Sheela Srivastava

Toxicity of some heavy metals in vivo and in vitro in Helianthus annuus.

To compare the toxicity of some heavy metals in vivo and in vitro, the effects of six metals, aluminum (Al), cadmium (Cd), copper (Cu), nickel (Ni), lead (Pb), and zinc (Zn) were studied in the oil-yielding plant Helianthus annuus. The percentage of seed germination and cytotoxic effects at different concentrations and durations of treatment as well as the growth rate of callus tissues in vitro were compared to ascertain the concentrations that can either support plant growth or cause lethality. Highest toxicity to the plant system was observed from the effects of Pb both at high and low concentrations whereas Zn was the least toxic; and similar effects were seen in vivo and in vitro. The clastogenic effects of Al, Cd, Cu, and Ni were dependent on concentration and length of treatment. Cu and Zn showed less severe cytotoxic damages than Al, Cd, Pb, and Ni. In vitro growth could be supported at 100-1000 times the diluted concentrations of the metals in comparison to in vivo treatment.

Plant biotechnology and molecular markers

In Vitro Androgenesis: Events Preceding Its Cytological Manifestation.- Doubled Haploids: A Powerful Biotechnological Tool for Genetic Enhancement in Oilseed Brassicas.- Double Fertilisation in vitro and Transgene Technology.- Polymorphism of Sexual and Somatic Embryos as Manifestation of Their Developmental Parallelism under Natural Conditions and in Tissue Culture.- Molecular Biology and Genetic Engineering of Polyamines in Plants.- Biotechnological Approaches Towards Improvement of Medicinal Plants.- Production of Phytochemicals in Plant Cell Bioreactors.- Development of Biotechnology for Commiphora wightii: A Potent Source of Natural Hypolipidemic and Hypocholesterolemic Drug.- Biotechnology in Quality Improvement of Oilseed Brassicas.- Role of Biotechnology for Incorporating White Rust Resistance in Brassica Species.- Current Trends in Forest Tree Biotechnology.- Cloning Forestry Species.- Micropropagation of Woody Plants.- Biotechnology in Mulberry (Morus spp.) Crop Improvement: Research Directions and Priorities.- Development of High Efficiency Micropropagation Protocol of an Adult Tree-Wrightia tomentosa.- In Vitro Regeneration and Improvement in Tropical Fruit Trees: An Assessment.- Tissue Culture of Cashewnut.- Changing Scenarios in Indian Horticulture.- Cryopreservation: A Potential Tool for Long-term Conservation of Medicinal Plants.- Molecular Mapping and Marker Assisted Selection of Traits for Crop Improvement.- Studies on Male Meiosis in Cultivated and Wild Vigna Species.- Transgenic Crops for Abiotic Stress Tolerance.- Cell Differentiation in Shoot Meristem: A Molecular Perspective.

Effect of cadmium and zinc interaction on metal uptake and regeneration of tolerant plants in linseed

Abstract The effect of cadmium and zinc interactions in lowering cadmium toxicity and in regeneration of tolerant plantlets from in vitro cultures of Linum usitatissimum (linseed) was monitored. Although zinc and cadmium are toxic at different concentrations, the interaction of these two ions at equimolar concentration can overcome the toxicity of cadmium. Tolerant plantlets can be regenerated in a culture medium containing both metals. The tolerant plantlets grew favourably in Cd 2+ and Zn 2+ separately. The regenerants showed greater uptake of cadmium in the roots than shoots, whereas zinc was found to be translocated from roots to shoots. Estimation of metal uptake inside the cells demonstrated the accumulation of cadmium into the cytoplasm in the regenerated roots, whereas more zinc was localised in the cell walls. In the regenerated shoots. however, an equal amount of zinc was found in the cell wall and the cytoplasm. The study of a stress-induced enzyme, peroxidase, in the regenerating tissues showed less activity in cadmium and zinc combination sets, indicating less stress in these tolerant regenerants.

