Revolutionizing Agriculture: Harnessing CRISPR/Cas9 for Crop Enhancement.

Plant crops serve as essential sources of nutritional sustenance, supplying vital nutrients to human diets. However, their productivity and quality are severely jeopardized by factors such as pests, diseases, and adverse abiotic conditions. Addressing these challenges using innovative biotechnological approaches is imperative for advancing sustainable agriculture. In recent years, genome editing technologies have emerged as pivotal genetic tools, revolutionizing plant molecular biology. Among these, the CRISPR-Cas9 system has gained prominence due to its unparalleled precision, streamlined design, and heightened success rates. This review article highlights the profound impact of CRISPR/Cas9 technology on crop improvement. The article critically examines the breakthroughs, ongoing enhancements, and future prospects associated with this cutting-edge technology. In conclusion, the utilization of CRISPR/Cas9 presents a transformative shift in agricultural biotechnology, holding the potential to mitigate longstanding agricultural challenges.

Ethrel-induced release of fresh seed dormancy causes remodelling of amylase activity, proteomics, phytohormone and fatty acid profile of groundnut (Arachis hypogaea L.).

It is important to have a short period of fresh seed dormancy in some of the groundnut species to counter pre-harvest sprouting (PHS). One of the main causes of PHS is the activation of ethylene-mediated pathways. To determine the effect of ethylene, the study was conducted and alterations in amylase, proteins and fatty acids were observed at the 0, 6, 12, and 24 h stages after ethrel administration. The result showed an increase in amylase activity, and the fatty acids profile showed a unique alteration pattern at different germination stages. Two-dimensional gel electrophoresis (2DGE) revealed differential expression of proteins at each stage. The trypsin digestion following spectral development through UPLC-MS/MS enabled identification of number of differentially expressed proteins. A total of 49 proteins were identified from 2DGE excised spots. The majority were belonged to seed storage-related proteins like Arah1, Arah2, AAI- domain containing protein, conglutin, Arah3/4, arachin, glycinin. Expression of lipoxygenase1, lipoxygenase9 and Arah2 genes were further confirmed by qRT-PCR which showed its involvement at transcript level. Up-regulation of lipoxygenase9 is correlated with decreased content of fatty acids during germination. Phytohormone detection revealed decrease in ABA, SA and JA content which are generally inhibitor of seed germination while GA, IAA and kinetin concentration increased revealing positive regulation of seed germination. We present an integrated view of proteomics, phytohormone profile, carbohydrate and lipid metabolism to unravel mechanism of fresh seed dormancy. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01332-6.

Transcriptome analysis of sesame-Macrophomina phaseolina interactions revealing the distinct genetic components for early defense responses.

Sesame (Sesamum indicum L.) is an oilseed crop challenged by many biotic stresses. Charcoal rot caused by Macrophomina phaseolina (MP) is one of the most devastating diseases of sesame. Till date, molecular mechanisms of resistance to charcoal rot in sesame is not yet reported. In this study, two sesame variety GT-10 (resistant) and RT-373 (susceptible) were identified with contrasting disease incidence when infected with MP. To get the molecular insight, root samples were collected at 0, 24, 48- and 72-h post inoculation (hpi) with the pathogen and generated RNAseq data was analyzed. A total of 1153 and 1226 differentially expressed genes (DEGS) were identified in GT-10 and RT-373, respectively. During the inoculation with MP, resistant genotype showed high number DEGs at early time point of 24 hpi and when compared to late expression in susceptible genotype at 48 hpi. Distinct clusters were represented for each time period represented by cytochrome P450 83B1-like, single anchor, hypothetical protein C4D60, kirola like and heat shock proteins in the resistant genotype contributing for resistance. Analysis of differentially expressed genes, catalogued the genes involved in synthesis of pathogenesis-related (PR) proteins, MYB, WRKY, leucine zipper protein, bHLH, bZIP and NAC transcription factors, ABC transporters (B, C and G subfamily), glutathione metabolism, secondary metabolites, fatty acid biosynthesis and phytohormones like auxin, abscisic acid, ethylene and gibberellic acid. Additionally, in the resistant response we have found three unique GO terms including ATP binding, ribonucleotide binding and nucleic acid binding in molecular function category. The molecular clues generated through this work will provide an important resource of genes contributing for disease resistance and could prioritize genes for functional validation in the important oil crop. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01039-6.

Transcriptomic signature reveals mechanism of flower bud distortion in witches'-broom disease of soybean (Glycine max).

