In vitro propagation of Vitis vinifera L. cv. ‘Monastrell’

BackgroundA protocol for the micropropagation of the grape (Vitis vinifera L.) cultivar ‘Monastrell’ was developed. Initial plant material was obtained from the sanitary selection of grapevine plants performed by real-time RT-PCR to confirm the absence of Grapevine fanleaf virus, Arabis mosaic virus, Grapevine leafroll-associated virus 1, Grapevine leafroll-associated virus 3, and Grapevine fleck virus.ResultsThe effects of the salt composition (comparing Lloyd and McCown woody plant medium and Murashige and Skoog medium 1/2 macronutrients) and the growth regulator benzylaminopurine (BAP), at 0 and 8.9 μM, on plant propagation were evaluated using nodes as explants. The most efficient procedure consisted of bud induction in the medium with Lloyd and McCown woody plant salts and 8.9 μM BAP for 30 d along with elongation in cytokinin-free medium for 60 d, which gave 22 nodes/explant (174 plants/initial plant). A second cycle of propagation in a medium without BAP for another 60 d could give approximately 10,000 nodes, which can be obtained after an additional 2 months of culture. All plants acclimatized after the second cycle of multiplication were successfully transferred to soil.ConclusionWe developed an optimal protocol for V. vinifera cv. ‘Monastrell’ micropropagation, the first described for this cultivar.     

Cloning,characterization and expression of Peking duck fatty acid synthase during adipocyte differentiation

BackgroundFatty acid synthase (FAS) is a key enzyme of de novo lipogenesis (DNL), which has been cloned from several species: Gallus gallus, Mus musculus, Homo sapiens, but not from Anas platyrhynchos. The current study was conducted to obtain the full-length coding sequence of Peking duck FAS and investigate its expression during adipocyte differentiation.ResultsWe have isolated a 7654 bp fragment from Peking duck adipocytes that corresponds to the FAS gene. The cloned fragment contains an open reading frame of 7545 bp, encodes a 2515 amino acid protein, and displays high nucleotide and amino acid homology to avian FAS orthologs. Twelve hour treatment of oleic acid significantly up-regulated the expression of FAS in duck preadipocytes (P < 0.05). However, 1000 μM treatment of oleic acid exhibited lipotoxic effect on cell viability (P < 0.05). In addition, during the first 24 h of duck adipocyte differentiation FAS was induced; however, after 24 h its expression level declined (P < 0.05).ConclusionWe have successfully cloned and characterized Peking duck FAS. FAS was induced during adipocyte differentiation and by oleic acid treatment. These findings suggest that Peking duck FAS plays a similar role to mammalian FAS during adipocyte differentiation.     

Effect of cytokinin types,concentrations and their interactions on in vitro shoot regeneration of Chlorophytum borivilianum Sant. & Fernandez

BackgroundChlorophytum borivilianum is a rare medicinal plant originally distributed throughout the forest of India. The tubers of C. borivilianum are used as an aphrodisiac and impotence supplement. The propagation of C. borivilianum is possible through seeds and tubers, but conventional methods may take several months. Hence in vitro technique of shoot regeneration could be an efficient alternative means of propagating the species. Latest study reported microtuberization of C. borivilianum but there is no sufficient study on a rapid method for shoot multiplication and elongation.ResultsYoung shoot buds of C. borivilianum were cultured on MS medium containing 6-benzylaminopurine (BAP) and Kinetin (Kn), both at 0, 8.88, 17.8 and 26.6 μM, either individually or in combinations. Proliferated shoots were subcultured on fresh medium of the same constituents on week 3 of culture for further shoot multiplication and elongation. BAP alone (8.88–26.6 μM) was significantly effective on shoot multiplication, while Kn alone (8.88–26.6 μM) was significantly effective on shoot elongation compared to the control containing MS basal medium without any plant growth regulator. However, combination of both cytokinins stimulated an interaction producing higher shoot number and shoot length compared to their individual application.ConclusionsThe most suitable combination was 8.88 μM BAP + 8.88 μM Kn, reaching a mean shoot number of 10.83 and shoot length of 6.85 cm.     

