Dark Incubation Research Articles

Production, Characterization of Indole Acetic Acid and its Bioactive Potential from Endophytic Fungi of Cymbidium aloifolium L.

The exploration of relationship between orchid and its endophytic fungi is significant as vast arrays of beneficial attributes are provided by the fungi to its host which includes the production of phytohormones like Indole acetic acid (IAA). The present investigation focused on production, characterization and applications of IAA from endophytic fungi of Cymbidium aloifolium, an epiphytic orchid. The Colletotrichum gloeosporioides (CAR4) isolated and characterized from C. aloifolium produced higher Indole acetic acid (10.7±1.97 ìM/mL). The CAR4 IAA produced was confirmed by UV/Vis spectrum, TLC, FTIR, HPLC, LCMS, NMR analysis. The CAR4 IAA treated Macrotyloma uniflorum and Vigna unguiculata seeds exhibited increased germination percentage by 3 - 8 %, root length by 1 - 14 mm, shoot length by 2 - 21 mm compared to standard IAA. The Murashige and Skoog’s Basal media (MSBM) with coconut milk (10 %), casein hydrolysate (250 mg/L) showed swelling of C. aloifolium seeds within 15 days and well developed protocorms after 60 days of incubation. The differentiation of protocorms into roots and shoots was higher in MSBM with CAR4 IAA (0.5 mg/L) and BAP (6-Benzylaminopurine) (1 mg/L) with average of 8.5 ± 0.83 shoots and 5.67 ± 0.81 roots. The culture parameters were optimized and maximum IAA production was in PDB with 4 mg/mL of ⟨-tryptophan, pH6, 30°C, 15 days incubation in dark. IAA production increased four folds with optimized parameters.

Incubation in light versus dark affects the vitality of UV-irradiated Tetraselmis suecica differently: A flow cytometric study.

In this study, we used flow cytometry to examine how incubation in dark versus light affects the vitality and viability of UV-irradiated Tetraselmis suecica. High UV doses (300 and 400 mJ/cm2) affected the esterase activity, membrane permeability, and chlorophyll content more when the subsequent incubation took place in light. For non- or low UV dose (100 and 200 mJ/cm2)-treated cells, incubation in light resulted in cell regrowth as compared to incubation in dark. Damaged cells (enzymatically active but with permeable membranes) did not recover when incubated under light or dark conditions. Exposure to light reduces the evaluation time of any given ballast water treatment, as viable cells will be detected at an earlier stage and the vitality is more affected. When evaluating the performance of UV-based ballast water treatment systems (BWTS), these results can be useful for type approval using T. suecica as a test organism in the test regime.

Roles of Direct Photoreception and the Internal Circadian Oscillator in the Regulation of Melatonin Secretion in the Pineal Organ of the Domestic Turkey: A Novel In Vitro Clock and Calendar Model.

The regulation of melatonin secretion in the avian pineal organ is highly complex and shows prominent interspecies differences. The aim of this study was to determine the roles of direct photoreception and the internal oscillator in the regulation of melatonin secretion in the pineal organ of the domestic turkey. The pineal organs were collected from 12-, 13- and 14-week-old female turkeys reared under a 12 L:12 D cycle with the photophase from 07.00 to 19.00, and were incubated in superfusion culture for 3–6 days. The cultures were subjected to different light conditions including 12 L:12 D cycles with photophases between 07.00 and 19.00, 13.00 and 01.00 or 01.00 and 13.00, a reversed cycle 12 D:12 L, cycles with long (16 L:8 D) and short (8 L:16 D) photophases, and continuous darkness or illumination. The pineal organs were also exposed to light pulses of variable duration during incubation in darkness or to periods of darkness during the photophase. The secretion of melatonin was determined by direct radioimmunoassay. The turkey pineal organs secreted melatonin in a well-entrained diurnal rhythm with a very high amplitude. Direct photoreception as an independently acting mechanism was able to ensure quick and precise adaptation of the melatonin secretion rhythm to changes in light-dark conditions. The pineal organs secreted melatonin in circadian rhythms during incubation in continuous darkness or illumination. The endogenous oscillator of turkey pinealocytes was able to acquire and store information about the light-dark cycle and then to generate the circadian rhythm of melatonin secretion in continuous darkness according to the stored data. The obtained data suggest that the turkey pineal gland is highly autonomous in the generation and regulation of the melatonin secretion rhythm. They also demonstrate that the turkey pineal organ in superfusion culture is a valuable model for chronobiological studies, providing a highly precise clock and calendar. This system has several features which make it an attractive alternative to other avian pineal glands for circadian studies.

