Detection and occurrence of genetically modified rice and potato in the Saudi food market.

The number of food products with genetically modified (GM) crops on the global market has increased due to advancements in genetic engineering technology. Legislation regulating the labeling and use of GM crops has increased considerably worldwide to provide consumers with health and safety assurance. It is still unclear whether genetically modified organisms (GMOs) are present in the food market of the Kingdom of Saudi Arabia due to a lack of scientific studies. This work was planned to detect GM rice and GM potatoes in the Saudi food market. One hundred non-labeled rice and rice product samples and 50 potato and potato samples were collected randomly from different market sites of Makkah, Riyadh and Jeddah during 2022-2023. The cetyl trimethyl ammonium bromide (CTAB) method was used to extract DNA. Viviants DNA extraction kit was used to extract DNA from rice starch and potato chips. To find GMOs in samples, CMOScreen 35S and NOS test kits were utilized. DNA-based qualitative and quantitative approaches were used to screen targets for PCR detection of GM rice sequences. The results indicated that 32 (32%) rice samples were positive for CaMV 35S promoter, while no positive result was detected for the NOS terminator. Besides, 30% of potato samples were positive for the CaMV 35S promoter, and the same samples were positive for the presence of the Cry V gene. It could be concluded that there were GM rice and potatoes in the Kingdom of Saudi Arabia's food markets. Establishing strong regulations and certified laboratories to monitor genetically modified foods (GMF) or crops in the Saudi market is recommended.

Resistance of Transgenic Maize Cultivars to Mycotoxin Production-Systematic Review and Meta-Analysis.

Approximately 25% of cereal grains present with contamination caused by fungi and the presence of mycotoxins that may cause severe adverse effects when consumed. Maize has been genetically engineered to present different traits, such as fungal or insect resistance and herbicide tolerance. This systematic review compared the observable quantities, via meta-analysis, of four mycotoxins (aflatoxins-AFL, fumonisins-FUM, deoxynivalenol-DON, zearalenone-ZEA) between genetically modified (GM) and conventional maize kernels. This study was conducted following the PRISMA guidelines, with searches performed using PubMed, Web of Science, Scopus, Google Scholar, and CAPES journals databases. Analyses were conducted using RevMan v.5.4 software. Transgenic maize showed a 58% reduction in total mycotoxins (p < 0.001) compared to conventional maize. FUM were the most impacted, with a 59% reduction (p < 0.001) in GM maize. AFL and ZEA levels were also lower in GM maize by 49% (p = 0.02) and 51% (p < 0.001), respectively. On the other hand, DON levels increased by 6% (p < 0.001) in GM maize compared to conventional maize. However, results for ZEA and DON were inconclusive due to the limited research and sample sizes. We conclude that transgenic maize reduces total mycotoxins by over 50%, primarily fumonisin and aflatoxin. Most studies presented maize varieties that were resistant to insects or herbicides, not fungal pathogens, showing a positive collateral effect of these genetic alterations. Therefore, transgenic maize appears to be a safer product for animal and human consumption from a toxicological point of view. Further studies with larger sample sizes are needed to confirm our findings for ZEA and DON in transgenic maize.

The Characterization of a Novel PrMADS11 Transcription Factor from Pinus radiata Induced Early in Bent Pine Stem.

A novel MADS-box transcription factor from Pinus radiata D. Don was characterized. PrMADS11 encodes a protein of 165 amino acids for a MADS-box transcription factor belonging to group II, related to the MIKC protein structure. PrMADS11 was differentially expressed in the stems of pine trees in response to 45° inclination at early times (1 h). Arabidopsis thaliana was stably transformed with a 35S::PrMADS11 construct in an effort to identify the putative targets of PrMADS11. A massive transcriptome analysis revealed 947 differentially expressed genes: 498 genes were up-regulated, and 449 genes were down-regulated due to the over-expression of PrMADS11. The gene ontology analysis highlighted a cell wall remodeling function among the differentially expressed genes, suggesting the active participation of cell wall modification required during the response to vertical stem loss. In addition, the phenylpropanoid pathway was also indicated as a PrMADS11 target, displaying a marked increment in the expression of the genes driven to the biosynthesis of monolignols. The EMSA assays confirmed that PrMADS11 interacts with CArG-box sequences. This TF modulates the gene expression of several molecular pathways, including other TFs, as well as the genes involved in cell wall remodeling. The increment in the lignin content and the genes involved in cell wall dynamics could be an indication of the key role of PrMADS11 in the response to trunk inclination.

