Accumulation of conjugated linoleic acid in <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> WU-P19 is enhanced by induction with linoleic acid and chitosan treatment

Production of conjugated linoleic acid (CLA) by the potential probiotic bacterium Lactobacillus plantarum WU-P19 was investigated with the aim of enhancing production. CLA produced using this bacterium may be used to supplement dietary intake. Cultures were fed linoleic acid for conversion to CLA and the CLA produced was measured. In some cases, chitosan was added to cultures to improve cellular uptake of linoleic acid. Under static conditions at 37 °C, the bacterium grew and produced CLA in the pH range of 5.5–6.5. At pH 6.0, a 36-h incubation period maximized the concentration of the dry biomass (0.82 g/L), the CLA content in the biomass (4.1 mg/g), and linoleic acid in the biomass (1.2 mg/g). In comparison with cultures grown without linoleic acid in the medium, supplementing the medium with linoleic acid at 600 μg/mL slowed the production of CLA, but the CLA content in the dry biomass increased to 12–14 mg/g and the linoleic acid content increased to 8–11 mg/g. Supplementing the culture medium with chitosan and linoleic acid enhanced production of CLA in the dry biomass to 21 mg/g within 36 h. Nearly 50% of the CLA was cis-9, trans-11-CLA, and the remainder was trans-10, cis-12-CLA. Linoleic acid content of the dry biomass was increased to 37 mg/g. Accumulation of CLA in the cells was enhanced by feeding linoleic acid. Supplementing the culture with linoleic acid and chitosan further increased accumulation of CLA.     

Production of volatile phenols by Lactobacillus plantarum in wine conditions

Some lactic acid bacteria produce volatile phenols in culture medium but this activity has not been extensively studied in wine conditions. Red and white wines were mixed with MRS medium at different ratios to study the influence of wine on the metabolism of p-coumaric and ferulic acids by Lactobacillus plantarum. In MRS broth supplemented with these precursors at 10 mg l^?1, only 4-ethylphenol was produced (1 mg l^?1) while, in the presence of wine, 4-vinylphenol was also obtained. Both volatile phenols are produced in nearly equal amounts (1 mg l^?1) or almost only 4-vinylphenol depending on the MRS:wine ratio. Thus, wine favours the accumulation of 4-vinylphenol. Ferulic acid was not or was weakly metabolized in the conditions studied.     

Selection of Potential Probiotic <Emphasis Type="Italic">Lactobacillus</Emphasis> with Inhibitory Activity Against <Emphasis Type="Italic">Salmonella</Emphasis> and Fecal Coliform Bacteria

Three hundred and sixty presumptive lactic acid bacteria (LAB) isolated from pregnant sows, newborn, suckling, and weaned piglets were preliminarily screened for anti-Salmonella activity. Fifty-eight isolates consisting of Lactobacillus reuteri (n = 32), Lactobacillus salivarius (n = 10), Lactobacillus mucosae (n = 8), Lactobacillus johnsonii (n = 5), and Lactobacillus crispatus (n = 3) were selected and further characterized for probiotic properties including production of antimicrobial substances, acid and bile tolerance, and cell adherence to Caco-2 cells. Eight isolates including Lact. johnsonii LJ202 and Lact. reuteri LR108 were identified as potential probiotics. LJ202 was selected for further use in co-culture studies of two-bacterial and multiple-bacterial species to examine its inhibitory activity against Salmonella enterica serovar Enteritidis DMST7106 (SE7106). Co-culture of LJ202 and SE7106 showed that LJ202 could completely inhibit the growth of SE7106 in 10 h of co-culture. In co-culture of multiple-bacterial species, culturable fecal bacteria from pig feces were used as representative of multiple-bacterial species. The study was performed to examine whether interactions among multiple-bacterial species would influence antagonistic activity of LJ202 against SE7106 and fecal coliform bacteria. Co-culture of SE7106 with different combinations of fecal bacteria and probiotic (LJ202 and LR108) or non-probiotic (Lact. mucosae LM303) strains revealed that the growth of SE7106 was completely inhibited either in the presence or in the absence of probiotic strains. Intriguingly, LJ202 exhibited notable inhibitory activity against fecal coliform bacteria while LR108 did not. Taken together, the results of co-culture studies suggested that LJ202 is a good probiotic candidate for further study its inhibitory effects against pathogen infections in pigs.     