Photoactivated Lysosomal Escape of a Monofunctional PtII Complex Pt‐BDPA for Nucleus Access

AbstractA photosensitizing monofunctional Pt complex, Pt‐BDPA, was prepared with a BODIPY chromophore. Apart from its DNA binding ability, this complex displays emission at ca. 578 nm and a singlet oxygen quantum yield of 0.133. Confocal imaging revealed that this complex was sequestered in lysosomes via endocytosis in the dark, preventing its access to the nucleus. Profiting from its photoinduced ROS generation ability, this complex undergoes lysosomal escape to access the nucleus upon photoirradiation. The photoinduced ROS still cause a drop in intracellular GSH, favoring the stability of Pt‐BDPA and contributing to its nuclear DNA accessibility. This complex displayed distinct cytotoxicity to all tested tumor cell lines upon photoirradiation, and the IC50 values were ca. 3–6 μm, which are distinctly lower than those found with only dark incubation (IC50>50 μm). These results are consistent with photoactivated lysosomal escape of this photosensitizing Pt complex to access the nucleus.

Photoactivated Lysosomal Escape of a Monofunctional PtII Complex Pt-BDPA for Nucleus Access.

A photosensitizing monofunctional Pt complex, Pt-BDPA, was prepared with a BODIPY chromophore. Apart from its DNA binding ability, this complex displays emission at ca. 578 nm and a singlet oxygen quantum yield of 0.133. Confocal imaging revealed that this complex was sequestered in lysosomes via endocytosis in the dark, preventing its access to the nucleus. Profiting from its photoinduced ROS generation ability, this complex undergoes lysosomal escape to access the nucleus upon photoirradiation. The photoinduced ROS still cause a drop in intracellular GSH, favoring the stability of Pt-BDPA and contributing to its nuclear DNA accessibility. This complex displayed distinct cytotoxicity to all tested tumor cell lines upon photoirradiation, and the IC50 values were ca. 3-6 μm, which are distinctly lower than those found with only dark incubation (IC50 >50 μm). These results are consistent with photoactivated lysosomal escape of this photosensitizing Pt complex to access the nucleus.

Extreme rates and diel variability of planktonic respiration in a shallow sub-arctic lake

Planktonic community respiration (CR) is a major component of aquatic biogeochemical cycling and food web energetics. Accurate, direct characterizations of short-term patterns and drivers of plankton CR are needed to understand aquatic biogeochemical processes and food web functioning. Recent work indicates CR may be commonly underestimated, and may undergo considerable diel changes that are missed using standard methodological approaches. To explore these possibilities, we applied an immediate, in situ, dark incubation approach at ~ 3 h intervals over 2.5 diel cycles in a shallow, productive, sub-arctic lake in interior Alaska, USA. Rates of CR varied 17-fold, strongly coupled to diel oscillations in water temperature. A weak inverse relationship to ~ 3 mg L−1 diel changes in dissolved organic carbon concentrations suggests CR partially modulated the standing stock of organic matter over short timescales. Average rates of CR were ~ 6 to 100-fold greater than published, conventional CR measurements, but comparable to existing free-water estimates of ecosystem respiration for nearby Alaskan lakes. Overall, this study places new weight on the importance of CR in whole-ecosystem biogeochemical transformations by supporting recent suggestions that planktonic CR may be commonly underestimated.

