Conventional Genetic Engineering Research Articles

Carotenoid-carbohydrate crosstalk: evidence for genetic and physiological interactions in storage tissues across crop species.

Carotenoids play essential roles in photosynthesis, photoprotection, and human health. Efforts to increase carotenoid content in several staple crops have been successful through both conventional selection and genetic engineering methods. Interestingly, in some cases, altering carotenoid content has had unexpected effects on other aspects of plant metabolism, impacting traits like sugar content, dry matter percentage, fatty acid content, stress tolerance, and phytohormone concentrations. Studies across several diverse crop species have identified negative correlations between carotenoid and starch contents, as well as positive correlations between carotenoids and soluble sugars. Collectively, these reports suggest a metabolic interaction between carotenoids and carbohydrates. We synthesize evidence pointing to four hypothesized mechanisms: (1) direct competition for precursors; (2) physical interactions in plastids; (3) influences of sugar or apocarotenoid signaling networks; and (4) nonmechanistic population or statistical sources of correlations. Though the carotenoid biosynthesis pathway is well understood, the regulation and interactions of carotenoids, especially in nonphotosynthetic tissues, remain unclear. This topic represents an underexplored interplay between primary and secondary metabolism where further research is needed.

An Integrated and Multi-Stakeholder Approach for Sustainable Phosphorus Management in Agriculture

Conventional agriculture relies on non-renewable rock phosphate as a source of phosphorus. The demand for food has led to increased phosphorus inputs, with a negative impact on freshwater biodiversity and food security. The importation of phosphorus fertilizers makes most food systems vulnerable to phosphorus supply risks. The geopolitical instability generated by the pandemic and the current Russia–Ukraine conflict, which has led to a 400% increase in phosphorus commodity prices, offers the international community and institutions an opportunity to embrace the global phosphorus challenge and move towards a more circular system. Here, we discuss an integrated and multi-stakeholder approach to improve phosphorus management in agriculture and increase the efficiency of the whole chain, highlighting the contribution of conventional breeding and genetic engineering, with a particular focus on low-phytic-acid (lpa) crops, whose grains may help in reducing phosphorus-management-related problems. In recent decades, the choice of short-term strategies—such as the use of phytase as a feed additive—rather than lpa mutants, has been carried out without considering the long-term money saving to be derived from lpa crops. Overall, lpa crops have the potential to increase the nutritional quality of foods and feeds, but more research is needed to optimize their performance.

A Sustainable Approach to Combat Micronutrient Deficiencies and Ensure Global Food Security through Biofortification

Micronutrient deficiencies, particularly in essential vitamins and minerals, pose a significant public health challenge, affecting over two billion people worldwide. These deficiencies contribute to various health issues, impaired cognitive development, and reduced productivity, ultimately hindering social and economic progress. Biofortification, a process of enhancing the nutritional content of staple crops through conventional breeding or genetic engineering, has emerged as a promising and sustainable approach to combat micronutrient deficiencies and ensure global food security. This review explores the potential of Biofortification as a cost-effective and sustainable solution to address hidden hunger and improve the nutritional status of vulnerable populations. Biofortification offers several advantages over traditional interventions, such as supplementation and food fortification. By targeting staple crops consumed by the majority of the population, Biofortification ensures a wide reach and sustained nutrient intake without requiring significant changes in dietary habits. Moreover, biofortified crops can be grown locally, reducing the reliance on external interventions and empowering farmers to improve their nutritional status and livelihoods. Numerous studies have demonstrated the efficacy of bio fortified crops in increasing micronutrient intake and improving health outcomes. For instance, iron-biofortified pearl millet has been shown to increase iron absorption and reduce anemia prevalence in children, while zinc-biofortified wheat has improved zinc status and reduced stunting. Additionally, vitamin A-biofortified sweet potato and cassava have significantly increased vitamin A intake and reduced vitamin A deficiency in various populations.
 Despite the promising results, the success of Biofortification relies on several factors, including the development of nutrient-dense varieties, consumer acceptance, and effective dissemination strategies. Collaboration among researchers, policymakers, and stakeholders is essential to scale up Biofortification efforts and ensure their long-term sustainability. By prioritizing Biofortification as a key strategy in combating micronutrient deficiencies, we can work towards a more nourished and food-secure world.

