Understanding Of Gene Function Research Articles

The putative prenyltransferase Nus1 is required for filamentation in the human fungal pathogen Candida albicans.

Candida albicans is a major fungal pathogen of humans that can cause serious systemic infections in vulnerable immunocompromised populations. One of its virulence attributes is its capacity to transition between yeast and filamentous morphologies, but our understanding of this process remains incomplete. Here, we analyzed data from a functional genomic screen performed with the C. albicans Gene Replacement And Conditional Expression collection to identify genes crucial for morphogenesis in host-relevant conditions. Through manual scoring of microscopy images coupled with analysis of each image using a deep learning-based method termed Candescence, we identified 307 genes important for filamentation in tissue culture medium at 37°C with 5% CO2. One such factor was orf19.5963, which is predicted to encode the prenyltransferase Nus1 based on sequence homology to Saccharomyces cerevisiae. We further showed that Nus1 and its predicted interacting partner Rer2 are important for filamentation in multiple liquid filament-inducing conditions as well as for wrinkly colony formation on solid agar. Finally, we highlight that Nus1 and Rer2 likely govern C. albicans morphogenesis due to their importance in intracellular trafficking, as well as maintaining lipid homeostasis. Overall, this work identifies Nus1 and Rer2 as important regulators of C. albicans filamentation and highlights the power of functional genomic screens in advancing our understanding of gene function in human fungal pathogens.

Hydrophilic/hydrophobic modified microchip for detecting multiple gene doping candidates using CRISPR-Cas12a and RPA

With significant advancements in understanding gene functions and therapy, the potential misuse of gene technologies, particularly in the context of sports through gene doping (GD), has come to the forefront. This raises concerns regarding the need for point-of-care testing of various GD candidates to counter illicit practices in sports. However, current GD detection techniques, such as PCR, lack the portability required for on-site multiplexed detection. In this study, we introduce an integrated microfluidics-based chip for multiplexed gene doping detection, termed MGD-Chip. Through the strategic design of hydrophilic and hydrophobic channels, MGD-Chip enables the RPA and CRISPR-Cas12a assays to be sequentially performed on the device, ensuring minimal interference and cross-contamination. Six potential GD candidates were selected and successfully tested simultaneously on the platform within 1 h. Demonstrating exceptional specificity, the platform achieved a detection sensitivity of 0.1 nM for unamplified target plasmids and 1 aM for amplified ones. Validation using mouse models established by injecting IGFI and EPO transgenes confirmed the platform's efficacy in detecting gene doping in real samples. This technology, capable of detecting multiple targets using portable elements, holds promise for real-time GD detection at sports events, offering a rapid, highly sensitive, and user-friendly solution to uphold the integrity of sports competitions.

Chromosome-level genome sequencing and multi-omics of the Hungarian White Goose (Anser anser domesticus) reveals novel miRNA-mRNA regulation mechanism of waterfowl feather follicle development

The Hungarian White Goose (Anser anser domesticus) is an excellent European goose breed, with high feather and meat production. Despite its importance in the poultry industry, no available genome assembly information has been published. This study aimed to present Chromosome-level and functional genome sequencing of the Hungarian White Goose. The results showed that the genome assembly has a total length of 1115.82 Mb, 39 pairs of chromosomes, 92.98% of the BUSCO index, and contig N50 and scaffold N50 were up to 2.32 Mb and 60.69 Mb, respectively. Annotation of the genome assembly revealed 19550 genes, 286 miRNAs, etc. We identified 235 expanded and 1,167 contracted gene families in this breed compared with the other 16 species. We performed a positive selection analysis between this breed and four species of Anatidae to uncover the genetic information underlying feather follicle development. Further, we detected the function of miR-199-x, miR-143-y, and miR-23-z on goose embryonic skin fibroblast. In summary, we have successfully generated a highly complete genome sequence of the Hungarian white goose, which will provide a great resource to improve our understanding of gene functions and enhance the studies on feather follicle development at the genomic level.

