KASP: a high-throughput genotyping system and its applications in major crop plants for biotic and abiotic stress tolerance.

Advances in plant molecular breeding have resulted in the development of new varieties with superior traits, thus improving the crop germplasm. Breeders can screen a large number of accessions without rigorous and time-consuming phenotyping by marker-assisted selection (MAS). Molecular markers are one of the most imperative tools in plant breeding programmes for MAS to develop new cultivars possessing multiple superior traits. Single nucleotide polymorphisms (SNPs) are ideal for MAS due to their low cost, low genotyping error rates, and reproducibility. Kompetitive Allele Specific PCR (KASP) is a globally recognized technology for SNP genotyping. KASP is an allele-specific oligo extension-based PCR assay that uses fluorescence resonance energy transfer (FRET) to detect genetic variations such as SNPs and insertions/deletions (InDels) at a specific locus. Additionally, KASP allows greater flexibility in assay design, which leads to a higher success rate and the capability to genotype a large population. Its versatility and ease of use make it a valuable tool in various fields, including genetics, agriculture, and medical research. KASP has been extensively used in various plant-breeding applications, such as the identification of germplasm resources, quality control (QC) analysis, allele mining, linkage mapping, quantitative trait locus (QTL) mapping, genetic map construction, trait-specific marker development, and MAS. This review provides an overview of the KASP assay and emphasizes its validation in crop improvement related to various biotic and abiotic stress tolerance and quality traits.

A novel professional-use synergistic peel technology to reduce visible hyperpigmentation on face: Clinical evidence and mechanistic understanding by computational biology and optical biopsy.

Topicals and chemical peels are the standard of care for management of facial hyperpigmentation. However, traditional therapies have come under recent scrutiny, such as topical hydroquinone (HQ) has some regulatory restrictions, and high concentration trichloroacetic acid (TCA) peel pose a risk in patients with skin of colour. The objective of our research was to identify, investigate and elucidate the mechanism of action of a novel TCA- and HQ-free professional-use chemical peel to manage common types of facial hyperpigmentation. Using computational modelling and in vitro assays on tyrosinase, we identified proprietary multi-acid synergistic technology (MAST). After a single application on human skin explants, MAST peel was found to be more effective than a commercial HQ peel in inhibiting melanin (histochemical imaging and gene expression). All participants completed the case study (N = 9) without any adverse events. After administration of the MAST peel by a dermatologist, the scoring and VISIA photography reported improvements in hyperpigmentation, texture and erythema, which could be linked to underlying pathophysiological changes in skin after peeling, visualized by non-invasive optical biopsy of face. Using reflectance confocal microscopy (VivaScope®) and multiphoton tomography (MPTflex™), we observed reduction in melanin, increase in metabolic activity of keratinocytes, and no signs of inflammatory cells after peeling. Subsequent swabbing of the cheek skin found no microbiota dysbiosis resulting from the chemical peel. The strong efficacy with minimum downtime and no adverse events could be linked to the synergistic action of the ingredients in the novel HQ- and TCA-free professional peel technology.

Hemibiotrophic Phytophthora infestans Modulates the Expression of SWEET Genes in Potato (Solanum tuberosum L.).

Sugar Efflux transporters (SWEET) are involved in diverse biological processes of plants. Pathogens have exploited them for nutritional gain and subsequently promote disease progression. Recent studies have implied the involvement of potato SWEET genes in the most devastating late blight disease caused by Phytophthora infestans. Here, we identified and designated 37 putative SWEET genes as StSWEET in potato. We performed detailed in silico analysis, including gene structure, conserved domains, and phylogenetic relationship. Publicly available RNA-seq data was harnessed to retrieve the expression profiles of SWEET genes. The late blight-responsive SWEET genes were identified from the RNA-seq data and then validated using quantitative real-time PCR. The SWEET gene expression was studied along with the biotrophic (SNE1) and necrotrophic (PiNPP1) marker genes of P. infestans. Furthermore, we explored the co-localization of P. infestans resistance loci and SWEET genes. The results indicated that nine transporter genes were responsive to the P. infestans in potato. Among these, six transporters, namely StSWEET10, 12, 18, 27, 29, and 31, showed increased expression after P. infestans inoculation. Interestingly, the observed expression levels aligned with the life cycle of P. infestans, wherein expression of these genes remained upregulated during the biotrophic phase and decreased later on. In contrast, StSWEET13, 14, and 32 didn't show upregulation in inoculated samples suggesting non-targeting by pathogens. This study underscores these transporters as prime P. infestans targets in potato late blight, pivotal in disease progression, and potential candidates for engineering blight-resistant potato genotypes.