Callus culture development of two varieties of Tagetes erecta and carotenoid production

BackgroundThe properties of natural pigments, such as antioxidants, functional, medical, and nutraceutical, have demonstrated the advantages of these natural compounds over synthetic ones. Some products are accepted only when they are pigmented with natural, food-quality colorants: for example poultry products (manly marigold flower extracts). Carotenoids such as β-carotene, β-criptoxanthin and lutein are very attractive as natural food colorants due to their antioxidant and pro-vitamin activities which provide additional value to the target products. Marigold (Tagetes erecta) is an Asteraceous ornamental plant native to Mexico, and it is also important as a carotenoid source for industrial and medicinal purposes but nowadays its production is destined mainly for ornamental purposes.ResultsFriable callus of T. erecta yellow flower (YF) and white flower (WF) varieties was induced from leaf explants on Murashige and Skoog (MS) medium supplemented with 9.0 μM 4-dichlorophenoxyacetic acid (2,4-D) and 8.8 μM benzyladenine (BA). Calluses developed from both varieties were different in pigmentation. Extract characterization from callus cultures was carried out by high-performance liquid chromatography (HPLC). This analytical process detected several carotenoids; the main pigments in extracts from YF callus were lutein and zeaxanthin, whereas in the extracts of the WF callus the main pigments were lutein, zeaxanthin, β-cryptoxanthin and β-carotene. Callus cultures of T. erecta accumulated pigments even after several rounds of subculture.ConclusionsWF callus appeared to be a suitable candidate as a source of different carotenoids, and tested varieties could represent an alternative for further studies about in vitro pigment production.     

Efficient regeneration and genetic transformation platform applicable to five Musa varieties

BackgroundBanana (Musa spp.) is an important staple food, economic crop, and nutritional fruit worldwide. Conventional breeding has been seriously hampered by their long generation time, polyploidy, and sterility of most cultivated varieties. Establishment of an efficient regeneration and transformation system for banana is critical to its genetic improvement and functional genomics.ResultsIn this study, a vigorous and repeatable transformation system for banana using direct organogenesis was developed. The greatest number of shoots per explant for all five Musa varieties was obtained using Murashige and Skoog medium supplemented with 8.9 μM benzylaminopurine and 9.1 μM thidiazuron. One immature male flower could regenerate 380–456, 310–372, 200–240, 130–156, and 100–130 well-developed shoots in only 240–270 d for Gongjiao, Red banana, Rose banana, Baxi, and Xinglongnaijiao, respectively. Longitudinal sections of buds were transformed through particle bombardment combined with Agrobacterium-mediated transformation using a promoterless β-glucuronidase (GUS) reporter gene; the highest transformation efficiency was 9.81% in regenerated Gongjiao plantlets in an optimized selection medium. Transgenic plants were confirmed by a histochemical assay of GUS, polymerase chain reaction, and Southern blot.ConclusionsOur robust transformation platform successfully generated hundreds of transgenic plants. Such a platform will facilitate molecular breeding and functional genomics of banana.     

Indole acetic acid and ACC deaminase from endophytic bacteria improves the growth of Solanum lycopersicum

