endocrine components of cultured fetal mouse pancreas

Summary Twenty to twenty-two days postcoitum mouse fetal pancreas organ bits were cultured on the dermal surface of irradiated pigskin as a substrate. The medium used for long term culture consisted of Eagle’s Minimum Essential Medium with the addition of 10% bovine serum, 0.02 U/ml insulin, 0.025 μg/ml glucagon, 3.63 μg/ml hydrocortisone, 100 μg/ml soybean trypsin inhibitor or 10⁻⁸ M atropine. When the medium lacked trypsin inhibitor or atropine but contained the three hormones, the pigskin support began to be destroyed after 2 to 4 wk in culture. Thereafter, the cultured cells could not grow and survive on the digested pigskin. When 10⁻⁶ M atropine was added to the medium, amylase secretion from cultured cells and destruction of pigskin were inhibited completely but pancreas cells could not grow or survive. In contrast, 100 μg/ml soybean trypsin inhibitor or 10⁻⁸ M atropine permitted cell growth, permitted amylase secretion from the cultured acinar cells, and prevented the destruction of pigskin. Under these conditions pancreas cells migrated or grew or both from the organ bits onto the surface of the pigskin dermis and organoid aggregations formed. Hydrocortisone was needed to permit growth for more than 2 wk. Glucagon and insulin had additive effects. Light and electron microscopic observations indicated the culture of at least five kinds of cells, i.e., duct, acinar, centroacinar, endocrine, and mesenchymal. The majority of cultured cells were duct cells and acinar cells. There were few mesenchymal cells. Mouse pancreas cells were cultured for at least 12 wk by this method. This investigation was supported by PHS Grant CA 30220 awarded by the National Cancer Institute, DHHS, Grant 1203M awarded by the Council for Tobacco Research, Inc., and Grant RD-65 (for equipment) awarded by the American Cancer Society. Nude mice were provided by Dr. Wendall M. Farrow of Life Sciences, Inc., Resource Laboratory N01, CP6-1005 of the National Cancer Institute.     

Interaction andin vivo growth inhibition of Ehrlich ascites tumor cells by jacalin

Jacalin has been found to agglutinate Ehrlich ascites cells. The agglutination was inhibited by α-glycosides of D-Gal and β -D-Gal(1 → 3)-D-GalNAc suggesting that the lectin-ascites interaction was carbohydrate-specific. There was 21.8% inhibition of tumour (ascites) cell growthin vivo in mice administered 50μg of jacalin by injection for 6 days following intraperitoneal injection of ascites cells. Administration of 100, 150 and 200μg jacalin resulted in 40.2, 57.5 and 83% inhibition respectively. Thein vivo inhibition of tumour cells growth by jacalin was due to its preferential binding with D-Gal-α -(1 → 6) present as terminal residues in the glycoprotein on tumour cell surface.     

Maintenance of milk protein gene expression in a subpopulation of 7,12-dimethylbenz (a) anthracene-induced rat mammary carcinoma cells grown on attached collagen gels

Summary Milk protein gene expression was studied in cell subpopulations of 7,12-dimethylbenz(a)anthracene-induced rat mammary carcinoma cells enriched or depleted for casein production grown on attached collagen gels. Culture of these cells in the presence of 10% fetal bovine serum, insulin (5 μg/ml), hydrocortisone (10 μg/ml), and prolactin (5 μg/ml) maintained α-, β-, and γ-casein and whey acidic protein mRNAs at levels identical to cells isolated from perphenazine-treated rats. Whey acidic protein mRNA levels in the tumor cells relative to the 14-d lactating gland were greater than those of the casein mRNAs. Withdrawal of prolactin from the casein-producing cells resulted in the loss of all four milk protein mRNAs. Subsequent addition of prolactin to the withdrawn cells caused a rapid accumulation of these mRNAs to prewithdrawal levels. Milk protein gene expression in this tumor cell subpopulation is modulated by prolactin (in the presence of insulin and hydrocortisone) in a similar manner to that observed in the normal mammary gland when these tumor cells are cultured on attached collagen gels. This work was supported by National Institutes of Health grant CA 16303. M. L. Johnson was the recipient of NIH Fellowship, HD 06157.     