Metagenomic Analysis of Rhizospheric Bacterial Community of Citrus Trees Expressing Phloem-Directed Antimicrobials.

Huanglongbing, also known as citrus greening, is currently the most devastating citrus disease with limited success in prevention and mitigation. A promising strategy for Huanglongbing control is the use of antimicrobials fused to a carrier protein (phloem protein of 16 kDa or PP16) that targets vascular tissues. This study investigated the effects of genetically modified citrus trees expressing Citrus sinensis PP16 (CsPP16) fused to human lysozyme and ß-defensin-2 on the soil microbiome diversity using 16S amplicon analysis. The results indicated that there were no significant alterations in alpha diversity, beta diversity, phylogenetic diversity, differential abundance, or functional prediction between the antimicrobial phloem-overexpressing plants and the control group, suggesting minimal impact on microbial community structure. However, microbiota diversity analysis revealed distinct bacterial assemblages between the rhizosphere soil and root environments. This study helps to understand the ecological implications of crops expressing phloem-targeted antimicrobials for vascular disease management, with minimal impact on soil microbiota.

Advances in Tissue Culture and Transformation Studies in Non-model Species: Passiflora spp. (Passifloraceae).

In this chapter, we report advances in tissue culture applied to Passiflora. We present reproducible protocols for somatic embryogenesis, endosperm-derived triploid production, and genetic transformation for such species knowledge generated by our research team and collaborators in the last 20 years. Our research group has pioneered the work on passion fruit somatic embryogenesis, and we directed efforts to characterize several aspects of this morphogenic pathway. Furthermore, we expanded the possibilities of understanding the molecular mechanism related to developmental phase transitions of Passiflora edulis Sims. and P. cincinnata Mast., and a transformation protocol is presented for the overexpression of microRNA156.

Advances in Tissue Culture and Transformation Studies in Non-model Species: Bixa orellana L. (Bixaceae).

Over the years, our team has dedicated significant efforts to studying a unique natural dye-producing species, annatto (Bixa orellana L.). We have amassed knowledge and established foundations that support the applications of gene expression analysis in comprehending in vitro morphogenic regeneration processes, phase transition aspects, and bixin biosynthesis. Additionally, we have conducted gene editing associated with these processes. The advancements in this field are expected to enhance breeding practices and contribute to the overall improvement of this significant woody species. Here, we present a step-by-step protocol based on somatic embryogenesis and an optimized transformation protocol utilizing Agrobacterium tumefaciens.

Direct Somatic Embryogenesis in Carica papaya L. Genotypes for Genetic Modification Purposes.

This chapter presents an efficient protocol for regenerating Carica papaya plants via somatic embryogenesis from immature zygotic embryos from economically important papaya genotypes. To achieve regenerated plants from somatic embryos, in the present protocol, four induction cycles are required, followed by one multiplication cycle and one regeneration cycle. With this optimized protocol, 80% of somatic embryos can be obtained in only 3.5 months. At this stage, calli containing more than 50% globular structures can be used for transformation (via agrobacterium, biobalistics, or any other transformation method). Once transformed, calli can be transferred to the following steps (multiplication, elongation, maturation, rooting, and ex vitro acclimatization) to regenerate a transformed somatic embryo-derived full plant.

The History of Agrobacterium Rhizogenes: From Pathogen to a Multitasking Platform for Biotechnology.

