Stable germline transgenesis using the Minos Tc1/mariner element in the sea urchin Lytechinus pictus.

Stable transgenesis is a transformative tool in model organism biology. Although the sea urchin is one of the oldest animal models in cell and developmental biology, studies in this animal have largely relied on transient manipulation of wild animals, without a strategy for stable transgenesis. Here, we build on recent progress to develop a more genetically tractable sea urchin species, Lytechinus pictus, and establish a robust transgene integration method. Three commonly used transposons (Minos, Tol2 and piggyBac) were tested for non-autonomous transposition, using plasmids containing a polyubiquitin promoter upstream of a H2B-mCerulean nuclear marker. Minos was the only transposable element that resulted in significant expression beyond metamorphosis. F0 animals were raised to sexual maturity, and spawned to determine germline integration and transgene inheritance frequency, and to characterize expression patterns of the transgene in F1 progeny. The results demonstrate transgene transmission through the germline, the first example of a germline transgenic sea urchin and, indeed, of any echinoderm. This milestone paves the way for the generation of diverse transgenic resources that will dramatically enhance the utility, reproducibility and efficiency of sea urchin research.

Overexpression of plant chitin receptors in wheat confers broad-spectrum resistance to fungal diseases.

Wheat (Triticum aestivum L.) is a globally staple crop vulnerable to various fungal diseases, significantly impacting its yield. Plant cell surface receptors play a crucial role in recognizing pathogen-associated molecular patterns (PAMPs) and activating PAMP-triggered immunity, boosting resistance against a wide range of plant diseases. Although the role of plant chitin receptor CERK1 in immune recognition and defense has been established in Arabidopsis and rice, its function and potential agricultural applications in enhancing resistance to crop diseases remain largely unexplored. Here, we identify and characterize TaCERK1 in Triticeae crop wheat, uncovering its involvement in chitin recognition, immune regulation, and resistance to fungal diseases. By a comparative analysis of CERK1 homologs in Arabidopsis and monocot crops, we demonstrate that AtCERK1 in Arabidopsis elicits the most robust immune response. Moreover, we show that overexpressing TaCERK1 and AtCERK1 in wheat confers resistance to multiple fungal diseases, including Fusarium head blight, stripe rust, and powdery mildew. Notably, transgenic wheat lines with moderately expressed AtCERK1 display superior disease resistance and heightened immune responses without adversely affecting growth and yield, compared to TaCERK1 overexpression transgenics. Our findings highlight the significance of plant chitin receptors across diverse plant species and suggest potential strategies for bolstering crop resistance against broad-spectrum diseases in agricultural production through the utilization of plant immune receptors.

Advancements in genetically modified insect pest-resistant crops in India.

MAIN CONCLUSION: The review offers insights into the current state of research on insect pest-resistant GM crops and the regulations governing the cultivation of GM crops in India. India has a rich crop diversity of more than 160 major and minor crops through its diverse agroclimatic conditions. Insect pests alone cause around USD 36 billion in crop loss annually in India. The last two decades witnessed considerable progress in managing insect pests by adopting innovative techniques including transgenics. In research, significant advancement has been brought in insect pest-resistant transgenics in India since its inception in 2002. However, any events have not been endorsed owing to biosafety impediments, except Bt cotton reaching the commercial release stage. A landmark decision to exempt certain types of gene-edited plants from genetically modified organism (GMO) regulations offers great promise for developing novel insect-resistant crops in India. The article reviews the current research on insect pest-resistant transgenics and its regulations in India.

Latin America: A hub for agrobiotechnological innovations.

BACKGROUND: Modern biotechnology is one of the last century's major advances in human science. Particularly in the agronomical field, the landscape of crop improvement technologies has witnessed a great expansion, driven by the integration of molecular and genetic engineering methodologies into the breeding toolbox. Latin America (LATAM) serves as a pioneering region in incorporating such techniques with several countries swiftly embracing these technologies. SCOPE: This review aims to give a comprehensive overview of the elements that influenced agrobiotech acceptance in LATAM countries and how such cases could provide support for upcoming technologies to be considered worldwide. CONCLUSIONS: Nearly 50 years of biotech breakthroughs have provided humankind with an impressive portfolio of tools already integrated into several life-sciences areas. The agronomical field has greatly progressed thanks to technologies derived from Genetically Modified Organisms (GMOs) and high promises are being made to also incorporate genome -editing products. LATAM's case is a prime example of how early introduction of novelties in the crop production chain can result in improved yields, paving the way for future developments to be easily integrated into the technological ecosystem of a region. The example set by LATAM can also be useful for the present gene-editing regulatory scenario. With several countries presently on the path to approving these methods in their current crop systems, basing their next steps on the southern continent's example, could represent a safe and practical pathway towards a new agronomical revolution.

