Efficient fertile plant regeneration from protoplasts of the Indica rice breeding line IR72 (Oryza sativa L.)

Protoplasts were isolated from embryogenic cell suspensions obtained from mature seed derived embryogenic callus of the advanced Indica type rice breeding line IR72 available from IRRI (International Rice Research Institute), Manila. Culture of protoplasts with the agarose bead type method without nurse culture led to sustained proliferation of protoplast derived clones. A simple culture protocol was developed which stimulated embryogenic development. Germination of somatic embryos has so far produced 277 green plants from 6 independent experiments. 117 plants have been transferred to soil and are growing in the greenhouse. A few of them have already flowered and set seeds.Abbreviations 2,4-D, 2,4 Dichlorophenoxyacetic acid - BAP Benzylaminopurine - CH Casein hydrolysate - ECS Embryogenie cell suspension - Kn Kinetin - NAA Napthaleneacetic acid; Media: - AA Muller and Grafe 1978 - MS Murashige and Skoog 1962 - N6 Chu et al. 1975 - R2 Ohira et al. 1973     

Growth and yield of lentil and wheat inoculated with three Glomus isolates from Saskatchewan soils

The vesicular-arbuscular mycorrhizal fungi (VAMF) Glomus clarum (Nicol. and Schenck) isolate NT4, G. mosseae (Nicol. and Gerd.) Gerd. and Trappe isolate NT6 and G. versiforme (Karst.) Berch isolate NT7 coexist in wheat field soils in Saskatchewan. This study assessed the response of lentil (Lens esculenta L.) and wheat (Triticum aestivum L.) to monospecific and mixed cultures of these VAMF isolates. Seedlings were inoculated with 100 spores of a VAMF isolate, or an equal mixture of spores of two isolates, and grown in a sterile soil mix in a growth chamber. Both crops responded differently to these different VAMF isolates. In the case of lentil, G. clarum NT4 was more effective than G. mosseae NT6 and G. versiforme NT7, and significantly increased (P<0.05) the shoot dry weight (43%) and grain yield (57%) compared with the uninoculated control. There was a significant positive correlation between the percentage of VAMF colonized roots and shoot dry weight (r=0.672^***) and shoot phosphorus concentration (r=0.608^***) of lentil. In the case of wheat, G. clarum NT4 had no effect on shoot dry weight, but produced significant (P<0.08) increases in grain yield (12%) and the phosphorus concentration of the shoot and grain. Although G. clarum NT4 and G. mosseae NT6 both produced similar levels of VAM colonization in wheat, the only response of wheat to isolate NT6 was an increase in plant height at harvest. The efficacy of G. clarum NT4 on both crops appeared to be related to its ability to produce more arbuscular colonization than G. mosseae NT6. Dual inoculation of seedlings with G. clarum NT4 and G. mosseae NT6 resulted in competition between these two isolates. This was evident from a comparison of plant shoot dry weight and grain yield, and VAMF spore production on the two crops inoculated either with isolate NT4 alone or in combination with NT6. G. mosseae NT6 reduced the efficacy of G. clarum NT4 by 16% when dual inoculated on lentil, but had no effect when the host was wheat. Based on spore production, it was found that G. clarum NT4 was more competitive than G. mosseae NT6 when dual inoculated on lentil or wheat. Isolate NT4 produced ca. 2000 and 500 spores/ 100 g substrate, respectively, in the lentil and wheat pots, which was approximately 2–3 times more spores than those produced by isolate NT6 with either crop. When the plants were dual inoculated, there was a 15–19% reduction in spore production by G. clarum NT4 and a 50–70% decrease in spore production by G. mosseae NT6. Our results show that G. clarum NT4 was more competitive and effective in its ability to colonize and increase the growth and yield of lentil and wheat than G. mosseae NT6 or G. versiforme NT7. The relative performance of isolate NT4 with different host plants suggests that this VAMF isolate exhibits a host preference for lentil.     

