Fruit crops are rich source of important vitamins, minerals, and dietary fibres. They are essential for global agriculture with respect to nutritional security. Globally, there is a rapid decline in the genetic base of fruit crops warranting breeding strategies to overcome the challenge. Applied mutagenesis has emerged as a viable approach for the focused enhancement of fruit crops utilizing precise genetic alterations to increase a variety of desirable characteristics. However, traditional mutagenesis using physical and chemical mutagens are majorly random in nature. Directed mutagenesis with advancements in genetic engineering and molecular technology allows precise manipulation of genes, which facilitates the efficient and precise knockout of target genes and the targeted insertion or modification of specific DNA sequences within the genome via homologous recombination (HR)-mediated gene replacement. This review presents an in-depth exploration of several directed mutagenesis techniques including CRISPR-Cas9, TILLING, TALEN, MutMap, and MutMap + emphasizing their transformative applications in fruit crops. It also discusses about space mutagenesis. These advanced techniques empower researchers to precisely introduce specific mutations into the genome, skilfully altering gene expression and reshaping protein function with remarkable precision. This review highlights successful examples of directed mutagenesis in a variety of fruit crops such as apples, grapes, citrus, and strawberries and elucidates the impact of directed mutagenesis on traits such as fruit size, colour, flavour, shelf-life, and resistance to diseases and environmental stresses.

Ethyl methanesulfonate (EMS)-induced mutagenesis is a prominent method for generating plant mutants, often resulting in chimera plants; however, their transcriptional and genetic characteristic remain elusive. In this investigation, chimera pea (Pisum sativum L.) specimens, labeled GY1 and GY2, exhibiting a distinctive phenotype with yellow and green leaves were meticulously cultivated via sequential double EMS mutagenesis. The observed color disparity between the yellow and green leaves was attributed to a significant reduction in chlorophyll content coupled with heightened lutein levels in both chimeric variants. Transcriptome profiling revealed the enrichment of differentially expressed genes in both GY1 and GY2, specifically implicating Kyoto Encyclopedia of Genes and Genomes pathways linked to amino acid biosynthesis and ribosome development, alongside Gene Ontology (GO) biological processes linked with stress response mechanisms. Few structural genes associated with chlorophyll and lutein biosynthesis exhibited discernible differential expression. Despite these functional similarities, distinctive nuances were evident between specimens, with GY1 exhibiting enrichment in GO pathways related to chloroplast development and GY2 showing enrichment for ribosome development pathways. Single-nucleotide polymorphism (SNP) analysis uncovered a shared pool of 599 and 598 polymorphisms in the yellow and green leaves of GY1 and GY2, respectively, likely stemming from the initial EMS mutagenesis step. Further investigation revealed an increased number of unique SNPs in the yellow leaves following the second EMS application, whereas the green leaves exhibited sparse and unique SNP occurrences, suggestive of potential evasion from secondary mutagenesis. This inherent genetic variability underpins the mechanism underlying the formation of chimera plants. Predominant base mutations induced by EMS were characterized by G/A and C/T transitions, constituting 74.1% of the total mutations, aligning with established EMS mutation induction paradigms. Notably, genes encoding the eukaryotic translation initiation factor eIIso4G and the ubiquitin ligase RKP, known to modulate leaf color in model plants, harbored two SNPs in the yellow leaves of both GY1 and GY2, implicating their putative role in the yellow leaf phenotype. Collectively, this study provides novel insights into the transcriptional and genetic characteristics of chimera plants via EMS-induced mutagenesis.

