Optimal Conditions For Induction Research Articles

Factors influencing induction and in vitro culture of hairy roots in Phytolacca americana L.

To use hairy roots for producing medicinal ingredients of Phytolacca americana L. we studied the factors influencing the induction and in vitro culture. Hairy roots could be incited from the veins of cut surface (morphological lower) of P. americana L. leaf explants around 18 days after infection with the strain of Agrobacterium rhizogenes ATCC15834. The highest rooting rate, 70%, was obtained when leaf explants were pre-cultured for 1 day, infected for 20 min, and co-cultured for 4 days. The transformation was confirmed by PCR amplification of rolC of Ri plasmid and silica gel thin-layer chromatography of opines from P. americana L. hairy roots. All the hairy root lines could grow rapidly on solid exogenous phytohormone-free MS medium. Among the 9 hairy root lines, the hairy root line 2 had most rapid growth, most branched lateral roots and most intensive root hair; the root surface of some hairy root lines seemed purple or red, while that of the other hairy root line appeared white. Among liquid media MS, 1/2MS, B5 and 6,7-V tested, the best growth for hairy root lines was attained in liquid exogenous phytohormone-free MS medium. Compared with exogenous phytohormone-free MS medium, 6,7-V medium was better for synthesis and accumulation of esculento side A in hairy roots. The established optimal conditions for induction and in vitro culture of P. americana hairy roots had laid an experimental and technological foundation for production of medicinal constituents esculento side A from large scale culture of hairy roots.

Zn2+-Inducible Expression Platform for Synechococcus sp. Strain PCC 7002 Based on the smtA Promoter/Operator and smtB Repressor.

This is the first metal-responsive expression system in cyanobacteria, to our knowledge, that does not exhibit low sensitivity for induction, which is one of the major hurdles for utilizing this class of genetic tools. In addition, high levels of expression can be generated that approximate those of established constitutive systems, with the added advantage of titratable control. Together, these properties establish this Zn2+-inducible system, which is based on the smtA7942 operator/promoter and smtB7942 repressor, as a versatile gene expression platform that expands the genetic toolbox of Synechococcus sp. strain PCC 7002.

Effects of controlled nutrient feeding and different temperatures during floral initiation on yield, berry size and drupelet numbers in red raspberry (Rubus idaeus L.)

The effect of fertility status and temperature conditions during floral induction on flowering, berry yield, and weight and drupelet numbers of individual berries were studied in ‘Glen Ample’ raspberries grown under controlled conditions. Withdrawal of normal fertilization prior to and at various stages during floral induction did not affect yield and berry size, but marginally advanced flowering and fruit ripening. The successive stages of floral initiation and differentiation were studied and identified by scanning electron microscopy of the uppermost lateral buds of plants grown for six weeks under naturally decreasing autumn photoperiods at temperatures of 9, 15 and 21°C. Low temperature advanced floral initiation, and advanced and enhanced flowering and berry yield in the following season. However, at variance from earlier studies, the plants eventually initiated flower primordia even at 21°C. Marginal low temperature and short day conditions during the last days before the temperature treatments were started on 17 September might possibly have reduced the subsequent induction requirements enough to explain this unexpected result. Correlation analyses revealed an over-all positive correlation between fruit weight and drupelet numbers (r=0.568, P=0.01). In berries from the early harvests, the number of drupelets per berry increased with decreasing temperature, while the numbers converged to the same level regardless of temperature in the later harvests. Based on the progress of the floral initiation process at the various temperatures, we interpret this to mean that only the early initiated flowers, that gave rise to the early maturing berries, were differentiated during the actual period of controlled temperature exposure, whereas the remaining flowers were differentiated afterwards when all plants were exposed to identical low temperature conditions. Increased femaleness under optimal floral induction conditions is in agreement with results in both monoecious and dioecious plants and circumstantial evidence suggest that, in the raspberry, this might be mediated by changes in gibberellin activity which acts as a male sexual hormone in plants and is known to inhibit growth cessation and floral initiation in raspberry.

