Development of an In Vitro Propagation Protocol and a Sequence Characterized Amplified Region (SCAR) Marker of Viola serpens Wall. ex Ging.

An efficient protocol of plant regeneration through indirect organogenesis in Viola serpens was developed in the present study. Culture of leaf explants on MS (Murashige and Skoog) medium supplemented with 2.0 mg/L 6-benzyladenine and 0.13 mg/L 2,4-dichloro phenoxy acetic acid. Adventitious shoot formation was observed when calli were transferred on to MS medium containing 0.5 mg/L α-naphthalene acetic acid and 2.25 mg/L kinetin, which showed the maximum 86% shoot regeneration frequency. The highest root frequency (80.92%) with the 5.6 roots per explant and 1.87 cm root length was observed on MS medium supplemented with 2 mg/L indole-3-butyric acid. The plantlets were transferred to the mixture of sand, coffee husk and soil in the ratio of 1:2:1 in a pot, and placed under 80% shade net for one month. It was then transferred to 30% shade net for another one month, prior to transplantation in the field. These plantlets successfully acclimatized under field conditions. A Sequence Characterized Amplified Region (SCAR) marker was also developed using a 1135 bp amplicon that was obtained from RAPD (Random Amplification of Polymorphic DNA) analysis of six accessions of V. serpens. Testing of several market samples of V. serpens using the SCAR marker revealed successful identification of the genuine samples of V. serpens. This study, therefore, provides a proficient in vitro propagation protocol of V. serpens using leaf explants and a SCAR marker for the authentic identification of V. serpens. This study will be helpful for conservation of authentic V. serpens.

Real-time monitoring of glutathione in living cells using genetically encoded FRET-based ratiometric nanosensor.

Reduced glutathione (GSH) level inside the cell is a critical determinant for cell viability. The level of GSH varies across the cells, tissues and environmental conditions. However, our current understanding of physiological and pathological GSH changes at high spatial and temporal resolution is limited due to non-availability of practicable GSH-detection methods. In order to measure GSH at real-time, a ratiometric genetically encoded nanosensor was developed using fluorescent proteins and fluorescence resonance energy transfer (FRET) approach. The construction of the sensor involved the introduction of GSH binding protein (YliB) as a sensory domain between cyan fluorescent protein (CFP; FRET donor) and yellow fluorescent protein (YFP; FRET acceptor). The developed sensor, named as FLIP-G (Fluorescence Indicator Protein for Glutathione) was able to measure the GSH level under in vitro and in vivo conditions. When the purified FLIP-G was titrated with different concentrations of GSH, the FRET ratio increased with increase in GSH-concentration. The sensor was found to be specific for GSH and also stable to changes in pH. Moreover, in live bacterial cells, the constructed sensor enabled the real-time quantification of cytosolic GSH that is controlled by the oxidative stress level. When expressed in yeast cells, FRET ratio increased with the external supply of GSH to living cells. Therefore, as a valuable tool, the developed FLIP-G can monitor GSH level in living cells and also help in gaining new insights into GSH metabolism.

Proteome Profiling of the Mutagen-Induced Morphological and Yield Macro-Mutant Lines of Nigella sativa L.

In the present investigation, the leaf proteome profile of the macro-mutant lines of Nigella sativa L. was analyzed to identify the key proteins involved in the expression of traits associated with the morphology, seed yield, and content of thymoquinone. In our earlier study, the macro-mutants were generated with contrasting morphological traits and seed yields through induced mutagenesis, using ethyl methyl sulfonate, gamma rays, and combinations of both. Analysis of the leaf proteome of the control and macro-mutant lines of N. sativa showed that twenty-three proteins were differentially expressed. These differentially expressed proteins were sequenced through mass spectrometry and identified using the MASCOT software. On the basis of their function, these proteins were categorized into several groups. Most proteins were found in the categories of signal transduction (18%) and carbon metabolism (18%). A total of 13% of proteins belonged to the categories of energy and metabolism. Proteins in the categories of secondary plant metabolism, stress defense, cytoskeleton, and protein synthesis were also found. The polycomb group protein (FIE1), transcription factor (PRE1), and geranyl diphosphate synthase were notable proteins, in addition to some proteins of signal transduction and carbon metabolism. Expression patterns of the differentially expressed proteins were also studied at the transcript level by using qRT-PCR. Transcriptomics analysis was consistent with the proteomics data. This study shows the changes that take place at the proteomic level through induced mutagenesis, as well as the involvement of some proteins in the expression traits associated with plant height, seed yield, and the thymoquinone content of N. sativa. The identified proteins might help elucidate the metabolic pathways involved in the expression of traits, including seed yield, and the active compounds of medicinal plants.

Responsive Proteins in Wheat Cultivars with Contrasting Nitrogen Efficiencies under the Combined Stress of High Temperature and Low Nitrogen.

Productivity of wheat (Triticumaestivum) is markedly affected by high temperature and nitrogen deficiency. Identifying the functional proteins produced in response to these multiple stresses acting in a coordinated manner can help in developing tolerance in the crop. In this study, two wheat cultivars with contrasting nitrogen efficiencies (N-efficient VL616 and N-inefficient UP2382) were grown in control conditions, and under a combined stress of high temperature (32 °C) and low nitrogen (4 mM), and their leaf proteins were analysed in order to identify the responsive proteins. Two-dimensional electrophoresis unravelled sixty-one proteins, which varied in their expression in wheat, and were homologous to known functional proteins involved in biosynthesis, carbohydrate metabolism, energy metabolism, photosynthesis, protein folding, transcription, signalling, oxidative stress, water stress, lipid metabolism, heat stress tolerance, nitrogen metabolism, and protein synthesis. When exposed to high temperature in combination with low nitrogen, wheat plants altered their protein expression as an adaptive means to maintain growth. This response varied with cultivars. Nitrogen-efficient cultivars showed a higher potential of redox homeostasis, protein stability, osmoprotection, and regulation of nitrogen levels. The identified stress-responsive proteins can pave the way for enhancing the multiple-stress tolerance in wheat and developing a better understanding of its mechanism.