Sulfate transport and metabolism: strategies to improve the seed protein quality.

Sulfur-containing amino acids (SAA), namely methionine, and cysteine are crucial essential amino acids (EAA) considering the dietary requirements of humans and animals. However, a few crop plants, especially legumes, are characterized with suboptimal levels of these EAA thereby limiting their nutritive value. Hence, improved comprehension of the mechanistic perspective of sulfur transport and assimilation into storage reserve, seed storage protein (SSP), is imperative. Efforts to augment the level of SAA in seed storage protein form an integral component of strategies to balance nutritive quality and quantity. In this review, we highlight the emerging trends in the sulfur biofortification approaches namely transgenics, genetic and molecular breeding, and proteomic rebalancing with sulfur nutrition. The transgenic 'push and pull strategy' could enhance sulfur capture and storage by expressing genes that function as efficient transporters, sulfate assimilatory enzymes, sulfur-rich foreign protein sinks, or by suppressing catabolic enzymes. Modern molecular breeding approaches that adopt high throughput screening strategies and machine learning algorithms are invaluable in identifying candidate genes and alleles associated with SAA content and developing improved crop varieties. Sulfur is an essential plant nutrient and its optimal uptake is crucial for seed sulfur metabolism, thereby affecting seed quality and yields through proteomic rebalance between sulfur-rich and sulfur-poor seed storage proteins.

Overexpression of rice lectin receptor-like kinase, OsLec-RLK, confers salinity stress tolerance and increases seed yield in pigeon pea (Cajanus cajan (L.) Millsp.).

KEY MESSAGE: OsLec-RLK overexpression enhances cell signalling and salt stress tolerance in pigeon pea, enhancing seed yield and harvest index and thus, enabling marginal lands to increase food and nutritional security. Lectin Receptor-like kinases (Lec-RLKs) are highly effective cell signaling molecules that counteract various stresses, including salt stress. We engineered pigeon pea by overexpressing OsLec-RLK gene for enhancing salt tolerance. The OsLec-RLK overexpression lines demonstrated superior performance under salt stress, from vegetative to reproductive phase, compared to wild types (WT). The overexpression lines had significantly higher K+/Na+ ratio than WT exposed to 100 mM NaCl. Under salt stress, transgenic lines showed higher levels of chlorophyll, proline, total soluble sugars, relative water content, and peroxidase and catalase activity than WT plants. Membrane injury index and lipid peroxidation were significantly reduced in transgenic lines. Analysis of phenological and yield attributes confirmed that the OsLec-RLK pigeon pea lines maintain plant vigor, with 10.34-fold increase in seed yield (per plant) and 4-5-fold increase in harvest index of overexpression lines, compared to wild type. Meanwhile, the overexpression of OsLec-RLK up-regulated the expression levels of histone deacetylase1, acyl CoA, ascorbate peroxidase, peroxidase, glutathione reductase and catalase, which were involved in the K+/Na+ homeostasis pathway. This study showed the potential of OsLec-RLK gene for increasing crop productivity and yields under salt stress and enabling the crops to be grown on marginal lands for increasing food and nutritional security.

From dysbiosis to neuropathologies: Toxic effects of glyphosate in zebrafish.