Engineering Bacteria to Catabolize the Carbonaceous Component of Sarin: Teaching E. coli to Eat Isopropanol.

We report an engineered strain of Escherichia coli that catabolizes the carbonaceous component of the extremely toxic chemical warfare agent sarin. Enzymatic decomposition of sarin generates isopropanol waste that, with this engineered strain, is then transformed into acetyl-CoA by enzymatic conversion with a key reaction performed by the acetone carboxylase complex (ACX). We engineered the heterologous expression of the ACX complex from Xanthobacter autotrophicus PY2 to match the naturally occurring subunit stoichiometry and purified the recombinant complex from E. coli for biochemical analysis. Incorporating this ACX complex and enzymes from diverse organisms, we introduced an isopropanol degradation pathway in E. coli, optimized induction conditions, and decoupled enzyme expression to probe pathway bottlenecks. Our engineered E. coli consumed 65% of isopropanol compared to no-cell controls and was able to grow on isopropanol as a sole carbon source. In the process, reconstitution of this large ACX complex (370 kDa) in a system naïve to its structural and mechanistic requirements allowed us to study this otherwise cryptic enzyme in more detail than would have been possible in the less genetically tractable native Xanthobacter system.

Abstract 35: Enhanced Reprogramming of Human Fibroblasts into Cardiomyocytes Using Minimal Transcription Factors

The adult human heart has limited regenerative capacity and is thus an important target for novel regenerative approaches to replenish lost cardiomyocytes after cardiac injury. Cardiac reprogramming that converts fibroblasts to contractile induced cardiomyocytes (iCMs) by overexpressing cardiac lineage specific transcription factors holds great promise as an alternative approach for cardiac regeneration and disease modeling. Significant advance has been made to generate mouse iCMs; however, human iCM (hiCM) generation remains challenging and the yield is low for clinical applications. Here, we leveraged the knowledge that we learned from studying mouse iCM reprogramming to define the optimal condition for hiCM induction. We titrated the dosage of the human reprogramming factors systematically and surprisingly found the minimal core components GATA4, MEF2C and TBX5 were sufficient to induce cardiac fate in human primary fibroblasts. This is in sharp contrast to what has been reported in the literature. Subsequently, we cloned these three factors into a polycistronic vector separated by 2A peptides for defined ratio of protein expression. By optimizing the growth condition, we further improved the efficiency of hiCM reprogramming. Mechanistically, we found the balanced expression of this minimal combination of transcription factors with tailored microenvironment enhanced the establishment of cardiac program in non-myocytes. In sum, our study demonstrates that the use of a single vector with only three transcription factors simplifies generation and improves the yield of hiCMs for potential future clinical applications.

Prokaryotic expression and characterization of receptor binding domain protein of the Middle East respiratory syndrome coronavirus

Objective To express the receptor binding domain (RBD) protein of the Middle East respiratory syndrome coronavirus (MERS-CoV) and to characterize the antigenicity of the purified recombinant protein. Methods The codon-optimized gene encoding the RBD protein of MERS-CoV was synthesized and then cloned into the pET30a (+ ) vector to construct the recombinant expression plasmid. The transformed E. coli BL21 (DE3) strains carrying expression plasmid were induced by IPTG under different conditions. The expressed products were purified by using nickel affinity chromatography and further analyzed by SDS-PAGE and Western blot assay. Indirect ELISA was performed to analyze the antigenicity and specificity of RBD proteins expressed in prokaryotic expression systems in human serological test. Results The recombinant RBD proteins were mainly expressed as conclusion body in an optimal induction condition of 37℃ and 0.5 mmol/L IPTG for 4 h. The high purified recombinant RBD proteins were obtained through denaturation and renaturation with a relative molecular mass of about 29×103. Results of the Western blot assay showed that the recombinant RBD proteins could have specific reaction with the serum samples collected form mice with MERS-CoV infection. Indirect ELISA revealed that the RBD proteins expressed in the prokaryotic expression system showed better sensitivity and specificity in the detection of antibodies against MERS-CoV in human serum samples. Conclusion This study reported the prokaryotic expression and purification of RBD protein of MERS-CoV for the first time, which might pave the way for further investigation on immunological detection of MERS-CoV and development of vaccines against MERS-CoV infection. Key words: Middle East respiratory syndrome; Coronavirus; Receptor binding domain; Prokaryotic expression; Purification; Identification

