Wricha Tyagi

In the North East Hill Region (NEHR) of India, acidic soils cause aluminum (Al³⁺) toxicity and phosphorus (Pi) deficiency, severely limiting crop productivity. Linseed offers potential for enhancing cropping intensity and diversification in this region. Contrasting oilseed‐type linseed genotypes identified for yield traits under acidic soils were further validated for root and shoot traits through hydroponic screening. Using an orthology‐based approach, eight gene families— MATE , ALMT1 , MGT1 , PT6 , PAP15 , WD40‐38 , SPL4 , and ACA3 —known to confer Al³⁺ toxicity and P deficiency tolerance in other plant species were targeted. Thirty‐one genic regions were identified. In roots of four genotypes (Lus 37, Lus 237, Lus 4, and Lus 49) exposed to Al³⁺ toxicity, UGT74 , MATE1365 , MGT1272 , and MGT1577 were upregulated in tolerant genotypes, indicating their probable roles in detoxification, transport, and ion homeostasis. Under low Pi conditions, eleven genes exhibited differential expression in shoots and roots of Lus 37, Lus 29, and Lus 4, with PT6826 and PT6635 upregulated in roots of tolerant Lus 37. Overall, LusMATE1365 , LusUGT74 , and LusPT6635 were identified as key candidate genes that could potentially confer tolerance to Al³⁺ toxicity and P deficiency. These insights will aid molecular breeding for linseed adaptation to acidic soils of NEHR.

Pearl millet and finger millet face challenges in adopting transgenic or editing approaches due to their inherent recalcitrance to genetic transformation protocols. To overcome these limitations, the current study aims to streamline the genetic transformation protocol for pearl millet and finger millet. We targeted mature embryos as explants to assess transformation efficiencies, gain in time, and overall feasibility. Notably, a transformation efficiency of 17.74% and 18.79%, for pearl millet and finger millet, respectively, was observed using a method that involved directly piercing the mature seeds at the embryonic region with a needle dipped in Agrobacterium suspension, followed by vacuum infiltration. After infection, the seeds were allowed to produce calli and differentiate into shoots and roots, resulting in the development of PCR-positive plants. The induction of infected explants to form calli and subsequent differentiation into shoots and roots, leading to putatively transformed plants, was achieved within 60–66 days. Chi-square analysis of hygromycin selection in T1 progeny showed a 3:1 segregation, indicating single-locus inheritance, and PCR of T1 plants with Cas9 and HptII primers confirmed intact T-DNA transmission. Furthermore, as a proof-of-concept for transformation leading to gene editing, a grain-specific phospholipase-d delta1 (PgPLD-delta1-7a), previously identified in our study, was successfully targeted in pearl millet using the CRISPR/Cas9 approach. This seed-piercing protocol has been successfully evaluated in two genotypes of pearl millet and one genotype of finger millet, resulting in the generation of putative-transformed plants.

Background: Gene editing has emerged as the most precise trait modification tool in plant breeding. However, an understanding of what to target and in which genetic background to obtain the intended phenotype is still emerging. This perspective presents an analytical overview of traits targeted, particularly in tomato and rice, where extensive data on gene editing are available in the public domain. Scope: The available gene editing data on allelic values for a given molecular pathway in crops like tomato and rice are revisited. The phenotypes of edits generated across genetic backgrounds were assessed and compared with available resequencing and phenotypic data. The traits evaluated in the current perspective were de novo domestication, grain quality, fruit colour, yield-related traits and stress tolerance to check whether the data available give significant leads to address these traits via editing in other crops. The rationale for editing a particular gene lies in the understanding of the diverse alleles generated, and in this perspective we attempt to discern both the advancements made and the bottlenecks encountered. Conclusions: The effectiveness of gene editing relies significantly on the roles of alleles generated in regulating specific genetic pathways. It is essential to conduct functional validation of the targeted allele across multiple distinct genetic backgrounds to ascertain its utility. The influence an allele exerts on a given trait is contingent upon factors like the nature of the trait, the position of the gene within a given pathway, and the genetic background in which it has been/will be tested. This perspective highlights how editing has led to a range of phenotypic variations influenced by the genetic background, with certain lines achieving the desired phenotype alongside pleiotropic effects, whereas others do not manifest the expected phenotype. This challenge may be addressed by prioritizing the identification of the right candidate and specific motifs in the regulatory regions as potential targets rather than directly intervening in coding sequences.