Gene mutations linked to diseases like cancer may be caused by exposure to environmental chemicals. The X-linked phosphatidylinositol glycan class A (PIG-A) gene, required for glycosylphosphatidylinositol (GPI) anchor biosynthesis, is a key target locus for in vitro genetic toxicity assays. Various organisms and cell lines may respond differently to genotoxic agents. Here, we compared the mutagenic potential of directly genotoxic ethyl methane sulfonate (EMS) to metabolically activated pro-mutagenic polycyclic aromatic hydrocarbons (PAHs). The two classes of mutagens were compared in an in vitro PIG-A gene mutation test using the metabolically active murine hepatoma Hepa1c1c7 cell line and the human TK6 cell line, which has limited metabolic capability. Determination of cell viability is required for quantifying mutagenicity. Two common cell viability tests, the MTT assay and propidium iodide (PI) staining measured by flow cytometry, were evaluated. The MTT assay overestimated cell viability in adherent cells at high benzo[a]pyrene (B[a]P) exposure concentrations, so PI-based cytotoxicity was used in calculations. The spontaneous mutation rates for TK6 and Hepa1c1c7 cells were 1.87 and 1.57 per million cells per cell cycle, respectively. TK6 cells exposed to 600 µM and 800 µM EMS showed significantly higher mutation frequencies (36 and 47 per million cells per cell cycle, respectively). Exposure to the pro-mutagen benzo[a]pyrene (B[a]P, 10 µM) did not increase mutation frequency in TK6 cells. In Hepa1c1c7 cells, mutation frequencies varied across exposure groups (50, 50, 29, and 81 per million cells per cell cycle when exposed to 10 µM B[a]P, 5-methylcholanthrene (5-MC), chrysene, or 16,000 µM EMS, respectively). We demonstrate that the choice of cytotoxicity assay and cell line can determine the outcome of the Pig-A mutagenesis assay when assessing a specific mutagen.

Chemical mutagenesis-driven forward genetic screens are pivotal in unveiling gene functions, yet identifying causal mutations behind phenotypes remains laborious, hindering their high-throughput application. Here, we reveal a non-uniform mutation rate caused by Ethyl Methane Sulfonate (EMS) mutagenesis in the C. elegans genome, indicating that mutation frequency is influenced by proximate sequence context and chromatin status. Leveraging these factors, we developed a machine learning enhanced pipeline to create a comprehensive EMS mutagenesis probability map for the C. elegans genome. This map operates on the principle that causative mutations are enriched in genetic screens targeting specific phenotypes among random mutations. Applying this map to Whole Genome Sequencing (WGS) data of genetic suppressors that rescue a C. elegans ciliary kinesin mutant, we successfully pinpointed causal mutations without generating recombinant inbred lines. This method can be adapted in other species, offering a scalable approach for identifying causal genes and revitalizing the effectiveness of forward genetic screens.

Quinoa (Chenopodium quinoa, 2n = 4x = 36), a super pseudocereal crop, has been introduced into China nearly 60 years. Many excellent varieties have been developed through massive selection; however, few are developed through mutagenesis breeding. In this study, the 'Longli-4' variety, locally cultivated in Gansu province, Northwest China, was selected for experimentation. The grains of 'Longli-4' were treated with ethyl methanesulfonate (EMS) at a concentration of 0.8% for 8 h. Nine plants from independent M2 families were randomly selected to investigate the mutagenesis effect of EMS on the quinoa genome. The results indicated that the single nucleotide polymorphisms (SNPs) induced by EMS were unevenly distributed across all 18 chromosomes, with an average mutation frequency of 91.2 SNPs/Mb, ranging from 4.5 to 203.5 SNPs/Mb. A significant positive correlation between the number of SNPs and chromosome length was identified through linear model analysis. Transitions from G/C to A/T were the most predominated in all variant categories, accounting for 34.4-67.2% of the mutations, and SNPs were significantly enriched in intergenic regions, representing 69.2-75.1% of the total mutations. This study provides empirical support for the application of low concentration EMS treatment in quinoa breeding.