Glyphosate, a globally prevalent herbicide known for its selective inhibition of the shikimate pathway in plants, is now implicated in physiological effects on humans and animals, probably due to its impacts in their gut microbiomes which possess the shikimate pathway. In this study, we investigate the effects of environmentally relevant concentrations of glyphosate on the gut microbiota, neurotransmitter levels, and anxiety in zebrafish. Our findings demonstrate that glyphosate exposure leads to dysbiosis in the zebrafish gut, alterations in central and peripheral serotonin levels, increased dopamine levels in the brain, and notable changes in anxiety and social behavior. While the dysbiosis can be attributed to glyphosate's antimicrobial properties, the observed effects on neurotransmitter levels leading to the reported induction of oxidative stress in the brain indicate a novel and significant mode of action for glyphosate, namely the impairment of the microbiome-gut-axis. While further investigations are necessary to determine the relevance of this mechanism in humans, our findings shed light on the potential explanation for the contradictory reports on the safety of glyphosate for consumers.

A glutathione-independent DJ-1/PfpI domain-containing tomato glyoxalaseIII2, SlGLYIII2, confers enhanced tolerance under salt and osmotic stresses.

In plants, glyoxalase enzymes are activated under stress conditions to mitigate the toxic effects of hyperaccumulated methylglyoxal (MG), a highly reactive carbonyl compound. Until recently, a glutathione-dependent bi-enzymatic pathway involving glyoxalase I (GLYI) and glyoxalase II (GLYII) was considered the primary MG-detoxification system. Recently, a new glutathione-independent glyoxalase III (GLYIII) mediated direct route was also reported in plants. However, the physiological significance of this new pathway remains to be elucidated across plant species. This study identified the full complement of 22 glyoxalases in tomato. Based on their strong induction under multiple abiotic stresses, SlGLYI4, SlGLYII2 and SlGLYIII2 were selected candidates for further functional characterisation. Stress-inducible overexpression of both glutathione-dependent (SlGLYI4 + SlGLYII2) and independent (SlGLYIII2) pathways led to enhanced tolerance in both sets of transgenic plants under abiotic stresses. However, SlGLYIII2 overexpression (OE) plants outperformed the SlGLYI4 + SlGLYII2 OE counterparts for their stress tolerance under abiotic stresses. Further, knockdown of SlGLYIII2 resulted in plants with exacerbated stress responses than those silenced for both SlGLYI4 and SlGLYII2. The superior performance of SlGLYIII2 OE tomato plants for better growth and yield under salt and osmotic treatments could be attributed to better GSH/GSSG ratio, lower reactive oxygen species levels, and enhanced antioxidant potential, indicating a prominent role of GLYIII MG-detoxification pathway in abiotic stress mitigation in this species.

Recent advances in arsenic mitigation in rice through biotechnological approaches.

Arsenic (As) is a major threat to the environment and human health due to its toxicity and carcinogenicity. Occurrence of alarming concentrations of As in water and soil leads to its bioaccumulation in crops which is a major health concern globally. Rice (Oryza sativa) is a staple food for a large population staying in As contaminated areas so, it is of utmost importance to reduce As levels in rice, especially grains. Amongst several strategies in practice, biotechnology may provide an effective option to reduce As accumulation in rice grains. Genetic engineering can be a viable approach to exploit potential genes playing roles in As metabolism pathway in plants. Besides, developing low As accumulating rice varieties through breeding is also an important area. Identifying genotypic variation in rice is a crucial step toward the development of a safe rice cultivar for growing in As-affected areas. Significant genotypic variation has been found in rice varieties for As accumulation in grains and that is attributable to differential expression of transporters, radial oxygen loss, and other regulators of As stress. This review provides recent updates on the research advances leading to transgenic and breeding approaches adopted to reduce As levels in rice, especially grains.

Arsenic (As) contamination in water, soil, and crops is creating a difficult situation for the large population across the globe. Various efforts are being made to reduce As levels in rice as it is a staple crop. This review presents recent biotechnological advances toward the development of low As accumulating rice. The review shall be an important information resource for the readers on the topic.

Gene and transgenics nomenclature for the laboratory axolotl-Ambystoma mexicanum.

The laboratory axolotl (Ambystoma mexicanum) is widely used in biological research. Recent advancements in genetic and molecular toolkits are greatly accelerating the work using axolotl, especially in the area of tissue regeneration. At this juncture, there is a critical need to establish gene and transgenic nomenclature to ensure uniformity in axolotl research. Here, we propose guidelines for genetic nomenclature when working with the axolotl.

