Demonstration of Aminopeptidase Activities Secreted by Amsonia tabernaemontana Walt. Cells

Using synthetic substrates, an uncomplicated and sensitive procedure for the determination of extracellular aminopeptidase was developed. The studied enzyme produced by the tested plant material (calli, cell suspension culture and roots of Amsonia tabernaemontana Walt. seedlings) hydrolyzed the substrates β‐naphthylamides (βNA) and 4‐(phenylazo) phenylamides (PAP‐amide) of the amino acids to β‐naphthylamine and 4‐(phenylazo) aniline, respectively, and amino acid. The β‐naphthylamides of the amino acids were applied for the identification of extracellular aminopeptidase, whereas the 4‐(phenylazo) phenylamides of the amino acids were used for the determination of intra‐ and extracellular aminopeptidase activity. By simultaneous azocoupling of β‐naphthol with Fast Garnet GBC salt on agar plates a corresponding brown‐red hardly water‐soluble azo‐dye was produced. The evaluation of dyed zones allowed the extracellular aminopeptidase activity to be assessed. No coloration of the agar medium was observed without inoculum, with heat‐inactivated cells (10 min at 100 °C) or in medium inoculated without substrate. On the agar plates with substrate and sterile Amsonia seedlings, changes in coloration were observed indicating a release of aminopeptidase from the roots during germination. The results show a 91.0 % intracellular and 9.0 % extracellular distribution of aminopeptidase activity, when a cell suspension culture of A. tabernaemontana Walt. as the plant material was used. The agar plate method described permits the rapid, uncomplicated and specific detection of plant producers of extracellular aminopeptidase, which could be particularly useful in future inhibitory and/or biotechnological studies.     

Protease from callus and cell suspension cultures of Onopordum turcicum (Compositae)

Summary Germinated seeds of Onopordum turcicum, which is a kind of thistle, were induced to develop callus on Murashige-Skoog medium. Both callus and suspension cultures of the plant synthesized extracellular and intracellular protease as a product of primary metabolism. The proteolytic activities of the enzyme complex from cell suspension and callus cultures were higher than those from the leaves and seeds of the plant.     

Peroxidase activity in calluses and cell suspension cultures of radishRaphanus sativus var. Cherry Bell

Calluses ofRaphanus sativus var. Cherry Bell were induced in a medium containing 2,4-dichlorophenoxyacetic acid and benzyladenine. The biomass and peroxidase activities were determined, both in agar cultures and cell suspension cultures. Different growth-regulator concentrations induced different responses measured as peroxidase activity in callus. The suspended-cell cultures showed the importance of selecting the cell line, in order to obtain an optimal response in extracellular peroxidase activity. The commercial production of this enzyme by utilizing plant cell tissue cultures is discussed.     

5‐Aminolevulinic acid promotes callus growth and paclitaxel production in light‐grown Taxus cuspidata suspension cultures

Cultured plant cells generally produce low levels of secondary metabolites, and elicitors of secondary metabolites usually inhibit callus growth. The aim of this study was to determine the effect of 5‐aminolevulinic acid (ALA), a chlorophyll precursor that promotes plant growth, on callus induction from leaves of Taxus cuspidata, and on callus growth on solid medium. ALA at 0.76, 7.6, and 76 μM had similar effects on callus induction and growth, while ALA at 760 μM had negative effects. Next, the effects of ALA concentrations on callus growth and paclitaxel production in suspension cultures in the dark were evaluated. The results showed that 0.76 and 7.6 μM ALA stimulated growth and paclitaxel production, while 76 μM ALA had negative effects. ALA is thought to promote cellular activity under light conditions. Therefore, the effects of light intensity on callus growth and paclitaxel production in the presence of ALA were evaluated. Our results showed that the best conditions for callus growth and paclitaxel production were 7.6 μM ALA under photosynthetically active radiation of 12 μmol photons m^?2 s^?1. Callus growth and paclitaxel production were inhibited under stronger light (24 μmol photons m^?2 s^?1). Together, these results show that ALA promoted callus growth and the production of paclitaxel by light‐grown cultured T. cuspidata cells.     