Genome-wide association mapping and genomic prediction for late blight and potato cyst nematode resistance in potato (Solanum tuberosum L.).

Potatoes are an important source of food for millions of people worldwide. Biotic stresses, notably late blight and potato cyst nematodes (PCN) pose a major threat to potato production worldwide, and knowledge of genes controlling these traits is limited. A genome-wide association mapping study was conducted to identify the genomic regulators controlling these biotic stresses, and the genomic prediction accuracy was worked out using the GBLUP model of genomic selection (GS) in a panel of 222 diverse potato accessions. The phenotype data on resistance to late blight and two PCN species (Globodera pallida and G. rostochiensis) were recorded for three and two consecutive years, respectively. The potato panel was genotyped using genotyping by sequencing (GBS), and 1,20,622 SNP markers were identified. A total of 7 SNP associations for late blight resistance, 9 and 11 for G. pallida and G. rostochiensis, respectively, were detected by additive and simplex dominance models of GWAS. The associated SNPs were distributed across the chromosomes, but most of the associations were found on chromosomes 5, 10 and 11, which have been earlier reported as the hotspots of disease-resistance genes. The GS prediction accuracy estimates were low to moderate for resistance to G. pallida (0.04-0.14) and G. rostochiensis (0.14-0.21), while late blight resistance showed a high prediction accuracy of 0.42-0.51. This study provides information on the complex genetic nature of these biotic stress traits in potatoes and putative SNP markers for resistance breeding.

Exogenous dsRNA trigger RNAi in Venturia inaequalis resulting in down regulation of target genes and growth reduction.

BACKGROUND: Venturia inaequalis is an apple scab causing fungal pathogen. It is a highly contagious and destructive pathogen which rapidly spreads infection in the surrounding orchards if not managed. The management and control of disease require multiple fungicides to be sprayed at different development stages of the apple. Persistent applications of fungicides also raises environmental concerns. Here, we demonstrate the potential of using spray induced gene silencing (SIGS) by developing target specific gene constructs for the synthesis of corresponding double-stranded RNA (dsRNA). METHODS AND RESULTS: The exogenous application of dsRNAs was found to reduce mycelial growth and spore formation of V. inaequalis on culture plates. Four genes of V. inaequalis viz. CIN1, CE5, VICE12 and VICE16 which get upregulated during infection, were selected as targets for the development of gene construct expressing the corresponding dsRNA. The effect of exogenously supplied in vitro synthesized dsRNA on V. inaequalis was assessed in culture bioassay experiments with respect to growth, and spore formation. The expression level of the target genes in treated and control fungus was evaluated using quantitative PCR. Fungus treated with VICE12 targeted dsRNA showed maximum reduction in colony size (~ 55%), conidia formation (~ 93%) and expression level of the corresponding gene (2.2 fold), which was followed by CIN1-dsRNA. VICE16-dsRNA treatment was least effective with 32% reduction in growth, the non-significant effect of conidial spore formation and 1.13 fold down regulation of corresponding target gene expression level. CONCLUSION: The result of this investigation validates the hypothesis that RNAi is evoked in V. inaequalis by exogenously supplied dsRNA and spray induced gene silencing (SIGS) based solutions may reduce burden of fungicide usage on apple crop against apple scab disease in future.

Identification of genes governing resistance to PCN (Globodera rostochiensis) through transcriptome analysis in Solanum tuberosum.