The Optimization of Soluble PTEN Expression in Escherichia coli.

As a vital tumor suppressor, PTEN (Phosphatase and tension homolog deleted on chromosome 10) is involved in inherited syndromes, and is among the most frequently inactivated tumor suppressor gene in sporadic cancers. PTEN loss-of-function widely occurs in human cancers via a variety of mechanisms, including genetic alterations and posttranslational modification. These suggest PTEN has a role of functional importance in a variety of cancers. In the present study, we constructed a prokaryotic expression vector that efficiently expresses GST-PTEN (the target protein in which PTEN is fused with glutathione S-transferase tag) in E. coli. We found that the target protein was partially soluble although major portions of the protein remained in the inclusion bodies. Furthermore, we explored the optimal induction temperature, isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration and induction time in a series of experiments. Expression level analysis indicated that PTEN reached its peak level at 36○C for 8 h with 1.5625mM IPTG, while solubility analysis revealed the optimal induction temperature was at 20○C, the optimal IPTG concentration was 0.1µM and the optimal induction time was up to 8 h. Taken together, we provide an optimal induction condition for expressing soluble fusion protein of PTEN in E. coli, facilitating further analysis of PTEN’s biological function in vitro.

A Stepwise Protocol for Induction and Selection of Prominent Coniferous Cell Cultures for the Production of β-Thujaplicin

In order to demonstrate the potential of plant cell culture systems to produce a target natural bioactive compound, we proposed a stepwise protocol for β-thujaplicin production as follows. 1. Induction phase: Characteristics of callus cultures originating from newly flushed shoots of 10 conifer species were evaluated on different basal media such as Murashige and Skoog (MS), Schenk and Hildebrandt (SH), and Lloyd and McCown's Woody Plant medium (WP) containing 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D) either alone or in combination with 1 μM of N6-benzyladenine (BA). The conifer species used were as follows: Chamaecyparis (C. obtusa Sieb. et Zucc. and C. pisifera Sieb. et Zucc.), Juniperus (J. chinensis L. 'Kaizuka', J. chinensis L. var. sargentii, and J. conferta Parlatore), Thuja (T. occidentalis L. and T. standishii (Gord.) Carr.), Thujopsis (T. dolabrata Sieb. et Zucc. and T. dolabrata Sieb. et Zucc. var. hondae), and Cryptomeria (C. japonica D. Don). We observed the phenotypes of each callus to determine the optimal conditions for callus induction and to infer biosynthetic activity of the calli over 4-8 weeks. 2. Habituation phase: Each of the cell cultures obtained was transferred to a modified MS medium containing 680 mg L(-1) KH2PO4 and 10 μM Picloram to select the habituated cells with synchronous growth pattern. The growth of each cell culture was highly improved in the habituation medium, except that of J. chinensis 'Kaizuka'. 3. Metabolite-production phase: The concentration of β-thujaplicin (known as hinokitiol in Japan) in the shoots of donor trees and the habituated cell cultures was analyzed via high-performance liquid chromatography (HPLC). Histochemical characteristics of the cells were also observed using laser scanning microscopy (LSM) imaging. After the third step, we tested the biosynthetic activity of two habituated calli (C. obtusa and J. conferta) on a 0.3%, w/v, yeast extract (YE)-containing medium. We found significant improvement in β-thujaplicin production in J. conferta callus (4600 μg g DW-1), which was up to 20-fold higher than in the habituation phase.

