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Summary: The shape of rice grains not only determines the thousand‐grain weight but also correlates closely with the grain quality.Here we identified an ultra‐large grain accession (ULG) with a thousand‐grain weight exceeding 60 g. The integrated analysis of QTL, BSA, de novo genome assembled, transcription sequencing, and gene editing was conducted to dissect the molecular basis of the ULG formation.The ULG pyramided advantageous alleles from at least four known grain‐shaping genes, OsLG3, OsMADS1, GS3, GL3.1, and one novel locus, qULG2‐b, which encoded a leucine‐rich repeat receptor‐like kinase. The collective impacts of OsLG3, OsMADS1, GS3, and GL3.1 on grain size were confirmed in transgenic plants and near‐isogenic lines. The transcriptome analysis identified 112 genes cooperatively regulated by these four genes that were prominently involved in photosynthesis and carbon metabolism. By leveraging the pleiotropy of these genes, we enhanced the grain yield, appearance, and stress tolerance of rice var. SN265.Beyond showcasing the pyramiding of multiple grain size regulation genes that can produce ULG, our study provides a theoretical framework and valuable genomic resources for improving rice variety by leveraging the pleiotropy of grain size regulated genes. [ABSTRACT FROM AUTHOR]

In plants, the pathogenesis-related (PR) proteins have been identified as important regulators of biotic and abiotic stresses. PR proteins branch out into 19 different classes (PR1–PR19). Basically, all PR proteins display a well-established method of action, with the notable exception of PR1, which is a member of a large superfamily of proteins with a common CAP domain. We have previously isolated and characterized the first PR1 from durum wheat, called TdPR-1.2. In the current research work, TdPR1.2 gene was used to highlight its functional activities under various abiotic (sodium chloride (100 mM NaCl) and oxidative stresses (3 mM H2O2), hormonal salicylic acid (SA), abscisic acid (ABA) and jasmonic acid (JA), and abiotic stresses (Botrytis cinerea and Alternaria solani). Enhancement survival index was detected in Arabidopsis transgenic plants expressing TdPR1.2 gene. Moreover, quantitative real-time reverse transcription PCR (qRT-PCR) analysis demonstrated induction of antioxidant enzymes such as catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD). It equally revealed a decrease of malondialdehyde (MDA) as well as hydrogen peroxide (H2O2) levels in transgenic Arabidopsis plants compared to control lines, confirming the role of TdPR1.2 in terms of alleviating biotic and abiotic stresses in transgenic Arabidopsis plants. Eventually, RT-qPCR results showed a higher expression of biotic stress-related genes (PR1 and PDF1.2) in addition to a downregulation of the wound-related gene (LOX3 and VSP2) in transgenic lines treated with jasmonic acid (JA). Notably, these findings provide evidence for the outstanding functions of PR1.2 from durum wheat which can be further invested to boost tolerance in crop plants to abiotic and biotic stresses. [ABSTRACT FROM AUTHOR]

Summary: Dinitrotoluene sulfonates (DNTSes) are highly toxic hazards regulated by the Resource Conservation and Recovery Act (RCRA) in the United States. The trinitrotoluene (TNT) red water formed during the TNT purification process consists mainly of DNTSes. Certain plants, including switchgrass, reed and alfalfa, can detoxify low concentrations of DNTS in TNT red water‐contaminated soils. However, the precise mechanism by which these plants detoxify DNTS remains unknown. In order to aid in the development of phytoremediation resources with high DNTS removal rates, we identified and characterized 1‐hydroxymethyl‐2,4‐dinitrobenzene sulfonic acid (HMDNBS) and its glycosylated product HMDNBS O‐glucoside as the degradation products of 2,4‐DNT‐3‐SO3Na, the major isoform of DNTS in TNT red water‐contaminated soils, in switchgrass via LC–MS/MS‐ and NMR‐based metabolite analyses. Transcriptomic analysis revealed that 15 UDP‐glycosyltransferase genes were dramatically upregulated in switchgrass plants following 2,4‐DNT‐3‐SO3Na treatment. We expressed, purified and assayed the activity of recombinant UGT proteins in vitro and identified PvUGT96C10 as the enzyme responsible for the glycosylation of HMDNBS in switchgrass. Overexpression of PvUGT96C10 in switchgrass significantly alleviated 2,4‐DNT‐3‐SO3Na‐induced plant growth inhibition. Notably, PvUGT96C10‐overexpressing transgenic switchgrass plants removed 83.1% of 2,4‐DNT‐3‐SO3Na in liquid medium after 28 days, representing a 3.2‐fold higher removal rate than that of control plants. This work clarifies the DNTS detoxification mechanism in plants for the first time, suggesting that PvUGT96C10 is crucial for DNTS degradation. Our results indicate that PvUGT96C10‐overexpressing plants may hold great potential for the phytoremediation of TNT red water‐contaminated soils. [ABSTRACT FROM AUTHOR]

