Agrobacterium-mediated transformation of cauliflower: optimization of protocol and development of Bt-transgenic cauliflower

A number of factors that are known to influence genetic transformation were evaluated to optimizeAgrobacterium-mediated transformation of hypocotyl explants of cauliflower variety Pusa Snowball K-1. The binary vector p35SGUSINT mobilized intoAgrobacterium strain GV2260 was used for transformation and transient GUS expression was used as the basis for identifying the most appropriate conditions for transformation. Explant age, preculture period, bacterial strain and density were found to be critical determinants of transformation efficiency. Using the optimized protocol, the syntheticcryIA(b) gene was mobilized into cauliflower. Molecular analyses of transgenics established the integration and expression of the transgene. Insect bioassays indicated the effectiveness of the transgene against infestation by diamondback moth (Plutella xylostella) larvae.     

Effect of ascorbic acid on stimulatory status of activated mouse peritoneal phagocytes

Mouse peritoneal macrophages (MPM) when elicited by the antioxidant ascorbic acid have been found to be significantly stimulatory, exhibiting marked alteration at the cellular and enzyme levels. Alterations recorded were as follows--cellular yield per mouse, their protein content, lysosomal acid hydrolase levels and capability to phagocyte, all were significantly enhanced. The new stimulant was observed to produce no synergistic action on MPM when thioglycollate, BCG or endotoxin along with the same stimulated the latter. Levels of antioxidants like ascorbic acid and glutathione were found to be enhanced in elicited macrophages whereas superoxide dismutase levels varied when the three above stimulators were administered. However, the ascorbic acid elicited cells showed an increase in glutathione levels and a decrease in SOD levels but no change in total intracellular ascorbic acid levels. Further, though ascorbic acid interaction enhanced the phagocytic capability of MPM as compared to resident cells, no significant boosting of phagocytic process could be observed when each of three stimulators coupled with ascorbic acid was used for macrophage elicitation.     

A set of cassettes and improved vectors for genetic and biochemical characterization of Pseudomonas genes

A set of broad-host-range vectors allowing direct selection of recombinant DNA molecules to facilitate subcloning and expression analyses of Pseudomonas genes was constructed using Bg/II lacZ alpha cassette. Controlled expression vectors pVDtac39 and pVDtac24 were shown to be useful for determination of enzymatic activities encoded by the cloned DNA fragments and Mr determination of the corresponding polypeptides. A set of Pseudomonas putida xylE gene cassettes truncated at the 5' end was constructed for translational (protein) fusion studies. A protein fusion of the Pseudomonas aeruginosa algD gene, coding for GDPmannose dehydrogenase, and the truncated xylE gene cassette was used to verify the putative coding region and translational signals predicted from the algD nucleotide sequence.     

An efficient direct induction of protocorm-like bodies from leaf subepidermal cells of Doritaenopsis hybrid using thin-section culture

High-frequency protocorm-like body (PLB) formation directly from thin leaf sections of Doritaenopsis hybrid was achieved in order to develop a mass-scale propagation system. Concentrated efforts were made to study the effects of different cytokinins on in vitro PLB induction from thin leaf sections. Among the cytokinins tested, thidiazuron (TDZ) was found to be a more effective inducer of PLBs than benzyladenine and zeatin. A modified Murashige and Skoog medium supplemented with 9.0 µM TDZ was found to be the optimum concentration for PLB development from thin leaf sections of Doritaenopsis hybrid. Of the two different explant types used in the present experiment, the highest percentage of PLB formation (72.3%) and highest number of PLBs (18) per explant were observed on thin leaf sections (1 mm thick), while only 20% (4.3 per explant) of comparatively large leaf segments (5 mm thick) were able to produce PLBs under the same culture conditions. Light microscopy observations indicated that the initial cell divisions for PLB formation occurred on the region near the cut surface and that an intact epidermal layer appeared to play an important role in PLB formation. Proembryo initiation occurred from several cells just beneath the intact epidermal cell, and globular PLBs were clearly visible after 3 weeks of culture and subsequently developed into mature PLBs.     