Elimination of the yeastCandida parapsilosis from lymphoid cells and monolayer cells in culture

Summary In our laboratory, airborne yeast contaminants of cell cultures have consistently been of the genusCandida (speciesCandida parapsilosis), which are difficult to control with fungicidal agents. To salvage cell lines that show the presence of this fungus, two effective methods may be employed. In early stages of infection, the addition of activated mouse peritoneal macrophages (5×10⁵ cells/ml) to the culture medium containing 5 μg Fungizone/ml eliminates all spores by phagocytosis. More heavily contaminated cultures can be depleted of fungi by density centrifugation on a layer of 38% Percoll. Remaining single spores, often not detectable by light microscopy, can be removed by the addition of macrophages (2×10⁵/ml) and Fungizone (5 μg/ml) to the culture medium. Contaminated monolayer cells can be freed of blastospores by several washes with balanced salt solution and subsequent culturing for 4 d in medium containing 10 μg Fungizone/ml without any toxic effects to the cells. These procedures can rescue valuable cell lines and hybridomas that would otherwise be lost. This work was supported by Veterans Administration Research Funds.     

Bioactive metabolites from <Emphasis Type="Italic">Penicillium</Emphasis> sp., an endophytic fungus residing in <Emphasis Type="Italic">Hopea hainanensis</Emphasis>

The metabolites of endophytic fungus Penicillium sp. from the leaf of Hopea hainanensis were reported for the first time. By bioassay-guided fractionation, the EtOAc extract of a solid-matrix steady culture of this fungus afforded six compounds, which were identified through a combination of spectral and chemical methods (IR, MS, ¹H- and ¹³C-NMR) to be monomethylsulochrin (1), rhizoctonic acid (2), asperfumoid (3), physcion (4), 7,8-dimethyl-iso-alloxazine (5) and 3,5-dichloro-p-anisic acid (6). Compounds 2, 3 and 6 were obtained from Penicillium sp. for the first time. All of the six isolates were subjected to in vitro bioactive assays including antifungal action against three human pathogenic fungi Candida albicans, Trichophyton rubrum and Aspergillus niger and cytotoxic activity against the human nasopharyngeal epidermoid tumor KB cell line and human liver cancer HepG2 cell line. As a result, compounds 2–4 and 6 inhibited the growth of C. albicans with MICs of 40.0, 20.0, 50.0 and 15.0 μg/ml, respectively and the compound 6 showed growth inhibition against A. niger with MICs of 40.0 μg/ml. In addition, compounds 1–3 and 6 exhibited cytotoxic activity against KB cell line with IC₅₀ value of 30.0, 20.0, 20.0, 5.0 μg/ml, respectively and against HepG2 cell line with IC₅₀ value of 30.0, 25.0, 15.0, 10.0 μg/ml, respectively.     

Antioxidant activities of cultured <Emphasis Type="Italic">Armillariella mellea</Emphasis>

This study aimed to evaluate the antioxidant activities of a cultured medicinal fungus—Armillariella mellea (Vahl. ex Fr.) Karst. (AM). Three antioxidant assay systems, namely cytochrome c, xanthine oxidase inhibition, and FeCl₂-ascorbic acid stimulated lipid peroxidation in rat tissue homogenate tests, were used. Total flavonoid and phenol contents of AM extracts were also analyzed. Results showed that both aqueous (AM-H₂O) and ethanolic (AM-EtOH) extracts of solid state cultured AM showed antioxidant activities in a concentration-dependent manner. At concentrations 1–100 μg/ml, the free radical scavenging activity was 73.7–92.1% for AM-H₂O, and 60.0–90.8% for AM-EtOH. These extracts also showed an inhibitory effect on xanthine oxidase activity, but with a lesser potency (IC₅₀ is 9.17 μg/ml for AM-H₂O and 7.48 μg/ml for AM-EtOH). In general, AM-H₂O showed a stronger antilipid peroxidation activity on different rat’s tissues than AM-EtOH. However, both AM extracts displayed a weak inhibitory effect on lipid peroxidation in plasma. Interestingly, the antilipid peroxidation activity of AM-H₂O (IC₅₀–6.66 μg/ml) in brain homogenate was as good as IC₅₀–5.42 μg/ml. AM-H₂O (80.0 mg/g) possessed a significantly higher concentration of total flavonoids than AM-EtOH (30.0 mg/g), whereas no difference was noted in the total phenol content between these two extracts. These results conclude that AM extracts possess potent free radical scavenging and antilipid peroxidation activities, especially the AM-H₂O in the brain homogenate. Published in Russian in Prikladnaya Biokhimiya i Mikrobiologiya, 2007, Vol. 43, No. 4, pp. 495–500. The text was submitted by the authors in English.     