Enhanced germination performance of dormant seeds of <i>Eragrostis tef</i> in the presence of light

Lack of germination or low germination due to seed dormancy prevents successful crop establishment in several important plants. To determine the presence of innate seed dormancy and effects of stress-related plant hormones on germination performance of Teff (Eragrostis tef) seeds, we primed seeds in 1% KNO3 for 24 hours in dark conditions at 21 ± 0.5 ºC along with varying concentrations of chemicals known to influence seed germination, including: acetyl salicylic acid (ASA); methyl jasmonate (JA-Me); giberellic acid (GA3); and indole acetic acid (IAA). Primed seeds were incubated either in constant light (210 µM/m2/s) or in darkness at 21 ± 0.5 ºC. The results indicated that primingsignificantly improved final germination percentage (FGP) in both light (92.5%) and dark (89.4%) conditions compared with untreated seeds. The inclusion of plant hormones in the priming media generally had limited effects, except for 10 mM ASA (94.5%) and 100 mM GA3 (92.5%). ASA generally provided faster seed germination than seeds primed in 1% KNO3 only, while the other plant hormones had no effect on the time required for 50% of FGP in the dark. Priming had no significant effect on time span of germination in either light or dark incubation conditions. The results demonstrate that E. tef has light-inducible seed germination and about half of freshly harvested seeds can be dormant, which can be eliminated to some extent by priming seeds in 1% KNO3.

Effects of the Photosystem II Inhibitors CCCP and DCMU on Hydrogen Production by the Unicellular Halotolerant Cyanobacterium Aphanothece halophytica.

The unicellular halotolerant cyanobacterium Aphanothece halophytica is a potential dark fermentative producer of molecular hydrogen (H2) that produces very little H2 under illumination. One factor limiting the H2 photoproduction of this cyanobacterium is an inhibition of bidirectional hydrogenase activity by oxygen (O2) obtained from splitting water molecules via photosystem II activity. The present study aimed to investigate the effects of the photosystem II inhibitors carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) on H2 production of A. halophytica under light and dark conditions and on photosynthetic and respiratory activities. The results showed that A. halophytica treated with CCCP and DCMU produced H2 at three to five times the rate of untreated cells, when exposed to light. The highest H2 photoproduction rates, 2.26 ± 0.24 and 3.63 ± 0.26 μmol H2 g−1 dry weight h−1, were found in cells treated with 0.5 μM CCCP and 50 μM DCMU, respectively. Without inhibitor treatment, A. halophytica incubated in the dark showed a significant increase in H2 production compared with cells that were incubated in the light. Only CCCP treatment increased H2 production of A. halophytica during dark incubation, because CCCP functions as an uncoupling agent of oxidative phosphorylation. The highest dark fermentative H2 production rate of 39.50 ± 2.13 μmol H2 g−1 dry weight h−1 was found in cells treated with 0.5 μM CCCP after 2 h of dark incubation. Under illumination, CCCP and DCMU inhibited chlorophyll fluorescence, resulting in a low level of O2, which promoted bidirectional hydrogenase activity in A. halophytica cells. In addition, only CCCP enhanced the respiration rate, further reducing the O2 level. In contrast, DCMU reduced the respiration rate in A. halophytica.

Role of auxin homeostasis and response in nitrogen limitation and dark stimulation of adventitious root formation in petunia cuttings.

Background and AimsAdventitious root (AR) formation in Petunia hybrida is inhibited by low nitrogen fertilization of stock plants but promoted by dark incubation of cuttings before planting. We investigated whether the plant hormone auxin is involved in nitrogen- and dark-mediated AR formation.MethodsConcentrations of indole-3-acetic acid (IAA) and RNA accumulation of genes controlling auxin homeostasis and function were monitored in the stem base in response to high versus low nitrogen supply to stock plants and to temporal dark vs. light exposure of cuttings by use of GC-MS/MS, a petunia-specific microarray and quantitative RT-PCR. Auxin source capacity, polar auxin transport in cuttings and auxin concentration in the rooting zone were manipulated to investigate the functional contribution of auxin homeostasis and response to the effects of nitrogen fertilization and dark exposure on rooting.Key ResultsThe nitrogen content of cuttings had only a marginal effect on IAA concentration in the stem base. Dark incubation enhanced the accumulation of IAA in the stem base during AR induction independent of nitrogen level. Early IAA accumulation in the dark depended on the upper shoot as an auxin source and was enhanced after apical IAA supply. Dark exposure stimulated RNA accumulation of auxin-related genes. In particular, expression of Ph-PIN1 and of genes controlling auxin signalling, including Ph-IAA14, Ph-ARF8, Ph-ARF10 and Ph-SAUR14, was enhanced, while the latter four were repressed in nitrogen-limited cuttings, particularly in the dark. Dark stimulation of rooting depended on polar auxin transport. Basal auxin application partially substituted the effect of dark exposure on rooting, whereas the auxin response of AR formation was strongly depressed by nitrogen limitation.ConclusionsIncreased auxin delivery from the upper shoot and enhanced auxin signalling in the stem base contribute to dark-stimulated AR formation, while nitrogen limitation inhibits AR formation downstream of the auxin signal.