Potato cyst nematodes (PCNs) are major pests worldwide that affect potato production. The molecular changes happening in the roots upon PCN infection are still unknown. Identification of transcripts and genes governing PCN resistance will help in the development of resistant varieties. Hence, differential gene expression of compatible (Kufri Jyoti) and incompatible (JEX/A-267) potato genotypes was studied before (0 DAI) and after (10 DAI) inoculation of Globodera rostochiensis J2s through RNA sequencing (RNA-Seq). Total sequencing reads generated ranged between 33 and 37 million per sample, with a read mapping of 48-84% to the potato reference genome. In the infected roots of the resistant genotype JEX/A-267, 516 genes were downregulated, and 566 were upregulated. In comparison, in the susceptible genotype Kufri Jyoti, 316 and 554 genes were downregulated and upregulated, respectively. Genes encoding cell wall proteins, zinc finger protein, WRKY transcription factors, MYB transcription factors, disease resistance proteins, and pathogenesis-related proteins were found to be majorly involved in the incompatible reaction after PCN infection in the resistant genotype, JEX/A-267. Furthermore, RNA-Seq results were validated through quantitative real-time PCR (qRT-PCR), and it was observed that ATP, FLAVO, CYTO, and GP genes were upregulated at 5 DAI, which was subsequently downregulated at 10 DAI. The genes encoding ATP, FLAVO, LBR, and GP were present in > 1.5 fold before infection in JEX-A/267 and upregulated 7.9- to 27.6-fold after 5 DAI; subsequently, most of these genes were downregulated to 0.9- to 2.8-fold, except LBR, which was again upregulated to 44.4-fold at 10 DAI.

Geophysical and geostatistical assessment of groundwater and soil quality using GIS, VES, and PCA techniques in the Jaipur region of Western India.

In present study, geophysical and geostatistical variability of ground water and agricultural soil investigated in the Jaipur region of Rajasthan (Western India) by applying the geographic information system (GIS), vertical electrical sounding (VES) ,and statistical analysis. Ground water and soil samples collected from different sites from the selected study area and variation pattern of quality parameters were assessed. A contour map analysis of distribution of metals and other contaminants in the samples was conducted using GIS. Maximum concentration of metals recorded in the soil samples in order of Fe, 11.25 mg kg-1 > Mn, 8.6 mg kg-1 > Zn, 7.2 mg kg-1 > Cu, 0.455 mg kg-1; however, maximum concentration of metals in the ground water samples was found as Zn, 2.64 mg L-1 > Cu, 0.86 mg L-1 > Fe, 0.39 mg L-1 > Mn, 0.18 mg L-1 > Pb, 0.065 mg L-1 > Ni, 0.016 mg L-1. Observed data emphasis variability in groundwater and soil quality parameter by PCA technique indicated 84.60% and 66.98% of variance, respectively. Soil quality index (SQI) value was observed as 0.482 indicating that 46% of soil sampling sites deteriorated and shown poor quality. Similarly, water quality index (WQI) value indicates good water quality at the sampling sites TW1, TW8, TW10, and TW12; however, TW3, TW4, TW6, TW19, TW20, and TW22 sites showed very poor water quality. The present study concludes that overexploitation of groundwater and unregulated discharge of wastewater leads to depletion of water and soil quality. Further, applying geographical and geostatistical techniques in assessing water and soil quality could be more effective tools in environmental monitoring and management for environmental and health safety.

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.

A comprehensive and conceptual overview of omics-based approaches for enhancing the resilience of vegetable crops against abiotic stresses.

MAIN CONCLUSION: Abiotic stresses adversely affect the productivity and production of vegetable crops. The increasing number of crop genomes that have been sequenced or re-sequenced provides a set of computationally anticipated abiotic stress-related responsive genes on which further research may be focused. Knowledge of omics approaches and other advanced molecular tools have all been employed to understand the complex biology of these abiotic stresses. A vegetable can be defined as any component of a plant that is eaten for food. These plant parts may be celery stems, spinach leaves, radish roots, potato tubers, garlic bulbs, immature cauliflower flowers, cucumber fruits, and pea seeds. Abiotic stresses, such as deficient or excessive water, high temperature, cold, salinity, oxidative, heavy metals, and osmotic stress, are responsible for the adverse activity in plants and, ultimately major concern for decreasing yield in many vegetable crops. At the morphological level, altered leaf, shoot and root growth, altered life cycle duration and fewer or smaller organs can be observed. Likewise different physiological and biochemical/molecular processes are also affected in response to these abiotic stresses. In order to adapt and survive in a variety of stressful situations, plants have evolved physiological, biochemical, and molecular response mechanisms. A comprehensive understanding of the vegetable's response to different abiotic stresses and the identification of tolerant genotypes are essential to strengthening each vegetable's breeding program. The advances in genomics and next-generation sequencing have enabled the sequencing of many plant genomes over the last twenty years. A combination of modern genomics (MAS, GWAS, genomic selection, transgenic breeding, and gene editing), transcriptomics, and proteomics along with next-generation sequencing provides an array of new powerful approaches to the study of vegetable crops. This review examines the overall impact of major abiotic stresses on vegetables, adaptive mechanisms and functional genomic, transcriptomic, and proteomic processes used by researchers to minimize these challenges. The current status of genomics technologies for developing adaptable vegetable cultivars that will perform better in future climates is also examined.