Maintenance of genome integrity is an essential process in all organisms. Mechanisms avoiding the formation of DNA lesions or mutations are well described in animals because of their relevance to human health and cancer. In plants, they are of growing interest because DNA damage accumulation is increasingly recognized as one of the consequences of stress. Although the cellular response to DNA damage is mostly studied in response to genotoxic treatments, the main source of DNA lesions is cellular activity itself. This can occur through the production of reactive oxygen species as well as DNA processing mechanisms such as DNA replication or transcription and chromatin dynamics. In addition, how lesions are formed and repaired is greatly influenced by chromatin features and dynamics and by DNA and RNA metabolism. Notably, actively transcribed regions or replicating DNA, because they are less condensed and are sites of DNA processing, are more exposed to DNA damage. However, at the same time, a wealth of cellular mechanisms cooperate to favour DNA repair at these genomic loci. These intricate relationships that shape the distribution of mutations along the genome have been studied extensively in animals but much less in plants. In this Review, we summarize how chromatin dynamics influence lesion formation and DNA repair in plants, providing a comprehensive view of current knowledge and highlighting open questions with regard to what is known in other organisms.

DNA repair proteins can be recruited by their histone reader domains to specific epigenomic features, with consequences on intragenomic mutation rate variation. Here, we investigated H3K4me1-associated hypomutation in plants. We first examined 2 proteins which, in plants, contain Tudor histone reader domains: PRECOCIOUS DISSOCIATION OF SISTERS 5 (PDS5C), involved in homology-directed repair, and MUTS HOMOLOG 6 (MSH6), a mismatch repair protein. The MSH6 Tudor domain of Arabidopsis (Arabidopsis thaliana) binds to H3K4me1 as previously demonstrated for PDS5C, which localizes to H3K4me1-rich gene bodies and essential genes. Mutations revealed by ultradeep sequencing of wild-type and msh6 knockout lines in Arabidopsis show that functional MSH6 is critical for the reduced rate of single-base substitution (SBS) mutations in gene bodies and H3K4me1-rich regions. We explored the breadth of these mechanisms among plants by examining a large rice (Oryza sativa) mutation data set. H3K4me1-associated hypomutation is conserved in rice as are the H3K4me1-binding residues of MSH6 and PDS5C Tudor domains. Recruitment of DNA repair proteins by H3K4me1 in plants reveals convergent, but distinct, epigenome-recruited DNA repair mechanisms from those well described in humans. The emergent model of H3K4me1-recruited repair in plants is consistent with evolutionary theory regarding mutation modifier systems and offers mechanistic insight into intragenomic mutation rate variation in plants.

Wx is the key gene that controls amylose content (AC), and various alleles have been found in rice populations. Wxb is the major allele in japonica and produces moderate AC (15~18%). It was recently found that editing the promoter of Wx could produce a series of alleles that have different Wx activities. Although some studies have edited the promoter, few studies have focused on the natural variations in Wx. Here, we used the Rice3K database to investigate variations in the Wx promoter and found that the allele Wx1764178 (A/G) has a higher LD (linkage disequilibrium) with the two key SNPs (1765751, T/G; 1768006, A/C), which could produce different Wx alleles and influence AC, as reported previously. Further study showed that the Wx1764178 allele (A/G) is functional and influences the expression of Wx positively. Editing the A allele using CRISPR‒Cas9 produced 36 and 3 bp deletions and caused a decrease in the expression of Wx. The apparent amylose content (AAC) in the edited lines was decreased by 7.09% and 11.50% compared with that of the wild type, which was the japonica variety Nipponbare with Wxb and the A allele at 1764178, while a complementary line with the G allele showed a lower AAC than the A allele with no effect on other agronomic traits. The AAC of the edited lines showed a higher increase than that of the wild type (Nipponbare, Wxb) in low-nitrogen conditions relative to high-nitrogen conditions. We also developed a dCAPS marker to identify the allele and found that the G allele has widely been used (82.95%) in japonica-bred varieties from Jiangsu Province, China. Overall, we found a functional allele (Wx1764178, A/G) in the Wx promoter that could affect AAC in japonica cultivars and be developed as markers for quality improvement in rice breeding programs.