Dissolved Organic Matter Composition in a Marsh‐Dominated Estuary: Response to Seasonal Forcing and to the Passage of a Hurricane

AbstractDissolved organic matter (DOM) is a large and complex mixture of compounds with source inputs that differ with location, season, and environmental conditions. Here, we investigated drivers of DOM composition changes in a marsh‐dominated estuary off the southeastern United States. Monthly water samples were collected at a riverine and estuarine site from September 2015 to September 2016, and bulk, optical, and molecular analyses were conducted on samples before and after dark incubations. Results showed that river discharge was the primary driver changing the DOM composition at the mouth of the Altamaha River. For discharge higher than ~150 m3/s, dissolved organic carbon (DOC) concentrations and the terrigenous character of the DOM increased approximately linearly with river flow. For low discharge conditions, a clear signature of salt marsh‐derived compounds was observed in the river. At the head of Sapelo Sound, changes in DOM composition were primarily driven by river discharge and possibly by summer algae blooms. Microbial consumption of DOC was larger during periods of high discharge at both sites, potentially due to the higher mobilization and influx of fresh material to the system. The Georgia coast was hit by Hurricane Matthew in October 2016, which resulted in a large input of carbon to the estuary. The DOC concentration was ~2 times higher and DOM composition was more aromatic with a stronger terrigenous signature compared to the seasonal maximum observed earlier in the year during peak river discharge conditions. This suggests that extreme events notably impact DOM quantity and quality in estuarine regions.

Rice DNA-Binding One Zinc Finger 24 (OsDOF24) Delays Leaf Senescence in a Jasmonate-Mediated Pathway

Leaf senescence is the final stage of leaf development and in cereal crops, the timing of senescence relative to grain filling has major effects on agronomic traits such as yield. Although many genetic factors are involved in the regulation of leaf senescence in cereals, the key regulators remain to be determined. Plant transcription factors with a conserved DOF (DNA-binding one zinc finger) domain play roles in multiple physiological processes. Here, we show a novel function for OsDOF24 as a repressor of leaf senescence in rice (Oryza sativa). In wild-type leaves, OsDOF24 expression rapidly decreased during natural senescence (NS) and dark-induced senescence (DIS). The gain-of-function mutant osdof24-D, which contains an enhancer-trap T-DNA in the OsDOF24 promoter, exhibited delayed leaf yellowing during NS and DIS. Transgenic plants overexpressing OsDOF24 showed the same phenotype during DIS. Reverse-transcription quantitative real-time PCR analysis revealed that senescence-associated genes (Osl85, Osl57 and OsNAP) and chlorophyll degradation genes (NYC1, NYC3 and SGR) were downregulated in the osdof24-D mutant during dark incubation. Among the phytohormones, only methyl jasmonate induced OsDOF24 expression. Furthermore, the reduced expression of jasmonate biosynthesis-related genes (OsLOX2, OsLOX8, OsHI-LOX, OsAOS1 and OsAOS2) in osdof24-D decreased endogenous jasmonate levels, resulting in delayed leaf senescence under DIS conditions. Yeast one-hybrid assays showed that OsDOF24 binds to the promoter region of OsAOS1. Taken together, our results demonstrate that OsDOF24 suppresses the induction of leaf senescence during vegetative growth by deactivating jasmonate biosynthetic pathways.