REGENERATION OF LIRIODENDRON TULIPIFERA (FAMILY MAGNOLIACEAE) FROM PROTOPLAST CULTURE

Protoplasts were isolated enzymatically from suspension cultures of two embryogenic lines of yellow-poplar (Liriodendron tulipifera L.) and cultured in droplets of a regeneration medium supplemented with 1 mg/1 2,4-D and 0.25 mg/1 6BA and solidified with agarose. Suspension cultures grown in the light yielded substantial numbers of protoplasts, while insignificant numbers were isolated from cultures grown in the dark. The protoplasts reformed cell walls within three days, and 75 percent of them divided at least once within one week in culture. Embryogenic suspension or callus cultures were regenerated by placing agarose droplets containing protoplast-derived microcalli directly into liquid conditioning medium or onto plates of conditioning medium solidified with agar. Embryogenesis was obtained by transferring the callus to a hormone-free induction medium.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of the rare medicinal plant <Emphasis Type="Italic">Ceropegia candelabrum</Emphasis> L. through somatic embryogenesis

Summary Efficient in vitro propagation of Ceropegia candelabrum L. (Asclepidaceae) through somatic embryogenesis was established. Somatic embryogenesis depended on the type of plant growth regulators in the callus-inducing medium. Friable callus, developed from leaf and internode explants grown on Murashige and Skoog (MS) medium supplemented with 4.52μM2,4-dichlorophenoxyacetic acid (2,4-D), underwent somatic embryogenesis. Compared to solid media, suspension culture was superior and gave rise to a higher number of somatic embryos. Transfer of the friable callus developed on MS medium containing 4.52μM 2,4-D to suspension cultures of half- or quarter-strength MS medium with lower levels of 2,4-D (0.23 or 0.45 μM) induced the highest number of somatic embryos, which developed up to the torpedo stage. Somatic embryogenesis was asynchronous with the dominance of globular embryos. About 100 mg of callus induced more than 500 embryos. Upon transfer to quarter-strength MS agar medium without growth regulators, 50% of the somatic embryos underwent maturation and developed into plantlets. Plantlets acclimatized under field conditions with 90% survival.     

Purification and characterization of intracellular and extracellular α‐glucosidases from Geobacillus toebii strain E134

Two different α‐glucosidase‐producing thermophilic E134 strains were isolated from a hot spring in Kozakli, Turkey. Based on the phenotypic, phylogenetic and chemotaxonomic evidence, the strain was proposed to be a species of G. toebii. Its thermostable exo‐α‐1,4‐glucosidases also were characterized and compared, which were purified from the intracellular and extracellular fractions with estimated molecular weights of 65 and 45 kDa. The intracellular and extracellular α‐glucosidases showed optimal activity at 65 °C, pH 7·0, and at 70 °C, pH 6·8, with 3·65 and 0·83 K_m values for the pNPG substrate, respectively. Both enzymes remained active over temperature and pH ranges of 35–70 °C and 4·5–11·0. They retained 82 and 84% of their activities when incubated at 60 °C for 5 h. Their relative activities were 45–75% and 45–60% at pH 4·5 and 11·0 values for 15 h at 35 °C. They could hydrolyse the α‐1,3 and α‐1,4 bonds on substrates in addition to a high transglycosylation activity, although the intracellular enzyme had more affinity to the substrates both in hydrolysis and transglycosylation reactions. Furthermore, although sodium dodecyl sulfate behaved as an activator for both of them at 60 °C, urea and ethanol only increased the activity of the extracellular α‐glucosidase. By this study, G. toebii E134 strain was introduced, which might have a potential in biotechnological processes when the conformational stability of its enzymes to heat, pH and denaturants were considered. Copyright © 2011 John Wiley & Sons, Ltd.     