Gene transfer by electroporation into intact scutellum cells of wheat embryos

Summary Gene transfer into intact cells was achieved by electroporating zygotic wheat embryos without any special pretreatment. Electroporation was tissue specific in so far as scutellum cells were found to be much more susceptible to gene transfer than other cell types of the embryo. The orientation of the embryos in the electroporation chamber also influenced the number of transformed scutellum cells; during electroporation, as in electrophoresis, the negatively charged plasmid DNA molecules seemed to move towards the positive electrode. Therefore, the embryos were arranged so that the scutella faced the negative electrode. The use of plasmids carrying either two chimeric anthocyanin regulatory genes or a chimeric gusA gene allowed clear identification of transformed cells in the scutellum. On some of the embryos, more than 100 transformed scutellum cells were found after electroporation with single electric pulses of 275 V/cm discharged from a 960-F capacitor and with 100 g DNA/ml electroporation buffer. Using the anthocyanin marker system, visibly transformed cells grew to produce red sectors.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - MES 2-N-morpholinoethane sulfonic acid     

Isolation of non-myelin plasma membranes unique to white matter

A procedure is described for isolating two membrane fractions from rabbit spina-cord white matter enriched with 5′-nucleotidase, a nonspecific plasma membrane marker, 2′, 3′-cyclic nucleotide phosphohydrolase, an oligodendroglial plasma membrane marker, and acetylcholinesterase, an axonal plasma membrane marker. While the two membrane fractions exhibited similar enrichments with respect to cyclic nucleotide phosphohydrolase, enrichments of 5′-nucleotidase and acetylcholinesterase were significantly greater in the heavier membranes were not detected. Moreover, gray matter did not yield homologous membrane fractions in the gradient when subjected to the identical procedure, indicating that the two membrane fractions were unique to white matter. While electronmicroscopic examination revealed that both membrane fractions were contaminated with myelin, the heavier fraction was least contaminated and exhibited a fair degree of homogeneity with respect to single membrane vesicular profiles. It was concluded that both membrane fractions were enriched with oligodendroglial and axonal plasma membranes, with the heavier fraction containing significantly more axolemma.     

Herbicide-resistant Indica rice plants from IRRI breeding line IR72 after PEG-mediated transformation of protoplasts

The commercially important Indica rice cultivar Oryza sativa cv. IR72 has been transformed using direct gene transfer to protoplasts. PEG-mediated transformation was done with two plasmid constructs containing either a CaMV 35S promoter/HPH chimaeric gene conferring resistance to hygromycin (Hg) or a CaMV 35S promoter/BAR chimaeric gene conferring resistance to a commercial herbicide (Basta) containing phosphinothricin (PPT). We have obtained so far 92 Hg^r and 170 PPT^r IR72 plants from protoplasts through selection. 31 Hg^r and 70 PPT^r plants are being grown in the greenhouse to maturity. Data from Southern analysis and enzyme assays proved that the transgene was stably integrated into the host genome and expressed. Transgenic plants showed complete resistance to high doses of the commercial formulations of PPT.     

Regulation of autophagy as a therapeutic option in glioblastoma

Apoptosis - Around three out of one hundred thousand people are diagnosed with glioblastoma multiforme, simply called glioblastoma, which is the most common primary brain tumor in adults. With a...     

Combination of LC3 shRNA Plasmid Transfection and Genistein Treatment Inhibited Autophagy and Increased Apoptosis in Malignant Neuroblastoma in Cell Culture and Animal Models

Malignant neuroblastoma is an extracranial solid tumor that usually occurs in children. Autophagy, which is a survival mechanism in many solid tumors including malignant neuroblastoma, deters the efficacy of conventional chemotherapeutic agents. To mimic starvation, we used 200 nM rapamycin that induced autophagy in human malignant neuroblastoma SK-N-BE2 and IMR-32 cells in cell culture and animal models. Combination of microtubule associated protein light chain 3 short hairpin RNA (LC3 shRNA) plasmid transfection and genistein (GST) treatment was tested for inhibiting rapamycin-induced autophagy and promoting apoptosis. The best synergistic efficacy caused the highest decrease in cell viability due to combination of 50 nM LC3 shRNA plasmid transfection and 25 µM GST treatment in rapamycin-treated SK-N-BE2 cells while combination of 100 nM LC3 shRNA plasmid transfection and 25 µM GST treatment in rapamycin-treated IMR-32 cells. Quantitation of acidic vesicular organelles confirmed that combination of LC3 shRNA plasmid transfection and GST treatment prevented rapamycin-induced autophagy due to down regulation of autophagy promoting marker molecules (LC3 II, Beclin 1, TLR-4, and Myd88) and upregulation of autophagy inhibiting marker molecules (p62 and mTOR) in both cell lines. Apoptosis assays showed that combination therapy most effectively activated mitochondrial pathway of apoptosis in human malignant neuroblastoma in cell culture and animal models. Collectively, our current combination of LC3 shRNA plasmid transfection and GST treatment could serve as a promising therapeutic strategy for inhibiting autophagy and increasing apoptosis in human malignant neuroblastoma in cell culture and animal models.     