Digitalization of potato breeding program: Improving data collection and management.

A plant breeding program involves hundreds of experiments, each having number of entries, genealogy information, linked experimental design, lists of treatments, observed traits, and data analysis. The traditional method of arranging breeding program information and data recording and maintenance is not centralized and is always scattered in different file systems which is inconvenient for retrieving breeding information resulting in poor data management and the loss of crucial data. Data administration requires a significant amount of manpower and resources to maintain nurseries, trials, germplasm lines, and pedigree records. Further, data transcription in scattered spreadsheets and files leads to nomenclature and typing mistakes, which affects data analysis and selection decisions in breeding programs. The accurate data recording and management tools could improve the efficiency of breeding programs. Recent interventions in data management using computer-based breeding databases and informatics applications and tools have made the breeder's life easier. Because of its digital nature, the data obtained is improved even further, allowing for the acquisition of images, voice recording and other specific data kinds. Public breeding programs are far behind the industry in the use of data management tools and softwares. In this article, we have compiled the information on available data recording tools and breeding data management softwares with major emphasis on potato breeding data management.

Generation of Asynaptic Mutants in Potato by Disrupting StDMC1 Gene Using RNA Interference Approach.

Fixing the genomic composition and multiplication through true potato seed (TPS) is an important challenge in autotetraploid potato. Disrupted meiotic cDNA (DMC1) is a meiotic gene that plays a central role in DNA recombination through crossing over in meiosis. Using the Arabidopsis DMC1 (AtDMC1) gene sequence, we retrieved Solanum tuberosum DMC1(StDMC1) from the diploid potato genome, and subsequently, sense and antisense regions of the StDMC1 gene were amplified in potato cv. Kufri Jyoti. The sense and antisense fragments were confirmed by Sanger-sequencing and cloned in the pRI101 vector. Agrobacterium-mediated transformation of the RNAi construct resulted in 44% transformation efficiency, and a total of 137 mutant lines were obtained. These mutant lines were further validated through pollen viability testing, and selected lines were used for gene expression analysis. The acetocarmine-based pollen staining showed reduced pollen viability ranging from 14 to 21% in four DMC1 mutant lines (DMC4-37, DMC4-41, DMC6-20, and DMC6-21), as compared to the Kufri Jyoti control plants, which on average exhibited 78% pollen viability. The phenotypic data was supported by the reduced expression of the StDMC1 gene in these four mutant lines compared to the control Kufri Jyoti. The results confirmed the generation of StDMC1 knockdown lines. This is the first report of StDMC1 mutant line generation in tetraploid potatoes and will be a step forward in generating non-recombinant mutants through sexual reproduction in potatoes.

Exploring genetic diversity and ascertaining genetic loci associated with important fruit quality traits in apple (Malus × domestica Borkh.).