Mutant populations are crucial for functional genomics and discovering novel traits for crop breeding. Sorghum, a drought and heat-tolerant C4 species, requires a vast, large-scale, annotated, and sequenced mutant resource to enhance crop improvement through functional genomics research. Here, we report a sorghum large-scale sequenced mutant population with 9.5 million ethyl methane sulfonate (EMS)-induced mutations that covered 98% of sorghum's annotated genes using inbred line BTx623. Remarkably, a total of 610 320 mutations within the promoter and enhancer regions of 18 000 and 11 790 genes, respectively, can be leveraged for novel research of cis-regulatory elements. A comparison of the distribution of mutations in the large-scale mutant library and sorghum association panel (SAP) provides insights into the influence of selection. EMS-induced mutations appeared to be random across different regions of the genome without significant enrichment in different sections of a gene, including the 5' UTR, gene body, and 3'-UTR. In contrast, there were low variation density in the coding and UTR regions in the SAP. Based on the Ka /Ks value, the mutant library (~1) experienced little selection, unlike the SAP (0.40), which has been strongly selected through breeding. All mutation data are publicly searchable through SorbMutDB (https://www.depts.ttu.edu/igcast/sorbmutdb.php) and SorghumBase (https://sorghumbase.org/). This current large-scale sequence-indexed sorghum mutant population is a crucial resource that enriched the sorghum gene pool with novel diversity and a highly valuable tool for the Poaceae family, that will advance plant biology research and crop breeding.

Induced mutations accelerate crop improvement by providing novel disease resistance and yield alleles. However, the alleles with no perceptible phenotype but have an altered function remain hidden in mutagenized plants. The whole-genome sequencing (WGS) of mutagenized individuals uncovers the complete spectrum of mutations in the genome. Genome-wide induced mutation resources can improve the targeted breeding of tomatoes and facilitate functional genomics. In this study, we sequenced 132 doubly ethyl methanesulfonate (EMS)-mutagenized lines of tomato and detected approximately 41 million novel mutations and 5.5 million short InDels not present in the parental cultivar. Approximately 97% of the genome had mutations, including the genes, promoters, UTRs, and introns. More than one-third of genes in the mutagenized population had one or more deleterious mutations predicted by Sorting Intolerant From Tolerant (SIFT). Nearly one-fourth of deleterious genes mapped on tomato metabolic pathways modulate multiple pathway steps. In addition to the reported GC>AT transition bias for EMS, our population also had a substantial number of AT>GC transitions. Comparing mutation frequency among synonymous codons revealed that the most preferred codon is the least mutagenic toward EMS. The validation of a potato leaf-like mutation, reduction in carotenoids in ζ-carotene isomerase mutant fruits, and chloroplast relocation loss in phototropin1 mutant validated the mutation discovery pipeline. Our database makes a large repertoire of mutations accessible to functional genomics studies and breeding of tomatoes.

Induced mutations have been an important tool for plant breeding and functional genomics for more than 80 years. Novel mutations can be induced by treating seed or other plant cells with chemical mutagens or ionizing radiation. The majority of released mutant crop varieties were developed using ionizing radiation. This has been shown to create a variety of different DNA lesions including large (e.g., >=10,000 bps) copy number variations (CNV). Detection of induced DNA lesions from whole genome sequence data is useful for choosing a mutagen dosage prior to committing resources to develop a large mutant population for forward or reverse-genetic screening. Here I provide a method for detecting large induced CNV from mutant plants that utilizes a new tool to streamline the process of obtaining read coverage directly from BAM files, comparing non-mutagenized controls and mutagenized samples, and plotting the results for visual evaluation. Example data is provided from low coverage sequence data from gamma-irradiated vegetatively propagated triploid banana.