Effects of salicylic acid on photosynthetic activity and chloroplast morphology under light and prolonged darkness

Salicylic acid (SA) is a key component of plant defence, which exerts a concentration-dependent effect on photosynthesis under multi-faceted influence of light. Photosynthetic activities and chloroplast morphology were studied in tomato plants after treatment with a sublethal, 0.1 mM, and a cell death-inducing, 1 mM concentrations of SA under normal photoperiod during light phase and after a prolonged dark phase. SA (1 mM) decreased the maximal (Fv/Fm) and effective quantum yields of PSII [Y(II)] and PSI [Y(I)] under both environmental conditions, however, the photoprotective processes were not significantly different between light and dark samples. Decrease in grana height, thylakoid dilation and deformation of lumen were also observed in the light. In contrast to illuminated samples, 0.1 mM SA decreased Y(II) and Y(I) after dark incubation, but nonphotochemical energy dissipation and cyclic electron flow increased, suggesting that the photoprotective mechanisms could be activated in plants exposed to prolonged darkness.

Investigation of the post-cryogenic regeneration ability of potato varieties under different cultivation conditions

Cryopreservation provides long-term storage of the gene pool of potato varieties in cryobanks at extremely low temperatures. Currently, droplet vitrification is the most widely used method for cryopreservation of potato varieties, which is constantly improving to increase the regeneration rates of the stored plant material. Different modifications of this method are used in the world’s leading potato genebanks. This paper presents the results of studying the effect of cultivation conditions after plunging into liquid nitrogen and thawing of shoots tips and axillary buds of in vitro plants on their postcryogenic recovery. The droplet-vitrification method modified at VIR was used for cryopreservation. The factor “prolonged dark incubation of explants” did not have a significant effect on the frequency of post-cryogenic regeneration of the studied varieties except for one variety (Krepysh), for which a significant increase in the regeneration rate was observed for the shoot tips cultivated in the darkness compared to the cultivation under the photoperiod 16/8 hours (light/darkness). The frequency of post-cryogenic regeneration of shoot tips was higher than that of the axillary buds for all varieties; however, these differences were significant (p < 0.05) only in two cases: for the variety Udacha (a photoperiod of 16/8 hours) and for the variety Krepysh (the dark incubation). The results of two-factor analysis of variance indicate that there is no effect of interaction of factor 1 (prolonged dark incubation) and factor 2 (explant type) on the ability of varieties to post-cryogenic recovery. Taking into account the obtained results, the further cryopreservation of an extended subset of 9 varieties was carried out using shoot tips, which, after freezing-thawing, were cultivated under the photoperiod of 16/8 hours. The frequency of post-cryogenic regeneration of these varieties varied from 30 to 60 %. A significant effect of genotype on postcryogenic recovery has been established. The ability of varieties to regenerate shoots after freezing and thawing was not related to the values of morphogenic indices of in vitro plants. The age of the meriklons (2–4 years) did not significantly affect either the morphogenic indices or the frequency of post-cryogenic regeneration.

Urea dynamics during Lake Taihu cyanobacterial blooms in China

Lake Taihu, the third largest freshwater lake in China, suffers from harmful cyanobacteria blooms caused by Microcystis spp., which do not fix nitrogen (N). Reduced N (i.e., NH4+, urea and other labile organic N compounds) is an important factor affecting the growth of Microcystis. As the world use of urea as fertilizer has escalated during the past decades, an understanding of how urea cycling relates to blooms of Microcystis is critical to predicting, controlling and alleviating the problem. In this study, the cycling rates of urea-N in Lake Taihu ranged from non-detectable to 1.37 μmol N L−1 h−1 for regeneration, and from 0.042 μmol N L−1 h−1 to 2.27 μmol N L−1 h−1 for potential urea-N removal. The fate of urea-N differed between light and dark incubations. Increased 15NH4+ accumulated and higher quantities of the removed urea-15N remained in the 15NH4+ form were detected in the dark than in the light. A follow-up incubation experiment with 15N-urea confirmed that Microcystis can grow on urea but its effects on urea dynamics were minor, indicating that Microcystis was not the major factor causing the observed fates of urea under different light conditions in Lake Taihu. Bacterial community composition and predicted functional gene data suggested that heterotrophic bacteria metabolized urea, even though Microcystis spp. was the dominant bloom organism.