Effects of thyroidal, gonadal and adrenal hormones on tissue respiration of streaked frog, Rana limnocharis, at low temperature

In vivo and in vitro effects of thyroidal, gonadal and adrenal hormones were studied on the rate of liver and skeletal muscle respiration in both the sexes of R. limnocharis during active and inactive phases of the annual activity cycle. Triiodothyronine (L-T3) and thyroxine (L-T4) did not stimulate tissue (liver and muscle) respiration in any of the experiments irrespective of season, sex and temperature. Testosterone, estradiol and corticosterone stimulated O2 uptake significantly irrespective of season, sex and temperature. Adrenaline and nor-adrenaline also stimulated tissue respiration significantly during the winter month. Since the ambient temperature was low even during the active phase (max. temperature 21 degrees C), it seems that the frog might have developed tissue sensitivity for gonadal and adrenal hormones at low temperatures when thyroid hormones are calorigenically ineffective.     

Real-time PCR assay for rapid,efficient and accurate detection of <Emphasis Type="Italic">Paramyrothecium roridum</Emphasis> a leaf spot pathogen of <Emphasis Type="Italic">Gossypium</Emphasis> species

Here we report a highly sensitive real-time PCR (qPCR) assay to detect Paramyrothecium roridum from pure culture and infected samples of cotton plants. A specific set of primer pair pMyro F/R is designed to target the 185 bp ITS region of rDNA of Paramyrothecium roridum species and validated using qPCR. The fluorescence signals were detected above the baseline threshold from samples containing Paramyrothecium roridum DNA, whereas other samples did not produce any fluorescence or produced fluorescence which did not reach detection threshold values. A single dissociation peak of increased fluorescence was obtained for the specific primers at 92.2 °C melting temperature. The limit of detection using SYBR Green dye in this assay was up to 0.1 pg per µL of DNA from pure culture of P. roridum. The assay is accurate, sensitive, less laborious and time saving for detection of P. roridum in infected tissues of cotton.     

Regulation of nucleoside diphosphate kinase and secretable virulence factors in Pseudomonas aeruginosa: roles of algR2 and algH.

Alginate is an important virulence factor for Pseudomonas aeruginosa during infection of the lungs of cystic fibrosis patients. The genes encoding enzymes for alginate production by P. aeruginosa are normally silent. They are activated in response to several environmental conditions, including high osmolarity, exposure to ethanol, or long-term growth under conditions of nutrient deprivation. Several genes which participate in the activation of alginate gene promoters have been identified; among these is the algR2 (algQ) gene. AlgR2 is an 18-kDa protein which has been shown to regulate the critical algD gene encoding GDP-mannose dehydrogenase as well as to regulate the levels of a tricarboxylic acid cycle enzyme, i.e., succinyl coenzyme A synthetase, and nucleoside diphosphate kinase (Ndk), an enzyme involved in nucleoside triphosphate synthesis. Succinyl coenzyme A synthetase and Ndk form a complex in P. aeruginosa. While algR2 is required for alginate synthesis at 37 degrees C, an algR2 insertion mutant was still able to make alginate slowly at 37 or at 30 degrees C. We used this observation to identify and clone a gene, termed algH. A strain with mutations in both algR2 and algH is unable to produce alginate at either 37 or 30 degrees C, and it is fully defective in Ndk production.     

Metabolism of Halophenols by 2,4,5-trichlorophenoxyacetic acid-degrading Pseudomonas cepacia

Resting cells of 2,4,5-trichlorophenoxyacetic acid-grown Pseudomonas cepacia AC1100 were able to completely and rapidly dechlorinate several chlorine-substituted phenols, including 2,4,5-trichlorophenol, 2,3,4,6-tetrachlorophenol, and pentachlorophenol. Several other trichlorophenols were only partially dechlorinated. The evidence suggests that 2,4,5-trichlorophenol is an intermediate in the degradation of 2,4,5-trichlorophenoxyacetic acid by strain AC1100. Moreover, although strain AC1100 was isolated by selection for growth on a chlorinated aromatic compound, brominated and fluorinated analogs were efficiently dehalogenated by strain AC1100 resting cells, whereas an iodinated analog was poorly dehalogenated.