Deciphering EGFP production via surface display and self-cleavage intein system in different hosts

In this study, six E. coli strains, which harboring enhanced green fluorescence protein (EGFP), an intein (INT) and ice nucleation protein (INP), were used to express the EGFP protein. The effect of E. coli strains on the surfaced displayed EGFP via INP-INT system was first investigated. Among these strains, E. coli JM109(DE3) was found the best strain for EGFP production (67.9 mg/L), two-fold as compared to that produced by E. coli DH1(DE3). It was concluded that lon mutation could stabilize recombinant proteins. Thus, the deletion of the gene coding for proteolysis might be a critical factor when using host for surface-expression production. The optimal conditions for cell cultivation, gene induction and intein cleavage for EGFP production were systematically studied and discussed. The objective function (O) value of 26.5 can be reached at a 12-h induction and 20-h cleavage process, where the EGFP yield is 58.6 mg/L with a purity of 45.3%. Furthermore, results indicated that the higher intein activity was obtained in the shorter cleavage duration; however, the shorter cleavage time sacrificed the production yield as expected. To compromise between the production yield and purity, the optimal conditions for the EGFP production were: 12-h induction and 20-h cleavage process is for the production of higher purity EGFP.

Enhanced extracellular expression of gene-optimized Thermobifida fusca cutinase in Escherichia coli by optimization of induction strategy

Our previous study demonstrated that when Thermobifida fusca cutinase was expressed in Escherichia coli without mediation of a signal peptide, it could release to the culture medium via the enhanced membrane permeability, which was based on its limited phospholipid hydrolysis activity. The goal of the present work was to achieve highly efficient extracellular production of the recombinant signal peptide-free cutinase in E. coli. The codons of the T. fusca cutinase gene were optimized for expression in E. coli, and a recombinant expression system was constructed using this optimized gene. After that, the induction strategy was optimized using high-cell-density cultivation in a 3-L fermentor. Results showed that the optimal induction condition was at a dry cell weight (DCW) of 13gL−1, and an IPTG/lactose combination induction strategy is proposed, in which IPTG is added once in a final concentration of 25.0μM, and lactose is fed at a rate of 0.5gL−1h−1. In this condition, an extracellular cutinase activity of 2258.5UmL−1 (5.1gL−1) was achieved, which represented the highest cutinase production ever reported, and demonstrated the potential of this system for the industrial production of cutinase.

Novel integration strategy coupling codon and fermentation optimization for efficiently enhancing sarcosine oxidase (SOX) production in recombinant Escherichia coli.

Sarcosine oxidase (SOX) was an important diagnostic enzyme in the renal function examination. An integrated strategy coupling codon and fermentation optimization was firstly proposed for improving SOX production from recombinant E. coli in 3-L fermentor. The expression suppression (gene phase) and poor balance between SOX expression and cell growth (fermentation phase) in the traditional SOX production were respectively improved by the multiple strategies. Based on the codon bias, the expression suppression was weakened via codon optimization and SOX activity reached 1,521 U/L. The induction toxicity was reduced with the optimal induction condition and SOX production increased to 4,015 U/L. Based on the kinetic analysis of μ x and μ p , a better balance between cell growth and expression was achieved by the two-stage pH-stat control strategy. The SOX activity was further improved to 8,490 U/L and fermentation cycle was also significantly shortened from 44 to 32 h. The substrate inhibition was weakened with a constant feeding fed-batch. With the assistance of integrated strategy, the activity and productivity reached 12,466 U/L and 389.6 U/(L h), respectively, or 3.1-fold and 4.3-fold of the uncontrolled fermentation. The strategy would be also useful in the industrial application of other similar enzymes.