Agrobacterium's journey has been a roller coaster, from being a pathogen to becoming a powerful biotechnological tool. While A. tumefaciens has provided the scientific community with a versatile tool for plant transformation, Agrobacterium rhizogenes has given researchers a Swiss army knife for developing many applications. These applications range from a methodology to regenerate plants, often recalcitrant, to establish bioremediation protocols to a valuable system to produce secondary metabolites. This chapter reviews its discovery, biology, controversies over its nomenclature, and some of the multiple applications developed using A. rhizogenes as a platform.

Exploratory comparative transcriptomic analysis reveals potential gene targets associated with Cry1A.105 and Cry2Ab2 resistance in fall armyworm (Spodoptera frugiperda).

Genetically modified (GM) crops, expressing Bacillus thuringiensis (Bt) insecticidal toxins, have substantially transformed agriculture. Despite rapid adoption, their environmental and economic benefits face scrutiny due to unsustainable agricultural practices and the emergence of resistant pests like Spodoptera frugiperda, known as the fall armyworm (FAW). FAW's adaptation to Bt technology in corn and cotton compromises the long-term efficacy of Bt crops. To advance the understanding of the genetic foundations of resistance mechanisms, we conducted an exploratory comparative transcriptomic analysis of two divergent FAW populations. One population exhibited practical resistance to the Bt insecticidal proteins Cry1A.105 and Cry2Ab2, expressed in the genetically engineered MON-89Ø34 - 3 maize, while the other population remained susceptible to these proteins. Differential expression analysis supported that Cry1A.105 and Cry2Ab2 significantly affect the FAW physiology. A total of 247 and 254 differentially expressed genes were identified in the Cry-resistant and susceptible populations, respectively. By integrating our findings with established literature and databases, we underscored 53 gene targets potentially involved in FAW's resistance to Cry1A.105 and Cry2Ab2. In particular, we considered and discussed the potential roles of the differentially expressed genes encoding ABC transporters, G protein-coupled receptors, the P450 enzymatic system, and other Bt-related detoxification genes. Based on these findings, we emphasize the importance of exploratory transcriptomic analyses to uncover potential gene targets involved with Bt insecticidal proteins resistance, and to support the advantages of GM crops in the face of emerging challenges.

Plant characterization of insect-protected soybean.

Insect-protected soybean (SIP) that produces the Cry1A.105 and Cry2Ab2 insecticidal crystal proteins has been developed to provide protection from feeding damage caused by targeted lepidopteran insect pests. Typically, as part of environmental risk assessment (ERA), plant characterization is conducted, and the data submitted to regulatory agencies prior to commercialization of genetically modified (GM) crops. The objectives of this research were to: (a) compare soybean with and without the SIP trait in plant characterization field trials designed to fulfill requirements for submissions to global regulatory agencies and address China-specific considerations and (b) compare risk assessment conclusions across regions and the methodologies used in the field trials. The soybean with and without the SIP trait in temperate, tropical, and subtropical germplasm were planted in replicated multi-location trials in the USA (in 2012 and 2018) and Brazil (in 2013/2014 and 2017/2018). Agronomic, phenotypic, plant competitiveness, and survival characteristics were assessed for soybean entries with and without the SIP trait. Regardless of genetic background, growing region, season, or testing methodology, the risk assessment conclusions were the same: the evaluated insect-protected soybean did not differ from conventional soybean in evaluated agronomic, phenotypic, competitiveness, and survival characteristics indicating no change in plant pest/weed potential. These results reinforce the concept of data transportability across global regions, different seasons, germplasm, and methodologies that should be considered when assessing environmental risks of GM crops.

Influence of Silver Nanoparticles on the Metabolites of Two Transgenic Soybean Varieties: An NMR-Based Metabolomics Approach.