Genetic diversity is the primary source of variability in any crop improvement program, and the diverse germplasm of any crop species represents an important genetic resource for gene or allele mining to meet future needs. Huge genetic and phenotypic diversity is present in the apple gene pool, even though, breeding programs have been mainly focused on a few traits of interests, which have resulted in the reduction of the diversity in the cultivated lines of apple. Therefore, the present study was carried out on 70 diverse apple genotypes with the objective of analyzing the genetic diversity and to identify the genetic loci associated with important fruit quality traits. A total of 140 SSR primers were used to characterize the 70 genotypes of apples, out of which only 88 SSRs were found to be polymorphic. The PIC values varied from 0.03 to 0.75. The value of MI, EMR, and RP varied from 0.03 to 3.5, 0.5 to 5.0, and 1.89 to 6.74, respectively. The dendrogram and structure analysis divided all the genotypes into two main groups. In addition to this, large phenotypic variability was observed for the fruit quality traits under study indicated the suitability of the genotypes for association studies. Altogether 71 novel MTAs were identified for 10 fruit quality traits, of which 15 for fruit length, 15 for fruit diameter, 12 for fruit weight, 2 for total sugar, 2 for TSS, 4 for reducing sugar, 5 for non-reducing sugar, 5 for fruit firmness, 5 for fruit acidity and 6 for anthocyanin, respectively. Consistent with the physicochemical evaluation of traits, there was a significant correlation coefficient among different fruit quality characters, and many common markers were found to be associated with these traits (fruit diameter, length, TSS, total sugar, acidity and anthocyanin, respectively) by using the different modeling techniques (GLM, MLM). The inferred genetic structure, diversity pattern and the identified MTAs will be serving as resourceful grounds for better predictions and understanding of apple genome towards efficient conservation and utilization of apple germplasm for facilitating genetic improvement of fruit quality traits. Furthermore, these findings also suggested that association mapping could be a viable alternative to the conventional QTL mapping approach in apple. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01382-w.

Characterizing the Potato Growing Regions in India Using Meteorological Parameters.

Currently, the multi-location testing of advanced hybrids in India is carried out at 25 centers under the All India Co-ordinated Research Project on Potato (AICRP-P), which is spread across the country. These centres have been chosen to represent different potato growing regions based on soil and agronomic features. However, the reliable deployment of the newly bred varieties in different regions requires a scientific delineation of potato growing zones with homogenous climates. The present study was undertaken to develop homogenous zones in the Indian sub-continent based on the environmental parameters of the potato growing season. A total of 1253 locations were identified across the country as having a plausible potato growing season of at least 70 days with suitable thermal limits. Six variables including five meteorological parameters including Physiological days (P days), Growing degree days (GDD), Mean daily temperature, Mean night temperature and Mean daily incident solar radiation, together with altitude as the sixth variable, were used for Agglomerative Hierarchical Clustering (AHC) and the Principal Component Analysis by Multidimensional Scaling (MDS) technique to derive identical classes. The thematic map of the classes was overlaid on potato growing districts of India using ArcGIS 9.1 software. The study clearly depicted that the clustering technique can effectively delineate the target population of environments (TPE) for potato genotypes performing well at different testing environments in India. The study also identifies target locations for future focus on breeding strategies, especially the high night temperature class having a large expanse in India. This is also vital in view of the impending climate change situation.

Demystifying the mechanism of action of professional facial peeling: In-vivo visualization and quantification of changes in inflammation, melanin and collagen using Vivascope® and ConfoScan®.

Professional peeling using chemicals (chemical peeling) is a popular non-surgical procedure commonly used for the treatment for photoaging, pigmentary disorders, scarring, fine lines, and wrinkles. The objective of our case study was to elucidate the mechanism of action of professional peels/peeling. For proof-of-concept, we used a commercial blended peel containing trichloroacetic acid and lactic acid. The facial peeling was performed by a physician on four subjects. These subjects were followed over time in the clinic to take clinical pictures and monitor surface and anatomical changes in inflammation, melanin, and collagen at regular intervals post-peel (5 min, 48 h, and day 9). Dermoscope and Vivascope® were used to image surface and subsurface anatomical changes, respectively, and ConfoScan® was used to quantify aforementioned anatomical changes. Based on Vivascope and ConfoScan analysis, we could see clear visual clinical evidence of controlled injury-healing mechanism of peel's action: immediate but transient onset of inflammation within 5 min (indicate injury response by skin), followed by melanin redistribution evident at 48 h (indicate activation of skin's defense system), and remodeled fibrous collagen network without any inflammatory cells on day 9 (healing response). To our knowledge, this is the first ever clinical study to deconvolute the mysterious mechanism of action of peels, in-vivo.

Genotypic variations for tuber nutrient content, dry matter and agronomic traits in tetraploid potato germplasm.