BackgroundEndophytic bacteria are ubiquitous in all plant species contributing in host plant's nutrient uptake and helping the host to improve its growth. Moringa peregrina which is a medicinal plant, growing in arid region of Arabia, was assessed for the presence of endophytic bacterial strains.ResultsPCR amplification and sequencing of 16S rRNA of bacterial endophytes revealed the 5 endophytic bacteria, in which 2 strains were from Sphingomonas sp.; 2 strains from Bacillus sp. and 1 from Methylobacterium genus. Among the endophytic bacterial strains, a strain of Bacillus subtilis LK14 has shown significant prospects in phosphate solubilization (clearing zone of 56.71 mm after 5 d), ACC deaminase (448.3 ± 2.91 nM α-ketobutyrate mg^- 1 h^- 1) and acid phosphatase activity (8.4 ± 1.2 nM mg^- 1 min^- 1). The endophytic bacteria were also assessed for their potential to produce indole-3-acetic acid (IAA). Among isolated strains, the initial spectrophotometry analysis showed significantly higher IAA production by Bacillus subtilis LK14. The diurnal production of IAA was quantified using multiple reactions monitoring method in UPLC/MS–MS. The analysis showed that LK14 produced the highest (8.7 μM) IAA on 14th d of growth. Looking at LK14 potentials, it was applied to Solanum lycopersicum, where it significantly increased the shoot and root biomass and chlorophyll (a and b) contents as compared to control plants.ConclusionThe study concludes that using endophytic bacterial strains can be bio-prospective for plant growth promotion, which might be an ideal strategy for improving growth of crops in marginal lands.     

Biomineralization of a calcifying ureolytic bacterium Microbacterium sp. GM-1

BackgroundBiomineralization is a significant process performed by living organisms in which minerals are produced through the hardening of biological tissues. Herein, we focus on calcium carbonate precipitation, as part of biomineralization, to be used in applications for environmental protection, material technology, and other fields. A strain GM-1, Microbacterium sp. GM-1, isolated from active sludge, was investigated for its ability to produce urease and induce calcium carbonate precipitation in a metabolic process.ResultsIt was discovered that Microbacterium sp. GM-1 resisted high concentrations of urea up to 60 g/L. In order to optimize the calcification process of Microbacterium sp. GM-1, the concentrations of Ni^2 + and urea, pH value, and culture time were analyzed through orthogonal tests. The favored calcite precipitation culture conditions were as follows: the concentration of Ni^2 + and urea were 50 μM and 60 g/L, respectively, pH of 10, and culture time of 96 h. Using X-ray diffraction analysis, the calcium carbonate polymorphs produced by Microbacterium sp. GM-1 were proven to be mainly calcite.ConclusionsThe results of this research provide evidence that Microbacterium sp. GM-1 can biologically induce calcification and suggest that strain GM-1 may play a potential role in the synthesis of new biominerals and in bioremediation or biorecovery.     

Effects of oligosaccharides from endophytic Fusarium oxysporum Dzf17 on activities of defense-related enzymes in Dioscorea zingiberensis suspension cell and seedling cultures

BackgroundThree oligosaccharides (EOS, WOS and SOS) were respectively prepared from the corresponding polysaccharides, namely exopolysaccharide (EPS), water-extracted mycelial polysaccharide (WPS) and sodium hydroxide-extracted mycelial polysaccharides (SPS) from the endophytic fungus Fusarium oxysporum Dzf17. In this study, the effects of EOS, WOS and SOS on the activities of the defense-related enzymes, namely phenylalanine ammonia lyase (PAL), polyphenoloxidase (PPO) and peroxidase (POD) in its host plant Dioscorea zingiberensis cultures were investigated.ResultsFor the suspension cell cultures of D. zingiberensis, the highest PAL activity was induced by 0.5 mg/mL of WOS at 48 h after treatment, which was 4.55-fold as that of control. Both PPO and POD activities were increased to the maximum values by 0.25 mg/mL of WOS at 48 h after treatment, which were respectively 3.74 and 3.45-fold as those of control. For the seedling cultures, the highest PAL activity was elicited by 2.5 mg/mL of EOS at 48 h after treatment, which was 3.62-fold as that of control. Both PPO and POD reached their maximum values treated with 2.5 mg/mL of WOS at 48 h after treatment, which were 4.61 and 4.19-fold as those of control, separately.ConclusionsBoth EOS and WOS significantly increased the activities of PAL, PPO and POD in the suspension cell and seedling cultures of D. zingiberensis. The results suggested that the oligosaccharides from the endophytic fungus F. oxysporum Dzf17 may be related to the activation and enhancement of the defensive mechanisms of D. zingiberensis suspension cell and seedling cultures.     