This study investigated the effect of AgNPs and AgNO3, at concentrations equivalent, on the production of primary and secondary metabolites on transgenic soybean plants through an NMR-based metabolomics. The plants were cultivated in a germination chamber following three different treatments: T0 (addition of water), T1 (addition of AgNPs), and T2 (addition of AgNO3). Physiological characteristics, anatomical analyses through microscopic structures, and metabolic profile studies were carried out to establish the effect of abiotic stress on these parameters in soybean plants. Analysis of the 1H NMR spectra revealed the presence of amino acids, organic acids, sugars, and polyphenols. The metabolic profiles of plants with AgNP and AgNO3 were qualitatively similar to the metabolic profile of the control group, suggesting that the application of silver does not affect secondary metabolites. From the PCA, it was possible to differentiate the three treatments applied, mainly based on the content of fatty acids, pinitol, choline, and betaine.

Agroinfiltration for Enhanced Transgene Expression in Coffee Leaves (Coffea arabica L.).

The Coffea spp. plant is a significant crop in Latin America, Africa, and Asia, and recent advances in genomics and transcriptomics have opened possibilities for studying candidate genes and introducing new desirable traits through genetic engineering. While stable transformation of coffee plants has been reported using various techniques, it is a time-consuming and laborious process. To overcome this, transient transformation methods have been developed, which avoid the limitations of stable transformation. This chapter describes an ex vitro protocol for transient expression using A. tumefaciens-mediated infiltration of coffee leaves, which could be used to produce coffee plants expressing desirable traits against biotic and abiotic stresses, genes controlling biochemical and physiological traits, as well as for gene editing through CRISPR/Cas9.

Coffee Cell Suspensions as a Platform for Transient Gene Expression Analysis.

Coffea arabica L. is a crucial crop globally, but its genetic homogeneity leads to its susceptibility to diseases and pests like the coffee berry borer (CBB). Chemical and cultural control methods are difficult due to the majority of the CBB life cycle taking place inside coffee beans. One potential solution is the use of the gene cyt1Aa from Bacillus thuringiensis as a biological insecticide. To validate candidate genes against CBB, a simple, rapid, and efficient transient expression system is necessary. This study uses cell suspensions as a platform for expressing the cyt1Aa gene in the coffee genome (C. arabica L. var. Catuaí) to control CBB. The Agrobacterium tumefaciens strain GV3101::pMP90 containing the bar and cyt1Aa genes are used to genetically transform embryogenic cell suspensions. PCR amplification of the cyt1Aa gene is observed 2, 5, and 7 weeks after infection. This chapter describes a protocol that can be used for the development of resistant varieties against biotic and abiotic stresses and CRISPR/Cas9-mediated genome editing.

CRISPR/Cas9-Mediated Genome Editing in Indica Rice (Oryza sativa L. subsp. indica var. CR-5272).

Tissue culture optimization protocols limit indica rice breeding. Such a challenge is vital because emergent techniques still rely on tissue culture methods and could allow the breeding of new varieties with higher production and toleration of adverse environmental effects caused by climate change. Genome editing technology, using CRISPR/Cas9, is a fast and precise method for accelerated plant breeding. It limited its use in indica subspecies because of the recalcitrant response to in vitro culture methods. This chapter describes a protocol for CRISPR/Cas9 editing in indica subspecies, specifically in the CR-5272 variety derived from parental lines IR-822, using Agrobacterium tumefaciens and biolistic transformation.

Unveiling the defensive role of Snakin-3, a member of the subfamily III of Snakin/GASA peptides in potatoes.