Nutrient deficiencies lead to various health issues and are common worldwide. Potato germplasm is a rich source of natural variations and genetic variability present in it can be exploited for developing nutrient-rich high-yielding potato varieties. In this study, variations in the yield, dry matter (DM) and mineral nutrients concentrations were evaluated in both peeled and unpeeled tubers of 243 highly diverse tetraploid potato accessions. These were raised under field conditions for two consecutive years. The germplasm studied has a wider range of variations in peeled tubers DM (13.71-27.80%), Fe (17.08-71.03 mg/kg), Zn (9.55-34.78 mg/kg), Cu (2.13-13.25 mg/kg), Mn (7.04-25.15), Ca (117.4-922.5 mg/kg), Mg (656.6-1510.6 mg/kg), S (1121.3-3765.8 mg/kg), K (1.20-3.09%), P (0.21-0.50%) and Mo (53.6-1164.0 ppb) concentrations compared to popular Indian potato varieties. Higher nutrient concentrations in whole tubers compared to tuber flesh suggest that these are present in high concentration in the tuber peripheral layers and peeling off the tubers results in the loss of nutrients. Highest loss due to peeling off the tubers was observed in Fe (35.63%) followed by Cu (22.80%), Mn (21.69%), Ca (21.27%), Mg (12.89%), K (12.75%), Zn (10.13%), and Mo (9.87%). The GCV and PCV for all the traits in peeled tubers ranged from 9.67 to 29.91%, and 13.84 to 43.32%, respectively. Several significant positive correlations were observed among the parameters and the first two principal components accounted for 39.37% of total variations. The results of this study will pave a way for the development of nutrient-rich high-yielding potato varieties. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01197-1.

Spraying of dsRNA molecules derived from Phytophthora infestans, along with nanoclay carriers as a proof of concept for developing novel protection strategy for potato late blight.

BACKGROUND: Phytophthora infestans is a late blight-causing oomycetes pathogen. It rapidly evolves and adapts to the host background and new fungicide molecules within a few years of their release, most likely because of the predominance of transposable elements in its genome. Frequent applications of fungicides cause environmental concerns. Here, we developed target-specific RNA interference (RNAi)-based molecules, along with nanoclay carriers, that when sprayed on plants are capable of effectively reducing late blight infection. RESULTS: Targeted the genes unique to sporulation, early satge infection and the metabolism pathway stages based on in an our own microarray data. We used nanoclay as a carrier for sorbitol dehydrogenase, heat shock protein 90, translation elongation factor 1-α, phospholipase-D like 3 and glycosylphosphatidylinositol-anchored acidic serine-threonine-rich HAM34-like protein double-stranded (ds)RNAs, which were assessed by culture bioassay, detached leaf assay and spray methods, and revealed a reduction in growth, sporulation and symptom expression. Plants sprayed with multigene targeted dsRNA-nanoclay showed enhanced disease resistance (4% disease severity) and less sporulation (<1 × 103 ) compared with plants sprayed with dsRNA alone. CONCLUSION: The use of nanoclay with multigene targeted dsRNA was assumed to be involved in effective delivery, protection and boosting the action of RNAi as a spray-induced gene silencing approach (SIGS). A significant reduction in growth, sporulation, disease severity and decreased gene expression authenticates the effects of SIGS on late blight progression. This study demonstrated as a proof of concept the dsRNA-nanoclay SIGS approach, which could be used as an alternative to chemical fungicides and transgenic approaches to develop an environmentally friendly novel plant protection strategy for late blight. © 2022 Society of Chemical Industry.

Holistic approach to visualize and quantify collagen organization at macro, micro, and nano-scale.

BACKGROUND: There is scarcity of imaging and image processing techniques for accurate discrimination and quantitation of the dermal extracellular matrix (ECM), primarily collagen. The aim of this study was to develop and demonstrate a holistic imaging and image processing approach to visualize and quantify collagen remodeling at the macro-, micro- and nano-scale using histochemical imaging, Reflectance Confocal Microscopy (RCM), and Atomic Force Microscopy (AFM), respectively. MATERIAL AND METHODS: For proof-of-concept, a commercial anti-aging product known to induce collagen neo-synthesis and re-organization was tested ex vivo on human skin biopsies from two aged females. RESULTS: Relative to untreated skin, collagen fibers (RCM) and fibrils (AFM) were longer and aligned after treatment. The content of collagen and elastin (histochemical imaging and ELISA) statistically improved after treatment. CONCLUSION: Based on our findings, we can conclude: (1) AFM, RCM, and histochemical imaging can accurately discriminate collagen from other ECM components in the skin and (2) the image processing methods can enable quantitation and hence capture small improvements in collagen remodeling after treatment (commercial cosmetic product with collagen organizer technology as proof-of-concept). The reported holistic imaging approach has direct clinical implications for scientists and dermatologists to make quick, real-time, and accurate decisions in skin research and diagnostics.