Efficiency of RAPD,ISSR, AFLP and ISTR markers for the detection of polymorphisms and genetic relationships in camote de cerro (Dioscorea spp.)

BackgroundAt present, species known as camote de cerro (Dioscorea spp.) are found only in the wilderness in Mexico, but their populations are extremely depleted because they are indiscriminately collected, it is urgent to evaluate the conservation status of these plants in order to design conservation genetics programs. In this study, genetic diversity parameters along with cluster analysis based on Jaccard's coefficient were estimated with the objective to assess the efficiency of Random Amplified Polymorphic DNA (RAPD), Inter Simple Sequence Repeat (ISSR), Amplified Fragment Length Polymorphism (AFLP) and Inverse Sequence Tagged Repeat (ISTR) molecular DNA markers in the Dioscorea genus.ResultsThe polymorphic information contents were quite similar for all markers (≈ 0.48). Genetic variation of Dioscorea spp., in terms of average heterozygosity was lower with ISTR (0.36), and higher when other markers were used (RAPD = 0.43; ISSR = 0.45 and AFLP = 0.47).ConclusionThis indicates an important level of genetic differences despite the fact that the plant is asexually propagated. Based on the diversity statistics, any marker tested in present work can be recommended for use in large-scale genetic studies of populations. However, the low correlations among different molecular marker systems show the importance of the complementarity of the information that is generated by different markers for genetic studies involving estimation of polymorphism and relationships.     

Kernel size and weight affected by three plant bioregulators applied at bloom to Non Pareil and Carmel almond cultivars

BackgroundThe yield of almonds [Prunus dulcis (Mill.) D.A. Webb] could be low due to climatic problems and any factor improving kernel size and weight, such as the use of plant bioregulators (PBRs), should be beneficial.ResultsThree plant bioregulators: 24-epibrassinolide (BL), gibberellic acid (GA₃) and kinetin (KN) were applied at three spray concentrations to Non Pareil and Carmel cultivars, at two phenological stages during bloom, in the 2014 and 2015 seasons. The results showed significant differences (P < 0.0001). For total dry weight of Non Pareil, the best treatment was BL (30 mg·L^-1), with an average of 1.45 g, while the control was 1.30 g, at pink button during 2015. For Carmel, the best dry weight was 1.23 g, achieved with BL (30 mg·L^-1) at fallen petals in both seasons. The average dry weight of the controls varied between 1.13 and 1.18 g. The greatest almond lengths and widths in Non Pareil were 24.98 mm and 15.05 mm, achieved with BL (30 mg·L^-1) and KN (50 μL·L^-1) treatments, respectively, applied at pink button in 2015. In Carmel, the greatest length and width were 24.38 and 13.44 mm, obtained with BL (30 mg·L^-1) applied at the stages of pink button and fallen petals, respectively, in 2015. The control reached lengths between 22.33 and 23.38 mm, and widths between 11.99 and 12.93 mm.ConclusionsThe use of the bioregulators showed significant favorable effects on dry weight, length and width of kernels at harvest, in both cultivars.     

Characterization of a hyperthermophilic sulphur-oxidizing biofilm produced by archaea isolated from a hot spring

BackgroundSulphur-oxidizing microorganisms are widely used in the biofiltration of total reduced sulphur compounds (odorous and neurotoxic) produced by industries such as the cellulose and petrochemical industries, which include high-temperature process steps. Some hyperthermophilic microorganisms have the capability to oxidize these compounds at high temperatures (> 60°C), and archaea of this group, for example, Sulfolobus metallicus, are commonly used in biofiltration technology.ResultsIn this study, a hyperthermophilic sulphur-oxidizing strain of archaea was isolated from a hot spring (Chillán, Chile) and designated as M1. It was identified as archaea of the genus Sulfolobus (99% homology with S. solfataricus 16S rDNA). Biofilms of this culture grown on polyethylene rings showed an elemental sulphur oxidation rate of 95.15 ± 15.39 mg S l^-1 d^-1, higher than the rate exhibited by the biofilm of the sulphur-oxidizing archaea S. metallicus (56.8 ± 10.91 mg l^-1 d^-1).ConclusionsThe results suggest that the culture M1 is useful for the biofiltration of total reduced sulphur gases at high temperatures and for other biotechnological applications.     