Triploidy Induction by Heat Shock in Mandarin Fish Siniperca chuatsi

Mandarin fish, Siniperca chuatsi, is an economically important fish due to its large size, fast growth, and delicious flesh. It is widely cultured in China. In this paper, triploidy in mandarin fish S. chuatsi was induced by heat shock. The most effective triploidy induction was achieved at 41°C, 8 min after fertilization for 2 min resulting in 40% triploid fish. There were no significant differences in the survival rates among the three treatment groups. Ploidy of fish was determined with a flow cytometer and chromosome counting. In conclusion, this paper presents optimal conditions for triploidy induction in mandarin fish with heat shock. The results will contribute to enhancement of its production in culture.

Production of a new non-specific nuclease from Yersinia enterocolitica subsp. palearctica: optimization of induction conditions using response surface methodology

A new non-specific nuclease from Yersinia enterocolitica subsp. palearctica (Y. NSN) was expressed in Escherichia coli (E. coli) BL 21 StarTM (DE3)plysS. Induction conditions, including isopropyl-β-D-thiogalactoside (IPTG) concentration, cell density (OD600), induction time and induction temperature, were optimized using response surface methodology. Statistical analysis of the results revealed that induction temperature and all the quadratic terms of variables had significant effects on enzyme activity of Y. NSN. The optimal induction conditions were as follows: 1.5 mmol/L IPTG, OD600 of 0.80, induction time of 20.5 h, and induction temperature of 32 °C. Under the optimized conditions, the highest enzyme activity could be obtained.

Production of Indirubin from Tryptophan by Recombinant Escherichia coli Containing Naphthalene Dioxygenase Genes from Comamonas sp. MQ

Indirubin, a red isomer of indigo, can be used for the treatment of various chronic diseases. However, the microbial production of indirubin did not receive much attention probably due to its low yield compared with indigo. In this study, the recombinant Escherichia coli containing the naphthalene dioxygenase (NDO) genes from Comamonas sp. MQ was used to produce indirubin from tryptophan. To enhance the production of indirubin, the induction conditions for NDO expression were optimized. The optimal induction conditions were carried out with 0.5 mM isopropyl-β-D-thiogalactopyranoside at 30 °C when cells were grown to OD600 ≈ 1.20. Subsequently, the effects of medium composition on indirubin production were investigated by response surface methodology, and 9.37 ± 1.01 mg/l indirubin was produced from 3.28 g/l tryptophan. Meanwhile, the indirubin production was further improved by adding 2-oxindole and isatin to the tryptophan medium after induction. About 57.98 ± 2.62 mg/l indirubin was obtained by the addition of 500 mg/l 2-oxindole after 1-h induction, which was approximately 6.2-fold to that without additional 2-oxindole. The present study provided a possible way to improve the production of indirubin and should lay the foundation for the application of microbial indirubin production.

Effect of Various Physiological Parameters and Different Carbon Sources on Cellulase and Xylanase Induction by Different Strains of Trichoderma Species

The recent interest in bioconversion of cellulosic wastes to value added chemicals has led to extensive studies on microorganisms capable of producing the enzyme. Cellulases are multi-enzyme complex proteins that catalyze the conversion of cellulose to glucose. Trichoderma is the main industrial source of cellulases and hemicellulases used to depolymerize plant biomass to simple sugars that are converted to chemical intermediates such as biofuels. The current study was carried out to isolate and characterize different Trichoderma species for successful applications. The strains were characterized for cellulase and xylanase assays to determine their extracellular hydrolytic activities. Expression of xylanase was induced maximally by xylan and repressed by glucose. Moreover, a comparative analysis was done between two different Trichoderma species which revealed that Trichoderma harzianum and Trichoderma harzianum Th-azad shows better enzyme activity than T. viride 01PP. Furthermore, optimal conditions for enzyme activity and induction of enzyme synthesis were also determined. The results revealed that the optimal pH, temperature and incubation time for cellulase production was found to be 5.5, 28° C and 120 h respectively. In the present study, we also evaluate the effects of wheat bran, cellulose powder as carbon sources for cellulase and xylanase production. The result showed that the enzyme production was highest with wheat bran as sole source ofcarbon.