BACKGROUND: Soybean (Glycine max L. Merril) crop is major source of edible oil and protein for human and animals besides its various industrial uses including biofuels. Phytoplasma induced floral bud distortion syndrome (FBD), also known as witches' broom syndrome (WBS) has been one of the major biotic stresses adversely affecting its productivity. Transcriptomic approach can be used for knowledge discovery of this disease manifestation by morpho-physiological key pathways. RESULTS: We report transcriptomic study using Illumina HiSeq NGS data of FBD in soybean, revealing 17,454 differentially expressed genes, 5561 transcription factors, 139 pathways and 176,029 genic region putative markers single sequence repeats, single nucleotide polymorphism and Insertion Deletion. Roles of PmbA, Zn-dependent protease, SAP family and auxin responsive system are described revealing mechanism of flower bud distortion having abnormalities in pollen, stigma development. Validation of 10 randomly selected genes was done by qPCR. Our findings describe the basic mechanism of FBD disease, right from sensing of phytoplasma infection by host plant triggering molecular signalling leading to mobilization of carbohydrate and protein, phyllody, abnormal pollen development, improved colonization of insect in host plants to spread the disease. Study reveals how phytoplasma hijacks metabolic machinery of soybean manifesting FBD. CONCLUSIONS: This is the first report of transcriptomic signature of FBD or WBS disease of soybean revealing morphological and metabolic changes which attracts insect for spread of disease. All the genic region putative markers may be used as genomic resource for variety improvement and new agro-chemical development for disease control to enhance soybean productivity.

Extremozymes and compatible solute production potential of halophilic and halotolerant bacteria isolated from crop rhizospheric soils of Southwest Saurashtra Gujarat.

Halophiles are one of the classes of extremophilic microorganisms that can flourish in environments with very high salt concentrations. In this study, fifteen bacterial strains isolated from various crop rhizospheric soils of agricultural fields along the Southwest coastline of Saurashtra, Gujarat, and identified by 16S rRNA gene sequencing as Halomonas pacifica, H. stenophila, H. salifodinae, H. binhaiensis, Oceanobacillus oncorhynchi, and Bacillus paralicheniformis were investigated for their potentiality to produce extremozymes and compatible solute. The isolates showed the production of halophilic protease, cellulase, and chitinase enzymes ranging from 6.90 to 35.38, 0.004-0.042, and 0.097-0.550 U ml-1, respectively. The production of ectoine-compatible solute ranged from 0.01 to 3.17 mg l-1. Furthermore, the investigation of the ectoine-compatible solute production at the molecular level by PCR showed the presence of the ectoine synthase gene responsible for its biosynthesis in the isolates. Besides, it also showed the presence of glycine betaine biosynthetic gene betaine aldehyde dehydrogenase in the isolates. The compatible solute production by these isolates may be linked to their ability to produce extremozymes under saline conditions, which could protect them from salt-induced denaturation, potentially enhancing their stability and activity. This correlation warrants further investigation.

Genome-Wide Identification, Quantification, and Validation of Differentially Expressed miRNAs in Eggplant (Solanum melongena L.) Based on Their Response to Ralstonia solanacearum Infection.

MicroRNAs (miRNAs), a type of short noncoding RNA molecule (21-23 nucleotides), mediate repressive gene regulation through RNA silencing at the posttranscriptional level and play an important role in the defense response to abiotic and biotic stresses. miRNAs of the plant system have been studied in model crops for their diverse regulatory role while less is known about their significance in other plants whose genome and transcriptome data are scarce in the database, including eggplant (Solanum melongena L.). In the present study, a next-generation sequencing platform was used for the sequencing of miRNA, and real-time quantitative PCR for miRNAs was used to validate the gene expression patterns of miRNAs in Solanum melongena plantlets infected with the bacterial wilt-causing pathogen Ralstonia solanacearum (R. solanacearum). Sequence analyses showed the presence of 375 miRNAs belonging to 29 conserved families. The miR414 is highly conserved miRNA across the plant system while miR5658 and miR5021 were found exclusively in Arabidopsis thaliana surprisingly, these miRNAs were found in eggplants too. The most abundant families were miR5658 and miR414. Ppt-miR414, hvu-miR444b, stu-miR8020, and sly miR5303 were upregulated in Pusa purple long (PPL) (susceptible) at 48 h postinfection, followed by a decline after 96 h postinfection. A similar trend was obtained in ath-miR414, stu-mir5303h, alymiR847-5p, far-miR1134, ath-miR5021, ath-miR5658, osa-miR2873c, lja-miR7530, stu-miR7997c, and gra-miR8741 but at very low levels after infection in the susceptible variety, indicating their negative role in the suppression of host immunity. On the other hand, osa-miR2873c was found to be slightly increased after 96 hpi from 48 hpi. Most of the miRNAs under study showed relatively lower expression in the resistant variety Arka Nidhi after infection than in the susceptible variety. These results shed light on a deeper regulatory role of miRNAs and their targets in regulation of the plant response to bacterial infection. The present experiment and their results suggested that the higher expression of miRNA leads to a decline in host mRNA and thus shows susceptibility.