Summary: Protein complexes from edible oyster mushrooms (Pleurotus sp.) composed of pleurotolysin A2 (PlyA2) and pleurotolysin B (PlyB) exert toxicity in feeding tests against Colorado potato beetle (CPB) larvae, acting through the interaction with insect‐specific membrane sphingolipid. Here we present a new strategy for crop protection, based on in planta production of PlyA2/PlyB protein complexes, and we exemplify this strategy in construction of transgenic potato plants of cv Désirée. The transgenics in which PlyA2 was directed to the vacuole and PlyB to the endoplasmic reticulum are effectively protected from infestation by CPB larvae without impacting plant performance. These transgenic plants showed a pronounced effect on larval feeding rate, the larvae feeding on transgenic plants being on average five to six folds lighter than larvae feeding on controls. Further, only a fraction (11%–37%) of the larvae that fed on transgenic potato plants completed their life cycle and developed into adult beetles. Moreover, gene expression analysis of CPB larvae exposed to PlyA2/PlyB complexes revealed the response indicative of a general stress status of larvae and no evidence of possibility of developing resistance due to the functional inactivation of PlyA2/PlyB sphingolipid receptors. [ABSTRACT FROM AUTHOR]

Summary: Fusarium head blight (FHB) and the presence of mycotoxin deoxynivalenol (DON) pose serious threats to wheat production and food safety worldwide. DON, as a virulence factor, is crucial for the spread of FHB pathogens on plants. However, germplasm resources that are naturally resistant to DON and DON‐producing FHB pathogens are inadequate in plants. Here, detoxifying bacteria genes responsible for DON epimerization were used to enhance the resistance of wheat to mycotoxin DON and FHB pathogens. We characterized the complete pathway and molecular basis leading to the thorough detoxification of DON via epimerization through two sequential reactions in the detoxifying bacterium Devosia sp. D6‐9. Epimerization efficiently eliminates the phytotoxicity of DON and neutralizes the effects of DON as a virulence factor. Notably, co‐expressing of the genes encoding quinoprotein dehydrogenase (QDDH) for DON oxidation in the first reaction step, and aldo‐keto reductase AKR13B2 for 3‐keto‐DON reduction in the second reaction step significantly reduced the accumulation of DON as virulence factor in wheat after the infection of pathogenic Fusarium, and accordingly conferred increased disease resistance to FHB by restricting the spread of pathogenic Fusarium in the transgenic plants. Stable and improved resistance was observed in greenhouse and field conditions over multiple generations. This successful approach presents a promising avenue for enhancing FHB resistance in crops and reducing mycotoxin contents in grains through detoxification of the virulence factor DON by exogenous resistance genes from microbes. [ABSTRACT FROM AUTHOR]

