RESUMEN
Organomercury is the most toxic biomagnifiable state of mercury, and to date, no natural organomercurial detoxification mechanism is encountered in plants. Bacterial merB gene encoding organomercury lyase show low expression in transgenic plants. For ideal expression, a synthetic merBps gene possessing143 out of 213 codons discrete from native merB gene from Escherichia. coli was fabricated based on codon usage in tobacco. Through Agrobacterium-mediated transformation, the merBps gene got successfully integrated into tobacco. Of several putative merBps transformants selected with 200 µg ml-1 kanamycin, only â¼45% were PCR positive for both nptII and merBps genes. Healthy and vigorously growing shoots of few PCR-positive putative transgenic lines were multiplied and rooted. After transplantation and acclimatization, the resultant plants flowered and fruited in pots. Southern analysis revealed the presence of a single copy of the merBps gene in four lines. RT-PCR and Western investigations established successful transcription and translation of the merBps gene in these transgenic lines, respectively. Fabrication of fully functional organomercury lyase in merBps transgenic lines was established based on the potential of their (i) seeds to germinate; (ii) shoots to grow and multiply; and (iii) leaf disc to remain green, even in the presence of 4 nmole ml-1 phenylmercuryacetate (PMA) while the wild type was susceptible to even 1 nmole ml-1 PMA. These findings confirmed that the synthetic merBps gene could be effectively expressed in plants and exploited for remediation of organomercurial contaminated sites.
Asunto(s)
Mercurio , Nicotiana , Escherichia coli/genética , Hojas de la Planta , Plantas Modificadas Genéticamente/genética , Nicotiana/genética , Transformación GenéticaRESUMEN
Pollen viability is crucial for wheat breeding programs. The unique potential of the protoplasm of live cells to turn brown due to the synthesis of silver nanoparticles (AgNPs) through rapid photoreduction of Ag+, was exploited for testing wheat pollen viability. Ag+-viability test medium (consisting of 0.5 mM AgNO3 and 300 mM KNO3) incubated with wheat pollen turned brown within 2 min under intense light (~600 µmol photon flux density m-2s-1), but not in dark. The brown medium displayed AgNPs-specific surface plasmon resonance band in its absorption spectrum. Light microscopic studies showed the presence of uniformly stained brown protoplasm in viable pollen incubated with Ag+-viability medium in the presence of light. Investigations with transmission electron microscope coupled with energy dispersive X-ray established the presence of distinct 5-35 nm NPs composed of Ag. Powder X-ray diffraction analysis revealed that AgNPs were crystalline and biphasic composed of Ag0 and Ag2O. Conversely, non-viable pollen and heat-killed pollen did not turn brown on incubation with Ag+-medium in light. We believe that the viable wheat pollen turn brown rapidly by bio-transforming Ag+ to AgNPs through photoreduction. Our findings furnish a novel simplest and rapid method for testing wheat pollen viability.
Asunto(s)
Citoplasma/metabolismo , Citoplasma/efectos de la radiación , Luz , Polen/fisiología , Plata/metabolismo , Coloración y Etiquetado , Supervivencia Tisular/fisiología , Triticum/fisiología , Polen/ultraestructuraRESUMEN
Owing to its essentiality for cellular metabolism, phosphate (PO43-) plays a pivotal role in ecosystem dynamics. Frequent testing of phosphate levels is necessary to monitor ecosystem health. Present investigations were aimed to identify the key factors that are essential for proper quantification of PO43-. Primarily, H+ levels played a critical role in the development of molybdenum blue complex by ammonium molybdate and PO43- with ascorbic acid as a reductant. Molybdenum blue complex formed in the presence of 8 to 12 mmol of H+ in 3 ml reaction mixture remained stable even after 72 h. Of different concentrations of ammonium molybdate and ascorbic acid tested, best molybdenum blue complex was formed when their concentrations were 24.3 and 5.68 µmol, respectively. More or less similar intensity of molybdenum blue complex (due to reduction of phosphomolybdic acid and not molybdic acid) was formed in the presence of H+ at levels ranging from 8 to 10 mmol in 3 ml reaction mixture. Our findings unequivocally demonstrated that (i) the reaction mixture containing 3% ammonium molybdate, 0.1% ascorbic acid and 5 M H2SO4 in the ratio of 1:1:1 is ideal for PO43- quantification; (ii) antimony (Sb) significantly curbs the formation of molybdenum blue under these ideal conditions; (iii) this fine-tuned protocol for PO43- quantification could be extended without any problem for determining the level of PO43- both in plant as well as soil samples; and (iv) Azotobacter possesses potential to enhance levels of total PO43- in leaves and grains and soluble/active PO43- in rhizosphere soils of wheat.
Asunto(s)
Ácido Ascórbico/farmacología , Fosfatos/metabolismo , Sustancias Reductoras/farmacología , Antimonio/metabolismo , Azotobacter/metabolismo , Concentración de Iones de Hidrógeno , Molibdeno/metabolismo , Reproducibilidad de los Resultados , Suelo/química , Triticum/metabolismoRESUMEN
Potential of root system of plants from wide range of families to effectively reduce membrane impermeable ferricyanide to ferrocyanide and blue coloured 2,6-dichlorophenol indophenol (DCPIP) to colourless DCPIPH2 both under non-sterile and sterile conditions, revealed prevalence of immense reducing strength at root surface. As generation of silver nanoparticles (NPs) from Ag+ involves reduction, present investigations were carried to evaluate if reducing strength prevailing at surface of root system can be exploited for reduction of Ag+ and exogenous generation of silver-NPs. Root system of intact plants of 16 species from 11 diverse families of angiosperms turned clear colorless AgNO3 solutions, turbid brown. Absorption spectra of these turbid brown solutions showed silver-NPs specific surface plasmon resonance peak. Transmission electron microscope coupled with energy dispersive X-ray confirmed the presence of distinct NPs in the range of 5-50 nm containing Ag. Selected area electron diffraction and powder X-ray diffraction patterns of the silver NPs showed Bragg reflections, characteristic of crystalline face-centered cubic structure of Ag(0) and cubic structure of Ag2O. Root system of intact plants raised under sterile conditions also generated Ag(0)/Ag2O-NPs under strict sterile conditions in a manner similar to that recorded under non-sterile conditions. This revealed the inbuilt potential of root system to generate Ag(0)/Ag2O-NPs independent of any microorganism. Roots of intact plants reduced triphenyltetrazolium to triphenylformazon and impermeable ferricyanide to ferrocyanide, suggesting involvement of plasma membrane bound dehydrogenases in reduction of Ag+ and formation of Ag(0)/Ag2O-NPs. Root enzyme extract reduced triphenyltetrazolium to triphenylformazon and Ag+ to Ag(0) in presence of NADH, clearly establishing potential of dehydrogenases to reduce Ag+ to Ag(0), which generate Ag(0)/Ag2O-NPs. Findings presented in this manuscript put forth a novel, simple, economically viable and green protocol for synthesis of silver-NPs under ambient conditions in aqueous phase, using root system of intact plants.