The study aimed to construct and evaluate an independently generated transgenic mouse model applied to the short-term carcinogenicity assessment. Mutated human HRAS fragment containing an intron point-mutation was inserted into C57BL/6JGpt mice via bacterial artificial chromosome transgenic technology, eventually generating BALB/c;B6J-Tg(hHRAS)16/Gpt mice, abbreviated as HRAS mice. The inserted human HRAS fragment in HRAS mice was characterized, revealing five tandem copies at chromosome 19. Baseline profiles, including biochemical, hematological, immunophenotypic, survival, and carcinogenic data of HRAS mice, were collected. To evaluate the tumor susceptibility in HRAS mice, we applied N-Nitroso-N-methylurea (MNU) to HRAS mice in a short-term carcinogenicity assessment conducted according to Good Laboratory Practice. The genetic characteristics of HRAS mice include five tandem arrays of mutated human HRAS fragments located in genomic coordinate 7,755,606 on chromosome 19 and the duplication of a 9-kilobase genome sequence (genomic coordinate 7,755,606–7,746,509) located on chromosome 19. HRAS mice showed a relatively lower incidence and range of spontaneous tumor formation during long-term observation compared to CByB6F1-Tg(HRAS)2Jic (Tg.rasH2) transgenic mice. The short-term carcinogenicity assessment showed a strong tumor response to MNU, with high incidences of lymphoma (≥ 90
This study reports the first isolation and characterization of the buffalo EF1α1 promoter, demonstrating its strong gene expression activity both in vitro across diverse cultured cell types and in vivo across multiple mouse organs. Although viral promoters, such as cytomegalovirus (CMV) and simian virus (SV40), are widely used for their strong expression in various cell lines in mammalian expression systems and in animal tissues, they are prone to methylation-induced transcriptional silencing and subsequent loss of exogenous gene expression. The most effective alternative to viral promoters is the synthetic hybrid CAG promoter (cytomegalovirus major immediate-early enhancer combined with the chicken beta-actin promoter) or mammalian cellular promoter such as human elongation factor 1 alpha (hEF1α), which drives strong gene expression but lacks consistency and is limited in their in vivo expression potential due to their vulnerability to epigenetic silencing. To overcome these challenges, the bbEF1α1 promoter was cloned and evaluated both in vitro and in vivo. It consistently drives higher levels of exogenous gene expression than CMV in diverse cell lines. Importantly, transgene expression was achieved in various organs of transgenic mice and in muscle tissue following in vivo electroporation. These findings establish the bbEF1α1 promoter as a powerful ubiquitous driver of gene expression, offering high stability with broad applications in gene therapy, biopharmaceutical production, and functional genomics.
Noccaea caerulescens is a metal hyperaccumulator plant species with the capacity to accumulate high concentrations of zinc (Zn), cadmium (Cd) and nickel (Ni). Several candidate genes have so far been associated with accumulation and tolerance of these metals, but gene function analysis has been cumbersome in the absence of an efficient stable plant transformation method for N. caerulescens. A previously identified mutation in the FLOWERING LOCUS C gene, conferring early flowering to the calamine accession St. Felix de Pallières, has been introgressed in the genetic background of five different genotypes, originating from the calamine populations Clough Wood (CLW) and Le Blémard (BLE), the ultramafic population Cira (CIR), and the non-metallicolous populations St. Baudille (SBD) and Werschmatt (WER). Agrobacterium tumefaciens mediated floral dipping transformation was employed in five introgression lines in these backgrounds and in Arabidopsis thaliana as control. Three introgression lines, of genetic backgrounds CLW, BLE and SBD, were successfully transformed, achieving an average transformation efficiency ≥ 0.29
This review evaluates CRISPR/Cas applications in agriculture from a global perspective with explicit reference to Türkiye. Using a literature gap-matrix approach organised around four analytical dimensions—environmental, economic, social and policy, and scientific and technological—we synthesize the primary evidence on water and input use, productivity, disease resistance, and product quality. The literature concentrates on water and fertilizer use, productivity, and off-target accuracy, whereas soil health, biodiversity, consumer acceptance, ethical considerations and regulatory frameworks remain systematically under-represented. Global deployment of CRISPR is already delivering measurable advantages in food security, shelf life and nutritional value, while in Türkiye the research base is at an early stage but has clear potential in wheat, barley, tomato and olive. Translating CRISPR into Turkish agricultural sustainability requires (i) a domestic biosafety framework aligned with the emerging European New Genomic Techniques approach, (ii) sustained investment in multi-location primary field trials, and (iii) inclusive deployment mechanisms—particularly through producer cooperatives—that allow smallholder farmers to benefit from edited varieties.