Strategy of oxygen transfer coefficient control on the l-erythrulose fermentation by newly isolated Gluconobacter kondonii

BackgroundThe effect of diverse oxygen transfer coefficient on the l-erythrulose production from meso-erythritol by a newly isolated strain, Gluconobacter kondonii CGMCC8391 was investigated. In order to elucidate the effects of volumetric mass transfer coefficient (k_La) on the fermentations, baffled and unbaffled flask cultures, and fed-batch cultures were developed in present work.ResultsWith the increase of the k_La value in the fed-batch culture, l-erythrulose concentration, productivity and yield were significantly improved, while cell growth was not the best in the high k_La. Thus, a two-stage oxygen supply control strategy was proposed, aimed at achieving high concentration and high productivity of l-erythrulose. During the first 12 h, k_La was controlled at 40.28 h^-1 to obtain high value for cell growth, subsequently k_La was controlled at 86.31 h^-1 to allow for high l-erythrulose accumulation.ConclusionsUnder optimal conditions, the l-erythrulose concentration, productivity, yield and DCW reached 207.9 ± 7.78 g/L, 6.50 g/L/h, 0.94 g/g, 2.68 ± 0.17 g/L, respectively. At the end of fermentation, the l-erythrulose concentration and productivity were higher than those in the previous similar reports.     

Improvement of hydrogen yield of ethanol-producing Escherichia coli recombinants in acidic conditions

BackgroundAn effective single culture with high glycerol consumption and hydrogen and ethanol coproduction yield is still in demand. A locally isolated glycerol-consuming Escherichia coli SS1 was found to produce lower hydrogen levels under optimized ethanol production conditions. Molecular approach was proposed to improve the hydrogen yield of E. coli SS1 while maintaining the ethanol yield, particularly in acidic conditions. Therefore, the effect of an additional copy of the native hydrogenase gene hycE and recombinant clostridial hydrogenase gene hydA on hydrogen production by E. coli SS1 at low pH was investigated.ResultsRecombinant E. coli with an additional copy of hycE or clostridial hydA was used for fermentation using 10 g/L (108.7 mmol/L) of glycerol with an initial pH of 5.8. The recombinant E. coli with hycE and recombinant E. coli with hydA showed 41% and 20% higher hydrogen yield than wild-type SS1 (0.46 ± 0.01 mol/mol glycerol), respectively. The ethanol yield of recombinant E. coli with hycE (0.50 ± 0.02 mol/mol glycerol) was approximately 30% lower than that of wild-type SS1, whereas the ethanol yield of recombinant E. coli with hydA (0.68 ± 0.09 mol/mol glycerol) was comparable to that of wild-type SS1.ConclusionsInsertion of either hycE or hydA can improve the hydrogen yield with an initial pH of 5.8. The recombinant E. coli with hydA could retain ethanol yield despite high hydrogen production, suggesting that clostridial hydA has an advantage over the hycE gene in hydrogen and ethanol coproduction under acidic conditions. This study could serve as a useful guidance for the future development of an effective strain coproducing hydrogen and ethanol.     