In Vitro Method for Synthesis of Large-Scale dsRNA Molecule as a Novel Plant Protection Strategy.

Double-stranded RNA (dsRNAs) molecules are the precursors and effective triggers of RNAi in most organisms. RNAi can be induced by the direct introduction of dsRNAs in plants, fungi, insects, and nematodes. Until now RNAi is usually established by transformation of the plant with a construct that produces hairpin RNAs. Alternatively, advances in RNA biology demonstrated efficiently the in vitro method of large-scale synthesis of dsRNA molecule. Here we describe the de novo synthesis of dsRNA molecule targeting the specific gene of interest for functional application. Selection of off-target effective siRNA regions, flanking of T7 promoter sequences, T7 polymerase reaction, and maintenance of the stability of dsRNA molecules are the main criteria of this method to obtain pure and effective yield for functional applications. IPTG (isopropyl-ß-D-thiogalactopyranoside) induced, T7 express E. coli cells, could be used for large scale synthesis of dsRNA molecule are also described in this method.

Salinity responses and tolerance mechanisms in underground vegetable crops: an integrative review.

MAIN CONCLUSION: The present review gives an insight into the salinity stress tolerance responses and mechanisms of underground vegetable crops. Phytoprotectants, agronomic practices, biofertilizers, and modern biotechnological approaches are crucial for salinity stress management. Underground vegetables are the source of healthy carbohydrates, resistant starch, antioxidants, vitamins, mineral, and nutrients which benefit human health. Soil salinity is a serious threat to agriculture that severely affects the growth, development, and productivity of underground vegetable crops. Salt stress induces several morphological, anatomical, physiological, and biochemical changes in crop plants which include reduction in plant height, leaf area, and biomass. Also, salinity stress impedes the growth of the underground organs, which ultimately reduces crop yield. Moreover, salt stress is detrimental to photosynthesis, membrane integrity, nutrient balance, and leaf water content. Salt tolerance mechanisms involve a complex interplay of several genes, transcription factors, and proteins that are involved in the salinity tolerance mechanism in underground crops. Besides, a coordinated interaction between several phytoprotectants, phytohormones, antioxidants, and microbes is needed. So far, a comprehensive review of salinity tolerance responses and mechanisms in underground vegetables is not available. This review aims to provide a comprehensive view of salt stress effects on underground vegetable crops at different levels of biological organization and discuss the underlying salt tolerance mechanisms. Also, the role of multi-omics in dissecting gene and protein regulatory networks involved in salt tolerance mechanisms is highlighted, which can potentially help in breeding salt-tolerant underground vegetable crops.

Potato biofortification: an effective way to fight global hidden hunger.

Hidden hunger is leading to extensive health problems in the developing world. Several strategies could be used to reduce the micronutrient deficiencies by increasing the dietary uptake of essential micronutrients. These include diet diversification, pharmaceutical supplementation, food fortification and crop biofortification. Among all, crop biofortification is the most sustainable and acceptable strategy to overcome the global issue of hidden hunger. Since most of the people suffering from micronutrient deficiencies, have monetary issues and are dependent on staple crops to fulfil their recommended daily requirements of various essential micronutrients. Therefore, increasing the micronutrient concentrations in cost effective staple crops seems to be an effective solution. Potato being the world's most consumed non-grain staple crop with enormous industrial demand appears to be an ideal candidate for biofortification. It can be grown in different climatic conditions, provide high yield, nutrition and dry matter in lesser time. In addition, huge potato germplasm have natural variations related to micronutrient concentrations, which can be utilized for its biofortification. This review discuss the current scenario of micronutrient malnutrition and various strategies that could be used to overcome it. The review also shed a light on the genetic variations present in potato germplasm and suggest effective ways to incorporate them into modern high yielding potato varieties.