Genome editing technologies are now available for many crop species, greatly enhancing our ability to investigate gene function and transforming the field of plant transgenesis. However, the capacity to regenerate whole plants from cell culture remains a major limiting factor in many crops. Even in species with regeneration potential, certain genotypes remain recalcitrant. The physiological state of plant cells plays a central role in growth and development and is closely associated with kinase-mediated signaling networks. Notably, several defense-related genes activated during cellular repair processes following transgenesis share significant homology with mammalian defense genes. In this study, we evaluated whether supplementation with three mammalian growth factors could enhance regeneration efficiency in tomato. We selected two cytokines and a pro-inflamatory factor showing homology with plant kinase genes. We compared the percentage of transgenic plants generated through CRISPR-Cas9-mediated mutagenesis of four genes involved in sugar and organic acid metabolism across six tomato lines exhibiting varying regeneration capacities. Over three years of transformation experiments, we demonstrated that the addition of mammalian growth factors during transgenesis significantly improved regeneration frequency, particularly in recalcitrant tomato genotypes. Furthermore, growth factor supplementation not only enhanced transformation efficiency in difficult-to-transform lines but also increased the production of stable secondary lines.
The immunodeficient mouse model offers a platform for evaluating therapeutic options in preclinical cancer research by enabling the growth of human xenografts. We previously developed BALB/c Rag2nullJak3null (BRJ), an immunodeficient mouse model useful for cancer studies. However, susceptibility to xenograft acceptance can be increased through signal regulatory protein alpha (SIRPα)-CD47 signaling, also known as the “don't eat me” signal. In this study, we generated BALB/c human-SIRPα BAC transgenic mice and crossed them with BRJ mice. The resulting human SIRPα-transgenic BRJ (BRJ-S) mice showed a significantly higher engraftment rate of human B-cell lymphoma than BRJ mice. Furthermore, we demonstrated better human tumor engraftment in BRJ-S mice by reducing phagocytosis. In summary, hSIRPα-transgenic BRJ mice (BRJ-S) serve as a promising immunodeficient model with enhanced capacity for human lymphoma engraftment. BRJ-S provides an advantageous and permissive model for xenografting and for studying cancer in research and drug development.
Various techniques are used to introduce new cultivars, especially in ornamental species like tuberose, which have limited genetic diversity. This study employed a completely randomized design with three replications to investigate the effects of different mutagens on mutation induction in tuberose bulbs. The mutagens tested included sodium azide at concentrations of 200, 300, and 400 mg L−1, ethyl methanesulfonate at 0.25, 0.5, and 0.75 mg L−1, and gamma rays at doses of 10, 20, and 30 Gy. After exposure to the mutagens, both mother and daughter bulbs were grown for two consecutive years under greenhouse conditions. Our results indicated an increase in the number of florets in bulbs treated with sodium azide, regardless of the concentration used. The largest floret diameter, measuring 47.82 mm, was recorded in samples treated with ethyl methanesulfonate at a concentration of 0.5 mg L−1. This same concentration also resulted in an increase in the reducing sugar content within the petals. The longest vase life, recorded at 8.66 days, was observed in samples exposed to gamma radiation at a dose of 10 Gy. Significant changes in enzymatic antioxidants were noted in bulbs treated with the different mutagens, both before and after harvest. In conclusion, all three mutagens had an influence on the growth of tuberose plants, affecting various morphological, physiological, and postharvest parameters.
The petunia genome contains an endogenous pararetrovirus, petunia vein clearing virus (PVCV). Previous analyses indicate that PVCV has suppressor activity against RNA silencing, but the suppressor protein has not been identified. Here we tested whether an open reading frame (ORF) of PVCV confers the activity that can suppress cosuppression of the CHS-A genes encoding chalcone synthase, which has a high rate of RNA turnover in the petal tissues of petunia. Petunia transformants that express PVCV ORF under the control of cauliflower mosaic virus 35S promoter were produced. The transgenic plants were crossed with those that have CHS-A cosuppression to produce plants that contain both the PVCV ORF transgene and CHS-A transgene. The coexistence of these transgenes resulted in phenotypic changes: pigmentation of various extents occurred on the originally white petals of CHS-A cosuppression phenotype. The generation of pigmented portions in flower petals coincided with higher transcript levels of CHS-A and PVCV ORF and less CHS-A short interfering RNA. These results indicate that the PVCV ORF can suppress CHS-A cosuppression and change the flower color phenotype when it is expressed as a transgene.