Improvement of ethanol production from sweet sorghum juice under batch and fed-batch fermentations: Effects of sugar levels,nitrogen supplementation,and feeding regimes

BackgroundFermentation process development has been very important for efficient ethanol production. Improvement of ethanol production efficiency from sweet sorghum juice (SSJ) under normal gravity (NG, 160 g/L of sugar), high gravity (HG, 200 and 240 g/L of sugar) and very high gravity (VHG, 280 and 320 g/L of sugar) conditions by nutrient supplementation and alternative feeding regimes (batch and fed-batch systems) was investigated using a highly ethanol-tolerant strain, Saccharomyces cerevisiae NP01.ResultsIn the batch fermentations without yeast extract, HG fermentation at 200 g/L of sugar showed the highest ethanol concentration (P_E, 90.0 g/L) and ethanol productivity (Q_E, 1.25 g/L·h). With yeast extract supplementation (9 g/L), the ethanol production efficiency increased at all sugar concentrations. The highest P_E (112.5 g/L) and Q_E (1.56 g/L·h) were observed with the VHG fermentation at 280 g/L of sugar. In the fed-batch fermentations, two feeding regimes, i.e., stepwise and continuous feedings, were studied at sugar concentrations of 280 g/L. Continuous feeding gave better results with the highest P_E and Q_E of 112.9 g/L and 2.35 g/L·h, respectively, at a feeding time of 9 h and feeding rate of 40 g sugar/h.ConclusionsIn the batch fermentation, nitrogen supplementation resulted in 4 to 32 g/L increases in ethanol production, depending on the initial sugar level in the SSJ. Under the VHG condition, with sufficient nitrogen, the fed-batch fermentation with continuous feeding resulted in a similar P_E and increased Q_P by 51% compared to those in the batch fermentation.     

Enhanced alkaline catalase production by Serratia marcescens FZSF01: Enzyme purification,characterization, and recombinant expression

BackgroundCatalase (CAT) is an important enzyme that degrades H₂O₂ into H₂O and O₂. To obtain an efficient catalase, in this study, a new strain of high catalase-producing Serratia marcescens, named FZSF01, was screened and its catalase was purified and characterized.ResultsAfter optimization of fermentation conditions, the yield of catalase produced by this strain was as high as 51,468 U/ml. This catalase was further purified using two steps: DEAE-fast flow and Sephedex-G150. The purified catalase showed a specific activity of 197,575 U/mg with a molecular mass of 58 kDa. This catalase exhibited high activity at 20–70°C and pH 5.0–11.0. Km of the catalase was approximately 68 mM, and Vmax was 1886.8 mol/min mg. This catalase was further identified by LC–MS/MS, and the encoding gene was cloned and expressed in Escherichia coli BL21 (DE3) with a production of 17,267 ± 2037 U/ml.ConclusionsTo our knowledge, these results represent one of the highest fermentation levels reported among current catalase-producing strains. This FZSF01 catalase may be suitable for several industrial applications that comprise exposure to alkaline conditions and under a wide range of temperatures.     

Effects of volatile fatty acids in biohydrogen effluent on biohythane production from palm oil mill effluent under thermophilic condition

BackgroundBiohydrogen effluent contains a high concentration of volatile fatty acid (VFA) mainly as butyric, acetic, lactic and propionic acids. The presence of various VFAs (mixture VFAs) and their cooperative effects on two-stage biohythane production need to be further studied. The effect of VFA concentrations in biohydrogen effluent of palm oil mill effluent (POME) on methane yield in methane stage of biohythane production was investigated.ResultsThe methane yield obtained in low VFA loading (0.9 and 1.8 g/L) was 15–20% times greater than that of high VFA loading (3.6 and 4.7 g/L). Butyric acid at high concentrations (8 g/L) has the individual significantly negative effect the methane production process (P < 0.05). Lactic, acetic and butyric acid mixed with propionic acid at a concentration higher than 0.5 g/L has an interaction significantly negative effect on the methanogenesis process (P < 0.05). Inhibition condition had a negative effect on both bacteria and archaea with inhibited on Geobacillus sp., Thermoanaerobacterium thermosaccharolyticum, Methanoculleus thermophilus and Methanothermobacter delfuvii resulting in low methane yield.ConclusionPreventing the high concentration of butyric acid, and propionic acid in the hydrogenic effluent could enhance methane production in two-stage anaerobic digestion for biohythane production.