An improved Agrobacterium-mediated transformation of recalcitrant indica rice (Oryza sativa L.) cultivars

Agrobacterium-mediated transformation of indica rice varieties has been quite difficult as these are recalcitrant to in vitro responses. In the present study, we established a high-efficiency Agrobacterium tumefaciens-mediated transformation system of rice (Oryza sativa L. ssp. indica) cv. IR-64, Lalat, and IET-4786. Agrobacterium strain EHA-101 harboring binary vector pIG121-Hm, containing a gene encoding for β-glucuronidase (GUS) and hygromycin resistance, was used in the transformation experiments. Manipulation of different concentrations of acetosyringone, days of co-culture period, bacterial suspension of different optical densities (ODs), and the concentrations of l-cysteine in liquid followed by solid co-culture medium was done for establishing the protocol. Among the different co-culture periods, 5 days of co-culture with bacterial cells (OD_600 nm?=?0.5–0.8) promoted the highest frequency of transformation (83.04 %) in medium containing l-cysteine (400 mg l^?1). Putative transformed plants were analyzed for the presence of a transgene through genomic PCR and GUS histochemical analyses. Our results also suggest that different cultural conditions and the addition of l-cysteine in the co-culture medium improve the Agrobacterium-mediated transformation frequencies from an average of 12.82 % to 33.33 % in different indica rice cultivars.     

Partially chemically defined liquid medium development for intensive propagation of industrial fermentation lactobacilli strains

An economic liquid growth medium was synthesised for high-rate production of cellular mass, lactic acid and bacteriocin in lactobacilli. Three lactobacilli that are applied extensively in industry—Lactobacillus casei NCIMB 11970, Lactobacillus plantarum NCIMB 8014, Lactobacillus lactis NCIMB 8586—were chosen to test the medium’s efficiency. These bacteria are chemoorganotrophs requiring rich, complex media for optimum growth. Contrary to the current practice of formulating a strain-specific medium, we attempted to prepare a universal broth that would allow easy formulation and optimisation. Man de Rogosa Sharp (MRS) medium, which can support the growth of lactobacilli, was found unsuitable for use in large quantities due to its high cost of preparation and its use of beef extract and peptone from poultry as nitrogen sources, which are not environmentally friendly and have potential health risks. The developed medium supported the growth of all the three bacteria equally, offering good maximum yields and incorporating only the chemical compounds needed, resulting in an improvement in the growth rate of the bacilli of between 50 % and 241 % compared to the same strains grown on MRS. Lactic acid production was between 28.6 and 35.74 g L^?1 and bacteriocin production ranged from 110 to 130 IU mL^?1.     

Growth conditions of clostridium perfringens type B for production of toxins used to obtain veterinary vaccines

The diseases caused for Clostridium perfringens are generically called enterotoxemias because toxins produced in the intestine may be absorbed into the general circulation. C. perfringens type B, grown in batch fermentation, produced toxins used to obtain veterinary vaccines. Glucose in concentrations of 1.4–111.1 mM was used to define the culture medium. The minimum concentration for a satisfactory production of vaccines against clostridial diseases was 55.6 mM. Best results were brought forth by meat and casein peptones, both in the concentration 5.0 g l^?1 in combination with glucose and a culture pH maintained at 6.5 throughout the fermentation process. The production of lactic, acetic and propionic organic acids was observed. Ethanol was the metabolite produced in the highest concentration when cultures maintained steady pH of 6.5 with exception of cultures with initial glucose concentration of 1.4 mM, where the highest production was of propionic acid. Maximal cell concentration and the highest toxin title concomitantly low yield coefficient to organic acids and ethanol were obtained using basal medium containing 111.1 mM glucose under a controlled pH culture (pH) 6.5 in batch fermentations of C. perfringens type B. These data contribute to improve process for industrial toxin production allowing better condition to produce a toxoid vaccine.     