Finger millet (Eleusine coracana) is a nutritionally important and climate-resilient cereal cultivated in rainfed regions of India and Eastern Africa, yet its genetic improvement has been limited by the lack of efficient and reproducible transformation systems. In this study, we developed a rapid and efficient Agrobacterium tumefaciens–mediated transformation and regeneration system using shoot apical meristem (SAM) explants, enabling direct, callus-free shoot organogenesis. Optimal regeneration and shoot elongation were achieved on Murashige and Skoog (MS) medium supplemented with 3.5 mg L⁻1 6-benzylaminopurine (BAP), 1.5 mg L⁻1 kinetin, 0.1 mg L⁻1 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.2 mg L⁻1 gibberellic acid (GA₃). Genotype-dependent responses were observed, with PR-202 requiring 2 mg L⁻1 AgNO3 to reduce phenolic browning, whereas VL-376 regenerated efficiently without AgNO3. Transformation efficiencies of 30–32
Cotton (Gossypium hirsutum L.) is a vital cash crop in India’s rainfed Central Zone, facing challenges from low productivity, erratic rainfall, and biotic (sucking pests, diseases) and abiotic (drought) stresses. While Bt cotton varieties with the Bacillus thuringiensis (Bt) gene have reduced bollworm losses, the need persists for high-yielding, stable varieties with broader tolerance. This study evaluates the newly released Bt cotton variety NH 1901 Bt (BG I), bred for rainfed regions of Maharashtra, Gujarat, and Madhya Pradesh. Evaluated over three years (2020–21 to 2022–23) in multi-location trials under the All India Coordinated Research Project (AICRP) on Cotton, NH 1901 Bt achieved a weighted mean seed cotton yield of 1474 kg/ha—a 17.32
Leucine aminopeptidases (LAPs) are multifunctional enzymes with roles in both defence and development. In plants, they are reported to be induced by wound-inflicting Lepidopteran insects and regulate wound response pathways leading to an effective defence response. Infestation by Hemipteran mustard aphid, Lipaphis erysimi (L.) Kaltenbach has been reported to induce wound response as well as a wound-responsive Arabidopsis thaliana Lap1 homologue (RI01; GenBank Accession: JK034053) in Rorippa indica (L.) Hiern. This is interesting as Hemipteran insects like aphids are assumed to inflict minimal wounding. In the present study, starting with the RI01 sequence information, we isolated the full length (1566 bp) sequence of a novel R. indica Lap (RiLap) gene, performed in silico analyses and developed transgenic R. indica plants with suppressed RiLAP activity by expressing a 565 bp antisense fragment of RiLap cDNA. We found that the isolated RiLAP is an acidic LAP of M17 family and suppressing it causes a significant increase in aphid herbivory but reduction in total chlorophyll content and possibly photosynthetic capacity in aphid infested transgenic plants of the T1 generation. These findings though preliminary suggest that RiLap could have a role in deterring aphids by acting as a regulatory protein simultaneously balancing defence response and photosynthetic capacity or plant growth. Noting the dearth of research in this area, this pilot study will be useful for designing future in depth analyses in understanding the role of Laps in defence response against Hemipteran insects. The study has implications in the development of sustainable pest management avenues.
Carotenoids are essential pigments in the plant photosynthetic apparatus, functioning in light harvesting, photoprotection, and signal transduction, and serving as precursors of vital nutrients such as vitamin A. Phytoene synthase (PSY) is the first rate-limiting enzyme in the plant carotenoid biosynthetic pathway, and its transcriptional regulation primarily depends on cis-acting promoter elements, associated transcription factors, and epigenetic status. The PSY promoter region contains core cis-elements as well as multiple light-, hormone-, and stress-responsive elements, which collectively function as key regulatory sites governing spatiotemporal expression. This review systematically summarizes recent advances in PSY promoter regulation by plant hormones (e.g., abscisic acid, ethylene, jasmonic acid), environmental factors (light signaling, temperature, salinity, and drought), and epigenetic mechanisms (DNA methylation, histone modifications, and chromatin remodeling). In addition, the application of transgenic and biotechnological approaches to PSY promoter regulation is further summarized. Including promoter sequence engineering with precise editing of cis-elements and promoter-targeted CRISPR activation/interference (CRISPRa/i) for tunable transcriptional control. Emphasis is placed on how these signals are integrated at the promoter level. Deeper insights into these mechanisms will provide both theoretical foundations and practical strategies for enhancing carotenoid accumulation and stress tolerance in crops through molecular design.
