Overexpression of coconut AINTEGUMENTA-like gene, CnANT, promotes in vitro regeneration in transgenic Arabidopsis

Knowledge of the molecular biological changes underlying the process of embryogenesis is important for the improvement of somatic embryogenesis of coconut. Among the transcription factors that control the transition from vegetative to embryogenic growth, members of APETALA2/Ethylene-responsive element binding protein domain family play an important role in promoting embryo development. Significant insights into the role of AP2 genes have been obtained by the ectopic expression of AP2 sub family genes in transgenic Arabidopsis. A homolog of the AINTEGUMENTA-like gene that encodes the two AP2 domains and the linker region was identified in the coconut genome. Phylogenetic analysis showed that this gene, CnANT, encodes a protein that branched with BABY BOOM/PLETHORA clade in the AINTEGUMENTA-like major clade and was similar to the oil palm EgAP2-1 protein. According to real time RT-PCR results, higher expression of CnANT was observed in more mature zygotic embryos. Also, high CnANT expression was recorded in embryogenic callus compared to other stages of somatic embryogenesis. We examined the effect of ectopic CnANT expression on the development and regenerative capacity of transgenic Arabidopsis. Overexpression of CnANT in Arabidopsis induced hormone free regeneration of explants. Furthermore, ectopic expression of CnANT enhanced regeneration in vitro and suggested a role for this gene in cell proliferation during in vitro culture.

Global scale transcriptome analysis of Arabidopsis embryogenesis in vitro

Background Somatic embryogenesis (SE) in plants is a process by which embryos are generated directly from somatic cells, rather than from the fused products of male and female gametes. Despite the detailed expression analysis of several somatic-to-embryonic marker genes, a comprehensive understanding of SE at a molecular level is still lacking. The present study was designed to generate high resolution transcriptome datasets for early SE providing the way for future research to understand the underlying molecular mechanisms that regulate this process. We sequenced Arabidopsis thaliana somatic embryos collected from three distinct developmental time-points (5, 10 and 15 d after in vitro culture) using the Illumina HiSeq 2000 platform. Results This study yielded a total of 426,001,826 sequence reads mapped to 26,520 genes in the A. thaliana reference genome. Analysis of embryonic cultures after 5 and 10 d showed differential expression of 1,195 genes; these included 778 genes that were more highly expressed after 5 d as compared to 10 d. Moreover, 1,718 genes were differentially expressed in embryonic cultures between 10 and 15 d. Our data also showed at least eight different expression patterns during early SE; the majority of genes are transcriptionally more active in embryos after 5 d. Comparison of transcriptomes derived from somatic embryos and leaf tissues revealed that at least 4,951 genes are transcriptionally more active in embryos than in the leaf; increased expression of genes involved in DNA cytosine methylation and histone deacetylation were noted in embryogenic tissues. In silico expression analysis based on microarray data found that approximately 5% of these genes are transcriptionally more active in somatic embryos than in actively dividing callus and non-dividing leaf tissues. Moreover, this identified 49 genes expressed at a higher level in somatic embryos than in other tissues. This included several genes with unknown function, as well as others related to oxidative and osmotic stress, and auxin signalling. Conclusions The transcriptome information provided here will form the foundation for future research on genetic and epigenetic control of plant embryogenesis at a molecular level. In follow-up studies, these data could be used to construct a regulatory network for SE; the genes more highly expressed in somatic embryos than in vegetative tissues can be considered as potential candidates to validate these networks.

In vitro fermentation of carbohydrates by porcine faecal inocula and their influence on Salmonella Typhimurium growth in batch culture systems

The aim of this study was to evaluate in vitro the influence of fermentable carbohydrates on the activity of porcine microbiota and survival of Salmonella Typhimurium in a batch culture system simulating the porcine hindgut. The carbohydrates tested were xylooligosaccharides, a mixture of fructooligosaccharides/inulin (FIN), fructooligosaccharides (FOS), gentiooligosaccharides (GEO) and lactulose (LAC). These ingredients stimulated the growth of selected Bifidobacterium and Lactobacillus species in pure cultures. In batch cultures, the carbohydrates influenced some fermentation parameters. For example, GEO and FIN significantly increased lactic acids compared with the control (no added carbohydrate). With the exception of LAC, the test carbohydrates increased the production of short-chain fatty acid (SCFA) and modified SCFA profiles. Quantitative analysis of bacterial populations by FISH revealed increased counts of the Bifidobacterium group compared with control and, with exception of FOS, increased Lactobacillus, Leuconostoc and Weissella spp. counts. Salmonella numbers were the lowest during the fermentation of LAC. This work has looked at carbohydrate metabolism by porcine microbiota in a pH-controlled batch fermentation system. It provides an initial model to analyse interactions with pathogens.