A small and efficient DNA mutation-inducing machine was constructed with an array of microplasma jet devices (7 × 1) that can be operated at atmospheric pressure for microbial mutagenesis. Using this machine, we report disruption of a plasmid DNA and generation of mutants of an oleaginous yeast Rhodosporidium toruloides. Specifically, a compact-sized microplasma channel (25 × 20 × 2 mm3) capable of generating an electron density of greater than 1013 cm-3 was constructed to produce reactive species (N2*, N2+, O, OH, and Hα) under helium atmospheric conditions to induce DNA mutagenesis. The length of microplasma channels in the device played a critical role in augmenting both the volume of plasma and the concentration of reactive species. First, we confirmed that microplasma treatment can linearize a plasmid by creating nicks in vitro. Second, we treated R. toruloides cells with a jet device containing 7 microchannels for 5 min; 94.8% of the treated cells were killed, and 0.44% of surviving cells showed different colony colors as compared to their parental colony. Microplasma-based DNA mutation is energy-efficient and can be a safe alternative for inducing mutations compared to conventional methods using toxic mutagens. This compact and scalable device is amenable for industrial strain improvement involving large-scale mutagenesis.

Plant-based foods may enhance the prevention of cancer. The present investigation aimed to assess the antigenotoxic effects of chitosan nanoparticles (CNPs) when loaded with the ethanol extract of C. cartilaginea (CNPs/Cc). Synthesis of CNPs and CNPs/Cc and their characterization were carried out using TEM, EDS, DSC, and Zeta potential. For in vivo experiments, animal groups were treated in the following groups: negative control, ethyl methanesulfonate (EMS) (240 mg/kg), CNPs (350 mg/kg), high and low doses of CNPs/Cc, CNPs plus EMS, high dose of CNPs/Cc plus EMS, and low dose of CNPs/Cc plus EMS. Bone marrow chromosomal aberrations and sperm shape abnormalities were examined. TEM results showed that CNPs and CNPs/Cc are spherical particles. CNPs' physical stability was observed to be lower than that of CNPs/Cc due to the presence of more positive charges on CNPs/Cc. EMS significantly enhanced chromosomal abnormalities and sperm shape abnormalities. CNPs showed powerful antigenotoxic properties. For the first time, it could be concluded that loading chitosan nanoparticles with C. cartilaginea extract significantly promotes its protective properties.

Generation of mutant populations with high genetic diversity is key for mutant screening and crop breeding. For this purpose, the single-seed descent method, in which one mutant line is established from a single mutagenized seed, is commonly used. This method ensures the independence of the mutant lines, but the size of the mutant population is limited because it is no greater than the number of fertile M1 plants. The rice mutant population size can be increased if a single mutagenized plant produces genetically independent siblings. Here, we used whole-genome resequencing to examine the inheritance of mutations from a single ethyl methanesulfonate (EMS)-mutagenized seed (M1 ) of Oryza sativa in its progeny (M2 ). We selected five tillers from each of three M1 plants. A single M2 seed was selected from each tiller, and the distributions of mutations induced by EMS were compared. Surprisingly, in most pairwise combinations of M2 siblings from the same parent, ≥85.2-97.9% of all mutations detected were not shared between the siblings. This high percentage suggests that the M2 siblings were derived from different cells of the M1 embryo and indicates that several genetically independent lines can be obtained from a single M1 plant. This approach should allow a large reduction in the number of M0 seeds needed to obtain a mutant population of a certain size in rice. Our study also suggests that multiple tillers of a rice plant originate from different cells of the embryo.

Genetic mutations provide the heritable material for plant adaptation to their environments. At the same time, the environment can affect the mutation rate across plant genomes. However, the extent to which environmental plasticity in mutation rates can facilitate or hinder adaptation remains a longstanding and unresolved question. Emerging discoveries of mechanisms affecting mutation rate variability provide opportunities to consider this question in a new light. Links between chromatin states, transposable elements, and DNA repair suggest cases of adaptive mutation rate plasticity could occur. Yet, numerous evolutionary and biological forces are expected to limit the impact of any such mutation rate plasticity on adaptive evolution. Persistent uncertainty about the significance of mutation rate plasticity on adaptation motivates new experimental and theoretical research relevant to understanding plant responses in changing environments.