The progeny of chlorsulfuron-resistant forage rape (Brassica napus L. subsp. napus, cv. Giant) and potato (Solanum tuberosum L. cv. Iwa) plants hemizygous for a single transgenic locus were screened for transgene segregation following an application of either water or chlorsulfuron. The transgenic locus contained three transgenes conferring kanamycin resistance (NOS-NPTII-NOS), β-glucuronidase (GUS) activity (35S-GUS-OCS), and chlorsulfuron resistance (a complete acetohydroxyacid synthase gene with a proline197 to serine substitution). In the absence of the herbicide application, plants segregated for GUS activity as expected for single locus inheritance. However, the progeny of plants sprayed with chlorsulfuron exhibited a highly distorted segregation for GUS activity, with a significant excess of transgenic progeny. Inducing such biased segregation provides a simple treatment to rapidly drive the fixation of transgenic alleles to homozygosity in open pollinated populations during seed increases of new cultivars.
Lemna minor (commonly known as duckweed) is a fast-growing aquatic plant recognized as a promising green bioreactor for recombinant protein production. Its rapid proliferation, high protein yield, environmental adaptability, and edibility make it highly attractive for biotechnological applications. It is essential to develop and expand genetic tools tailored to this species to maximize these advantages and further unlock its biotechnological potential. A key strategy for achieving this goal is the implementation of advanced genome editing technologies, such as the CRISPR/Cas9 system. Although multiplex CRISPR/Cas9 gene editing has previously been successfully applied in Lemna aequinoctialis, the capability of the endogenous plant tRNA processing system for multiplex editing in L. minor using the polycistronic tRNA–sgRNA (PTG)/Cas9 system has not yet been explored. In this study, a PTG construct was engineered to include four sgRNAs designed to simultaneously target two plant-specific glycosyltransferase genes: α-1,3-fucosyltransferase (FucT) and β-1,2-xylosyltransferase (XylT). As anticipated, the PTG-Cas9 system successfully induced frameshift mutations, characterized by insertions and deletions (indels), in regenerated L. minor plants derived from transformed calli. Validation via PCR and RT-PCR analysis, followed by sequencing of the target loci, confirmed the presence of indels at the target sites. Furthermore, western blot analyses utilizing antibodies specific to XylT and FucT in two homozygous lines (lines 44 and 217) revealed truncated XylT proteins in both lines. Moreover, an in-frame FucT protein was detected in line 217, whereas FucT expression was absent in line 44. This study marked the first successful demonstration of PTG-Cas9 system for multiplex genome editing in L. minor, paving the way for advanced genetic engineering in this species.
Interleukin-11 (Il11) is a cytokine that belongs to the IL-6 family and is involved in inflammation, fibrosis, cancer, and ageing. Here, we generated an Il11-Cre knock-in mouse line and crossed it with the R26-tdTomato reporter to label and track of Il11-positive cells and their descendants under both developmental and pathological conditions. Using multicolor immunofluorescence staining, we identified tdTomato cells in various cell types, including fibroblasts and osteoblasts in bone, neurons in brain, enteroendocrine cells and fibroblasts in the gastrointestinal tract, pancreatic β-cells, and serous acinar cells in the submandibular glands. Notably, we observed a significant increase in fibroblasts labelled by tdTomato in a colitis model. These results indicate that this Il11-Cre line can serve as a useful genetic tool for genetic manipulation studies based on the Cre-loxp system in the aforementioned cells.
