CRISPR/Cas12a ribonucleoprotein mediated editing of tryptophan 2,3-dioxygenase of Spodoptera frugiperda.

In insect genome editing CRISPR/Cas9 is predominantly employed, while the potential of several classes of Cas enzymes such as Cas12a largely remain untested. As opposed to Cas9 which requires a GC-rich protospacer adjacent motif (PAM), Cas12a requires a T-rich PAM and causes staggered cleavage in the target DNA, opening possibilities for multiplexing. In this regard, the utility of Cas12a has been shown in only a few insect species such as fruit flies and the silkworm, but not in non-model insects such as the fall armyworm, Spodoptera frugiperda, a globally important invasive pest that defies most of the current management methods. In this regard, a more recent genetic biocontrol method known as the precision-guided sterile insect technique (pgSIT) has shown successful implementation in Drosophila melanogaster, with certain thematic adaptations required for application in agricultural pests. However, before the development of a controllable gene drive for a non-model species, it is important to validate the activity of Cas12a in that species. In the current study we have, for the first time, demonstrated the potential of Cas12a by editing an eye color gene, tryptophan 2,3-dioxygenase (TO) of S. frugiperda by microinjecting ribonucleoprotein complex into pre-blastoderm (G0) eggs. Analysis of G0 mutants revealed that all five mutants (two male and three female) exhibited distinct edits consisting of both deletion and insertion events. All five edits were further validated through in silico modeling to understand the changes at the protein level and further corroborate with the range of eye-color phenotypes observed in the present study.

RiceMetaSys: Drought-miR, a one-stop solution for drought responsive miRNAs-mRNA module in rice.

MicroRNAs are key players involved in stress responses in plants and reports are available on the role of miRNAs in drought stress response in rice. This work reports the development of a database, RiceMetaSys: Drought-miR, based on the meta-analysis of publicly available sRNA datasets. From 28 drought stress-specific sRNA datasets, we identified 216 drought-responsive miRNAs (DRMs). The major features of the database include genotype-, tissue- and miRNA ID-specific search options and comparison of genotypes to identify common miRNAs. Co-localization of the DRMs with the known quantitative trait loci (QTLs), i.e., meta-QTL regions governing drought tolerance in rice pertaining to different drought adaptive traits, narrowed down this to 37 promising DRMs. To identify the high confidence target genes of DRMs under drought stress, degradome datasets and web resource on drought-responsive genes (RiceMetaSys: DRG) were used. Out of the 216 unique DRMs, only 193 had targets with high stringent parameters. Out of the 1081 target genes identified by Degradome datasets, 730 showed differential expression under drought stress in at least one accession. To retrieve complete information on the target genes, the database has been linked with RiceMetaSys: DRG. Further, we updated the RiceMetaSys: DRGv1 developed earlier with the addition of DRGs identified from RNA-seq datasets from five rice genotypes. We also identified 759 putative novel miRNAs and their target genes employing stringent criteria. Novel miRNA search has all the search options of known miRNAs and additionally, it gives information on their in silico validation features. Simple sequence repeat markers for both the miRNAs and their target genes have also been designed and made available in the database. Network analysis of the target genes identified 60 hub genes which primarily act through abscisic acid pathway and jasmonic acid pathway. Co-localization of the hub genes with the meta-QTL regions governing drought tolerance narrowed down this to 16 most promising DRGs. Database URL: http://14.139.229.201/RiceMetaSys_miRNA Updated database of RiceMetaSys URL: http://14.139.229.201/RiceMetaSysA/Drought/.

Delineation of loci governing an extra-earliness trait in lentil (Lens culinaris Medik.) using the QTL-Seq approach.