Ethyl methanesulfonate (EMS)-induced mutagenesis is a powerful tool to generate genetic resource for identifying untapped genes and characterizing the function of genes to understand the molecular basis of important agronomic traits. This review focuses on application of contemporary EMS mutagenesis in the field of plant development and abiotic stress tolerance research, with particular focuses on reviewing the mutation types, mutagenesis site, mutagen concentration, mutagenesis duration, the identification and characterization of mutations responsible for altered stress tolerance responses. The application of EMS mutation breeding combined with genetic engineering in the future plant breeding and fundamental research was also discussed. The collective information in this review will provide good insight on how EMS mutagenesis is efficiently applied to improve abiotic stress tolerance of crops with the utilization of Next-generation sequencing (NGS) for mutation identification.

Physical mutagens are a powerful tool used for genetic research and breeding for over eight decades. Yet, when compared to chemical mutagens, data sets on the effect of different mutagens and dosages on the spectrum and density of induced mutations remain lacking. To address this, we investigated the landscape of mutations induced by gamma and X-ray radiation in the most widely cultivated crop species: rice. A mutant population of a tropical upland rice, Oryza sativa L., was generated and propagated via self-fertilization for seven generations. Five dosages ranging from 75 Gy to 600 Gy in both X-ray and gamma-irradiated material were applied. In the process of a forward genetic screens, 11 unique rice mutant lines showing phenotypic variation were selected for mutation analysis via whole-genome sequencing. Thousands of candidate mutations were recovered in each mutant with single base substitutions being the most common, followed by small indels and structural variants. Higher dosages resulted in a higher accumulation of mutations in gamma-irradiated material, but not in X-ray-treated plants. The in vivo role of all annotated rice genes is yet to be directly investigated. The ability to induce a high density of single nucleotide and structural variants through mutagenesis will likely remain an important approach for functional genomics and breeding.

Epigenomics has become a significant research interest at a time when rapid environmental changes are occurring. Epigenetic mechanisms mainly result from systems like DNA methylation, histone modification, and RNA interference. Epigenetic mechanisms are gaining importance in classical genetics, developmental biology, molecular biology, cancer biology, epidemiology, and evolution. Epigenetic mechanisms play important role in the action and interaction of plant genes during development, and also have an impact on classical plant breeding programs, inclusive of novel variation, single plant heritability, hybrid vigor, plant-environment interactions, stress tolerance, and performance stability. The epigenetics and epigenomics may be significant for crop adaptability and pliability to ambient alterations, directing to the creation of stout climate-resilient elegant crop cultivars. In this review, we have summarized recent progress made in understanding the epigenetic mechanisms in plant responses to biotic and abiotic stresses and have also tried to provide the ways for the efficient utilization of epigenomic mechanisms in developing climate-resilient crop cultivars, especially in chickpea, and other legume crops.

Glucose is both the favourite carbon and energy source and acts as a hormone that plays a regulating role in many biological processes. Calorie restriction extends the lifespan in many organisms, including Schizosaccharomyces pombe, while uptake of high glucose leads to undesired results, such as diabetes and aging. In this study, sequence analysis of Schizosaccharomyces pombe ird5 and ird11 mutants was performed using next-generation sequencing techniques and a total of 20 different mutations were detected. ird11 is resistant to oxidative stress without calorie restriction, whereas ird5 displays an adaptive response against oxidative stress. We selected nine candidate mutations located in the non-coding (6) and coding (3) region among a total of 20 different mutations. The nine candidate mutations, which are thought to be responsible for ird5 and ird11 mutant phenotypes, were investigated via forward and backward mutations by using various cloning techniques. The results of this study provide report-like information that will contribute to understanding the relationship between glucose sensing/ signalling and oxidative stress response components.