Development and application of reverse transcription-loop mediated isothermal amplification assay for sensitive detection of groundnut bud necrosis virus infecting potato.

Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detection of groundnut bud necrosis virus (GBNV) causing potato stem necrosis disease. The isothermal temperatures, reaction periods and concentrations of reaction mixture were optimized where, the assay worked well at 65 °C for 50 min, 6 U of WarmStart Bst 2.0 DNA polymerase, 1.4 mM dNTPs and 2.0 mM MgSO4. The optimized assay proved to be specific to GBNV with no cross reactivity to other viruses infecting potato in India. The specificity of RT-LAMP assay was found to be 100 fold more sensitive than that of RT-PCR. The developed assay was applied for the detection of GBNV from 80 potato leaf samples where 24 samples were found infected which was confirmed by RT-PCR. It was concluded that the RT-LAMP assay developed for detection of GBNV was specific, sensitive and suitable for its use in virus indexing under potato seed production programme.

CRISPR/Cas9-mediated editing of phytoene desaturase (PDS) gene in an important staple crop, potato.

The gene editing using the CRISPR/Cas9 system has become an important biotechnological tool for studying gene function and improving crops. In this study, we have used CRISPR/Cas9 system for editing the phytoene desaturase gene (PDS) in popular Indian potato cultivar Kufri Chipsona-I. A construct (pHSE401) carrying two target gRNAs with glycine tRNA processing system under the control of Arabidopsis U6 promoter and the Cas9 protein was constructed and transformed in potato plants using Agrobacterium-mediated genetic transformations. The regeneration efficiency of 45% was observed in regenerated plants, out of which 81% of the putative transformants shoot lines exhibited mutant or bleached phenotype (albinism). The deletion mutations were detected within the StPDS gene in the genotyped plants and a mutation efficiency of 72% for gRNA1 and gRNA2 has been detected using Sanger sequencing. Hence, we set up a CRISPR/Cas9-mediated genome editing protocol which is efficient and generates mutations (deletions) within StPDS gene in potato. The bleached phenotype is easily detectable after only few weeks after Agrobacterium-mediated transformation. This is the first report as a proof of concept for CRISPR/Cas9-based editing of PDS gene in Indian potato cv. Kufri Chipsona-I. This study demonstrates that CRISPR/Cas9 can be used to edit genes at high frequency within the genome of the potato for various traits. Therefore, this study will aid in creating important mutants for modifying molecular mechanisms controlling traits of agronomic importance.

Purification and characterization of novel exopolysaccharides produced from Lactobacillus paraplantarum KM1 isolated from human milk and its cytotoxicity.

BACKGROUND: Microbial origin polysaccharides have gained popularity due to lesser toxicity, better degradability and selectivity as compared to their synthetic counterparts and can be used as emulsifier, stabilizer, thickener, texturizer, flocculating and gelling agent. Here main emphasis on exopolysaccharide production from potential lactic acid bacteria that has GRAS status. RESULTS: This work was aimed at isolating, purifying and characterizing an extracellular polysaccharide (EPS) produced by a foodgrade lactic acid bacteria Lactobacillus paraplantarum KM1. L. paraplantarum KM1 was isolated from human milk and identified by conventional and molecular techniques. The 16S rRNA sequence of the isolate was registered in National Centre for Biotechnology Information (NCBI) under accession number KX671558. L. paraplantarum KM1 was found to produce EPSs in lactose containing MRS medium, and the maximum yield (47.4 mg/ml) was achieved after 32-h incubation. As evident from TLC and HPLC analyses, the polysaccharide was found to be a heteropolymer-containing glucose, galactose and mannose as main sugars. Different oligosaccharides namely hexoses were obtained after partial hydrolysis of the polymer using MALDI-ToF-MS. The total molecular weight of all polysaccharides present was 348.7 kDa with 100 °C thermal stability as well as water soluble in nature. Cell cytotoxicity revealed that the purified EPS was safe for consumption; thus, it can be used in various food industries as emulsifying and texture agent. CONCLUSIONS: The present study highlighted that exopolysaccharides could be harnessed to improve food products in terms of texture, emulsifying agents, pharmaceutical industry (antioxidants, antitumour, anti-inflammatory and antiviral agents) and as safety purposes.

Development and application of fluorescent loop mediated isothermal amplification technique to detect Phytophthora infestans from potato tubers targeting ITS-1 region.