Dopamine Polymerization-Mediated Surface Functionalization toward Advanced Bacterial Therapeutics.

Bacteria-based therapy has spotlighted an unprecedented potential in treating a range of diseases, given that bacteria can be used as both drug vehicles and therapeutic agents. However, the use of bacteria for disease treatment often suffers from unsatisfactory outcomes, due largely to their suboptimal bioavailability, dose-dependent toxicity, and low targeting colonization. In the past few years, substantial efforts have been devoted to tackling these difficulties, among which methods capable of integrating bacteria with multiple functions have been extensively pursued. Different from conventional genetic engineering and modern synthetic bioengineering, surface modification of bacteria has emerged as a simple yet flexible strategy to introduce different functional motifs. Polydopamine, which can be easily formed via in situ dopamine oxidation and self-polymerization, is an appealing biomimetic polymer that has been widely applied for interfacial modification and functionalization. By virtue of its catechol groups, polydopamine can be efficiently codeposited with a multitude of functional elements on diverse surfaces.In this Account, we summarize the recent advances from our group with a focus on the interfacial polymerization-mediated functionalization of bacteria for advanced microbial therapy. First, we present the optimized strategy for bacterial surface modification under cytocompatible conditions by in situ dopamine polymerization. Taking advantage of the hydrogen bonding, π-π stacking, Michael addition, and Schiff base reaction with polydopamine, diverse functional small molecules and macromolecules are facilely codeposited onto the bacterial surface. Namely, monomodal, dual-modal, and multimodal surface modification of bacteria can be achieved by dopamine self-deposition, codeposition with a unitary composition, and codeposition with a set of multiple components, respectively. Second, we outline the regulation of bacterial functions by surface modification. The formed polydopamine surface endows bacteria with the ability to resist in vivo insults, such as gastrointestinal tract stressors and immune clearance, resulting in greatly improved bioavailability. Integration with specific ligands or therapeutic components enables the modified bacteria to increase targeting accumulation and colonization at lesion sites or play synergistic effects in disease treatment. Bacteria codeposited with different bioactive moieties, such as protein antigens, antibodies, and immunoadjuvants, are even able to actively interact with the host, particularly to elicit immune responses by either suppressing immune overactivation to promote the reversion of pathological inflammations or provoking protective innate and/or adaptive immunity to inhibit pathogenic invaders. Third, we highlight the applications of surface-modified bacteria as multifunctional living therapeutics in disease treatment, especially alleviating inflammatory bowel diseases via oral delivery and intervening in different types of cancer through systemic or intratumoral injection. Finally, we discuss the challenges and prospects of dopamine polymerization-mediated multifunctionalization for preparing advanced bacterial therapeutics as well as their bench to bedside translation. We anticipate that this Account can provide an insightful overview of bacterial therapy and inspire innovative thinking and new efforts to develop next-generation living therapeutics for treating various diseases.

Enhancing Nutrition, Crop Resilience, and Food Security through Biofortification

Biofortification is a process of enhancing the nutritional quality of food crops through conventional plant breeding, genetic engineering, or agronomic practices. It has emerged as an important agricultural strategy to improve public health by increasing the micronutrient density in staple crops and vegetables. Biofortification provides a cost-effective and sustainable approach to combat micronutrient deficiencies, also known as hidden hunger, which affects over 2 billion people worldwide. This review provides an overview of biofortification efforts targeting major micronutrients such as iron, zinc, vitamin A, and folate. The genetic and molecular mechanisms underlying elevated micronutrient accumulation are discussed. The review also summarizes the impacts of biofortification in enhancing micronutrient intake, nutritional status, and health outcomes based on results from efficacy and effectiveness studies. The role of biofortification in building climate resilience and food security is also examined. Overall, biofortification has shown considerable promise in tackling malnutrition sustainably in developing countries. However, continued research and policy support are needed to maximize its impact on nutrition security worldwide.