Developing early maturing lentil has the potential to minimize yield losses, mainly during terminal drought. Whole-genome resequencing (WGRS) based QTL-seq identified the loci governing earliness in lentil. The genetic analysis for maturity duration provided a good fit to 3:1 segregation (F2), indicating earliness as a recessive trait. WGRS of Globe Mutant (late parent), late-flowering, and early-flowering bulks (from RILs) has generated 1124.57, 1052.24 million raw and clean reads, respectively. The QTL-Seq identified three QTLs (LcqDTF3.1, LcqDTF3.2, and LcqDTF3.3) on chromosome 3 having 246244 SNPs and 15577 insertions/deletions (InDels) and 13 flowering pathway genes. Of these, 11 exhibited sequence variations between bulks and validation (qPCR) revealed a significant difference in the expression of nine candidate genes (LcGA20oxG, LcFRI, LcLFY, LcSPL13a, Lcu.2RBY.3g060720, Lcu.2RBY.3g062540, Lcu.2RBY.3g062760, LcELF3a, and LcEMF1). Interestingly, the LcELF3a gene showed significantly higher expression in late-flowering genotype and exhibited substantial involvement in promoting lateness. Subsequently, an InDel marker (I-SP-383.9; LcELF3a gene) developed from LcqDTF3.2 QTL region showed 82.35% PVE (phenotypic variation explained) for earliness. The cloning, sequencing, and comparative analysis of the LcELF3a gene from both parents revealed 23 SNPs and InDels. Interestingly, a 52 bp deletion was recorded in the LcELF3a gene of L4775, predicted to cause premature termination of protein synthesis after 4 missense amino acids beyond the 351st amino acid due to the frameshift during translation. The identified InDel marker holds significant potential for breeding early maturing lentil varieties.

Dataset on double mutation in PGIP of Glycine max improves defense to PG of Sclerotinia sclerotiorum.

The cell wall of the Glycine max altered by the polygalacturonases (PGs) secreted by the fungus Sclerotinia sclerotiorum, causes disease and quality losses. In soybeans, a resistance protein called polygalacturonases-inhibiting proteins (PGIPs) binds to the PG to block fungal infection. The active site residues of PGIP3, VAL170 and GLN242 are mutated naturally by various amino acids in different types of PGIPs. Therefore, the mutation of VAL170 to GLY is ineffective but the GLN242 amino acid mutation by LYS significantly alters the structure and is crucial for interacting with the PG protein. Docking and Molecular Dynamics simulation provide a comprehensive evaluation of the interactions between gmPGIP and ssPG. By elucidating the structural basis of the interaction between gmPGIP and ssPG, this investigation lays a foundation for the development of targeted strategies in-order to enhance soybean resistance against Sclerotinia sclerotiorum. By leveraging this knowledge, researchers can potentially engineer soybean varieties with improved resistance to the fungus, thereby reducing disease incidence and improving crop yields.

A review of artificial intelligence-assisted omics techniques in plant defense: current trends and future directions.

Plants intricately deploy defense systems to counter diverse biotic and abiotic stresses. Omics technologies, spanning genomics, transcriptomics, proteomics, and metabolomics, have revolutionized the exploration of plant defense mechanisms, unraveling molecular intricacies in response to various stressors. However, the complexity and scale of omics data necessitate sophisticated analytical tools for meaningful insights. This review delves into the application of artificial intelligence algorithms, particularly machine learning and deep learning, as promising approaches for deciphering complex omics data in plant defense research. The overview encompasses key omics techniques and addresses the challenges and limitations inherent in current AI-assisted omics approaches. Moreover, it contemplates potential future directions in this dynamic field. In summary, AI-assisted omics techniques present a robust toolkit, enabling a profound understanding of the molecular foundations of plant defense and paving the way for more effective crop protection strategies amidst climate change and emerging diseases.

Pushing for Self-sufficiency in Edible Oils in India in the Aftermath of Recent Global Events.

India is largely import dependent in meeting its domestic demand of edible oils. This study aims to discuss the consequences of recent global events such as COVID-19 and the Russia-Ukraine war on edible oil imports. Due to prevailing supply chain disruptions and local shortages in significant supplier countries, international prices became highly volatile, and import volumes were hit severely. This led to an almost doubling of the cost of imports from US $ billion 9.52 in 2019-20 to US $18.70 billion in 2021-22, putting an enormous burden on the Indian exchequer. Overall, an increase in the price of all edible oils has been recorded since the later parts of 2021, exerting inflationary pressure on the food price index. As edible oils are part of staple diets, the import dependency of such a large magnitude makes India extremely vulnerable to external shocks. This calls for immediate attention to the issue of self-sufficiency (atma nirbharata) in edible oils production by emphasizing long-term measures.