Interaction of benzo[c]phenanthridine and protoberberine alkaloids with animal and yeast cells

We compared the effects of four quaternary benzo[c]phenanthridine alkaloids – chelerythrine, chelilutine, sanguinarine, and sanguilutine – and two quaternary protoberberine alkaloids – berberine and coptisine – on the human cell line HeLa (cervix carcinoma cells) and the yeastsSaccharomyces cerevisiae andSchizosaccharomyces japonicus var. versatilis. The ability of alkaloids to display primary fluorescence, allowed us to record their dynamics and localization in cells. Cytotoxic, anti-microtubular, and anti-actin effects in living cells were studied. In the yeasts, neither microtubules nor cell growth was seriously affected even at the alkaloid concentration of 100 μg/ml. The HeLa cells, however, responded to the toxic effect of alkaloids at concentrations ranging from 1 to 50 μg/ml. IC₅₀ values for individual alkaloids were: sanguinarine IC₅₀ = 0.8 μg/ml, sanguilutine IC₅₀ = 8.3 μg/ml, chelerythrine IC₅₀ = 6.2 μg/ml, chelilutine IC₅₀ = 5.2 μg/ml, coptisine IC₅₀ = 2.6 μg/ml and berberine IC₅₀ >10.0 μg/ml. In living cells, sanguinarine produced a decrease in microtubule numbers, particularly at the cell periphery, at a concentration of 0.1 μg/ml. The other alkaloids showed a similar effect but at higher concentrations (5–50 μg/ml). The strongest effects of sanguinarine were explained as a consequence of its easy penetration through the cell membrane owing to nonpolar pseudobase formation and to a high degree of molecular planarity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Neurotoxicity caused by didanosine on cultured dorsal root ganglion neurons

The purpose of the present study was to investigate whether didanosine (ddI) directly causes morphological and ultrastructural abnormalities of dorsal root ganglion (DRG) neurons in vitro. Dissociated DRG cells and organotypic DRG explants from embryonic 15-day-old Wistar rats were cultured for 3 days and then exposed to ddI (1 μg/ml, 5 μg/ml, 10 μg/ml, and 20 μg/ml) for another 3 days and 6 days, respectively. Neurons cultured continuously in medium served as normal controls. The diameter of the neuronal cell body and neurite length were measured in dissociated DRG cell cultures. Neuronal ultrastructural changes were observed in both culture models. ddI induced dose-dependent decreases in neurite number, length of the longest neurite in each neuron, and total neurite length per neuron in dissociated DRG cell cultures with 3 days treatment. There were no morphological changes seen in organotypic DRG cultures even with longer exposure time (6 days). But ddI induced ultrastructural changes in both culture models. Ultrastructural abnormalities included loss of cristae in mitochondria, clustering of microtubules and neurofilaments, accumulation of glycogen-like granules, and emergence of large dense particles between neurites or microtubules. Lysosome-like large particles emerged inconstantly in neurites. ddI induced a neurite retraction or neurite loss in a dose-dependent manner in dissociated DRG neurons, suggesting that ddI may partially contribute to developing peripheral neuropathy. Cytoskeletal rearrangement and ultrastructural abnormalities caused by ddI in both culture models may have a key role in neurite degeneration.     