Conventional Breeding vs. Genetic Engineering in Maize: A Comparative Study

Conventional Breeding vs. Genetic Engineering in Maize: A Comparative Study

Estimating the Genetic Diversity of Oranges Citrus spp. in South Sulawesi, Indonesia, Using RAPD Markers

Oranges hold significant economic importance, being cultivated extensively worldwide and having a large global market. Indonesia, ranked eighth globally as a producer of oranges, is one of the countries with high genetic diversity of oranges. This diversity is distributed across various regions of Indonesia, including South Sulawesi. Despite the advancements in DNA-based molecular marker techniques for assessing genetic diversity, information on orange diversity in South Sulawesi is currently unavailable and under-researched. In this study, random amplified polymorphic DNA (RAPD) markers were utilized to analyze the genetic diversity of oranges in five production centers in South Sulawesi. Leaf samples of 13 orange varieties were collected from the five production centers: Pangkep, Sidrap, Bantaeng, North Luwu, and Selayar in South Sulawesi, Indonesia. Genomic DNA extraction from the orange leaves followed the protocol of the DNA Mini Kit Geneaid. DNA amplification was carried out using the RAPD method with 14 primers: OPE-04, OPH-04, OPH-15, OPN-14, OPN-16, OPR-08, OPR-20, OPW-06, OPW-09, OPX-07, OPX-11, OPX-17, UBC-18, and UBC-51. The RAPD primers yielded 109 amplified fragments ranging in size from 200 to 2000 base pairs (bp), and all RAPD primers showed 100% polymorphism. The genetic diversity value (He) of oranges in South Sulawesi was moderate (0.236). Cluster analysis based on a similarity coefficient of 77% divided the 175 orange genotypes into five groups. The most closely related genotypes were SB6 and SB7, exhibiting 100% similarity, followed by genotypes JS8 and JS9 and JS13 and JS17, with genetic similarities exceeding 99% for each pair. Genotypes P9 and SI5 displayed the highest genetic distance, with a similarity coefficient of 57%. The dendrogram diagram can serve as a basis for selecting desired plant traits in the improvement of plant characteristics through both conventional breeding and genetic engineering activities.

Non-homologous end-joining-deficient filamentous fungal strains mitigate the impact of off-target mutations during the application of CRISPR/Cas9.

CRISPR/Cas9 genome editing technology has been implemented in almost all living organisms. Its editing precision appears to be very high and therefore could represent a big change from conventional genetic engineering approaches. However, guide RNA binding to nucleotides similar to the target site could result in undesired off-target mutations. Despite this, evaluating whether mutations occur is rarely performed in genome editing studies. In this study, we generated CRISPR/Cas9-derived filamentous fungal strains and analyzed them for the occurrence of mutations, and to which extent genome stability affects their occurrence. As a test case, we deleted the (hemi-)cellulolytic regulator-encoding gene xlnR in two Aspergillus niger strains: a wild type (WT) and a non-homologous end-joining (NHEJ)-deficient strain ΔkusA. Initial phenotypic analysis suggested a much higher prevalence of mutations in the WT compared to NHEJ-deficient strains, which was confirmed and quantified by whole-genome sequencing analysis. Our results clearly demonstrate that CRISPR/Cas9 applied to an NHEJ-deficient strain is an efficient strategy to avoid unwanted mutations. IMPORTANCE Filamentous fungi are commonly used biofactories for the production of industrially relevant proteins and metabolites. Often, fungal biofactories undergo genetic development (genetic engineering, genome editing, etc.) aimed at improving production yields. In this context, CRISPR/Cas9 has gained much attention as a genome editing strategy due to its simplicity, versatility, and precision. However, despite the high level of accuracy reported for CRISPR/Cas9, in some cases unintentional cleavages in non-targeted loci-known as off-target mutations-could arise. While biosafety should be a central feature of emerging biotechnologies to minimize unintended consequences, few studies quantitatively evaluate the risk of off-target mutations. This study demonstrates that the use of non-homologous end-joining-deficient fungal strains drastically reduces the number of unintended genomic mutations, ensuring that CRISPR/Cas9 can be safely applied for strain development.

General guidelines for CRISPR/Cas-based genome editing in plants.

CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) technology is a versatile genome editing tool that has been used to improve agriculturally important plant traits. Due to its precision, CRISPR/Cas9 is more effective than either conventional plant breeding methods or standard genetic engineering approaches for the rapid development of new varieties resilient to climate change. In addition to knowledge in tissue culture-based plant transformation, effective gene-specific single guide RNA (sgRNA) design, prediction of its off-target effect and utilization of vectors, promoters, Cas proteins and terminators is required for CRISPR/Cas9. Various bioinformatics tools are available for the best sgRNA design and screening of the off-targets. Various tools are used in the delivery of CRISPR/Cas components into cells and the genome. Moreover, some recent studies proved the simultaneous silencing of different paralogs in the same family or several genes working in the same pathway by using multiple-target sgRNA designs. This review summarizes the type of promoters, Cas proteins, recognition sequences, and terminators available for the development of knock-out and overexpression plant lines. It also provides a general guideline for the development of genome-edited plants from the design of sgRNAs to the selection of non-transgenic genome-edited T2 generation.

Applications of Genomic Tools in Plant Breeding: Crop Biofortification.

Crop breeding has mainly been focused on increasing productivity, either directly or by decreasing the losses caused by biotic and abiotic stresses (that is, incorporating resistance to diseases and enhancing tolerance to adverse conditions, respectively). Quite the opposite, little attention has been paid to improve the nutritional value of crops. It has not been until recently that crop biofortification has become an objective within breeding programs, through either conventional methods or genetic engineering. There are many steps along this long path, from the initial evaluation of germplasm for the content of nutrients and health-promoting compounds to the development of biofortified varieties, with the available and future genomic tools assisting scientists and breeders in reaching their objectives as well as speeding up the process. This review offers a compendium of the genomic technologies used to explore and create biodiversity, to associate the traits of interest to the genome, and to transfer the genomic regions responsible for the desirable characteristics into potential new varieties. Finally, a glimpse of future perspectives and challenges in this emerging area is offered by taking the present scenario and the slow progress of the regulatory framework as the starting point.

Biofortified Crops – Boon for Nutritional Security

Micronutrient deficiencies of iron, zinc, and vitamin-A are such serious global health issues, that it affects one out of every three people worldwide. The intensity of this “hidden hunger” compels us to acknowledge global nutritional security issues. Plant based food are the most popular and trending choices for people of all rungs of the social ladder. Biofortification is a sustainable and promising process of improving nutrition in plant based food through different agronomic approaches, conventional plant breeding and genetic engineering. Biofortified crops have been developed with high iron, high zinc, vitamin-A, with other nutritional quality enhancements and these crops have already proved to be a ‘boon for nutritional security’. This review highlights some selected Biofortified crops with special reference to rice (Oryza sativa), as 50% of the global population relies on it.

Targeted T cell receptor gene editing provides predictable T cell product function for immunotherapy

SummaryAdoptive transfer of T cells expressing a transgenic T cell receptor (TCR) has the potential to revolutionize immunotherapy of infectious diseases and cancer. However, the generation of defined TCR-transgenic T cell medicinal products with predictable in vivo function still poses a major challenge and limits broader and more successful application of this “living drug.” Here, by studying 51 different TCRs, we show that conventional genetic engineering by viral transduction leads to variable TCR expression and functionality as a result of variable transgene copy numbers and untargeted transgene integration. In contrast, CRISPR/Cas9-mediated TCR replacement enables defined, targeted TCR transgene insertion into the TCR gene locus. Thereby, T cell products display more homogeneous TCR expression similar to physiological T cells. Importantly, increased T cell product homogeneity after targeted TCR gene editing correlates with predictable in vivo T cell responses, which represents a crucial aspect for clinical application in adoptive T cell immunotherapy.

Plant Genetics as a Tool for Manipulating Crop Microbiomes: Opportunities and Challenges.

Growing human population size and the ongoing climate crisis create an urgent need for new tools for sustainable agriculture. Because microbiomes have profound effects on host health, interest in methods of manipulating agricultural microbiomes is growing rapidly. Currently, the most common method of microbiome manipulation is inoculation of beneficial organisms or engineered communities; however, these methods have been met with limited success due to the difficulty of establishment in complex farm environments. Here we propose genetic manipulation of the host plant as another avenue through which microbiomes could be manipulated. We discuss how domestication and modern breeding have shaped crop microbiomes, as well as the potential for improving plant-microbiome interactions through conventional breeding or genetic engineering. We summarize the current state of knowledge on host genetic control of plant microbiomes, as well as the key challenges that remain.

Over-Expression of a Melon Y3SK2-Type LEA Gene Confers Drought and Salt Tolerance in Transgenic Tobacco Plants.