Optimal conditions for cold-shock induction of triploidy in red tilapia

Production of sterile triploid red tilapia [Oreochromis mossambicus (Mozambique tilapia); Peters, 1852 × Oreochromis niloticus (Nile tilapia); Linnaeus, 1758] is an effective strategy to overcome their prolific breeding. Optimal conditions for cold-shock induction of triploidy in red tilapia were investigated by experimentally examining two variables: appropriate temperature of the shock and duration of shock treatment. A constant time after insemination of 4 min was used to determine the best combination of temperature (6, 7, 8, 9, 11, 13, 15 °C) with different durations of shock (10, 20, 30, 40, 50 min) with resultant ploidy level verified karyotypically. Shock duration for 30 min at a temperature of 9 °C was found most effective in producing maximum triploidy (98.7 %) with higher rates of hatching (63.2 %) and survival up to yolk-sac stage (75.8 %). The chromosome count confirmed that triploid percentages were higher when cold shock was used for longer durations at each temperature; however, hatching rates were generally decreased. The maximum triploid yield (82.1 %) obtained was higher than the yield obtained using heat shock (72.7 %) in red tilapia previously. The application of the results of this study has the potential to greatly improve the production of triploid red tilapia in commercial aquaculture.

English

The objective of this study was to establish an efficient and reproducible in vitroplant regeneration for Citrullus lanatus cv. Zaojia. To achieve optimal conditions for adventitious shoot induction, five explants (entire cotyledons, distal cotyledons, proximal cotyledons, cotyledonary node A and cotyledonary node B) were tested on MS medium supplemented with different concentrations and combinations of growth regulators (0 to 0.2 mg/L IAA and 1.0 to 5.0 mg/L BA), the results showed that entire cotyledons cultured in MS + BA (2.0mg/L) + IAA(0.2mg/L) achieved the highest regenerated rate (89.67%) and the optimal protocol screened in this experiment had 7.69 ± 0.10 shoots per explants. Adventitious shoots were able to elongate both on MS medium with 0.2 mg/L KT and 0.2 mg/L NAA; IBA 0.3mg/L was found to be effective in the production of root. Acclimatized plantlets transferredto pot resumed growth, and their stems and leaves elongated and expanded in one month.   Key words: Watermelon (Citrullus lanatus Thumb.), optimized system, regeneration, cotyledon explants, cotyledonary node.

Growth Promotion and Flowering Induction in Mango (Mangifera indica L. cv “Ataulfo”) Trees by Burkholderia and Rhizobium Inoculation: Morphometric, Biochemical, and Molecular Events

Inoculation of mango trees with Burkholderia caribensis XV and Rhizobium sp. XXV led to mango growth promotion (dry biomass increased in root 89 %, stem 34 %, leaves 51 %, and foliar area 53 %), floral fate (floral buds 100 %), and increased number of flowers (100 %). Nitrogen content in leaves was similar in inoculated and noninoculated trees, around 1.4 % (optimal condition for floral induction). The total foliar nitrogen content increased significantly (56 %) when the microbial inoculation treatment was applied. In addition, the initial content of sucrose, glucose, and fructose in leaves was higher in the microbial inoculation treatment trees but decreased during the evaluated period. The sucrose content in the noninoculated trees presented similar dynamics compared to the microbial inoculation treatment trees, but glucose and fructose showed increased values compared to those of the microbial inoculation treatment. FLOWERING LOCUS T (FT) expression profiles normalized to ACTIN showed similar dynamics but different expression levels: RQ values of 0.03 and 0.05 for noninoculated and microbial inoculation treatments, respectively. In addition, FT expression profiles in microbial inoculation, normalized to the noninoculated treatment, showed an increased FT expression dynamic over time (up to RQ = 2.2), although a drastic decrease in the last sampling date, when all trees presented developed panicles and flowers, was observed. This FT upregulation was in accordance with the flowering induction in that treatment. Temperature had an important influence on mango flowering induction, which was observed for a 1-month period (~10 °C at night and 20 °C during the day). Bud growth that occurred during that period generated mixed and floral buds depending on the exposure time to these inductive temperatures, less than 2 weeks and more than 3 weeks, respectively. Data indicate that inoculation of mango trees with plant growth-promoting rhizobacteria (associated with this crop) is a potential alternative way to promote growth and induce flowering in mango, greatly reducing the high economical costs and environmental contamination associated with traditional agricultural practices.