The use of transgenics in the laboratory axolotl.

The ability to generate transgenic animals sparked a wave of research committed to implementing such technology in a wide variety of model organisms. Building a solid base of ubiquitous and tissue-specific reporter lines has set the stage for later interrogations of individual cells or genetic elements. Compared to other widely used model organisms such as mice, zebrafish and fruit flies, there are only a few transgenic lines available in the laboratory axolotl (Ambystoma mexicanum), although their number is steadily expanding. In this review, we discuss a brief history of the transgenic methodologies in axolotl and their advantages and disadvantages. Next, we discuss available transgenic lines and insights we have been able to glean from them. Finally, we list challenges when developing transgenic axolotl, and where further work is needed in order to improve their standing as both a developmental and regenerative model.

Origin and development of circumventricular organs in living vertebrate.

The circumventricular organs (CVOs) function by mediating chemical communication between blood and brain across the blood-brain barrier. Their origin and developmental mechanisms involved are not understood in enough detail due to a lack of molecular markers common for CVOs. These rather small and inconspicuous organs are found in close vicinity to the third and fourth brain ventricles suggestive of ancient evolutionary origin. Recently, an integrated approach based on analysis of CVOs development in the enhancer-trap transgenic zebrafish led to an idea that almost all of CVOs could be highlighted by GFP expression in this transgenic line. This in turn suggested that an enhancer along with a set of genes it regulates may illustrate the first common element of developmental regulation of CVOs. It seems to be related to a mechanism of suppression of the canonical Wnt/ ß-catenin signaling that functions in development of fenestrated capillaries typical for CVOs. Based on that observation the common molecular elements of the putative developmental mechanism of CVOs will be discussed in this review.

Advances in the regulation of plant salt-stress tolerance by miRNA.

Salt stress significantly affects the growth, development, yield, and quality of plants. MicroRNAs (miRNAs) are involved in various stress responses via target gene regulation. Their role in regulating salt stress has also received significant attention from researchers. Various transcription factor families are the common target genes of plant miRNAs. Thus, regulating the expression of miRNAs is a novel method for developing salt-tolerant crops. This review summarizes plant miRNAs that mediate salt tolerance, specifically miRNAs that have been utilized in genetic engineering to modify plant salinity tolerance. The molecular mechanism by which miRNAs mediate salt stress tolerance merits elucidation, and this knowledge will promote the development of miRNA-mediated salt-tolerant crops and provide new strategies against increasingly severe soil salinization.

Unravelling the multi-faceted regulatory role of polyamines in plant biotechnology, transgenics and secondary metabolomics.

Polyamines (PAs) are ubiquitous low-molecular-weight, aliphatic compounds with wide as well as complex application in fundamental areas of plant growth and development. PAs are mediator of basic metabolism of organisms which include cell division and differentiation, biotic and abiotic stress tolerance, reversal of oxidative damage, stabilization of nucleic acids, and protein and phospholipid binding. In plants, it attributes in direct and indirect organogenesis, endogenous phytohormone regulation, cellular compartmentalization, fruit and flower development, senescence, and secondary metabolite production which are highly tuned as first line of defense response. There are several aspects of polyamine-directed mechanism that regulate overall plant growth in vitro and in vivo. In the present review, we have critically discussed the role played by polyamine on the enhanced production of bioactive natural products and how the same polyamines are functioning against different environmental stress conditions, i.e., salinity, drought, high CO2 content, herbivory, and physical wounding. The role of polyamines on elicitation process has been highlighted previously, but it is important to note that its activity as growth regulator under in vitro condition is correlated with an array of intertwined mechanism and physiological tuning. Medicinal plants under different developmental stages of micropropagation are characterized with different functional aspects and regulatory changes during embryogenesis and organogenesis. The effect of precursor molecules as well as additives and biosynthetic inhibitors of polyamines in rhizogenesis, callogenesis, tuberization, embryogenesis, callus formation, and metabolite production has been discussed thoroughly. The beneficial effect of exogenous application of PAs in elicitation of secondary metabolite production, plant growth and morphogenesis and overall stress tolerance are summarized in this present work. KEY POINTS: • Polyamines (PAs) play crucial roles in in vitro organogenesis. • PAs elicitate bioactive secondary metabolites (SMs). • Transgenic studies elucidate and optimize PA biosynthetic genes coding SMs.