A new method for the robust expression and single-step purification of dCas9 for CRISPR interference/activation (CRISPRi/a) applications

CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated enzyme 9) is known for its simplicity, versatility, and scalability in genome editing applications. In vitro Cas9, when complexed with sgRNA, binds and cleaves the complementary target sequences with almost perfect precision. The enzyme is exploited for various applications in understanding and changing gene function. dCas9 (deactivated or dead Cas9) is a double mutated version of Cas9 that bears mutations in the nuclease domains of the enzyme and thus cannot cleave the target DNA. dCas9 is equally advantageous since it can alter gene expression using various transcriptional activators CRISPRa and repressors CRISPRi. Additionally, dCas9 can bind to the desired target gene without cleaving it, making it a unique reagent to study the kinetics and stability of RNA-protein-DNA interactions required to design more efficient and specific gene-editing nucleases. An appreciable quantity of pure and homogeneous protein is needed to characterise dCas9 for its structural and functional understanding. This study used an N-terminal acidic tag to express the dCas9 in an E. coli-bacterial host. A simple single-step protocol for robust and efficient production of dCas9 has been described. The study and methods are distinctive as the purification is performed in a single step using inexpensive multi-modal hydroxyapatite chromatography. The purified protein can be used in different in vitro and in vivo studies.

Modelling human genetic disorders in Xenopus tropicalis.

Recent progress in human disease genetics is leading to rapid advances in understanding pathobiological mechanisms. However, the sheer number of risk-conveying genetic variants being identified demands in vivo model systems that are amenable to functional analyses at scale. Here we provide a practical guide for using the diploid frog species Xenopus tropicalis to study many genes and variants to uncover conserved mechanisms of pathobiology relevant to human disease. We discuss key considerations in modelling human genetic disorders: genetic architecture, conservation, phenotyping strategy and rigour, as well as more complex topics, such as penetrance, expressivity, sex differences and current challenges in the field. As the patient-driven gene discovery field expands significantly, the cost-effective, rapid and higher throughput nature of Xenopus make it an essential member of the model organism armamentarium for understanding gene function in development and in relation to disease.

Advancements in genetic techniques and functional genomics for enhancing crop traits and agricultural sustainability.

Genetic variability is essential for the development of new crop varieties with economically beneficial traits. The traits can be inherited from wild relatives or induced through mutagenesis. Novel genetic elements can then be identified and new gene functions can be predicted. In this study, forward and reverse genetics approaches were described, in addition to their applications in modern crop improvement programs and functional genomics. By using heritable phenotypes and linked genetic markers, forward genetics searches for genes by using traditional genetic mapping and allele frequency estimation. Despite recent advances in sequencing technology, omics and computation, genetic redundancy remains a major challenge in forward genetics. By analyzing close-related genes, we will be able to dissect their functional redundancy and predict possible traits and gene activity patterns. In addition to these predictions, sophisticated reverse gene editing tools can be used to verify them, including TILLING, targeted insertional mutagenesis, gene silencing, gene targeting and genome editing. By using gene knock-down, knock-up and knock-out strategies, these tools are able to detect genetic changes in cells. In addition, epigenome analysis and editing enable the development of novel traits in existing crop cultivars without affecting their genetic makeup by increasing epiallelic variants. Our understanding of gene functions and molecular dynamics of various biological phenomena has been revised by all of these findings. The study also identifies novel genetic targets in crop species to improve yields and stress tolerances through conventional and non-conventional methods. In this article, genetic techniques and functional genomics are specifically discussed and assessed for their potential in crop improvement.

Clover: An unbiased method for prioritizing differentially expressed genes using a data-driven approach.

Identifying key genes from a list of differentially expressed genes (DEGs) is a critical step in transcriptome analysis. However, current methods, including Gene Ontology analysis and manual annotation, essentially rely on existing knowledge, which is highly biased depending on the extent of the literature. As a result, understudied genes, some of which may be associated with important molecular mechanisms, are often ignored or remain obscure. To address this problem, we propose Clover, a data-driven scoring method to specifically highlight understudied genes. Clover aims to prioritize genes associated with important molecular mechanisms by integrating three metrics: the likelihood of appearing in the DEG list, tissue specificity, and number of publications. We applied Clover to Alzheimer's disease data and confirmed that it successfully detected known associated genes. Moreover, Clover effectively prioritized understudied but potentially druggable genes. Overall, our method offers a novel approach to gene characterization and has the potential to expand our understanding of gene functions. Clover is an open-source software written in Python3 and available on GitHub at https://github.com/G708/Clover.

"Next top" mouse models advancing CTCL research.