Synthesis of distinctly different sets of antimicrobial activities by elicited plant cell suspension cultures

The aim of this study was to evaluate the potential of plant suspension cultures for the production of antimicrobial activities. The extracellular, intracellular and cell wall bound fractions of 16 heterotrophic, photoautotrophic and photomixotrophic plant cell suspension cultures each treated with nine different elicitors were tested for the elicitor dependent production of antimicrobial activities. Distinctly different patterns of bioactivities directed against a panel of human isolates including Gram-positive (Bacillus subtilis, Staphylococcus aureus) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa) bacteria as well as fungi (Candida maltosa) were identified for all except the two autotrophic cell cultures. The intracellular fractions of elicited cell cultures were more active than extracellular fractions while cell wall bound fractions showed almost no activities. The intracellular fraction of heterotrophic Lavendula angustifolia cells elicited with a preparation of Pseudomonas syringae was the most active fraction against Candida maltosa. The intracellular fraction of photomixotrophic Arabidopsis thaliana cells elicited with salicylic acid was active against all test isolates. An antimicrobial protein could be identified and partially purified from this culture. Our findings suggest that elicited plant cell cultures may present a new promising alternative source of antimicrobial proteins.     

Antifeedant effects of in vitro culture extracts of the neem tree,Azadirachta indica against the desert locust (Schistocerca gregaria (Forskål))

Callus and micropropagated shoots were initiated from leaf explants of the neem tree, Azadirachta indica A. Juss. A variety of whole plant and in vitro cell cultures from neem seedlings of Ghanian origin were tested for insect antifeedant compounds using the desert locust (Schistocerca gregaria (Forskål)). Feeding suppression occurred when whole extracts of seed, leaf, callus, suspension and shoot cultures were tested in no-choice feeding bioassays. Controls of sucrose, carrot callus and the plant growth medium showed no feeding deterrence. Azadirachtin, the main known antifeedant in neem seed kernels, was quantified from a seed extract by HPLC but was not detected in any of the other extracts. Antifeedancy was determined during batch growth of a suspension culture which had been in culture for 5 months; results indicated that antifeedants were still being formed and that levels increased after maximum biomass was attained.     

Influence of endophytic fungal elicitation on production of inophyllum in suspension cultures of <Emphasis Type="Italic">Calophyllum inophyllum</Emphasis> L.

The influence of dried cell powder and culture filtrates of endophytic fungi on production of inophyllum in cell suspension cultures of leaf- and stem-derived callus of Calophyllum inophyllum was investigated. Two fungi, Nigrospora sphaerica and Phoma spp., endophytic to C. inophyllum, were isolated from leaf tissues, and were identified by both 18S rRNA gene amplification and sequencing. Elicitation of suspension cultures of both callus types of C. inophyllum with dried cell powder and culture filtrates of both fungi consistently elicited production of inophyllum A, B, C, and P. In comparison to stem-derived callus, suspension cultures of leaf-derived callus enhanced production of most inophyllum. Of the four inophyllum studied, the highest production of inophyllum A, C, and P was achieved in elicited suspension cultures of leaf-derived callus. Suspension cultures of stem-derived callus enhanced production only of inophyllum B. When suspension cultures of leaf-derived callus were elicited with 40 mg dried cell powder of Phoma spp., a level of 751-fold (6.84 mg/100 g elicited biomass) of inophyllum A was produced, compared to control. Whereas, a level of 414-fold (6.22 mg/100 g elicited biomass) of inophyllum B was produced when suspension cultures of stem-derived callus were elicited with 20 mg dried cell powder of N. sphaerica. When compared to control, a 10% culture filtrate of N. sphaerica in suspension cultures of leaf-derived callus elicited inophyllum C and P production by 928-fold (7.43 mg/100 g elicited biomass) and 750-fold (1.5 mg/100 g elicited biomass), respectively.     

2H‐Pyran‐2‐one and 2H‐Furan‐2‐one Derivatives from the Plant Endophytic Fungus Pestalotiopsis fici

Two new α‐pyrones (=2H‐pyran‐2‐ones), ficipyrones A and B ( 1 and 2 , resp.), and two new α‐furanones (=2H‐furan‐2‐ones), ficifuranones A and B ( 3 and 4 , resp.), together with three known metabolites, antibiotic F 0368 ( 5 ), hydroxyseiridin ( 6 ), and hydroxyisoseiridin ( 7 ), were isolated from solid cultures of the plant endophytic fungus Pestalotiopsis fici. Their structures were elucidated primarily by NMR spectroscopy, and the absolute configuration of 1 was deduced from the circular‐dichroism (CD) data. Compound 1 showed antifungal activity against the plant pathogen Gibberella zeae (CGMCC 3.2873) with an IC₅₀ value of 15.9 μM .     