In vitro fermentation of oat bran obtained by debranning with a mixed culture of human fecal bacteria

The prebiotic potential of oat samples was investigated by in vitro shaker-flask anaerobic fermentations with human fecal cultures. The oat bran fraction was obtained by debranning and was compared with other carbon sources such as whole oat flour, glucose, and fructo-oligosaccharide. The oat bran fraction showed a decrease in culturable anaerobes and clostridia and an increase in bifidobacteria and lactobacilli populations. A similar pattern was observed in fructo-oligosaccharide. Butyrate production was higher in oat bran compared to glucose and similar to that in fructo-oligosaccharide. Production of propionate was higher in the two oat media than in fructo-oligosaccharide and glucose, which can be used as energy source by the liver. This study suggests that the oat bran fraction obtained by debranning is digested by the gut ecosystem and increases the population of beneficial bacteria in the indigenous gut microbiota. This medium also provides an energy source preferred by colonocytes when it is metabolized by the gut flora.

Response of porcine intestinal in vitro organ culture tissues following exposure to Lactobacillus plantarum JC1 and Salmonella Typhimurium SL1344.

The development of novel intervention strategies for the control of zoonoses caused by bacteria such as Salmonella spp. in livestock requires appropriate experimental models to assess their suitability. Here, a novel porcine intestinal in vitro organ culture (IVOC) model utilizing cell crown (CC) technology (CCIVOC) (Scaffdex) was developed. The CCIVOC model was employed to investigate the characteristics of association of S. enterica serovar Typhimurium strain SL1344 with porcine intestinal tissue following exposure to a Lactobacillus plantarum strain. The association of bacteria to host cells was examined by light microscopy and electron microscopy (EM) after appropriate treatments and staining, while changes in the proteome of porcine jejunal tissues were investigated using quantitative label-free proteomics. Exposure of porcine intestinal mucosal tissues to L. plantarum JC1 did not reduce the numbers of S. Typhimurium bacteria associating to the tissues but was associated with significant (P < 0.005) reductions in the percentages of areas of intestinal IVOC tissues giving positive staining results for acidic mucins. Conversely, the quantity of neutrally charged mucins present within the goblet cells of the IVOC tissues increased significantly (P < 0.05). In addition, tubulin- was expressed at high levels following inoculation of jejunal IVOC tissues with L. plantarum. Although L. plantarum JC1 did not reduce the association of S. Typhimurium strain SL1344 to the jejunal IVOC tissues, detection of increased acidic mucin secretion, host cytoskeletal rearrangements, and proteins involved in the porcine immune response demonstrated that this strain of L. plantarum may contribute to protecting the pig from infections by S. Typhimurium or other pathogens.

Rifaximin modulates the colonic microbiota of patients with Crohn's disease: an in vitro approach using a continuous culture colonic model system.

Rifaximin, a rifamycin derivative, has been reported to induce clinical remission of active Crohn's disease (CD), a chronic inflammatory bowel disorder. In order to understand how rifaximin affects the colonic microbiota and its metabolism, an in vitro human colonic model system was used in this study. We investigated the impact of the administration of 1800 mg/day of rifaximin on the faecal microbiota of four patients affected by colonic active CD [Crohn's disease activity index (CDAI > 200)] using a continuous culture colonic model system. We studied the effect of rifaximin on the human gut microbiota using fluorescence in situ hybridization, quantitative PCR and PCR–denaturing gradient gel electrophoresis. Furthermore, we investigated the effect of the antibiotic on microbial metabolic profiles, using 1H-NMR and solid phase microextraction coupled with gas chromatography/mass spectrometry, and its potential genotoxicity and cytotoxicity, using Comet and growth curve assays. Rifaximin did not affect the overall composition of the gut microbiota, whereas it caused an increase in concentration of Bifidobacterium, Atopobium and Faecalibacterium prausnitzii. A shift in microbial metabolism was observed, as shown by increases in short-chain fatty acids, propanol, decanol, nonanone and aromatic organic compounds, and decreases in ethanol, methanol and glutamate. No genotoxicity or cytotoxicity was attributed to rifaximin, and conversely rifaximin was shown to have a chemopreventive role by protecting against hydrogen peroxide-induced DNA damage. We demonstrated that rifaximin, while not altering the overall structure of the human colonic microbiota, increased bifidobacteria and led to variation of metabolic profiles associated with potential beneficial effects on the host.