This review systematically describes the application of in vivo mouse models in studying cutaneous T-cell lymphoma (CTCL), a complex hematological neoplasm. It highlights the diverse research approaches essential for understanding CTCL's intricate pathogenesis and evaluating potential treatments. The review categorizes various mouse models, including xenograft, syngeneic transplantation, and genetically engineered mouse models (GEMMs), emphasizing their contributions to understanding tumor-host interactions, gene functions, and studies on drug efficacy in CTCL. It acknowledges the limitations of these models, particularly in fully replicating human immune responses and early stages of CTCL. The review also highlights novel developments focusing on the potential of skin-targeted GEMMs in studying natural skin lymphoma progression and interactions with the immune system from onset. In conclusion, a balanced understanding of these models' strengths and weaknesses are essential for accelerating the deciphering of CTCL pathogenesis and developing treatment methods. The GEMMs engineered to target specifically skin-homing CD4+ T cells can be the next top mouse models that pave the way for exploring the effects of CTCL-related genes.

HortGenome Search Engine, a universal genomic search engine for horticultural crops.

Horticultural crops comprising fruit, vegetable, ornamental, beverage, medicinal and aromatic plants play essential roles in food security and human health, as well as landscaping. With the advances of sequencing technologies, genomes for hundreds of horticultural crops have been deciphered in recent years, providing a basis for understanding gene functions and regulatory networks and for the improvement of horticultural crops. However, these valuable genomic data are scattered in warehouses with various complex searching and displaying strategies, which increases learning and usage costs and makes comparative and functional genomic analyses across different horticultural crops very challenging. To this end, we have developed a lightweight universal search engine, HortGenome Search Engine (HSE; http://hort.moilab.net), which allows for the querying of genes, functional annotations, protein domains, homologs, and other gene-related functional information of more than 500 horticultural crops. In addition, four commonly used tools, including 'BLAST', 'Batch Query', 'Enrichment analysis', and 'Synteny Viewer' have been developed for efficient mining and analysis of these genomic data.

Exposure of chimaeric embryos to exogenous FGF4 leads to the production of pure ESC-derived mice

Producing chimaeras constitutes the most reliable method of verifying the pluripotency of newly established cells. Moreover, forming chimaeras by injecting genetically modified embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) into the embryo is part of the procedure for generating transgenic mice, which are used for understanding gene function. Conventional methods for generating transgenic mice, including the breeding of chimaeras and tetraploid complementation, are time-consuming and cost-inefficient, with significant limitations that hinder their effectiveness and widespread applications. In the present study, we modified the traditional method of chimaera generation to significantly speed up this process by generating mice exclusively derived from ESCs. This study aimed to assess whether fully ESC-derived mice could be obtained by modulating fibroblast growth factor 4 (FGF4) levels in the culture medium and changing the direction of cell differentiation in the chimaeric embryo. We found that exogenous FGF4 directs all host blastomeres to the primitive endoderm fate, but does not affect the localisation of ESCs in the epiblast of the chimaeric embryos. Consequently, all FGF4-treated chimaeric embryos contained an epiblast composed exclusively of ESCs, and following transfer into recipient mice, these embryos developed into fully ESC-derived newborns. Collectively, this simple approach could accelerate the generation of ESC-derived animals and thus optimise ESC-mediated transgenesis and the verification of cell pluripotency. Compared to traditional methods, it could speed up functional studies by several weeks and significantly reduce costs related to maintaining and breeding chimaeras. Moreover, since the effect of stimulating the FGF signalling pathway is universal across different animal species, our approach can be applied not only to rodents but also to other animals, offering its utility beyond laboratory settings.

Abstract 6621: Development of a melanocyte lineage-specific knockout system in zebrafish

Abstract Zebrafish have emerged as an ideal model organism for investigating melanocyte and melanoma biology. While CRISPR-Cas9 technology has significantly advanced our understanding of gene functions in zebrafish, two challenges remain: 1) how do we knock out embryonic lethal genes in adult melanocytes? and 2) how do we knock out tumor promoting genes (rather than tumor suppressors)? To address this, we have developed a new transgenic line in which Cas9 is knocked into the melanocyte-specific endogenous mitfa locus. To test this system in normal melanocytes, Tg(mitfa:Cas9) embryos were injected with a plasmid containing albino sgRNA. Cells with integration of this plasmid exhibited almost complete loss of pigmentation, demonstrating the high efficiency of our approach. This system can now be rapidly used to knock out embryonic essential genes specifically in melanocytes and evaluate the phenotype of adult fish. In melanoma, the commonly available zebrafish lines overexpress the oncogene BRAFV600E in melanocytes with germline knockout of tp53. This is not comparable to the situation in humans, where global p53 loss is rare. By utilizing our mitfa:Cas9 system, we have generated tumors with melanoma-specific loss of tp53 or ptena/b, thereby eliminating non-specific tumors that originate from knockout of tumor suppressors in other cell types. Using this system, we are now testing whether knockout of tumor promoting genes delays melanoma onset and progression, something that has previously been difficult to efficiently achieve in zebrafish. In addition to specificity, an additional advantage of our system is that we can create nearly complete knockout of genes in the melanocytes by the F1, rather than the F2 generation. This saves several months of time and reduces the number of uninformative animals without the relevant genotype. Collectively, our system provides a highly efficient mechanism to study essential genes in melanocytes and melanoma in the zebrafish system. Citation Format: Sarah Perlee, Yilun Ma, Jacob Swanson, Zhitao Ming, Julia Xia, Timothée Lionnet, Maura McGrail, Richard White. Development of a melanocyte lineage-specific knockout system in zebrafish [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6621.