Plant regeneration via somatic embryogenesis from protoplasts of six explants in Coker 201 (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>)

Fertile regenerated plants were obtained from protoplasts via somatic embryogenesis in Coker 201 (Gossypium hirsutum L.). Protoplasts were isolated from six different explantsleaves, hypocotyls, young roots, embryogenic callus, immature somatic embryos and suspension cultures and cultured in liquid thin layer KM8P medium. Callus-forming percentage of 20–50% was obtained in protoplast cultures from embryogenic callus, immature embryos and suspension cultures, and visible callus formed within 2 months. Callus-forming percentage of 5–20% in protoplast cultures from young roots, hypocotyls and leaves, and visible callus formed in 3 months. NAA 5.371 μM/kinetin 0.929 μM was effective to stimulate protoplast division and callus formation from six explants. Percentage of callus formation in the medium with 2,4-D 0.452 μM/kinetin 0.465 μM was over 40% from suspension cultures and immature embryos, 25% from embryogenic callus and 10% from hypocotyls. Callus from protoplasts developed into plantlets via somatic embryogenesis. Over 100 plantlets were obtained from protoplasts derived from 6 explants. Ten plants have been transferred to the soil, where they all have set seeds.     

<Emphasis Type="Italic">Cupressus</Emphasis> callus and cell suspension cultures: Effect of seiridins on their growth and sensitivity

Summary Cupressus macrocarpa and C. arizonica were examined for callus and cell culture production in vitro. Both species produced callus on agar-solidified MSCY medium supplemented with vitamins, antioxidants, 0.14 μM kinetin (KIN), and 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Suspension cultures of both species were established in liquid MSCY medium. Seiridin (SE) and iso-seiridin (ISE), two phytotoxic butenolides produced by Seiridium cardinale, S. cupressi, and S. unicorne, the causal agents of many canker diseases of cypress, were tested on callus or cell suspension cultures. In the medium without other plant growth regulators (PGR), SE promoted cell proliferation of cypress better than ISE, for callus initiation, callus maintenance, and cell suspension cultures. The growth rates of cypress callus tissues and suspension cultures of both cypress species on media containing 50–150 μM SE or ISE were measured. At concentrations of 50 μM and higher, growth rates increased exponentially with the SE concentration. A comparison with KIN and 2,4-D indicated that 50 μM SE promoted growth of callus tissues and cell suspension cultures more than 100 μM ISE. SE can also interact with, or counteract, KIN and 2,4-D. It was demonstrated that SE could replace KIN in the medium for C. arizonica. SE could be involved in cell enlargement and proliferation processes. The less susceptible cypress species (C. arizonica) had a high content of terpenoids than that of the more susceptible species (C. macrocarpa). SE could be a useful tool as a phytohormonal-like regulator to manipulate physiological changes at the cellular level and as an elicitor of sensitivity or tolerance of cypress germplasm to the phytotoxin.     

1α,25-Dihydroxyvitamin D3-induced changes in intracellular pH in osteoblast-like cells modulate gene expression

1α,25-Dihydroxyvitamin D₃ exerts rapid nongenomic effects on rat osteoblast-like cells independent of the classic nuclear receptor. These effects include changes in phospholipid metabolism and cell calcium. Intracellular calcium itself has been proposed to regulate intracellular pH in osteoblast cell lines. The purpose of this study was to determine the effect of 1α,25-dihydroxyvitamin D₃ on intracellular pH, the relationship of changes in calcium to changes in pH, and the role of pH changes in genomic activation. 1α,25-Dihydroxyvitamin D₃ increased intracellular pH within 10 min in rat osteoblast-like cells, an effect that was inhibited by removal of extracellular sodium and by the biologically inactive epimer 1β,25-dihydroxyvitamin D₃. The hormone increased intracellular calcium in Quin 2 loaded cells in the presence and absence of extracellular sodium. The 1α,25-dihydroxyvitamin D₃-induced increments in osteocalcin and osteopontin mRNA levels were abolished in sodium-free medium. The results indicate that 1α,25-dihydroxyvitamin D₃-induced increments in cellular calcium precede cell alkalinization and that these changes in intracellular pH may modulate steady-state mRNA levels of genes induced by vitamin D.     