KEY MESSAGE: The first in-depth characterization of a subfamily III Snakin/GASA member was performed providing experimental evidence on promoter activity and subcellular localization and unveiling a role of potato Snakin-3 in defense Snakin/GASA proteins share 12 cysteines in conserved positions in the C-terminal region. Most of them were involved in different aspects of plant growth and development, while a small number of these peptides were reported to have antimicrobial activity or participate in abiotic stress tolerance. In potato, 18 Snakin/GASA genes were identified and classified into three groups based on phylogenetic analysis. Snakin-1 and Snakin-2 are members of subfamilies I and II, respectively, and were reported to be implicated not only in defense against pathogens but also in plant development. In this work, we present the first in-depth characterization of Snakin-3, a member of the subfamily III within the Snakin/GASA gene family of potato. Transient co-expression of Snakin-3 fused to the green fluorescent protein and organelle markers revealed that it is located in the endoplasmic reticulum. Furthermore, expression analyses via pSnakin-3::GUS transgenic plants showed GUS staining mainly in roots and vascular tissues of the stem. Moreover, GUS expression levels were increased after inoculation with Pseudomonas syringae pv. tabaci or Pectobacterium carotovorum subsp. carotovorum and also after auxin treatment mainly in roots and stems. To gain further insights into the function of Snakin-3 in planta, potato overexpressing lines were challenged against P. carotovorum subsp. carotovorum showing enhanced tolerance to this bacterial pathogen. In sum, here we report the first functional characterization of a Snakin/GASA gene from subfamily III in Solanaceae. Our findings provide experimental evidence on promoter activity and subcellular localization and reveal a role of potato Snakin-3 in plant defense.

Drought-tolerant transgenic wheat HB4®: a hope for the future.

Drought-tolerant transgenic [genetically modified (GM)] HB4® wheat carrying the drought-responsive sunflower gene Hahb4 was first developed in Argentina in 2019 and has already been approved for marketing and consumption as food/feed in at least ten countries. It has also been approved in Argentina and Brazil for commercial cultivation.

Identification and Profiling Analysis of microRNAs in Guava Fruit (Psidium guajava L.) and Their Role during Ripening.

The guava (Psidium guajava L.) is a climacteric fruit with an accelerated post-harvest overripening. miRNAs are small RNA sequences that function as gene regulators in eukaryotes and are essential for their survival and development. In this study, miRNA libraries were constructed, sequenced and analyzed from the breaker and ripe stages of guava fruit cv. Siglo XXI. One hundred and seventy-four mature miRNA sequences from 28 miRNA families were identified. The taxonomic distribution of the guava miRNAs showed a high level of conservation among the dicotyledonous plants. Most of the predicted miRNA target genes were transcription factors and genes involved in the metabolism of phytohormones such as abscisic acid, auxins, and ethylene, as revealed through an ontology enrichment analysis. The miRNA families miR168, miR169, miR396, miR397, and miR482 were classified as being directly associated with maturation, whereas the miRNA families miR160, miR165, miR167, miR3930, miR395, miR398, and miR535 were classified as being indirectly associated. With this study, we intended to increase our knowledge and understanding of the regulatory process involved in the ripening process, thereby providing valuable information for future research on the ripening of guava fruit.

Development of a bioassay method to test activity of cry insecticidal proteins against Diatraea spp. (Lepidoptera: Crambidae) sugarcane stem borers.

The genus Diatraea (Lepidoptera: Crambidae) includes stem borers representing the most critical sugarcane pests in the Americas. Colombia's most widely distributed and damaging Diatraea species include Diatraea saccharalis, D. indigenella, D. busckella, and D. tabernella. The reduced efficacy of biological tools commonly used in controlling several species highlights the importance of evaluating alternative management strategies, such as transgenic plants expressing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt). The selection of optimal Bt insecticidal proteins for Diatraea control depends on bioassays with purified Bt proteins. Because there is no described artificial diet for borer species other than D. saccharalis and availability of most purified Bt toxins is restricted, this study aimed at developing a bioassay method using fresh corn tissue and providing proof of concept by testing susceptibility to the Cry1Ac insecticidal protein from Bt. Toxicity was evaluated with a single Cry1Ac dose applied directly to corn discs. Stem borer mortality after seven days was higher than 90% for all four tested Diatraea species, while control mortality was below 8%. In addition, we observed that Cry1Ac caused more than 90% weight inhibition in all survivors and delayed development. These results validate the use of this method to determine mortality and growth inhibition due to the consumption of the Cry1Ac protein in each of the Diatraea species. Furthermore, this method could be used to assess other entomopathogenic substances to control these insect pests.