RNAi-Mediated Silencing of Laccase 2 in Culex pipiens Pupae via Dehydration and Soaking Results in Multiple Defects in Cuticular Development.

Mosquitoes transmit a range of pathogens, causing devastating effects on human health. Population genetic control strategies have been developed and successfully used for several mosquito species. The most important step in identifying potential targets for mosquito control is the understanding of gene function. RNA interference (RNAi) is a powerful tool for gene silencing which has been widely used to study gene function in insects via knockdown of expression. The success of RNAi in insects depends on the efficient delivery of dsRNA into the cells, with microinjections being the most commonly used to study mosquito gene function. However, microinjections in the pupal stage lead to significant mortality in Aedes and Culex species, and few studies have performed microinjections in Culicinae pupae. Advanced techniques, such as CRISPR/Cas9 knockout, require establishing individual mosquito lines for each gene studied, and maintaining such lines may be limited by the insect-rearing capacity of a laboratory. Moreover, at times gene knockout during early development (embryo stage) has a deleterious effect on mosquito development, precluding the analysis of gene function in the pupal and adult stages and its potential for mosquito control. There is a need for a simple procedure that can be used for the fast and reliable examination of adult gene function via RNAi knockdown. Here, we focus on the aquatic stages of the mosquito life cycle and suggest a quick and easy assay for screening the functional role of genes in Culex pipiens mosquitoes without using microinjections. By dehydration of early stage pupae and subsequent rehydration in highly concentrated dsRNA, we achieved a moderate knockdown of laccase 2, a gene that turns on in the pupal stage and is responsible for melanization and sclerotization of the adult cuticle.

A nuclease-dead Cas9-derived tool represses target gene expression.

Manipulation of gene expression is central to understanding gene function, engineering cell behavior, and altering biological traits according to production demands. Nuclease-dead Cas9 (dCas9), a variant of active Cas9, offers a versatile platform for the precise control of genome function without DNA cleavage. Notably, however, an effective and universal dCas9-based transcriptional repression system remains unavailable in plants. The non-canonical histone acetyltransferase TENDRIL-LESS (CsTEN) is responsible for chromatin loosening and histone modification in cucumber (Cucumis sativus). In this study, we engineered a gene regulation tool by fusing TEN and its truncated proteins with dCas9. The full-length dCas9-TEN protein substantially repressed gene expression, with the N-terminal domain identified as the core repression domain. We subsequently validated the specificity and efficacy of this system through both transient infection and genetic transformation in cucumber and Arabidopsis (Arabidopsis thaliana). Electrophoretic mobility shift assay (EMSA) revealed the ability of the N-terminal domain of TEN to bind to chromatin, which may promote target binding of the dCas9 complex and enhance the transcriptional repression effect. Our tool enriches the arsenal of genetic regulation tools available for precision breeding in crops.

Non-invasive dsRNA delivery via feeding for effective gene silencing in teleost fish: A novel approach in the study of gene function analysis

RNA interference (RNAi) is a powerful technique for gene silencing, involving post-transcriptional regulation of gene expression. Successful application of RNAi has been demonstrated in various organisms like nematodes, insects, and oysters by ingesting double-stranded RNA (dsRNA)-expressing bacteria. In this study, we attempted a non-invasive feeding method in the marine economy teleost fish, Sebastes schlegelii. To validate the feasibility of this approach in S. schlegelii, we utilized rotifers, Artemia nauplii, and commercial pellet feed as vectors to deliver Escherichia coli strain HT115 engineered to express dsRNA, targeting dead end (dnd) gene, known to be important for primordial germ cells (PGCs) migration and proliferation in fish. Our experimental results consistently showed that knockdown of the dnd gene effectively inhibited the development of PGCs in S. schlegelii, resulting in gonadal dysplasia and even sex reversal. This study marks a significant advancement in understanding gene function in teleost fish, laying the groundwork for future large-scale research in this field.