Effects of conjugated linoleic acids on growth performance,serum lysozyme activity,lymphocyte proliferation,and antibody production in broiler chicks

Abstract

This study was conducted to investigate the effect of dietary conjugated linoleic acids (CLA) on growth performance and immune responses in broiler chicks. A total of 240 day-old Arbor Acre male broiler chicks were randomly allotted into four dietary treatments with different inclusion levels of CLA (0, 2.5, 5.0 or 10.0 g/kg) for six weeks. Growth performance, peripheral blood lymphocyte (PBL) proliferation, lysozyme activity, phagocytic activity (carbon clearance) and serum antibody titers against Newcastle disease virus (NDV) vaccine were examined. There were no significant differences in growth performance among treatments (p > 0.05). Chicks fed CLA diets produced more lysozyme activity in serum than the control group at 2 and 6 weeks of age (p < 0.05). Dietary CLA enhanced the PBL proliferation in response to concanavalin A (ConA) at the age of 42 d (p < 0.05). Phagocytic ability was also affected by dietary CLA and chicks fed CLA diets had faster carbon clearance rate (p < 0.05), but antibody titers to NDV was not influenced by dietary CLA. The results of the study suggested that dietary CLA could enhance innate and cellular immune response in broiler chicks, and not affect the growth performance.     

Linoleate isomerase activity occurs in lactic acid bacteria strains and is affected by pH and temperature

Aims: To investigate the ability of lactic acid bacteria (LAB) to convert linoleic acid (LA) and α‐linolenic acid (α‐LNA) to conjugated linoleic acid (CLA) and conjugated linolenic acid (CLNA), respectively. To assess pH and temperature influences on CLA and CLNA production by Lactobacillus sakei LMG 13558. Methods and Results: A screening of 48 LAB yielded one Lactobacillus curvatus, five Lactobacillus plantarum and four Lact. sakei strains displaying linoleate isomerase (LAI) activity. CLNA conversion percentages varied largely (1–60%). CLA conversion, occurring in three strains, was lower (2–5%). The LAI gene sequences of the ten LAI‐positive strains shared 75–99% identity with the LAI gene sequence of a Lact. plantarum AS1.555. At pH 6·2, CLA and CLNA production by Lact. sakei LMG 13558 was higher at 30°C than at 20 and 25°C. At pH 5·5 (30°C) or 37°C (pH 6·2), LA was not converted and α‐LNA only slightly converted. Conclusions: LAB show strain‐dependent LAI activity. Production of CLA and CLNA is affected by pH and temperature, as shown for Lact. sakei LMG 13558. Significance and Impact of the Study: Several LAB produce CLA and/or CLNA, as shown for Lact. sakei and Lact. curvatus for the first time. These findings offer potential for the manufacturing of fermented functional foods.     

One-pot conjugated linoleic acid production from castor oil by Rhizopus oryzae lipase and resting cells of Lactobacillus plantarum

Conjugated linoleic acid (CLA) has attracted as novel type of fatty acids having unusual health-promoting properties such as anticarcinogenic and antiobesitic effects. The present work employed castor oil as substrate for one-pot production of CLA using washed cells of Lactobacillus plantarum (L. plantarum) and lipases as catalysts. Among the screened lipases, the lipase Rhizopus oryzae (ROL) greatly assisted resting cells to produce CLA. Mass spectral analysis of the product showed that two major isomers of CLA were produced in the reaction mixture i.e. cis-9, trans-11 56.55% and trans-10, cis-12 43.45%. Optimum factors for CLA synthesis were found as substrate concentration (8 mg/mL), pH (6.5), washed cell concentration (12% w/v), and incubation time of 20 h. Hence, the combination of ROL with L. plantarum offers one pot production of CLA selectively using castor oil as a cost-effective substrate.     