An ipdC gene knock-out of Azospirillum brasilense strain SM and its implications on indole-3-acetic acid biosynthesis and plant growth promotion

The indole-3-pyruvate decarboxylase gene (ipdC), coding for a key enzyme of the indole-3-pyruvic acid pathway of IAA biosynthesis in Azospirillum brasilense SM was functionally disrupted in a site-specific manner. This disruption was brought about by group II intron-based Targetron gene knock-out system as other conventional methods were unsuccessful in generating an IAA-attenuated mutant. Intron insertion was targeted to position 568 on the sense strand of ipdC, resulting in the knock-out strain, SMIT568s10 which showed a significant (∼50%) decrease in the levels of indole-3-acetic acid, indole-3-acetaldehyde and tryptophol compared to the wild type strain SM. In addition, a significant decrease in indole-3-pyruvate decarboxylase enzyme activity by ∼50% was identified confirming a functional knock-out. Consequently, a reduction in the plant growth promoting response of strain SMIT568s10 was observed in terms of root length and lateral root proliferation as well as the total dry weight of the treated plants. Residual indole-3-pyruvate decarboxylase enzyme activity, and indole-3-acetic acid, tryptophol and indole-3-acetaldehyde formed along with the plant growth promoting response by strain SMIT568s10 in comparison with an untreated set suggest the presence of more than one copy of ipdC in the A. brasilense SM genome.

Antifungal effect of antimicrobial peptides (AMPs LR14) derived from Lactobacillus plantarum strain LR/14 and their applications in prevention of grain spoilage

The concern for food safety has led to an increased interest in the development of novel antimicrobials. Keeping this aim in mind, we have investigated the antifungal effect of antimicrobial peptides (AMPs LR14) produced by Lactobacillus plantarum strain LR/14 against four spoilage fungi, namely, Aspergillus niger, Rhizopus stolonifer, Mucor racemosus and Penicillium chrysogenum. Interestingly, all the four fungi were inhibited, suggesting that AMPs LR14 exhibited anti-fungal property. The peptides inhibited both, the spore germination and hyphal growth, however, the former stage was found to be more susceptible. The hyphal extensions were also inhibited in a dose-dependent manner. Viability test of treated spores confirmed the fungicidal activity of AMPs LR14. AMPs LR14 were also studied for the prevention of wheat grain spoilage under storage. Unhygienic conditions in damp godowns and store-houses, lead to loss of food grains and make them unfit for human consumption due to microbial deterioration. The treatment of wheat seeds with AMPs LR14 prevented fungal growth even after a prolonged storage under laboratory conditions for ∼2.5 years. The carbohydrate and protein content of the AMPs LR14-treated seeds denoted no significant loss, but the seed viability was affected as germination was retarded. Such studies have not been reported for any bacteriocin/AMP to the best of our knowledge.

Indole-3-acetic acid biosynthesis in the biocontrol strain Pseudomonas fluorescens Psd and plant growth regulation by hormone overexpression.

Pseudomonas fluorescens is an important biological component of agricultural soils that bestows a number of direct and indirect beneficial attributes to the plants. We analyzed the biocontrol strain P. fluorescens Psd for indole-3-acetic acid (IAA) biosynthesis and studied the effect of its consequent manipulation on its plant-growth-promoting (PGP) potential. While the indole pyruvic acid (IPyA) pathway commonly associated with PGP bacteria was lacking, the indole acetamide (IAM) pathway generally observed in phytopathogens was expressed in strain Psd. Overexpression of IAM pathway genes iaaM-iaaH, from Pseudomonas syringae subsp. savastanoi drastically increased IAA levels and showed a detrimental effect on sorghum root development. On the other hand, heterologous expression of the indole-3-pyruvate decarboxylase/phenylpyruvate decarboxylase gene (ipdC/ppdC) of the IPyA pathway from the PGP bacterium Azospirillum brasilense SM led to enhancement of the IAA level. A more favorable effect of this recombinant strain on sorghum root growth and development suggests that metabolic engineering could be used to generate strains with improved PGP function.