Climate change, with its attendant negative effects, is expected to hamper agricultural production in the coming years. To counteract these negative effects, breeding of environmentally resilient plants via conventional means and genetic engineering is necessary. Stress defense genes are valuable tools by which this can be achieved. Here we report the successful cloning and functional characterization of a melon Y3SK2-type dehydrin gene, designated as CmLEA-S. We generated CmLEA-S overexpressing transgenic tobacco lines and performed in vitro and in vivo drought and salt stress analyses. Seeds of transgenic tobacco plants grown on 10% polyethylene glycol (PEG) showed significantly higher germination rates relative to wild-type seeds. In the same way, transgenic seeds grown on 150 mM sodium chloride (NaCl) recorded significantly higher germination percentages compared with wild-type plants. The fresh weights and root lengths of young transgenic plants subjected to drought stress were significantly higher than that of wild-type plants. Similarly, the fresh weights and root lengths of transgenic seedlings subjected to salt stress treatments were also significantly higher than wild-type plants. Moreover, transgenic plants subjected to drought and salt stresses in vivo showed fewer signs of wilting and chlorosis, respectively. Biochemical assays revealed that transgenic plants accumulated more proline and less malondialdehyde (MDA) compared with wild-type plants under both drought and salt stress conditions. Finally, the enzymatic activities of ascorbate peroxidase (APX) and catalase (CAT) were enhanced in drought- and salt-stressed transgenic lines. These results suggest that the CmLEA-S gene could be used as a potential candidate gene for crop improvement.

Micronutrient Biofortification in Rice through New Breeding Techniques (NBTs): Bangladesh Perspective

Background: Micronutrient deficiencies are serious health issues in developing countries of Asia and Africa, where millions of people are suffering from inadequate micronutrient intake. In Bangladesh, micronutrient deficiencies are found severe due to low income, food habits, and rice-based staple food consumption, (rice has an insufficiency of different types of vitamins and minerals). To lessen micronutrient malnutrition, supplementation has been employed but has not yet reached the goal. Agronomic and genetic biofortification has the potential to address micronutrient deficiencies. Biofortification in Rice grain is a convenient and affordable way to supply the desired micronutrients. The development of micronutrient-rich popular rice cultivars through conventional breeding is currently being harnessed for the limitation of natural resources of the related donor rice cultivars containing the required amount of micronutrients. To overcome these hurdles of conventional breeding, genetic engineering and genome editing have emerged as promising tools of micronutrient biofortification in rice. Methods: Identify the needs and explore the potential strategies by the search for relevant literature known to the authors was carried out to complete this review. Results: Highlighted here the sources, functions, and requirements of iron, zinc, vitamin A, vitamin B1, vitamin B9, and betanin in rice and their biofortification through conventional breeding, genetic engineering, and genome editing including their promises and hindrances. Conclusion: New breeding techniques are timely alternatives for developing nutrient-rich rice cultivars to eliminate hidden hunger and poverty in Bangladesh.

Saccharomyces cerevisiae in making halal products based on conventional biotechnology and genetic engineering

Indonesia is the nation with the world's largest Muslim population. Halal products is very important for the life of a Muslim. This includes the fulfillment of halal food, especially food-processed materials. Saccharomyces cerevisiae has contributed a lot in biotechnological processes in both conventional and modern biotechnology genetic engineering. Conventional biotechnology has been around since time immemorial by utilizing microbes for the manufacture of low-carbohydrate, long-lasting foods and drinks. The products produced also vary, some are halal or haram. Halal products such as bread, tape and bioethanol are used as fuel. Illegal products include intoxicating drinks such as sake. The development of modern biotechnology technologies that are currently developing such as genetic engineering in the manufacture of bioethanol which can be used as an alternative fuel that is environmentally friendly.

Genetic diversity and SNP's from the chloroplast coding regions of virus-infected cassava.