Transcriptional loss of domestication-driven cytoskeletal GhPRF1 gene causes defective floral and fiber development in cotton (Gossypium).

KEY MESSAGE: Constitutive- and fiber-specific RNAi of GhPRF1 gene illustrated strong correlation between domestication-driven profilin genes and floral/fiber architecture in cotton. During morpho-transformation of short-fuzz of wild cotton into the elongating spinnable fibers under the millennia of human selection, actin-polymerizing cytoskeletal profilin genes had undergone significant sequence alterations and spatiotemporal shift in their transcription levels. To comprehend the expression dynamics of profilin genes with their phenotypic implications, transgenic expression modulation of cotton profilin 1 (GhPRF1) gene was performed in the constitutive- and fiber-specific manner in Coker 310FR cotton cultivar. The constitutive GhPRF1-RNAi lines (35S:GhPRF1-RNAi) exhibited distorted 'monadelphous' staminal-tube, reduced pollen-viability and poorly developed fibers, whereas floral and fiber development of fiber-specific GhPRF1-RNAi lines showed no abnormalities. Moreover, the fiber-specific GhPRF1 overexpression lines (FBP7:GhPRF1-Ox) showed increased emergence of fiber-initials on the ovule surface, on the contrary to no fiber-initials in fiber-specific RNAi lines (FBP7:GhPRF1-RNAi). Interestingly, the average seed weight and fiber weight of FBP7:GhPRF1-Ox lines increased > 60% and > 38%, respectively, compared with FBP7:GhPRF1-RNAi lines and untransformed control seeds. On a molecular basis, the aberrant floral and fiber development of 35S:GhPRF1-RNAi lines was largely associated with sugar metabolism and hormone-signaling mechanisms. These observations illustrated the strong correlation between domestication-driven GhPRF genes, and floral/fiber development in cotton. Also, the enhanced agronomic traits in GhPRF1-Ox lines of cotton empowered us to recognize their imperative roles, and their future deployment for the sustainable cotton crop improvement.

SLAM-ITseq: sequencing cell type-specific transcriptomes without cell sorting.

Cell type-specific transcriptome analysis is an essential tool for understanding biological processes in which diverse types of cells are involved. Although cell isolation methods such as fluorescence-activated cell sorting (FACS) in combination with transcriptome analysis have widely been used so far, their time-consuming and harsh procedures limit their applications. Here, we report a novel in vivo metabolic RNA sequencing method, SLAM-ITseq, which metabolically labels RNA with 4-thiouracil in a specific cell type in vivo followed by detection through an RNA-seq-based method that specifically distinguishes the thiolated uridine by base conversion. This method has successfully identified the cell type-specific transcriptome in three different tissues: endothelial cells in brain, epithelial cells in intestine and adipocytes in white adipose tissue. As this method does not require isolation of cells or RNA prior to the transcriptomic analysis, SLAM-ITseq provides an easy yet accurate snapshot of the transcriptional state in vivo.

Biofortification and bioavailability of Zn, Fe and Se in wheat: present status and future prospects.

KEY MESSAGE: Knowledge of genetic variation, genetics, physiology/molecular basis and breeding (including biotechnological approaches) for biofortification and bioavailability for Zn, Fe and Se will help in developing nutritionally improved wheat. Biofortification of wheat cultivars for micronutrients is a priority research area for wheat geneticists and breeders. It is known that during breeding of wheat cultivars for productivity and quality, a loss of grain micronutrient contents occurred, leading to decline in nutritional quality of wheat grain. Keeping this in view, major efforts have been made during the last two decades for achieving biofortification and bioavailability of wheat grain for micronutrients including Zn, Fe and Se. The studies conducted so far included evaluation of gene pools for contents of not only grain micronutrients as above, but also for phytic acid (PA) or phytate and phytase, so that, while breeding for the micronutrients, bioavailability is also improved. For this purpose, QTL interval mapping and GWAS were carried out to identify QTLs/genes and associated markers that were subsequently used for marker-assisted selection (MAS) during breeding for biofortification. Studies have also been conducted to understand the physiology and molecular basis of biofortification, which also allowed identification of genes for uptake, transport and storage of micronutrients. Transgenics using transgenes have also been produced. The breeding efforts led to the development of at least a dozen cultivars with improved contents of grain micronutrients, although land area occupied by these biofortified cultivars is still marginal. In this review, the available information on different aspects of biofortification and bioavailability of micronutrients including Zn, Fe and Se in wheat has been reviewed for the benefit of those, who plan to start work or already conducting research in this area.