Saccharomyces cerevisiae and Candida tropicalis as starter cultures for the alcoholic fermentation of tchapalo, a traditional sorghum beer

Starter cultures of Candida tropicalis and Saccharomyces cerevisiae isolated from tchapalo were tested in pure culture and co-culture of four ratios [2:1, 25:4, 1:4, 2:3 (cells/cells)] for their ability to ferment sorghum wort to produce tchapalo. All the starters showed means growth rate between 0.043 and 0.101 h^?1. Only C. tropicalis in pure culture showed growth rate lower than that of S. cerevisiae in single culture. During fermentation, according to total soluble solids depletion, yeast starters could be grouped in four different profiles. But in the beer produced, total soluble solids contents were statistically identical. The lowest values were obtained with co-culture C. tropicalis + S. cerevisiae in the ratios of 2:1 and 2:3. Starter cultures with large ratio of C. tropicalis produced a higher organic acids and 2-butanone than S. cerevisiae in pure culture. However, co-culture C. tropicalis + S. cerevisiae (2:1) was the alone starter which produced higher ethanol than S. cerevisiae in pure culture. The beers produced with C. tropicalis + S. cerevisiae (25:4), C. tropicalis + S. cerevisiae (1:4) and C. tropicalis were widely different from those produced with the others starter cultures.     

Probiotic-mediated biotransformation of monosodium glutamate to γ-aminobutyric acid: differential production in complex and minimal media and kinetic modelling

Lactic cultures were screened for their ability to accumulate γ-aminobutyric acid (GABA) in the culture medium. Lactobacillus bulgaricus CFR 2028 produced the highest yields of GABA (22.7 mM) from the substrate monosodium glutamate (MSG). Instrumental characterization by high-performance liquid chromatography and structural characterization by mass spectrometry confirmed GABA production by L. bulgaricus CFR 2028. The differential production of GABA in complex and minimal media indicated that minimal medium (modified tryptone, yeast extract and glucose; TYG) supported the highest production of GABA (37 mM), thereby signifying that the acidic pH alone could have a significant bearing on the yield of GABA. This observation opens up avenues for the optimization of the medium components for yield maximization. The biotransformation kinetics of MSG was examined in batch experiments by varying initial MSG concentration (1–5 %). The Monod model was fitted to determine the kinetic parameters under the MSG uninhibited domain, and the MSG inhibited domain was represented well by Briggs–Haldane model.     

Enhancement of ABE fermentation through regulation of ammonium acetate and D–xylose uptake from acid-pretreated corncobs

Clostridium acetobutylicum TISTR 1462 and Clostridium beijerinckii TISTR 1461 were chosen to optimize acetone–butanol–ethanol (ABE) fermentation by using glucose as a carbon source. The enhancement in its productivity by adding various concentrations of ammonium acetate was studied. Then, the variation of glucose/xylose ratios in the pre-grown medium was investigated. The results showed that both increased ammonium acetate in the production medium and D–xylose in the pre-grown medium could produce more ABE. With these conditions, using corncob hydrolysate as a substrate, 20.58 g/L ABE was produced from C. beijerinckii TISTR 1461 with 0.44 g/L/h and 0.45 of ABE productivity and yield, respectively.     