Acquisition of pig intramuscular preadipocytes through dedifferentiation of mature adipocytes and establishment of optimal induction conditions

Intramuscular fat deposition is a major contributing factor to variations in pork quality. Understanding the mechanisms driving the differentiation and metabolism of muscle-derived adipocytes is important for regulating the fat deposition in muscle. Studies on intramuscular adipocytes commonly involve stromal-vascular (SV) cell cultures, which contain preadipocytes but also several other types of primordial cells. Hence, it is crucial to obtain pure intramuscular preadipocytes for investigating adipocyte differentiation and metabolism in muscle tissue. In this study, we established cultures of pure intramuscular preadipocytes that were derived from mature adipocytes of newborn pigs. Pure mature adipocytes were isolated from the longissimus dorsi (LD) muscle and allowed to dedifferentiate into fibroblast-like cells in ceiling culture. These fibroblast-like cells turned out to be preadipocytes; they exhibited the ability to redifferentiate into mature adipocytes when adipogenically induced in vitro. The redifferentiation process was confirmed by lipid accumulation in the cytoplasm and expression patterns of peroxisome proliferator-activated receptor gamma 2 (PPARγ2), CCAAT/enhancer binding protein alpha (C/EBPα), lipoprotein lipase (LPL), and adiponectin genes, which were all similar to those observed in previous preadipocyte studies. We optimized the induction conditions for intramuscular preadipocytes by adding 0.25 nM dexamethasone (DEX), 5 μg/mL insulin (INS), and 0.1 mM 3-isobutyl-1-methylxanthine (IBMX). Therefore, this study provides a new model for studying the mechanisms of intramuscular preadipocyte differentiation and metabolism.

In vitro callus-induction in globe artichoke (Cynara cardunculus L. var. scolymus) as a system for the production of caffeoylquinic acids

SummaryGlobe artichoke (Cynara cardunculus L. var. scolymus) provides a rich dietary source of bio-active compounds derived from phenylpropanoid metabolism, notably caffeoylquinic acids (CQAs) and flavonoids. Micropropagation techniques have been established for this species, but in vitro cultures have not yet been extended to generate an efficient system for the induction of callus tissue. In this study, we compared more than 100 combinations of media supplements (e.g., phytohormones, absorbers of polyphenols, and inhibitors of polyphenol oxidase), along with various light regimes, and three different genotypes of globe artichoke to define the optimal conditions for callus induction from leaf explants. This led to the elaboration of an in vitro culture protocol which resulted in a high frequency of callus induction after just 1 week in culture. The procedure used leaf explants from virus-free, meristem culturederived plantlets. Quantitative HPLC analysis revealed that, as in globe artichoke leaves, the predominant phenolic esters present in callus were mono- and di-caffeoylquinic acids (diCQA). The concentration of diCQA was three- to five-fold higher in calli than in leaves. The exposure of calli to UV-C light further enhanced the levels of CQAs. In vitro callus culture combined with UV-C irradiation may thus represent a viable production system for diCQA that is suitable for the synthesis of pharmacologically-active compounds.