Pea (Pisum sativum L.) symbiosis with nodule bacteria supplying plants with additional nitrogen is a very specific plant-microbial interaction. Mutual recognition of the partners occurs through perception of bacterial signal molecules (Nod factors) by plant receptors, enabling bacterial entry via root hairs and formation of nitrogen-fixing nodules. The pea gene Sym2, described but not yet cloned, exists in different allelic forms defining the symbiotic specificity, and is therefore thought to encode a Nod factor receptor. The PsLykX gene is a strong candidate for the Sym2, since its alleles coincide with high or low symbiotic specificity; however, to date, no genetic evidence has been obtained for a role of PsLykX in symbiosis. Here, we knocked-out the PsLykX in European pea cultivar Caméor using Agrobacterium-mediated hairy root transformation and CRISPR-Cas9 editing. The roots with editing events confirmed by sequencing lost the ability to form nodules, providing direct functional evidence that PsLykX is essential, at least, for the symbiosis between pea cultivar Caméor and Rhizobium ruizarguesonis RCAM1026.
Low temperature is a common abiotic stress, which can seriously restrict the normal growth and development of crops. In this study, wheat cold-regulated gene 80 (Wcor80) was isolated from “Dongnongdongmai 1” (Dn1) material and characterized. The expression of Wcor80 was induced by brassinolide (BR) and low temperature (LT) treatments, and it was preferentially expressed in the leaves under abscisic acid (ABA) treatment and in the tillering nodes under BR treatment. Overexpression of Wcor80 in transgenic Arabidopsis significantly increased tolerance to LT treatment. Overall, our findings suggest that Wcor80 functions as a positive regulator of resistance to LT stress, providing a theoretical basis for the molecular breeding and cultivation regulation of winter wheat.
Despite being a main protein supplier in the human diet, wheat proteins represent a health challenge for some people. Besides the well-known celiac disease caused by gluten proteins, there is an occupational illness known as baker´s asthma. Amylase/trypsin inhibitors (ATIs) have been reported to be the major group of wheat proteins responsible for bakers´ asthma. As part of the characterization of stress-tolerant (HB4® technology) transgenic wheat (event IND-ØØ412-7, HB4 wheat), the level of the seven ATIs (0.28, 0.19 + 0.53, CM2, CM3, CM16, and CM17) was determined and compared to non-transgenic varieties. The materials tested in this study included the transgenic event in two different genetic backgrounds, their conventional counterparts (cv. Algarrobo and cv. Basilio), and five additional commercial varieties. Grain samples were obtained from field trials in Argentina in 2020 at six different locations. No significant differences were found in the ATIs levels between HB4 wheat and its isolines. ATIs levels in HB4 wheat were then analyzed within the natural variation given by the varieties and locations included in this study. Altogether, this study confirmed that ATIs levels, as previously reported for other allergens, are mainly affected by the genetic background and the environmental conditions, and that the ATIs levels measured in HB4 transgenic wheat are within the range of natural variability observed in the non-transgenic counterparts.
Isoflavonoids are key secondary metabolites in leguminous plants that play essential roles in plant physiology and provide significant health benefits to humans. In the isoflavone biosynthetic pathway, isoflavone synthase (IFS) catalyzes the conversion of naringenin and liquiritigenin into the bioactive isoflavones genistein and daidzein. This study aimed to enhance genistein and daidzein accumulation in soybean seeds through genetic engineering. Agrobacterium tumefaciens strain EHA105 harboring the binary vector pCAMBIA1301 containing GmIFS under the control of a seed-specific promoter (Gmβ-conglycinin) was used to transform modified half-seed explants of soybean cv. JS335. Hygromycin-B-resistant plants were regenerated, hardened, and confirmed by histochemical GUS assay. Molecular analysis by PCR validated the presence of the GmIFS transgene, yielding a 700 bp amplicon. Biochemical analysis revealed that seeds of T₀ transgenic plants showed a 1.53-fold increase in total phenolic content and a 3.67-fold increase in flavonoid content compared to non-transformed controls. Antioxidant assays demonstrated significantly higher DPPH radical-scavenging activity and ferric-reducing antioxidant power (FRAP) in GmIFS-overexpressing plants. HPLC analysis further indicated that transgenic seeds accumulated, on average, 4.07-fold higher daidzein and 1.75-fold higher genistein levels relative to control plants. qRT-PCR analysis showed significantly elevated GmIFS expression in immature cotyledons, mature cotyledons, and seeds of transgenic plants. Overall, these results demonstrate that GmIFS overexpression effectively enhances isoflavone production in soybean seeds, highlighting the potential of metabolic engineering of biosynthetic pathway genes to improve nutritional quality.