Plasmid-encoded sequestration of copper by Pseudomonas pickettii strain US321

Abstract.Pseudomonas pickettii strain US321 appeared to elicit a copper resistance mechanism upon exposure to copper. The bacterial colonies turned blue in the presence of this metal in a chemically defined medium, suggesting accumulation of copper. Prolonged exposure to copper resulted in the characteristic “copper sink” morphology of the colonies. Atomic absorption spectrophotometric analysis confirmed that this organism can accumulate copper. The strain US321 exhibited a high-molecular-weight plasmid, pUS321. The plasmid-cured strain, PC25, is highly sensitive to copper owing to a poor copper management. A plasmid-encoded sequestration mechanism operating in the strain US321 is suggested.

Mechanism of Copper Resistance in a Copper Mine Isolate Pseudomonas putida Strain S4

The mechanism of copper resistance in a multiple-metal-resistant natural isolate Pseudomonas putida strain S4 is based on inducible efflux. Active extrusion of copper ions occurs from the cytoplasm during the exponential phase of growth. Involvement of ATPase in the efflux of copper ions has been demonstrated by employing specific inhibitors. The effluxed copper is not thrown out of the cell, but remains in a bound form (to a protein) in the periplasm. Thus, a balance between the intracellular level, to fulfill the metabolic requirements, and the periplasmic sequestration, to evade toxicity, is maintained by this isolate.

Characterization of a Bacteriocin from Lactobacillus plantarum Strain LR/14

A rhizospheric isolate of a lactic acid bacterium, identified as Lactobacillus plantarum strain LR/14, was characterized to produce a bacteriocin. A supernatant from 20h culture growth was used as the source of bacteriocin. The antimicrobial compound showed remarkable stability at high temperatures (100°C for 30 min and 121°C for 15 min under 15 psi pressure) and to the presence of organic solvents, detergents and surfactants. It was also active in the pH regime of pH 2.0–6.0. Moreover, the compound was stable under different storage temperatures as tested up to 24 months. While antimicrobial function was not lost by catalase or β-glycerophosphate treatment, the same was sensitive to a number of proteolytic enzymes. The crude preparation inhibited not only related strains but also other gram-positive and gram-negative bacteria, such as Staphylococcus aureus, Listeria monocytogenes and urogenic E. coli. Bacteriocinogenic activity co-migrated as a single protein band on tricine-SDS-PAGE with molecular mass of ∼3.6 kDa.

Phenazine-1-carboxylic acid is a more important contributor to biocontrol Fusarium oxysporum than pyrrolnitrin in Pseudomonas fluorescens strain Psd.

Phenazines and pyrrolnitrin (Prn) are broad spectrum antibiotics, produced by bacteria, more so by the biocontrol strains to kill the phytopathogens in soil. We have studied a rhizospheric soil isolate of Pseudomonas fluorescens strain Psd producing both phenazine-1-carboxylic acid (PCA) and Prn. In order to study the contribution of these antibiotics, the phzD and prnC genes involved in PCA and Prn biosynthesis, were disrupted in a site-specific manner using a group II intron-based Targetron gene-knockout system, and gene disruption followed by allelic exchange through homologous recombination, respectively. The resulting knockout strains Psdphz122s-34 and PsdprnC::gen did not produce PCA and Prn, respectively. In fact, by combining these two strategies, a Psdphz122s-34prnC::gen double mutant could also be generated. Identification and lack of PCA production was corroborated by HPLC/APCI-MS analysis, and TLC detection for both the antibiotics in these mutants. Loss of antifungal activity against the phytopathogenic fungus Fusarium oxysporum was observed using in vitro growth assays on plates or growth chamber experiments with tomato seedling on an artificial substrate. Based on the characterization of these gene knockout mutants, we propose that PCA and Prn have a major role in antifungal activity of strain Psd.