Optimising protocol for successful development of haploids in marigold (Tagetes spp.) through in vitro androgenesis

For the first time, we have developed a protocol for rapid and successful induction of haploids in Tagetes spp. using in vitro androgenesis techniques. Various factors affecting the in vitro androgenesis response in Tagetes spp. were optimised. At first, a correlation was established between floret size/bud size and stage of microspore development in different genotypes of Tagetes erecta and Tagetes patula. The floret size of 3.5–4.0 mm (French marigold) and 3.0–3.5 mm (African marigold) contained the highest percent of microspores at early-uninucleate to early-binucleate stage. The frequency of shoot formation from anthers ranged from 0 to 78.6% in T. patula cv. Pusa Arpita and 0 to 63.9% in T. erecta cv. Pusa Basanti Gainda. The French marigold cultivars exhibited higher androgenic response over the African marigold cultivars. The effects of basal media, plant growth regulator combinations and sucrose concentration for direct differentiation of shoot buds and haploid induction were studied. Marigold anthers cultured on newly formulated EMS1 basal media (enriched with coconut water, AgNO3, PVP etc.) exhibited a very high androgenic response over the other commercially available media. We have also optimised the cold temperature pre-treatments and culture condition for high frequency androgenesis in marigold. Ten days cold pre-treated anthers along with 20 days dark incubation of cultures proved highly beneficial for haploid induction. Among a total of 424 anther-regenerants, 56 plants were randomly selected for flow cytometry and cytological analysis. The ploidy analysis revealed 14.3% of anther regenerants as dihaploids, 66.1% as tetraploid (similar to donor mother plant) and 19.6% as polyploid. The determination of ploidy level by counting the number of chloroplast in stomatal guard cells of marigold was also established for rapid screening of haploids. The identified haploids were successfully diploidised and are being utilised in the hybrid breeding programme at our institute. The developed protocol will facilitate doubled haploids based breeding in Tagetes spp. This is the first report of successful induction of haploids in Tagetes spp. using in vitro androgenesis techniques. We have studied various factors affecting the in vitro androgenesis response in Tagetes spp. A correlation was established between floret size/ bud size and stage of microspore development in different genotypes of Tagetes erecta and Tagetes patula. Basal media and cultural conditions were optimised for high frequency androgenesis in marigold. Haploids were successfully induced and their ploidy level was assessed through flowcytometry and cytological analysis.

Using mRNA to investigate the effect of low-pressure ultraviolet disinfection on the viability of E. coli

The efficacy of ultraviolet (UV) disinfection has been analyzed and validated by numerous studies using culture-based methods, yet the discovery of the viable but nonculturable state necessitates the investigation of UV disinfection based on viability parameters. Paired regulators of the SOS response system, recA-lexA, and the programmed cell death system, mazEF, in Escherichia coli were chosen as the target genes, and the effect of UV irradiation on the mRNAs of the four genes was studied. This research showed that, after UV irradiation, the responses of the mRNAs were highly consistent, with reduction percentages of approximately 60% at 20 mJ/cm2, 70% at 40 mJ/cm2, and 90% at 80 mJ/cm2, and these reductions were believed to be the result of direct UV damage to nucleic acids. After 24 h of dark incubation, recA and lexA were both upregulated but to a lesser extent for repressor lexA; and mazE and mazF were both downregulated. This result implies that UV irradiation induces the dark repair system more actively, and the cells will proceed to death at a rate similar to that associated with natural decay.

Contrasting Effects of an Alien Worm on Benthic N Cycling in Muddy and Sandy Sediments

The North American oligochaete Sparganophilus tamesis is widespread in European freshwaters. Its ecological effects on benthic nitrogen (N) biogeochemistry were studied in two contrasting environments: the organic-rich muddy sediments of the eutrophic Mincio River (Italy) and the organic-poor sandy sediments of the oligotrophic Cazaux-Sanguinet Lake (France). Oxygen and inorganic N fluxes and denitrification rates (IPT) were measured by dark incubation of intact cores with different worm biomass. Sediment oxygen demand and denitrification were higher in muddy than in sandy sediments; however, at the two sites, bioturbation by the oligochaetes stimulated differing microbial O2 and NO3− respiration and NH4+ production. In particular, the relative effect of S. tamesis on sediment metabolism was greater in Cazaux-Sanguinet Lake than in the Mincio River. As a result, S. tamesis favored net N loss in the Mincio River, whereas it increased NH4+ recycling and lowered denitrification efficiency in the Cazaux-Sanguinet Lake. Our results suggest that the effects of S. tamesis on N biogeochemistry might differ depending on local trophic settings. These results have implications for the conservation of isoetids in the French Lake, whose persistence can be menaced by oligochaete-induced nutrient mobilization.