Molecular and histological validation of modified in ovulo nucellus culture based high-competency direct somatic embryogenesis and amplitude true-to-the-type plantlet recovery in Kinnow mandarin.

Kinnow (Citrus nobilis Lour. × Citrus deliciosa Ten.) needs to be genetically improved for traits such as seedlessness using biotechnological tools. Indirect somatic embryogenesis (ISE) protocols have been reported for citrus improvement. However, its use is restricted due to frequent occurrences of somaclonal variation and low recovery of plantlets. Direct somatic embryogenesis (DSE) using nucellus culture has played a significant role in apomictic fruit crops. However, its application in citrus is limited due to the injury caused to tissues during isolation. Optimization of the explant developmental stage, explant preparation method, and modification in the in vitro culture techniques can play a vital role in overcoming the limitation. The present investigation deals with a modified in ovulo nucellus culture technique after the concurrent exclusion of preexisting embryos. The ovule developmental events were examined in immature fruits at different stages of fruit growth (stages I-VII). The ovules of stage III fruits (>21-25 mm in diameter) were found appropriate for in ovulo nucellus culture. Optimized ovule size induced somatic embryos at the micropylar cut end on induction medium containing Driver and Kuniyuki Walnut (DKW) basal medium with kinetin (KIN) 5.0 mg L-1 and malt extract (ME) 1,000 mg L-1. Simultaneously, the same medium supported the maturation of somatic embryos. The matured embryos from the above medium gave robust germination with bipolar conversion on Murashige and Tucker (MT) medium + gibberellic acid (GA3) 2.0 mg L-1 + ά-naphthaleneacetic acid (NAA) 0.5 mg L-1 + spermidine 100 mg L-1 + coconut water (CW) 10% (v/v). The bipolar germinated seedlings established well upon preconditioning in a plant bio regulator (PBR)-free liquid medium under the light. Consequently, a cent percent survival of emblings was achieved on a potting medium containing cocopeat:vermiculite:perlite (2:1:1). Histological studies confirmed the single nucellus cell origin of somatic embryos by undergoing normal developmental events. Eight polymorphic Inter Simple Sequence Repeats (ISSR) markers confirmed the genetic stability of acclimatized emblings. Since the protocol can induce rapid single-cell origin of genetically stable in vitro regenerants in high frequency, it has potential for the induction of solid mutants, besides crop improvement, mass multiplication, gene editing, and virus elimination in Kinnow mandarin.

Genetic Dissection of Seedling Root System Architectural Traits in a Diverse Panel of Hexaploid Wheat through Multi-Locus Genome-Wide Association Mapping for Improving Drought Tolerance.

Cultivars with efficient root systems play a major role in enhancing resource use efficiency, particularly water absorption, and thus in drought tolerance. In this study, a diverse wheat association panel of 136 wheat accessions including mini core subset was genotyped using Axiom 35k Breeders' Array to identify genomic regions associated with seedling stage root architecture and shoot traits using multi-locus genome-wide association studies (ML-GWAS). The association panel revealed a wide variation of 1.5- to 50-fold and were grouped into six clusters based on 15 traits. Six different ML-GWAS models revealed 456 significant quantitative trait nucleotides (QTNs) for various traits with phenotypic variance in the range of 0.12-38.60%. Of these, 87 QTNs were repeatedly detected by two or more models and were considered reliable genomic regions for the respective traits. Among these QTNs, eleven were associated with average diameter and nine each for second order lateral root number (SOLRN), root volume (RV) and root length density (RLD). A total of eleven genomic regions were pleiotropic and each controlled two or three traits. Some important candidate genes such as Formin homology 1, Ubiquitin-like domain superfamily and ATP-dependent 6-phosphofructokinase were identified from the associated genomic regions. The genomic regions/genes identified in this study could potentially be targeted for improving root traits and drought tolerance in wheat.