Timely gene detection assay and reliable screening of genetically engineered plants using an improved direct PCR-based technology.

Engineered plants have been widely produced for fundamental and practical use. Several methods have been developed for genetically modified crop detection and quantification; however; they still laborious and expensive. Efforts are needed to set-up diagnosis-oriented techniques as alternatives to overcome DNA extraction which remains a tedious and time-consuming procedure. Here, we established a standard direct PCR workflow using a regular Taq polymerase without prior DNA purification over a wide range of plant species. Only a small amount of fresh tissue allowed direct amplification of target gene sequences. Evaluation of accuracy, sensitivity, and reproducibility of direct PCR assay was investigated for proof-of-concept, and subsequently applied to gene detection assays and rapid transgenic revealing. The newly established method achieved full success and has amplified constitutive housekeeping genes from several plant specimens in a reproducible manner with high-quality sequencing profiles. In our case, the screening of transgenic plants confirmed that both the gfp-ER reporter gene and the npt II selectable marker were integrated into the plant genome. This direct PCR approach provides a powerful tool for large-scale PCR-based gene detection making DNA purification irrelevant. It could be easily implemented for downstream applications in the field of genetic fingerprinting, plant biotechnology, and functional genomics.

Emerging Molecular Strategies for Improving Rice Drought Tolerance.

Rice occupies a pre-eminent position as a food crop in the world. Its production, how- ever, entails up to 3000 liters of water per kilogram of grain produced. Such high demand makes rice prone to drought easily. Sustainable rice cultivation with limited water resources requires the deployment of a suitable strategy for better water use efficiency and improved drought tolerance. Several drought-related genes have been evaluated in rice for their mode of action in conferring drought tolerance. Manipulation of components of abscisic acid signal transduction, stomatal density, deposition of cuticular wax, and protein modification pathways are emerging as priority targets. Gene reprogramming by microRNAs is also being explored to achieve drought tolerance. Genetically dissected Quantitative Trait Loci (QTLs) and their constituent genes are being deployed to develop drought-tolerant rice varieties. Progressive research and challenges include a better understanding of crucial components of drought response and search for new targets and the deployment of improved varieties in the field.

Overexpression of AtDREB1 and BcZAT12 genes confers drought tolerance by reducing oxidative stress in double transgenic tomato (Solanum lycopersicum L.).

KEY MESSAGE: Double transgenic tomato developed by AtDREB1A and BcZAT12 genes pyramiding showed significant drought tolerance by reducing oxidative stress with enhanced yield. Although a large number of efforts have been made by different researchers to develop abiotic stress tolerance tomato for improving yield using single gene, however, no reports are available which targets AtDREB1 and BcZAT12 genes together. Hence, in the present study, double transgenic plants were developed using AtDREB1 and BcZAT12 genes to improve yield potential with better drought tolerance. Double transgenic (DZ1-DZ5) tomato lines showed enhanced drought tolerance than their counterpart non-transgenic and single transgenic plants at 0, 07, 14, and 21 days of water deficit, respectively. Double transgenic plants showed increased activity of antioxidant enzymes, like catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR) and guaiacol peroxidase (POD), and accumulation of non-enzymatic antioxidants like ascorbic acid, glutathione as compared to non-transgenic and single transgenic. Additionally, the transcript analysis of antioxidant enzymes revealed the increased level of gene expression in double transgenic tomato lines. Developed double-transgenic tomato plants co-over-expressing both genes exhibited more enzymatic and non-enzymatic anti-oxidative activities as compared to the non-transgenic and single transgenic control, respectively. This is the preliminary report in tomato, which forms the basis for a multigene transgenic approach to cope with drought stress.