Cassava is a staple food crop in sub-Saharan Africa; it is a rich source of carbohydrates and proteins which currently supports livelihoods of more than 800 million people worldwide. However, its continued production is at stake due to vector-transmitted diseases such as Cassava mosaic disease and Cassava brown streak disease. Currently, the management and control of viral diseases in cassava relies mainly on virus-resistant cultivars of cassava. Thus, the discovery of new target genes for plant virus resistance is essential for the development of more cassava varieties by conventional breeding or genetic engineering. The chloroplast is a common target for plant viruses propagation and is also a potential source for discovering new resistant genes for plant breeding. Non-infected and infected cassava leaf samples were obtained from different locations of East Africa in Tanzania, Kenya and Mozambique. RNA extraction followed by cDNA library preparation and Illumina sequencing was performed. Assembling and mapping of the reads were carried out and 33 partial chloroplast genomes were obtained. Bayesian phylogenetic analysis from 55 chloroplast protein-coding genes of a dataset with 39 taxa was performed and the single nucleotide polymorphisms for the chloroplast dataset were identified. Phylogenetic analysis revealed considerable genetic diversity present in chloroplast partial genome among cultivated cassava of East Africa. The results obtained may supplement data of previously selected resistant materials and aid breeding programs to find diversity and achieve resistance for new cassava varieties.

A review on corn breeding for insect pest resistance

Significant yield reduction and effect on almost every aspect of the plants by insect pests have been a mega problem in agricultural crops. Scientist tackle with many challenges to develop highly efficient techniques either through conventional breeding or modern genetic engineering to understand the mechanism of resistance and its application for benefit of human kind. Antibiosis, antixenosis and tolerance are the resistance mechanisms which have been developed for successful control of economically important insect pests in corn. Plant morphology and allelochemicals, induced resistance, callus tissue culture and genetic transformation were used as major tools to advance resistance by corn breeders. Insect pest resistant corn has been attributed for social, economical as well as environmental benefits. However, outcome of these achievements are not reflected due to low use of insect resistant corn by farmers in many developing countries of the world.

Animal transgenesis now and beyond in the era of genome editing: Snapshots from the 15th Transgenic Technology Meeting (TT2019) in Kobe, Japan.

The 15th Transgenic Technology (TT) Meeting of the International Society for Transgenic Technologies (ISTT) was held for the first time in Japan in April of 2019 (TT2019). Delegates from around the world, including researchers, technicians and trainees, spent an exciting 4days, engaging in scientific and technical discussions, information sharing and networking. In the era of genome editing, CRISPR technologies prevailed as popular subjects of discussion throughout the meeting on topics ranging from their applications in the mouse and growing number of other research and industrial animal species, technical challenges in the production of genome-edited animals, to ethical considerations in biomedical research. In particular, impressive progress was reported on the use of CRISPR technologies in nonconventional animal models and large mammalian species in biomedical and industrial settings, indicating areas of expanding frontiers in animal transgenesis. Amid growing excitement with genome editing technologies, reports on conventional genetic engineering approaches and reproductive biology techniques demonstrated that these techniques remain essential to meet the demanding needs of generating complex genome modifications, complementing genome editing approaches. TT2019 was a great success, concluding with a widely shared appreciation of the current field and hints for future directions of animal transgenesis.

Generation of selectable marker-free soft transgenic rice with transparent kernels by downregulation of SSSII-2

The amylose content (AC) of rice endosperm starch varies from 0 to 35%, and is associated with rice cooking and eating quality. Soft rice has low AC, generally between 6% and 15%, and its eating quality is high whether it is consumed hot or cold. However, the appearance quality of current soft rice cultivars needs to be improved, especially opaque endosperm. Conventional genetic engineering has improved some agronomic traits of soft rice varieties, but not endosperm appearance. In the present study, a RNAi construct of the soluble starch synthase II-2 (SSSII-2) and the hygromycin phosphotransferase (HPT) gene were introduced into an elite japonica rice variety, Kangtiaowuyunjing (KWY8) by co-transformation. Several selectable marker-free (SMF) transgenic lines were obtained, and SSSII-2 expression was significantly downregulated in selected transgenic lines, resulting in lower AC of the endosperm. The physicochemical properties of the transgenic rice kernels, including gel consistency (GC) and rapid visco analyzer (RVA) profile, differed significantly from those of wild-type rice and were similar to those of a soft rice variety, Nanjing 46 (NJ46). These findings indicate that the cooking, eating, and processing qualities of transgenic rice are comparable to those of NJ46. However, the transgenic rice endosperm retained a transparent appearance under low-moisture conditions. Thus, SMF SSSII-2 RNAi rice provides a resource for breeding soft rice with transparent endosperm.