Lipid metabolism in pigs fed supplemental conjugated linoleic acid and/or dietary arginine

We proposed that the combination of conjugated linoleic acid (CLA) and arginine would decrease adiposity by depressing lipid synthesis in liver and adipose tissues of growing pigs. Pigs were allotted to treatments in a 2 × 2 factorial design with two lipids (CLA or canola oil) and two amino acids [l-arginine or l-alanine (isonitrogenous control)]; supplements were provided from 80 to 110 kg body weight (approximately 4 weeks). Treatment groups (n = 4) were: control (2.05% l-alanine plus 1% canola oil); CLA (2.05% l-alanine plus 1% CLA); arginine (1.0% l-arginine plus 1.0% canola oil); arginine plus CLA (1.0% arginine plus 1.0% CLA). Arginine increased backfat thickness (P = 0.07) in the absence or presence of CLA, and arginine supplementation increased subcutaneous and retroperitoneal adipocyte volume, especially in combination with dietary CLA (interaction P = 0.001). Arginine increased palmitate incorporation into total lipids by over 60% in liver (P = 0.07). Dietary CLA increased palmitate incorporation into lipids in longissimus muscle by over 100% (P = 0.01), and CLA increased longissimus muscle lipid by nearly 20%. CLA increased glucose oxidation to CO₂ by over 80% in retroperitoneal and subcutaneous adipose tissues (P = 0.04), and doubled palmitate oxidation to CO₂ in intestinal duodenal mucosal cells (P = 0.07). Arginine supplementation decreased muscle pH at 45 min postmortem (P = 0.001), indicating elevated early postmortem glycolysis, and CLA and arginine independently increased PGC-1α gene expression in longissimus muscle. CLA but not arginine depressed mTOR gene expression in intestinal duodenal mucosal cells. CLA decreased serum insulin by 50% (P = 0.02) but increased serum triacylglycerols by over 40%. CLA supplementation increased (P ≤ 0.01) total saturated fatty acids in liver and adipose tissue. In conclusion, neither CLA nor arginine depressed tissue lipid synthesis in growing/finishing pigs, and in fact dietary CLA promoted elevated intramuscular lipid and arginine increased carcass adiposity.     

Homo-fermentative production of d-lactic acid by Lactobacillus sp. employing casein whey permeate as a raw feed-stock

Casein whey permeate (CWP), a lactose-enriched dairy waste effluent, is a viable feed stock for the production of value-added products. Two lactic acid bacteria were cultivated in a synthetic casein whey permeate medium with or without pH control. Lactobacillus lactis ATCC 4797 produced d-lactic acid (DLA) at 12.5 g l^?1 in a bioreactor. The values of Leudking–Piret model parameters suggested that lactate was a growth-associated product. Batch fermentation was also performed employing CWP (35 g lactose l^?1) with casein hydrolysate as a nitrogen supplement in a bioreactor. After 40 h, L. lactis produced 24.3 g lactic acid l^?1 with an optical purity >98 %. Thus CWP may be regarded as a potential feed-stock for DLA production.     

Encapsulation of Lactobacillus plantarum 423 and its Bacteriocin in Nanofibers

Plantaricin 423, produced by Lactobacillus plantarum 423, was encapsulated in nanofibers that were produced by the electrospinning of 18% (w/v) polyethylene oxide (200 000 Da). The average diameter of the nanofibers was 288 nm. Plantaricin 423 activity decreased from 51 200 AU/ml to 25 600 AU/ml and from 204 800 AU/ml to 51 200 AU/ml after electrospinning, as determined against Lactobacillus sakei DSM 20017 and Enterococcus faecium HKLHS, respectively. Cells of L. plantarum 423 encapsulated in nanofibers decreased from 2.3 × 10¹⁰ cfu/ml before electrospinning to 4.7 × 10⁸ cfu/ml thereafter. Cells entrapped in the nanofibers continued to produce plantaricin 423. This is the first report on the encapsulation of a bacteriocin and cells of L. plantarum in nanofibers. The method may be used to design a drug delivery system for bacteriocins and the encapsulation of probiotic lactic acid bacteria. The technology is currently being optimized.     

