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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 Ag0 and cubic structure of Ag2O. Root system of intact plants raised under sterile conditions also generated Ag0/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 Ag0/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 Ag0/Ag2O-NPs. Root enzyme extract reduced triphenyltetrazolium to triphenylformazon and Ag+ to Ag0 in presence of NADH, clearly establishing potential of dehydrogenases to reduce Ag+ to Ag0, which generate Ag0/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.

We discovered that Yeast Extract Mannitol (YEM) medium possessed immense potential to generate silver nanoparticles from AgNO3 upon autoclaving, which was evident from (i) alteration in color of the medium; (ii) peak at ∼410 nm in UV-Vis spectrum due to surface plasmon resonance specific to silver nanoparticles; and (iii) TEM investigations. TEM coupled with EDX confirmed that distinct nanoparticles were composed of silver. Yeast extract and mannitol were key components of YEM medium responsible for the formation of nanoparticles. PXRD analysis indicated crystalline geometry and Ag/Ag2O phases in nanoparticles generated with YEM medium, yeast extract and mannitol. Our investigations also revealed that both mannitol and yeast extract possessed potential to convert ∼80% of silver ions in 0.5 mM AgNO3 to nanoparticles, on autoclaving for 30 min at 121°C under a pressure of 1.06 kg/cm2. Addition of filter sterilized AgNO3 under ambient conditions to pre-autoclaved YEM medium and yeast extract brought about color change due to the formation of silver nanoparticles, but required prolonged duration. In general, even after 72 h intensity of color was significantly less than that recorded following autoclaving. Silver nanoparticles formed at room temperature were more heterogeneous compared to that obtained upon autoclaving. In summary, our findings demonstrated that (i) YEM medium and its constituents promote synthesis of silver nanoparticles; and (ii) autoclaving enhances rapid synthesis of silver nanoparticles by YEM medium, yeast extract and mannitol.

In this communication, a novel, green, efficient and economically viable light mediated protocol for generation of Au-nanoparticles using most vital organelle, chloroplasts, of the plant system is portrayed. Thylakoids/chloroplasts isolated from Potamogeton nodosus (an aquatic plant) and Spinacia oleracea (a terrestrial plant) turned Au3+ solutions purple in presence of light of 600 µmol m−2 s−1 photon flux density (PFD) and the purple coloration intensified with time. UV-Vis spectra of these purple colored solutions showed absorption peak at ∼545 nm which is known to arise due to surface plasmon oscillations specific to Au-nanoparticles. However, thylakoids/chloroplasts did not alter color of Au3+ solutions in dark. These results clearly demonstrated that photosynthetic electron transport can reduce Au3+ to Au0 which nucleate to form Au-nanoparticles in presence of light. Transmission electron microscopic studies revealed that Au-nanoparticles generated by light driven photosynthetic electron transport system of thylakoids/chloroplasts were in range of 5–20 nm. Selected area electron diffraction and powder X-ray diffraction indicated crystalline nature of these nanoparticles. Energy dispersive X-ray confirmed that these nanoparticles were composed of Au. To confirm the potential of light driven photosynthetic electron transport in generation of Au-nanoparticles, thylakoids/chloroplasts were tested for their efficacy to generate Au-nanoparticles in presence of light of PFD ranging from 60 to 600 µmol m−2 s−1. The capacity of thylakoids/chloroplasts to generate Au-nanoparticles increased remarkably with increase in PFD, which further clearly demonstrated potential of light driven photosynthetic electron transport in reduction of Au3+ to Au0 to form nanoparticles. The light driven donation of electrons to metal ions by thylakoids/chloroplasts can be exploited for large scale production of nanoparticles.

Proline, a stress marker, is routinely quantified by a protocol that essentially uses hazardous toluene. Negative impacts of toluene on human health prompted us to develop a reliable alternate protocol for proline quantification. Absorbance of the proline-ninhydrin condensation product formed by reaction of proline with ninhydrin at 100 °C in the reaction mixture was significantly higher than that recorded after its transfer to toluene, revealing that toluene lowers sensitivity of this assay. λ of the proline-ninhydrin complex in the reaction mixture and toluene were 508 and 513 nm, respectively. Ninhydrin in glacial acetic acid yielded higher quantity of the proline-ninhydrin condensation product compared to ninhydrin in mixture of glacial acetic acid and HPO, indicating negative impact of HPO on proline quantification. Further, maximum yield of the proline-ninhydrin complex with ninhydrin in glacial acetic acid and ninhydrin in mixture of glacial acetic acid and HPO was achieved within 30 and 60 min, respectively. This revealed that HPO has negative impact on the reaction rate and quantity of the proline-ninhydrin complex formed. In brief, our proline quantification protocol involves reaction of a 1-ml proline sample with 2 ml of 1.25 % ninhydrin in glacial acetic acid at 100 °C for 30 min, followed by recording absorbance of the proline-ninhydrin condensation product in the reaction mixture itself at 508 nm. Amongst proline quantification protocols known till date, our protocol is the most simple, rapid, reliable, cost-effective, and eco-friendlier.