Aluminium toxicity and phosphorus deficiency are co-existing characteristics of low pH stress that significantly affect the grain yield of crops. The increasing acidity of Indian soils potentially limits the cultivable area for lentil (Lens culinaris), the third most widely consumed pulse. Breeding for tolerance requires an understanding of interdependent biological responses, but the molecular characterization of integrated tolerance remains elusive. Therefore, this study aimed to integrate high aluminium and low phosphorus stress responsive associations across the genomics, transcriptomics, proteomics, and metabolomics of multiple crop species. The overlapping molecular signatures were fine mapped to 23 candidates that serve multiple regulatory roles crucial for cellular homeostasis. Most of these genes have not been adequately discussed in the context of soil acidity tolerance. Thus, a multi-omics guided regulatory framework was developed to provide new insights into tolerance mechanisms. In silico genotyping of 29 lentil genotypes across 588 genes related to transomics loci yielded seven nonsynonymous and three synonymous variants likely associated with their differential response to stress. The results suggest comprehensive genotyping of multi-omics specific targets to identify potential candidates for marker-trait association studies. In conclusion, data-driven exploratory analysis of multi-omics variants highlights potential biomarkers as targets for genetically improving complex biological traits.

Chillies from Northeast India exhibit wide variability in fruit morphology, pungency, bearing habit and crop duration. An untargeted metabolite profiling using LC-HRMS of four 'heirloom' pungent landraces viz. Naga chilli (AL-1), Dalle khursani (AL-2), Sohmynken khnai (AL-3), and J-41(B) was performed and compared with Kashi anmol (KA). While AL-2, J-41(B) and KA belong to C. annuum species, AL-1 is categorised as C. chinense and AL-3 is C. frutescens. A total of 6990 consistent peaks of monoisotopic masses were detected, out of which 2702 metabolites were identified using accurate mass error < 10 ppm. A higher number of differentially accumulated metabolites were seen in J-41(B) versus AL-3 (1376), followed by J-41(B) versus AL-2 (1365), J-41(B) versus AL-1 (1257), KA versus AL-2 (649), AL-3 versus KA (616), KA versus AL-1 (594) and J-41(B) versus KA (413). Variation among species was higher than variation within species. Pathway analysis identified fatty acid, carotenoid, flavonoid and capsaicinoid as key pathways. We identified eight major categories of metabolites, including fatty acids, sterol lipids, and flavonoids, which together account for over 70% of the significantly expressed metabolites across the genotypes. This study explores untargeted metabolites in various chilli species, offering insights into the biochemical and molecular mechanisms which may play a role in governing important fruit traits. Identification of key metabolites and underlying alleles for twenty-one genes across three pathways (flavonoid, capsaicinoid and carotenoid) suggests that the metabolites and associated alleles identified in this study can be used as biomarkers for further characterization of these heirloom chilli and could provide distinct parameter(s) in distinguishing improved cultivars from landraces. This will contribute towards breeding programs in aiding selection of fruits of the desirable traits. The online version contains supplementary material available at 10.1007/s12298-025-01576-4.

The process of generating stable transformants is time-consuming, labor-intensive, and genotype-dependent. In contrast, transient gene expression techniques, such as agroinfiltration, offer a rapid assessment of gene function and expression. Agroinfiltration, widely employed for studying gene function, has been extensively applied in leaf tissues of Nicotiana benthamiana and various other plant species. Despite its broad utility in various plants, to our knowledge, no prior investigation has been reported in pigeonpea. In this study, we developed an agroinfiltration method for transiently expressing a green fluorescent protein (mGFP5) reporter gene in four pigeonpea genotypes using syringe infiltration at the seedling stage under greenhouse conditions. The expression of the reporter gene mGFP5 was assessed at 72-, 96-, and 120 h post-infiltration (hpi). Additionally, we assessed the effect of morphogenic genes, specifically growth-regulating factor 4 (GRF4) and GRF-interacting factor 1 (GIF1), from both rice and pigeonpea on the expression of mGFP5 in four pigeonpea genotypes. Our findings demonstrate that OsGRF4-GIF1 led to enhanced mGFP5 expression compared to CcGRF4-GIF1 in four diverse pigeonpea genotypes. Fluorescence could be detected till 120 hpi. Furthermore, PCR, RT-PCR, and fluorescence quantification confirmed the presence and expression of mGFP5 at 72 hpi. Our results highlight the efficacy of agroinfiltration in quickly evaluating candidate genes in four genetically diverse pigeonpea genotypes, thereby reducing the time required for the initial assessment of constructs suitable for diverse molecular biology analyses.