A soybean trypsin inhibitor reduces the resistance to transgenic maize in a population of Spodoptera frugiperda (Lepidoptera: Noctuidae).

Lepidopteran pests have been successfully managed by the adoption of insect resistant transgenic plants expressing Cry and/or Vip insecticidal proteins derived from Bacillus thuringiensis (Bt plants). Among such pests, Spodoptera frugiperda (Smith, 1797) (Lepidoptera: Noctuidae) is highlighted for its destructive potential in maize crops and for cases of field-evolved resistance to Bt plants. Cry insecticidal proteins expressed in Bt plants are known for their interaction with insect midgut receptors and subsequent midgut cell disruption that leads to target pest death. In the midgut of lepidopteran larval pests such as S. frugiperda, serine proteases are important in dietary protein digestion and activation or degradation of insecticidal proteins. This work was conducted to evaluate if the use of a soybean trypsin inhibitor (SBTI) could disrupt the development of a Bt-susceptible and a Bt-resistant population of S. frugiperda ingesting Bt (expressing Cry1F, Cry1A.105, and Cry2Ab2 Cry proteins) and non-Bt maize plants. The SBTI was produced and purified using recombinant expression in E. coli followed by purification in Ni-Sepharose. Bioassays using non-Bt maize leaves indicated that the development of susceptible and resistant populations of S. frugiperda was not influenced by the ingestion of SBTI. However, when the resistant population consumed Bt maize plants amended with SBTI, high mortality along with a reduction in larval weight and reduced activity of digestive trypsins were observed. Although the mode of action was not elucidated, it is possible that the consumption of SBTI increased susceptibility to Bt maize in the resistant population of S. frugiperda.

Performance of cotton expressing Cry1Ac, Cry1F and Vip3Aa19 insecticidal proteins against Helicoverpa armigera, H. zea and their hybrid progeny, and evidence of reduced susceptibility of a field population of H. zea to Cry1 and Vip3Aa in Brazil.

The genetically modified cotton DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 expressing Cry1Ac, Cry1F and Vip3Aa19 from Bacillus thuringiensis Berliner (Bt) has been cultivated in Brazil since the 2020/2021 season. Here, we assessed the performance of DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 cotton expressing Cry1Ac, Cry1F and Vip3Aa19 against Helicoverpa armigera (Hübner), Helicoverpa zea (Boddie), and their hybrid progeny. We also carried out evaluations with DAS-21023-5 × DAS-24236-5 cotton containing Cry1Ac and Cry1F. In leaf-disk bioassays, DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 was effective in controlling neonates from laboratory colonies of H. armigera, H. zea and the hybrid progeny (71.9%-100% mortality). On floral bud bioassays using L2 larvae, H. zea presented complete mortality, whereas H. armigera and the hybrid progeny showed <55% mortality. On DAS-21023-5 × DAS-24236-5 cotton, the mortality of H. armigera on leaf-disk and floral buds ranged from 60% to 73%, whereas mortality of hybrids was <46%. This Bt cotton caused complete mortality of H. zea larvae from a laboratory colony in the early growth stages, but mortalities were <55% on advanced growth stages and on floral buds. In field studies conducted from 2014 to 2019, DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 cotton was also effective at protecting plants against H. armigera. In contrast, a population of H. zea collected in western Bahia in 2021/2022 on Bt cotton expressing Cry1 and Vip3Aa proteins, showed 63% mortality after 30 d, with insects developing into fifth and sixth instars, on DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 cotton. We conclude that H. armigera, H. zea, and their hybrid progeny can be managed with DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 cotton; however we found the first evidence in Brazil of a significant reduction in the susceptibility to DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 cotton of a population of H. zea collected from Bt cotton in Bahia in 2021/2022.