In vitro evaluation of the fermentation properties and potential prebiotic activity of caprine cheese whey oligosaccharides in batch culture systems

The prebiotic effect of oligosaccharides recovered and purified from caprine whey, was evaluated by in vitro fermentation under anaerobic conditions using batch cultures at 37ºC with human faeces. Effects on key gut bacterial groups were monitored over 24h by fluorescence in situ hybridisation (FISH), which was used to determine a quantitative prebiotic index score. Production of short-chain fatty acids (SCFAs) as fermentation end products was analysed by high-performance liquid chromatography (HPLC). Growth of Bifidobacterium spp was significantly higher (p ≥ 0.05) with the purified oligosaccharides compared to the negative control. Lactic and propionic acids were the main SCFAs produced. Antimicrobial activity of the oligosaccharides was also tested, revealing no inhibition though a decrease in Staphylococcus aureus and Escherichia coli growth. These findings indicate that naturally extracted oligosaccharides from caprine whey could be used as new and valuable source of prebiotics.

In Vitro Fermentation of NUTRIOSE® FB06, a wheat dextrin soluble fibre, in a continuous culture human colonic model system

Wheat dextrin soluble fibre may have metabolic and health benefits, potentially acting via mechanisms governed by the selective modulation of the human gut microbiota. Our aim was to examine the impact of wheat dextrin on the composition and metabolic activity of the gut microbiota. We used a validated in vitro three-stage continuous culture human colonic model (gut model) system comprised of vessels simulating anatomical regions of the human colon. To mimic human ingestion, 7 g of wheat dextrin (NUTRIOSE® FB06) was administered to three gut models, twice daily at 10.00 and 15.00, for a total of 18 days. Samples were collected and analysed for microbial composition and organic acid concentrations by 16S rRNA-based fluorescence in situ hybridisation and gas chromatography approaches, respectively. Wheat dextrin mediated a significant increase in total bacteria in vessels simulating the transverse and distal colon, and a significant increase in key butyrate-producing bacteria Clostridium cluster XIVa and Roseburia genus in all vessels of the gut model. The production of principal short-chain fatty acids, acetate, propionate and butyrate, which have been purported to have protective, trophic and metabolic host benefits, were increased. Specifically, wheat dextrin fermentation had a significant butyrogenic effect in all vessels of the gut model and significantly increased production of acetate (vessels 2 and 3) and propionate (vessel 3), simulating the transverse and distal regions of the human colon, respectively. In conclusion, wheat dextrin NUTRIOSE® FB06 is selectively fermented in vitro by Clostridium cluster XIVa and Roseburia genus and beneficially alters the metabolic profile of the human gut microbiota.

An assessment of the prebiotic potential of single and blended substrates in anaerobic in vitro batch culture fermentations using canine faecal samples as inocula

Previously, using an in vitro static batch culture system, it was found that rice bran (RB), inulin, fibersol, mannanoligosaccharides (MOS), larch arabinogalactan and citrus pectin elicited prebiotic effects (in terms of increased numbers of bifidobacteria and lactic acid bacteria) on the faecal microbiota of a dog. The aim of the present study was to confirm the prebiotic potential of each individual substrate using multiple faecal donors, as well as assessing the prebiotic potential of 15 substrate blends made from them. Anaerobic static and stirred, pH-controlled batch culture systems inoculated with faecal samples from healthy dogs were used for this purpose. Fluorescence in situ hybridization (FISH) analysis using seven oligonucleotide probes targeting selected bacterial groups and DAPI (total bacteria) was used to monitor bacterial populations during fermentation runs. High-performance liquid chromatography was used to measure butyrate produced as a result of bacterial fermentation of the substrates. RB and a MOS/RB blend (1:1, w/w) were shown to elicit prebiotic and butyrogenic effects on the canine microbiota in static batch culture fermentations. Further testing of these substrates in stirred, pH-controlled batch culture fermentation systems confirmed the prebiotic and butyrogenic effects of MOS/RB, with no enhancement of Clostridium clusters I and II and Escherichia coli populations.

In vitro evaluation of the fermentation properties and potential probiotic activity of Lactobacillus plantarum C4 in batch culture systems

Lactobacillus plantarum C4 has been tested in in vitro pH-controlled anaerobic faecal batch cultures as compared to Lactobacillus rhamnosus GG to determine changes caused to the composition of faecal bacteria. Effects upon major groups of the microbiota and levels of short-chain fatty acids (SCFA) were assessed over 24 h. Concomitantly, hydrophobic character and ability of both bacterial cells to adhere in vitro to Caco-2 cells were investigated. Quantitative analysis of bacterial populations revealed that there was a significant increase in Lactobacillus/Enterococcus numbers in vessels with probiotic supplemented with fructooligosaccharides (FOS), compared to the negative control. L. plantarum C4 showed to have more hydrophilic behaviour and fulfilled better adhesive properties, compared to L. rhamnosus GG. Thus, L. plantarum C4 can modulate the intestinal microbiota in vitro, promoting changes in some numerically and metabolically relevant microbial populations and shifts in the production of SCFA.