Conjugated Linoleic Acid Accumulation via 10-Hydroxy-12-Octadecaenoic Acid during Microaerobic Transformation of Linoleic Acid by Lactobacillus acidophilus

Specific isomers of conjugated linoleic acid (CLA), a fatty acid with potentially beneficial physiological and anticarcinogenic effects, were efficiently produced from linoleic acid by washed cells of Lactobacillus acidophilus AKU 1137 under microaerobic conditions, and the metabolic pathway of CLA production from linoleic acid is explained for the first time. The CLA isomers produced were identified as cis-9, trans-11- or trans-9, cis-11-octadecadienoic acid and trans-9, trans-11-octadecadienoic acid. Preceding the production of CLA, hydroxy fatty acids identified as 10-hydroxy-cis-12-octadecaenoic acid and 10-hydroxy-trans-12-octadecaenoic acid had accumulated. The isolated 10-hydroxy-cis-12-octadecaenoic acid was transformed into CLA during incubation with washed cells of L. acidophilus, suggesting that this hydroxy fatty acid is one of the intermediates of CLA production from linoleic acid. The washed cells of L. acidophilus producing high levels of CLA were obtained by cultivation in a medium containing linoleic acid, indicating that the enzyme system for CLA production is induced by linoleic acid. After 4 days of reaction with these washed cells, more than 95% of the added linoleic acid (5 mg/ml) was transformed into CLA, and the CLA content in total fatty acids recovered exceeded 80% (wt/wt). Almost all of the CLA produced was in the cells or was associated with the cells as free fatty acid.     

Lactobacillus plantarum 299v Prevents Caspase-Dependent Apoptosis In Vitro

Selective microbes used as probiotics can enhance epithelial cell protection. We have previously shown that a Lactobacillus plantarum strain 299v (Lp299v) has the ability to induce mucin genes. In the current study, we utilized a cytokine model of inflammation in cell culture to study the modulation of apoptosis by this probiotic. HT-29 cells were pre-incubated with the Lp299v or L. plantarum strain adh- (Lpadh-), a non-adherent derivative of Lp299v. Cells were challenged with a mixture of cytokines (TNF-α, IFN-γ, and IL-1a) to imitate conditions of inflammation. To assess for cell death, we evaluated TUNEL, multi-caspase, and caspase-3 and caspase-7 activity assays. There was a marked decrease in apoptosis as measured by TUNEL⁺ cells in samples pre-treated with Lp299v (18.7 ± 4.1%, p < 0.01) and Lpadh- (16.6 ± 3.2%, p < 0.05) prior to cytokine exposure when compared to cells (43.6 ± 6.2%) exposed to the cytokine mixture. Lp299v pre-incubation with HT-29 cells reduced caspase⁺ cells in the multi-caspase activity assay (3.6 ± 0.6%, p < 0.05) compared to cells exposed to cytokines (68.9 ± 5.1%) whereas Lpadh- did not (46.8 ± 17.5%, p > 0.05). Similarly, caspase-3, caspase-7 activity was also reduced by Lp299v. Selected probiotics may confer an exogenous protective effect at the mucosal–luminal interface for intestinal epithelial cells via alteration of caspase-dependent apoptotic pathways.     

Growth of <Emphasis Type="Italic">Dehalococcoides mccartyi</Emphasis> species in an autotrophic consortium producing limited acetate

The dechlorinating Dehalococcoides mccartyi species requires acetate as carbon source, but little is known on its growth under acetate limiting conditions. In this study, we observed growth and dechlorination of a D. mccartyi-containing mixed consortium in a fixed-carbon-free medium with trichloroethene in the aqueous phase and H₂/CO₂ in the headspace. Around 4 mM formate was produced by day 40, while acetate was constantly below 0.05 mM. Microbial community analysis of the consortium revealed dominance by D. mccartyi and Desulfovibrio sp. (57 and 22% 16S rRNA gene copies, respectively). From this consortium, Desulfovibrio sp. strain F1 was isolated and found to produce formate and acetate (1.2 mM and 48 µM, respectively, by day 24) when cultivated alone in the above mentioned medium without trichloroethene. An established co-culture of strain F1 and D. mccartyi strain 195 demonstrated that strain 195 could grow and dechlorinate using acetate produced by strain F1; and that acetate was constantly below 25 µM in the co-culture. To verify that such low level of acetate is utilizable by D. mccartyi, we cultivated strain 195 alone under acetate-limiting conditions and found that strain 195 consumed acetate to below detection (5 µM). Based on the acetate consumption and cell yield of D. mccartyi, we estimated that on average 1.2?×?10⁸ acetate molecules are needed to supply carbon for one D. mccartyi cell. Our study suggests that Desulfovibrio may supply a steady but low amount of fixed carbon to dechlorinating bacteria, exhibiting important implications for natural bio-attenuation when fixed carbon is limited.     