The goal of this study was to develop a fluorescent based loop-mediated isothermal amplification (LAMP) assay for a simple, sensitive and visual detection of P. infestans from tubers targeting a novel internal transcribed spacer 1 (ITS-1) region of ribosomal DNA. The ITS-1 LAMP primers were designed using the Primer Explorer V4 software. The optimization of LAMP reaction conditions and reagents concentrations were carried out with time, temperature, MgSO4, dNTPs and WarmStart Bst DNA polymerase. The amplified products were analysed using SYBR Green I dye and by agarose gel electrophoresis. We optimized reaction conditions included reagent mix, incubated at 65 °C for 60 min. The target specificity of primers was assessed with PCR, restriction digestion and sequence analysis. The developed LAMP assay was evaluated for its analytical specificity, sensitivity and validation in field tuber samples. The analytical specificity of LAMP primers indicates positive reaction with P. infestans and closely related species except P. erythosepctica. We were able to detect down to 1 pg/µl of DNA using the newly developed LAMP primers whereas the minimal amount detectable for conventional PCR was 0.1 ng/µl. Further, the samples with positive reaction developed a characteristic fluorescent green color. The detection of LAMP assay for inoculum of P. infestans was determined in the artificially inoculated leaves and tubers. In 98 field tuber samples, 54 (55.10%) were confirmed as positive by LAMP while 39 (39.79%) positive by PCR. The LAMP assay developed in this study has a potential to be a beneficial tool in early detection of P. infestans in low cost laboratory. Because the LAMP assay performed well in aspects of sensitivity, repeatability, target specificity, reliability, and visibility, it is suitable for detection of P. infestans in infected potato tubers.

Utilization of horticultural waste (Apple Pomace) for multiple carbohydrase production from Rhizopus delemar F2 under solid state fermentation.

The brown rot fungus Rhizopus delemar F2 was shown to produce extracellular thermostable and multiple carbohydrase enzymes. The potential of Rhizopus delemar F2 in utilizing apple pomace under solid state fermentation (SSF) is the purpose of the study. Solid state fermentation (SSF) is a very effective technique opposed to submerged fermentation in various aspects. Enhanced production of multiple carbohydrases 18.20 U g-1 of cellulose, 158.30 U g-1 of xylanase, 61.50 U g-1 of pectinase and amylase 21.03 U g-1 was released by microwave pretreatment of apple pomace at 450 W for 1 min and then by incubation the culture thus obtained at 30 °C for 6 days with moisture content of 1:4.5. Apple pomace can serve as a potential source of raw material for the production of multiple carbohydrases. Besides, it can find great commercial significance in production of bioethanol and various industries like textile, fruit juice, paper and pulp industry.

Purification and characterization of lipase by Bacillus methylotrophicus PS3 under submerged fermentation and its application in detergent industry.

Lipase production bacterial isolate was isolated from soil of service station and identified as Bacillus methylotrophicus PS3 by 16SrRNA with accession number |LN999829.1|. Lipase enzyme was purified by sequential methods of ammonium sulfate precipitation and Sephadex G-100 gel column chromatography. The molecular weight of purified enzyme was 31.40 kDa on SDS-PAGE. This purification procedure resulted in 2.90-fold purification of lipase with a 24.10% final yield. The purified lipase presented maximal hydrolytic activity at a temperature of 55 °C, and pH of 7.0. Lipase activity was stimulated by Triton X-100 and SDS with Mg2+ and Ca2+ metals employ a positive effect and outlast its stable in organic solvent i.e. methanol and ethanol.

Evaluation of health potential of nutritionally enriched Kodo millet (Eleusine coracana) grown in Himachal Pradesh, India.

In this study, Kodo millet grains were phytochemically investigated for their nutritional and antioxidant potential for their use as functional foods. Methanolic extracts of grains showed higher phenolic content and antioxidant activity. TLC studies of the extracted polyphenols from kodo millet showed the predominant presence of ferulic acid and cinnamic acid in the millet. Further quantification of these polyphenols was done by using HPLC, analysing ferulic acid and cinnamic acid. Antagonistic spectrum of the polyphenols extracted showed inhibition against four bacterial test indicators viz. Staphylococcus aureus, Leuconostoc mesenteroides, Bacillus cereus and Enterococcus faecalis proving its antimicrobial action. The grains of kodo millet grains taken under study were found to posses' high protein, carbohydrates, minerals, crude fibers, polyphenols and antioxidants thus can be used as a good source of nutrition with additional health benefits.