Cardiotoxicity of Jamesoni's mamba (Dendroaspis jamesoni) venom and its fractionated components in primary cultures of rat myocardial cells

Summary Primary cultures of spontaneously beating myocardial cells isolated from neonatal rat hearts were used to screen the cardiotoxic effects of Jamesoni's mamba (Dendroaspis jamesoni) venom and components isolated from the venom by gel filtration and ion exchange chromatography. Cardiotoxicity was evaluated on the basis of leakage of lactate dehydrogenase (LDH), changes in morphology, cell membrane lysis, cellular viability, and alterations in spontaneous beating activity. The whole venom caused dose- and time-dependent leakage of LDH, disruption of the cell monolayer, decreases in viability, and inhibition of beating activity. Gel filtration of the venom yielded eight fractions (DjI to DjVIII). DjI (30 μg/ml), DjII (20 μg/ml), and DjV (20 μg/ml) caused significant (P<0.001) leakage of LDH, extensive morphologic damage, and decreases in viability. At lower concentrations DjI to DjVIII caused progressive inhibition of spontaneous beating activity. The main fraction (DjV), which was the most toxic, was further separated into 14 polypeptides (Dj1 to Dj14) by ion-exchange chromatography using Bio-Rex 70. Based on the ability to induce LDH leakage, produce morphologic damage, lyse cell membranes, and arrest beating activity, four categories of polypeptides were identified: cardiotoxins, Dj1 and Dj2; cardiotoxinlike polypeptides, Dj3 to Dj8; less active membrane lytic polypeptides, Dj9 to Dj13; and membrane lytic polypeptide, Dj14. This study was supported in part by the Fulbright Scholar Program (1986–1987) and the Burroughs Wellcome Fund. D. A. is a Burroughs Wellcome Scholar in Toxicology.     

Micropropagation of Lepidium virginicum (Brassicaceae), a plant with antiprotozoal activity

Summary Micropropagation is a technique to ensure a constant and uniform source of medicinal plants. In this report, we describe the micropropagation of Lepidium virginicum L. (Brassicaceae), a wild plant used as an antiamoebic in traditional Mexican medicine. In vitro-germinated seeds were cultured in Murashige and Skoog (MS) medium to obtain pathogen-free cotyledons, hypocotyls, and apical bud (AB) explants. For induction of morphogenesis, the effect of cytokinins, benzyladenine (BA) and kinetin (KN), combined with auxin, indole-3-acetic acid (IAA) was evaluated. The best rate of shoot proliferation was induced 15 d after culture on MS mineral medium supplemented with IAA∶KN (0.57∶13.94 μM) from AB explants. Maximum shoot elongation was achieved without plant growth regulators. The effect of indole-3-butyric acid (IBA) (14.76 μM) was evaluated for in vitro root induction; 60 d after culture all the shoots had developed roots. All rooted plants were successfully transferred to pots and 100% acclimatized in ex vitro conditions. The methanol extracts from the micropropagated active explants of L. virginicum showed and IC₅₀ antiprotozoal value between 141.90 and 268.53 μg ml⁻¹.     

Regeneration of pineapple plants via somatic embryogenesis and organogenesis

Summary We have developed efficient methods for plant regeneration, via both embryogenesis and organogenesis, of Smooth Cayenne pineapple, Ananas comosus (L.) Merr. Leaf bases and core (stem) sections of in vitro shoots, produced from culture of crown tip meristem, were used as explants for plant regeneration as follows: (1) Leaf base and core section explants cultured on Murashige and Skoog (MS) medium containing 41 μM 4-amino-3,5,6-trichloropicolinic acid (picloram, P) or thidiazuron (T)/P combinations produced embryogenic tissues. Different types of embryogenic tissues (friable emryogenic tissue, embryogenic cell cluster, and chunky embryogenic tissue) have been developed with varying properties in terms of growth rate and state of development. The embryogenic tissues regenerated shoots upon culture on MS medium containing 13 μM 6-benzylaminopurine (BA) and 1μM α-naphthaleneacetic acid (NAA) followed by culture on MS medium containing 4 μM BA. (2) Crown tip meristems cultured on MS medium containing 13 μM BA followed by leaf explants cultured on MS medium with 27 μM NAA and 1 μM BA produced shoots via direct organogenesis. (3) Explants cultured on MS medium containing 5 μM T and 0.5 μM indole-3-butyric acid (IBA) produced nodular globular structures, which produced shoots upon culture on MS medium containing 1 μM BA and 1 μM gibberellic acid. Shoots obtained from all of the above methods were rooted in half-strength MS medium containing 3 μM NAA and 2.5 μM IBA. Plants were transferred to the greenhouse or shipped to Costa Rica for field trials. Somatic embryo-derived plants exhibited 21 % spininess, and organogenic-derived plants exhibited 5% spininess in the field trials.