Peptide Extracts from Cultures of Certain Lactobacilli Inhibit Helicobacter pylori

Helicobacter pylori inhibition by probiotic lactobacilli has been observed in vitro and in vivo. Carefully selected probiotic Lactobacillus strains could therefore play an important role in the treatment of H. pylori infection and eradication. However, the underlying mechanism for this inhibition is not clear. The aim of this study was to examine if peptide extracts, containing bacteriocins or other antibacterial peptides, from six Lactobacillus cultures (Lactobacillus acidophilus La1, Lactobacillus amylovorus DCE 471, Lactobacillus casei YIT 9029, Lactobacillus gasseri K7, Lactobacillus johnsonii La1, and Lactobacillus rhamnosus GG) contribute to the inhibition of H. pylori. Peptide extracts from cultures of Lact. amylovorus DCE 471 and Lact. johnsonii La1 were most active, reducing the viability of H. pylori ATCC 43504 with more than 2 log units within 4 h of incubation (P < 0.001). The four other extracts were less or not active. When six clinical isolates of H. pylori were tested for their susceptibility towards five inhibitory peptide extracts, similar observations were made. Again, the peptide extracts from Lact. amylovorus DCE 471 and Lact. johnsonii La1 were the most inhibitory, while the three other extracts resulted in a much lower inhibition of H. pylori. Protease-treated extracts were inactive towards H. pylori, confirming the proteinaceous nature of the inhibitory substance.     

Somatic embryogenesis and synthetic seed production in Rhinacanthus nasutus (L.) Kurz.

Young healthy cotyledon and leaf explants of Rhinacanthus nasutus (L.) Kurz. were incubated on Murashige and Skoog (MS) medium supplemented with 1.0–5.0 mg/l 2, 4-dichlorophenoxyacetic acid (2,4-D) either alone or in combination with 0.3–1.5 mg/l indole-3-butyric acid (IBA). The optimum callus induction (100 %) was observed from cotyledon explants on MS medium supplemented with 4 mg/l 2, 4-D and 0.5 mg/l IBA. The friable, embryogenic callus when subcultured on half strength MS medium supplemented with IBA (3.0–5.0 mg/l) produced several somatic embryos at various stages of development (globular, heart, torpedo) after 45 days of culture. The highest frequency of callus embryogenesis was observed on ½MS medium supplemented with 4.0 mg/l IBA. Moreover, 47 % of incubated callus responded with a mean number of 16.3 somatic embryos per gram callus. For germination, somatic embryos at the torpedo stage were isolated and subcultured on ½MS medium supplemented with 0.5 mg/l each of 6-benzyladenine and indole-3-acetic acid. After 45 days of culture, plantlets developed with mean lengths of 3.8 cm. Somatic embryos at the torpedo stage were collected and suspended in a matrix of MS medium containing sodium alginate (3 % W/V), dropped into 100 mM calcium chloride (CaCl₂·2H₂O) solution for the production of synthetic seeds. Optimum growth ability of synthetic seed was obtained on MS medium supplemented with 0.2 mg/l gibberellic acid (GA₃). Well developed healthy plantlets derived from somatic embryos and synthetic seeds were hardened and successfully transplanted to soil.