Plant growth promoting characteristics of halophilic and halotolerant bacteria isolated from coastal regions of Saurashtra Gujarat.

Halophiles are a class of microorganisms that thrive in environments with very high salt concentrations. The coastal regions of Saurashtra Gujarat host a diverse group of microorganisms including halophilic and halotolerant bacteria that may have plant growth promoting characteristics. Microorganisms with plant growth promoting characteristics are of immense importance in the field of agriculture and the present study was conducted to investigate the characteristics of halophilic and halotolerant bacteria isolated from agricultural soils of coastal regions of Junagadh and Porbandar districts of Saurashtra, Gujarat. A total of 15 isolated strains showed indole acetic acid production, solubilization of phosphate and potash, and nitrogen fixing capacity ranging from 18.77-33.48 µg ml-1, 50.10-106.10%, 180.42-239.92% and 0.170-0.480 g kg-1 of Jensen's agar medium, respectively, while two isolates were also found positive for siderophore production. Besides, nine out of fifteen isolates also showed positive ACC deaminase activity ranging from 0.92-5.78 µM α-ketobutyrate mg-1 h-1. The isolates were further characterized by physiological, microscopic, and biochemical tests. The halophilic and halotolerant bacterial isolates were identified by 16S rRNA gene sequencing as belonging to Halomonas pacifica, H. stenophila, and Bacillus haynesii, B. licheniformis and Oceanobacillus aidingensis respectively. The 16S rRNA partial gene sequence of two isolates belonging to H. pacifica and H. stenophila were submitted to NCBI with accession number MK955347 and MK961217 respectively. The findings of the present investigation showed that isolated bacterial halophiles possess promising plant growth promoting characteristics. Their potential as bioinoculants to alleviate salinity stress in crops and for bioremediation deserves further investigation.

Fungal Genomic Resources for Strain Identification and Diversity Analysis of 1900 Fungal Species.

Identification and diversity analysis of fungi is greatly challenging. Though internal transcribed spacer (ITS), region-based DNA fingerprinting works as a "gold standard" for most of the fungal species group, it cannot differentiate between all the groups and cryptic species. Therefore, it is of paramount importance to find an alternative approach for strain differentiation. Availability of whole genome sequence data of nearly 2000 fungal species are a promising solution to such requirement. We present whole genome sequence-based world's largest microsatellite database, FungSatDB having >19M loci obtained from >1900 fungal species/strains using >4000 assemblies across globe. Genotyping efficacy of FungSatDB has been evaluated by both in-silico and in-vitro PCR. By in silico PCR, 66 strains of 8 countries representing four continents were successfully differentiated. Genotyping efficacy was also evaluated by in vitro PCR in four fungal species. This approach overcomes limitation of ITS in species, strain signature, and diversity analysis. It can accelerate fungal genomic research endeavors in agriculture, industrial, and environmental management.

VigSatDB: genome-wide microsatellite DNA marker database of three species of Vigna for germplasm characterization and improvement.

Genus Vigna represented by more than 100 species is a source of nutritious edible seeds and sprouts that are rich sources of protein and dietary supplements. It is further valuable because of therapeutic attributes due to its antioxidant and anti-diabetic properties. A highly diverse and an extremely ecological niche of different species can be valuable genomic resources for productivity enhancement. It is one of the most underutilized crops for food security and animal feeds. In spite of huge species diversity, only three species of Vigna have been sequenced; thus, there is a need for molecular markers for the remaining species. Computational approach of microsatellite marker discovery along with evaluation of polymorphism utilizing available genomic data of different genotypes can be a quick and an economical approach for genomic resource development. Cross-species transferability by e-PCR over available genomes can further prioritize the potential SSR markers, which could be used for genetic diversity and population differentiation of the remaining species saving cost and time. We present VigSatDB-the world's first comprehensive microsatellite database of genus Vigna, containing >875 K putative microsatellite markers with 772 354 simple and 103 865 compound markers mined from six genome assemblies of three Vigna species, namely, Vigna radiata (Mung bean), Vigna angularis (Adzuki bean) and Vigna unguiculata (Cowpea). It also contains 1976 validated published markers. Markers can be selected on the basis of chromosomes/location specificity, and primers can be generated using Primer3core tool integrated at backend. Efficacy of VigSatDB for microsatellite loci genotyping has been evaluated by 15 markers over a panel of 10 diverse genotype of V. radiata. Our web genomic resources can be used in diversity analysis, population and varietal differentiation, discovery of quantitative trait loci/genes, marker-assisted varietal improvement in endeavor of Vigna crop productivity and management.