CyanoCyc cyanobacterial web portal.

CyanoCyc is a web portal that integrates an exceptionally rich database collection of information about cyanobacterial genomes with an extensive suite of bioinformatics tools. It was developed to address the needs of the cyanobacterial research and biotechnology communities. The 277 annotated cyanobacterial genomes currently in CyanoCyc are supplemented with computational inferences including predicted metabolic pathways, operons, protein complexes, and orthologs; and with data imported from external databases, such as protein features and Gene Ontology (GO) terms imported from UniProt. Five of the genome databases have undergone manual curation with input from more than a dozen cyanobacteria experts to correct errors and integrate information from more than 1,765 published articles. CyanoCyc has bioinformatics tools that encompass genome, metabolic pathway and regulatory informatics; omics data analysis; and comparative analyses, including visualizations of multiple genomes aligned at orthologous genes, and comparisons of metabolic networks for multiple organisms. CyanoCyc is a high-quality, reliable knowledgebase that accelerates scientists' work by enabling users to quickly find accurate information using its powerful set of search tools, to understand gene function through expert mini-reviews with citations, to acquire information quickly using its interactive visualization tools, and to inform better decision-making for fundamental and applied research.

Advances in RNA Interference for Plant Functional Genomics: Unveiling Traits, Mechanisms, and Future Directions.

RNA interference (RNAi) is a conserved molecular mechanism that plays a critical role in post-transcriptional gene silencing across diverse organisms. This review delves into the role of RNAi in plant functional genomics and its applications in crop improvement, highlighting its mechanistic insights and practical implications. The review begins with the foundational discovery of RNAi's mechanism, tracing its origins from petunias to its widespread presence in various organisms. Various classes of regulatory non-coding small RNAs, including siRNAs, miRNAs, and phasiRNAs, have been uncovered, expanding the scope of RNAi-mediated gene regulation beyond conventional understanding. These RNA classes participate in intricate post-transcriptional and epigenetic processes that influence gene expression. In the context of crop enhancement, RNAi has emerged as a powerful tool for understanding gene functions. It has proven effective in deciphering gene roles related to stress resistance, metabolic pathways, and more. Additionally, RNAi-based approaches hold promise for integrated pest management and sustainable agriculture, contributing to global efforts in food security. This review discusses RNAi's diverse applications, such as modifying plant architecture, extending shelf life, and enhancing nutritional content in crops. The challenges and future prospects of RNAi technology, including delivery methods and biosafety concerns, are also explored. The global landscape of RNAi research is highlighted, with significant contributions from regions such as China, Europe, and North America. In conclusion, RNAi remains a versatile and pivotal tool in modern plant research, offering novel avenues for understanding gene functions and improving crop traits. Its integration with other biotechnological approaches such as gene editing holds the potential to shape the future of agriculture and sustainable food production.

EDITING CDKN1A AND RASD1 IN A CELL CULTURE SYSTEM

Abstract As an individual ages, there is a higher incidence of cancer and infertility. Testicular germ cell cancer rates have been rising year after year. According to the American Cancer Society 1 of every 250 males will develop testicular cancer during their lifetime. In this research project, we aim to develop a molecular toolbox to analyze gene function in male germinal cells using a CRISPR/Cas9 cell culture system. This biotechnology allows for the targeted alteration and cutting of specific DNA strands. To achieve this, we will utilize cancer cell lines (SUSA and NT2/D1 testicular cancer cell lines) and knock out two genes of interest: cyclin-dependent kinase inhibitor 1 (CDKN1A) and ras-related dexamethasone-induced 1 (RASD1). Our objective is to investigate the effects of gene knockout on cell proliferation. For this purpose, we designed primers and single-guide RNAs using bioinformatic databases: Benchling and Chop Chop. The tools we used were employed to amplify human genomic DNA and assess the effectiveness of the guide RNAs through in vitro cutting of our genes of interest, followed by gel electrophoresis observations. Based on the results obtained from these experiments, our next step is to electroporate the guide RNA and Cas9 complex to alter gene function in our cell culture system. Through a better understanding of gene function in testicular cancer cell lines, we hope to inform future therapeutic targets for both testicular cancer and age-related infertility.

Identification of QTLs and allelic effect controlling lignan content in sesame (Sesamum indicum L.) using QTL-seq approach.