Antimicrobial Resistance,Virulence Factors and Genetic Diversity of Escherichia coli Isolates from Household Water Supply in Dhaka,Bangladesh

Background

Unsafe water supplies continue to raise public health concerns, especially in urban areas in low resource countries. To understand the extent of public health risk attributed to supply water in Dhaka city, Bangladesh, Escherichia coli isolated from tap water samples collected from different locations of the city were characterized for their antibiotic resistance, pathogenic properties and genetic diversity.

Methodology/Principal Findings

A total of 233 E. coli isolates obtained from 175 tap water samples were analysed for susceptibility to 16 different antibiotics and for the presence of genes associated with virulence and antibiotic resistance. Nearly 36% (n = 84) of the isolates were multi-drug(≥3 classes of antibiotics) resistant (MDR) and 26% (n = 22) of these were positive for extended spectrum β-lactamase (ESBL). Of the 22 ESBL-producers, 20 were positive for bla _CTX-M-15, 7 for bla _OXA-1-group (all had bla _OXA-47) and 2 for bla _CMY-2. Quinolone resistance genes, qnrS and qnrB were detected in 6 and 2 isolates, respectively. Around 7% (n = 16) of the isolates carried virulence gene(s) characteristic of pathogenic E. coli; 11 of these contained lt and/or st and thus belonged to enterotoxigenic E. coli and 5 contained bfp and eae and thus belonged to enteropathogenic E. coli. All MDR isolates carried multiple plasmids (2 to 8) of varying sizes ranging from 1.2 to >120 MDa. Ampicillin and ceftriaxone resistance were co-transferred in conjugative plasmids of 70 to 100 MDa in size, while ampicillin, trimethoprim-sulfamethoxazole and tetracycline resistance were co-transferred in conjugative plasmids of 50 to 90 MDa. Pulsed-field gel electrophoresis analysis revealed diverse genetic fingerprints of pathogenic isolates.

Significance

Multi-drug resistant E. coli are wide spread in public water supply in Dhaka city, Bangladesh. Transmission of resistant bacteria and plasmids through supply water pose serious threats to public health in urban areas.     

Pancreatic stellate cell-potentiated insulin secretion from Min6 cells is independent of interleukin 6-mediated pathway

Pancreatic stellate cells (PSCs) secrete various factors, which can influence the β-cell function. The identification of stellate cell infiltration into the islets in pancreatic diseases suggests possible existence of cross-talk between these cells. To elucidate the influence of PSCs on β-cell function, mouse PSCs were cocultured with Min6 cells using the Transwell inserts. Glucose-stimulated insulin secretion from Min6 cells in response to PSCs was quantified by enzyme-linked immunosorbent assay and insulin gene expression was measured by quantitative polymerase chain reaction. Upon cytometric identification of IL6 in PSC culture supernatants, Min6 cells were cultured with IL6 to assess its influence on the insulin secretion and gene expression. PLC-IP₃ pathway inhibitors were added in the cocultures, to determine the influence of PSC-secreted IL6 on Glucose-stimulated insulin secretion from Min6 cells. Increased insulin secretion with a concomitant decrease in total insulin content was noticed in PSC-cocultured Min6 cells. Although increased GSIS was noted from IL6-treated Min6 cells, no change in the total insulin content was noted. Coculture of Min6 cells with PSCs or their exposure to IL6 did not alter either the expression of β-cell-specific genes or that of miRNA-375. PSC-cocultured Min6 cells, in the presence of PLC-IP₃ pathway inhibitors (U73122, Neomycin, and Xestospongin C), did not revoke the observed increase in GSIS. In conclusion, the obtained results indicate that augmented insulin secretion from Min6 cells in response to PSC secretions is independent of IL6-mediated PLC-IP₃ pathway.