Influence of abiotic elicitation on production of dipyranocoumarins in suspension cultures of <Emphasis Type="Italic">Calophyllum inophyllum</Emphasis> L.

Effects of elicitation with heavy metals such as copper, cadmium, chromium (abiotic elicitation) and supplementation of CaCl₂ on production of dipyranocoumarins (inophyllums) in suspension cultures of leaf and stem callus of Calophyllum inophyllum were studied. The optimum timing for elicitor introduction was found to be the 10th day after initiating the suspension cultures. Cadmium as abiotic elicitor in suspension cultures of stem callus was found best to elicit maximum production of inophyllums A, C, and calophyllolide while cadmium in suspension cultures of leaf callus was found best for eliciting maximum production of inophyllums B and P. Inophyllum D was the only dipyranocoumarin whose highest production was achieved when 1.0 mM chromium was used as abiotic elicitor in suspension cultures of stem callus. Out of the three abiotic elicitors used, none could result biomass growth. Only incorporation of CaCl₂ in suspension cultures resulted biomass growth. A maximum of 35.26-fold biomass growth was achieved when suspension cultures of stem callus were incorporated with 2.0 mM CaCl₂. CaCl₂ was noted to have no positive influence on production of most of the dipyranocoumarins under study.     

Establishment of callus,cell suspension and shoot cultures of Leonurus cardiaca L. and diterpene analysis

Summary Callus cultures, cell suspension cultures and shoot cultures of Leonurus cardiaca L. (Motherwort) were established and growth conditions optimized. Shoot cultures showed constant growth whether in the dark or under continuous light, accumulating varying amounts of the furanic labdane diterpenes leosibiricin, preleosibirin, leosibirin and isoballotenol acetate, which are also present in the soil-grown plants. Only traces of leosibiricin were detected in callus cultures, while cell suspension cultures did not produce any furanic diterpenes. A small amount of furanic labdane diterpenes was found in the medium of shoot cultures. Callus and shoot culture induction of several other Lamiaceae species is also described.Abbreviations 2 4-D 2,4-dichlorophenoxyacetic acid - BAP benzylaminopurine - GA₃ gibberellic acid - HPLC high pressure liquid chromatography - NAA naphthyl acetic acid - TLC thin layer chromatography     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of embryogenic cell suspension cultures of <Emphasis Type="Italic">Santalum album</Emphasis> L.

An efficient method for Agrobacterium-mediated genetic transformation of embryogenic cell suspension cultures of Santalum album L. is described. Embryogenic cell suspension cultures derived from stem internode callus were transformed with Agrobacterium tumefaciens harbouring pCAMBIA 1301 plant expression vector. Transformed colonies were selected on medium supplemented with hygromycin (5 mg/l). Continuously growing transformed cell suspension cultures were initiated from these colonies. Expression of β-glucuronidase in the suspension cultures was analysed by RT-PCR and GUS histochemical staining. GUS specific activity in the transformed suspension cultures was quantified using a MUG-based fluorometric assay. Expression levels of up to 105,870 pmol 4-MU/min/mg of total protein were noted in the transformed suspension cultures and 67,248 pmol 4-MU/min/mg of total protein in the spent media. Stability of GUS expression over a period of 7 months was studied. Plantlets were regenerated from the transformed embryogenic cells. Stable insertion of T-DNA into the host genome was confirmed by Southern blot analysis. This is the first report showing stable high-level expression of a foreign protein using embryogenic cell suspension cultures in S. album. U. K. S. Shekhawat and T. R. Ganapathi contributed equally to this work.     

Tissue culture and <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of hemp (<Emphasis Type="Italic">Cannabis sativa</Emphasis> L.)