Sesame (Sesamum indicum L.), an oilseed crop, is gaining worldwide recognition for its healthy functional ingredients as consumption increases. The content of lignans, known for their antioxidant and anti-inflammatory effects, is a key agronomic trait that determines the industrialization of sesame. However, the study of the genetics and physiology of lignans in sesame is challenging, as they are influenced by multiple genes and environmental factors, therefore, the understanding of gene function and synthetic pathways related to lignan in sesame is still limited. To address these knowledge gaps, we conducted genetic analyses using F7 recombinant inbred line (RIL) populations derived from Goenbaek and Gomazou as low and high lignin content variants, respectively. Using the QTL-seq approach, we identified three loci, qLignan1-1, qLignan6-1, and qLignan11-1, that control lignan content, specifically sesamin and sesamolin. The allelic effect between loci was evaluated using the RIL population. qLignan6-1 had an additive effect that increased lignan content when combined with the other two loci, suggesting that it could be an important factor in gene pyramiding for the development of high-lignan varieties. This study not only highlights the value of sesame lignan, but also provides valuable insights for the development of high-lignan varieties through the use of DNA markers in breeding strategies. Overall, this research contributes to our understanding of the importance of sesame oil and facilitates progress in sesame breeding for improved lignan content.

Genotype andphenotype data standardization, utilization andintegration inthe big data era foragricultural sciences.

Large-scale genotype and phenotype data have been increasingly generated to identify genetic markers, understand gene function and evolution and facilitate genomic selection. These datasets hold immense value for both current and future studies, as they are vital for crop breeding, yield improvement and overall agricultural sustainability. However, integrating these datasets from heterogeneous sources presents significant challenges and hinders their effective utilization. We established the Genotype-Phenotype Working Group in November 2021 as a part of the AgBioData Consortium (https://www.agbiodata.org) to review current data types and resources that support archiving, analysis and visualization of genotype and phenotype data to understand the needs and challenges of the plant genomic research community. For 2021-22, we identified different types of datasets and examined metadata annotations related to experimental design/methods/sample collection, etc. Furthermore, we thoroughly reviewed publicly funded repositories for raw and processed data as well as secondary databases and knowledgebases that enable the integration of heterogeneous data in the context of the genome browser, pathway networks and tissue-specific gene expression. Based on our survey, we recommend a need for (i) additional infrastructural support for archiving many new data types, (ii) development of community standards for data annotation and formatting, (iii) resources for biocuration and (iv) analysis and visualization tools to connect genotype data with phenotype data to enhance knowledge synthesis and to foster translational research. Although this paper only covers the data and resources relevant to the plant research community, we expect that similar issues and needs are shared by researchers working on animals. Database URL: https://www.agbiodata.org.

METHODS OF GENETIC TRANSFORMATION: MAJOR EMPHASIS TO CROP PLANTS

Advancements in gene transfer technology have indeed opened up exciting possibilities for more effectively manipulating the genetic makeup of live organisms, ranging from microorganisms to plants and animals. Direct and indirect transformations are the two basic types of gene transfer techniques. Indirect method comprises Agrobacterium mediated method as it involves intermediate host between gene of interest and target and this method is most opted one out of all present. Direct gene transformation methods, on the other hand, do not involve the use of an intermediate host organism. Instead, they rely on physical means to transfer genes between cells. Biolistic transformation uses high-velocity particles to deliver DNA into target cells, while microinjection and macroinjection involve the direct injection of DNA into cells. Protoplast fusion combines the genetic material of two different cells by fusing their protoplasts. Natural methods for gene transfer encompass mechanisms that occur naturally in various organism, includes transposition, conjugation, phage and retroviral transductions and bacterial transformation. Chemical techniques utilize chemical agents to facilitate gene transfer, such as calcium phosphate-mediated transformation, polyethylene glycol (PEG)-mediated transformation, DEAE (Diethylethanolamine)-Dextran-mediated transformation. Genes can be also being transferred using electrical techniques such as electroporation and electrofusion. Crop improvement and trait improvement are now being hastened by the fast-rising number of sequenced plant genomes, information from functional genomics data to understand gene function, innovative gene cloning, and tissue culture techniques. Despite being indispensable, its progress is still hindered by the fact that many plant species and agricultural genotypes exhibit low transformability or are resistant to established tissue culture and regeneration conditions. Here, we review the techniques employed in plant transformation and provide a concise overview of their evolution in agricultural crops, from their first inception to present time.