Microbial Synthesis and Characterization of Silver Nanoparticles using Alternaria alternata

In this study, extracellular biosynthesis of silver nanoparticles (AgNPs) was carried out using Alternaria alternata. AgNPs were synthesized using vegetative and cell-free filtrate methods. Silver nitrate (AgNO3) solution was reduced with fungal mycelial mass and cell-free filtrate at a ratio of 1:4 (v/v). Further, the effects of biosynthesis parameters (reaction time, reaction temperature, light and dark incubation, and static and agitated conditions) weredetermined. The structural integrity of the synthesized AgNPs was investigated through UV-visible spectrophotometry, X-ray diffraction (XRD) analysis, and transmission electron microscopy (TEM). Parameter optimization revealed that cell-free filtrate AgNP synthesis at 40 °C with constant agitation and incubation in darkness for 120 h using 1 mM AgNO3resulted in the highest absorbance value (at 400 nm). Further, TEM images showed that the synthesized AgNPs were spherical, homogeneous, and well dispersed while the XRD peaks confirmed the purity of the AgNPs obtained. The diameters of the AgNPs were found to range from 7.48 to 12.15 nm. This study identifies Alternaria alternata as a potential candidate for use in the industrial biosynthesis of AgNPs.

Genetic Transformation of Oat Mediated by Agrobacterium is enhanced with Sonication and Vacuum Infiltration

BackgroundOat (Avena sativa) with high nutritive value and fiber content is used as the main food grain in many countries for human diet as well as animal feed. Recently, it became difficult to transfer new genes through the conventional breeding due to the lack of desirable traits.ObjectivesThe current study aimed at achieving a standardized protocol for Agrobacterium-mediated transformation in oat.Materials and MethodsFor oat transformation, mature seeds were sterilized, germinated, and used for explants generation. Agrobacterium tumefaciens GV3101 with the binary vector pCAMBIA 1305.1, which carries gus as reporter gene, was utilized in the transformation. The co-cultivation treatment assisted with sonication, and vacuum infiltration, and their combination was employed for transformation with different incubation periods of 48, 72, and 96 hours under the dark conditions.ResultsAmong the different transformation treatments, the vacuum treatment with 72 hours dark incubation had the best results. Vacuum infiltration of the cultures from leaf base produced a maximum of 25% hygromycin-resistant explants. These explants upon GUS assay and PCR analysis revealed 21.85% and 19.04% transformation efficiency, respectively.ConclusionsIt could be concluded that vacuum infiltration assisted Agrobacterium-mediated transformation is the most efficient method to conduct the genetic improvement of the oat using transformation protocol.

Overexpression of SrDXS1 and SrKAH enhances steviol glycosides content in transgenic Stevia plants

BackgroundStevia rebaudiana produces sweet-tasting steviol glycosides (SGs) in its leaves which can be used as natural sweeteners. Metabolic engineering of Stevia would offer an alternative approach to conventional breeding for enhanced production of SGs. However, an effective protocol for Stevia transformation is lacking.ResultsHere, we present an efficient and reproducible method for Agrobacterium-mediated transformation of Stevia. In our attempts to produce transgenic Stevia plants, we found that prolonged dark incubation is critical for increasing shoot regeneration. Etiolated shoots regenerated in the dark also facilitated subsequent visual selection of transformants by green fluorescent protein during Stevia transformation. Using this newly established transformation method, we overexpressed the Stevia 1-deoxy-d-xylulose-5-phosphate synthase 1 (SrDXS1) and kaurenoic acid hydroxylase (SrKAH), both of which are required for SGs biosynthesis. Compared to control plants, the total SGs content in SrDXS1- and SrKAH-overexpressing transgenic lines were enhanced by up to 42–54% and 67–88%, respectively, showing a positive correlation with the expression levels of SrDXS1 and SrKAH. Furthermore, their overexpression did not stunt the growth and development of the transgenic Stevia plants.ConclusionThis study represents a successful case of genetic manipulation of SGs biosynthetic pathway in Stevia and also demonstrates the potential of metabolic engineering towards producing Stevia with improved SGs yield.