Summary Hemp (Cannabis sativa L.) is cultivated in many parts of the world for ils fiber, oil, and seed. The development of new hemp cultivars with improved traits could be facilitated through the application of biotechnological strategies. The purpose of this study was to investigate the propagation of hemp in tissue culture and to establish a protocol for Agrobacterium-mediated transformation for foreign gene introduction. Stem and leaf segments from seedlings of four hemp varieties were placed on Murashige and Skoog medium with Gamborg B5 vitamins (MB) supplemented with 5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 μM kinetin, 3% sucrose, and 8 gl⁻¹ agar. Large masses of callus were produced within 4 wk for all cultivars. Suspension cultures were established in MB medium containing 2.5 μM 2,4-D. To promote embryogenesis or organogenesis, explants, callus, and suspension cultures derived from a range of explant sources and seedling ages were exposed to variations in the culture medium and changes to the culture environment. None of the treatments tested were successful in promoting plantlet regeneration. Suspension cells were transformed with Agrobacterium tumefaciens strain EHA101 carrying the binary vector pNOV3635 with a gene encoding phosphomannose isomerase (PMI). Transformed callus was selected on medium containing 1–2% mannose. A chlorophenol red assay was used to confirm that the PMI gene was expressed. Polymerase chain reaction and Southern hybridization detected the presence of the PMI gene. Copy number in different lines ranged from one to four.     

Induction and genetic identification of a callus‐like growth developed in the brown alga Fucus vesiculosus

Induction of an axenic filamentous‐like callus growth from the brown algae Fucus vesiculosus is described. Different treatments were investigated in various combinations to develop axenic cultures based on identification of surface symbionts via 18S ribosomal RNA. Moreover, viability was confirmed after such processes by 2,3,5‐triphenyl tetrazolium chloride assay that demonstrated an average viability of 29%, relative to nonsterilized explants. After six weeks of a phototrophic cultivation on artificial sea water‐12‐nitrilotriacetic acid (0.5% w/v agar), a filamentous‐like callus growth was observed, which was identified genetically through its mitochondrial DNA after subculturing. Achievement of confirmed marine callus cultures might enrich old previously established blue biotechnology techniques and open new chances for cultivation of brown algae for production of good manufacturing practice‐compliant bioproducts.     

A TLC bioautographic method for the detection of α‐ and β‐glucosidase inhibitors in plant extracts

Introduction – Bioautographic assays using TLC play an important role in the search for active compounds from plants. A TLC assay has previously been established for the detection of β‐glucosidase inhibitors but not for α‐glucosidase. Nonetheless, α‐glucosidase inhibition is an important target for therapeutic agents against of type 2 diabetes and anti‐viral infections. Objective – To develop a TLC bioautographic method to detect α‐ and β‐glucosidase inhibitors in plant extracts. Methodology – The enzymes α‐ and β‐d ‐glucosidase were dissolved in sodium acetate buffer. After migration of the samples, the TLC plate was sprayed with enzyme solution and incubated at room temperature for 60 min in the case of α‐d ‐glucosidase, and 37°C for 20 min in the case of β‐d ‐glucosidase. For detection of the active enzyme, solutions of 2‐naphthyl‐α‐D‐glucopyranoside or 2‐naphthyl‐β‐D‐glucopyranoside and Fast Blue Salt were mixed at a ratio of 1 : 1 (for α‐d ‐glucosidase) or 1 : 4 (for β‐d ‐glucosidase) and sprayed onto the plate to give a purple background colouration after 2–5 min. Results – Enzyme inhibitors were visualised as white spots on the TLC plates. Conduritol B epoxide inhibited α‐d ‐glucosidase and β‐d ‐glucosidase down to 0.1 µg. Methanol extracts of Tussilago farfara and Urtica dioica after migration on TLC gave enzymatic inhibition when applied in amounts of 100 µg for α‐glucosidase and 50 µg for β‐glucosidase. Conclusion – The screening test was able to detect inhibition of α‐ and β‐glucosidases by pure reference substances and by compounds present in complex matrices, such as plant extracts. Copyright © 2009 John Wiley & Sons, Ltd.