Difference in competence for in vitro proliferation and ex vitro growth of genetically identical mature and juvenile clones of apomictic Malus species

The apomictic system in Malus wits Used Is a model to examine rejuvenation by generating genetically identical tissue culture lines that had two entirely different developmental origins: either embryo-derived tissues (juvenile clones) or somatic tissue from the adult/mature tree (mature clones). These two lines were then subsequently used to examine in vitro difference between mature (M) and juvenile (J) tissues in potential for shoot, root proliferation and ex vitro (glasshouse) growth. The M clones of M. hupehensis and M. toringoides in vitro had significantly fewer total shoots and shoot more than 2 cm in length per proliferating explant than the J clones and also rooted less efficiently. Ex vitro (glasshouse) juvenile clones had shorter internodes, a greater number of leaves and more dry weight compared to their mature counterparts.

Oestrogenic activity of benzyl salicylate, benzyl benzoate and butylphenylmethylpropional (Lilial) in MCF7 human breast cancer cells in vitro

Benzyl salicylate, benzyl benzoate and butylphenylmethylpropional (Lilial) are added to bodycare cosmetics used around the human breast. We report here that all three compounds possess oestrogenic activity in assays using the oestrogen-responsive MCF7 human breast cancer cell line. At 3 000 000-fold molar excess, they were able to partially displace [H-3]oestradiol from recombinant human oestrogen receptors ER alpha and ER beta, and from cytosolic ER of MCF7 cells. At concentrations in the range of 5 x 10(-5) to 5 x 10(-4) M, they were able to increase the expression of a stably integrated oestrogen-responsive reporter gene (ERE-CAT) and of the endogenous oestrogen-responsive pS2 gene in MCF7 cells, albeit to a lesser extent than with 10(-8) M 17 beta-oestradiol. They increased the proliferation of oestrogen-dependent MCF7 cells over 7 days, which could be inhibited by the antioestrogen fulvestrant, suggesting an ER-mediated mechanism. Although the extent of stimulation of proliferation over 7 days was lower with these compounds than with 10(-8) M 17 beta-oestradiol, given a longer time period of 35 days the extent of proliferation with 10(-4) M benzyl salicylate, benzyl benzoate or butylphenylmethylpropional increased to the same magnitude as observed with 10(-8) M 17 beta-oestradiol over 14 days. This demonstrates that benzyl salicylate, benzyl benzoate and butylphenylmethylpropional are further chemical components of cosmetic products which give oestrogenic responses in a human breast cancer cell line in culture. Further research is now needed to investigate whether oestrogenic responses are detectable using in vivo models and the extent to which these compounds might be absorbed through human skin and might enter human breast tissues. Copyright (C) 2009 John Wiley & Sons, Ltd.

Recent advances in the application of in vitro systems to plant improvement

The horticultural industry was instrumental in the early development and exploitation of genetic techniques over a century ago. This review will describe recent advances in a range of in vitro methods and their application to plant breeding, with especial emphasis on horticultural crops. These methods include improvements in the efficiency of haploid breeding techniques in many fruit and vegetable species using either microspore-derived or ovule-derived plants. Significant molecular information is now available to supplement these essentially empirical approaches and this may enable the more predictable application of these technologies in previously intransigent crops. Similarly there are now improved techniques for isolation of somatic hybrids, by application of either in vitro fertilisation or the culture of excised ovules from interspecific crosses. In addition to examples taken from the traditional scientific literature, emphasis will also be given to the use of patent databases as a valuable source of information on recent novel technologies developed in the commercial world.

Seasonal variations in the developmental competence of bovine oocytes matured in vitro

Ovaries were collected over a period of two years from heifers slaughtered at under 30 months of age and used to harvest 1757 oocytes. After in vitro maturation, fertilisation and culture, the proportions of oocytes and cleaved embryos that developed to blastocysts were significantly higher (P < 0.01) in the autumn, from September to November, than in the spring, from March to May. In contrast, embryo development, as assessed by oocytes that developed to eight or more cells and blastocysts, was lowest (P < 0.01) in the spring. These results were consistent during the two-year study, indicating a seasonal fluctuation in oocyte competence.