In our endeavor to improve upon nitrogen fixation efficiency of a soil diazotroph that would be unaffected by synthetic nitrogenous fertilizers, we have deleted a part of the negative regulatory gene and constitutively expressed the positive regulatory gene in the chromosome of CBD15, a strain isolated from the local field soil. No antibiotic resistance gene or other foreign gene was there in the chromosome of the engineered strain. Wheat seeds inoculated with this engineered strain, which we have named HKD15, were tested for three years in pots and one year in the field. Yield of wheat was enhanced by ∼60% due to inoculation of seeds by HKD15 in the absence of any urea application. Ammonium only marginally affected acetylene reduction by the engineered strain. When urea was also applied, the same wheat yield could be sustained by using seeds inoculated with HKD15 and using ∼85kg less urea (∼40kg less nitrogen) than the usual ∼257 kg urea (∼120 kg nitrogen) per hectare. Wheat plants arising from the seeds inoculated with the engineered strain, exhibited far superior overall performance, had much higher dry weight and nitrogen content and assimilated molecular N much better. A nitrogen balance experiment also revealed much higher total nitrogen content. IAA production by the wild type and the engineered strain was about the same. Inoculation of the wheat seeds with HKD15 did not adversely affect the microbial population in the field rhizosphere soil.IMPORTANCE Application of synthetic nitrogenous fertilizers is a standard agricultural practice to augment crop yield. Plants, however, utilize only a fraction of the applied fertilizers, while the unutilized fertilizers cause grave environmental problems. Wild type soil diazotrophic microrganisms cannot replace synthetic nitrogenous fertilizers, as these reduce atmospheric nitrogen very inefficiently and almost none at all in presence of added nitrogenous fertilizers. If the nitrogen fixing ability of soil diazotrophs could be improved upon and sustained even in the presence of synthetic nitrogenous fertilizers, then a mixture of the bacterial and reduced quantity of chemical nitrogenous fertilizers could be employed to obtain the same grain yield, but at much reduced environmental cost. The engineered that we have reported here has considerably enhanced nitrogen fixation and excretion abilities and can replace ∼85 kg of urea per hectare, but sustain same wheat yield, if the seeds are inoculated with it before sowing.

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The unique potential of chloroplasts/thylakoids to harness light energy to transport electrons from H2O to various entities was exploited for reduction of Ag+ to generate nanoparticles (NPs). Spinach thylakoids/chloroplasts turned AgNO3 solutions brown in light, but not in dark. Besides showing Ag-NPs specific surface plasmon resonance band, these brown solutions showed presence of 5–30 nm crystalline NPs composed of Ag. Powder X-ray diffraction (PXRD) analysis revealed that Ag-NPs were biphasic composed of face-centered cubic Ag0 and cubic Ag2O. X-ray photoelectron spectroscopy (XPS) data further corroborated the presence of Ag2O in Ag-NPs. Limited formation of Ag-NPs in dark and increased generation of Ag0/Ag2O–NPs with increase in light intensity (photon flux density) by thylakoids/chloroplasts, established the role of light-harvesting photosynthetic machinery in generation of Ag0/Ag2O-NPs. Potential of thylakoids/chloroplasts to generate Ag-NPs from Ag+ on exposure to red and blue wavelength regions of visible light of electromagnetic spectrum, further confirmed the involvement of photosynthetic electron transport in reduction of Ag+ and generation of Ag-NPs. While light energy mediated photosynthetic electron transport donates energized electrons extracted from H2O to Ag+ to form Ag0-NPs, O2 released as a by-product during photolysis of H2O oxidizes Ag0 to form Ag2O-NPs. Our findings furnish a novel, simple, economic and green method that can be exploited for commercial production of Ag0/Ag2O-NPs.