Background: PSEUDO RESPONSE REGULATOR (PRR) genes are essential components of circadian clock, playing vital roles in multiple processes including plant growth, flowering and stress response. Nonetheless, little is known about the evolution and function of PRR family in Rosaceae species. Results: In this study, a total of 43 PRR genes in seven Rosaceae species were identified through comprehensive analysis. The evolutionary relationships were analyzed with phylogenetic tree, duplication events and synteny. PRR genes were classified into three groups (PRR1, PRR5/9, PRR3/7). The expansion of PRR family was mainly derived from dispersed and whole-genome duplication events. Purifying selection was the major force for PRR family evolution. Synteny analysis indicated the existence of multiple orthologous PRR gene pairs between pear and other Rosaceae species. Moreover, the conserved motifs of eight PbPRR proteins supported the phylogenetic relationship. PRR genes showed diverse expression pattern in various tissues of pear (Pyrus bretschneideri). Transcript analysis under 12-h light/ dark cycle and constant light conditions revealed that PRR genes exhibited distinct rhythmic oscillations in pear. PbPRR59a and PbPRR59b highly homologous to AtPRR5 and AtPRR9 were cloned for further functional verification. PbPRR59a and PbPRR59b proteins were localized in the nucleus. The ectopic overexpression of PbPRR59a and PbPRR59b significantly delayed flowering in Arabidopsis transgenic plants by repress the expression of AtGI, AtCO and AtFT under long-day conditions. Conclusions: These results provide information for exploring the evolution of PRR genes in plants, and contribute to the subsequent functional studies of PRR genes in pear and other Rosaceae species. [ABSTRACT FROM AUTHOR]

SUMMARY 12‐Oxo‐phytodienoic acid reductases (OPRs) perform vital functions in plants. However, few studies have been reported in sugarcane (Saccharum spp.), and it is of great significance to systematically investigates it in sugarcane. Here, 61 ShOPRs, 32 SsOPRs, and 36 SoOPRs were identified from R570 (Saccharum spp. hybrid cultivar R570), AP85‐441 (Saccharum spontaneum), and LA‐purple (Saccharum officinarum), respectively. These OPRs were phylogenetically classified into four groups, with close genes similar structures. During evolution, OPR gene family was mainly expanded via whole‐genome duplications/segmental events and predominantly underwent purifying selection, while sugarcane OPR genes may function differently in response to various stresses. Further, ScOPR2, a tissue‐specific OPR, which was localized in cytoplasm and cell membrane and actively response to salicylic acid (SA), methyl jasmonate, and smut pathogen (Sporisorium scitamineum) stresses, was cloned from sugarcane. In addition, both its transient overexpression and stable overexpression enhanced the resistance of transgenic plants to pathogen infection, most probably through activating pathogen‐associated molecular pattern/pattern‐recognition receptor‐triggered immunity, producing reactive oxygen species, and initiating mitogen‐activated protein kinase cascade. Subsequently, the transmission of SA and hypersensitive reaction were triggered, which stimulated the transcription of defense‐related genes. These findings provide insights into the function of ScOPR2 gene for disease resistance. [ABSTRACT FROM AUTHOR]

Introduction: Despite the absence of definitive evidence indicating that the cp4-epsps gene and its resultant recombinant proteins have significant harmful effects on either human or animal health, the safety assessment of genetically modified (GM) crops expressing the CP4-EPSPS proteins has been controversial. This study endeavor was aimed at evaluating the potential risks posed by the CP4- EPSPS protein in transgenic crops, thereby contributing to the advancement of risk assessment methodologies in the context of genetically engineered crops. Methods: To ascertain the appropriate daily dosages for oral gavage administration, the expression levels of the CP4-EPSPS protein in a recombinant yeast were quantified. Subsequently, physiological and biochemical analysis, metabolomics, and metagenomic analysis were conducted based on a 90-day Sprague-Dawley (SD) rats feeding experiment, respectively, thereby enhancing the depth and precision of our risk assessment framework. Results: The results from the physiological and biochemical analysis, organ pathological, blood metabolism, gut microbiota, and correlation analysis of metabolites and gut microbiota revealed several biomarkers for further risk assessment. These biomarkers include clinical biochemical indexes such as total bilirubin (TBIL), direct bilirubin (DBIL), creatine kinase (CK), and lactate dehydrogenase (LDH); metabolites like Methionine, 2-Oxovaleric acid, and LysoPC (16:0); and gut microbiota including Blautia wexlerae, Holdemanella biformis, Dorea sp. CAG 317, Coriobacteriaceae and Erysipelotrichaceae. Conclusion: In conclusion, the risk can be significantly reduced by directly consuming inactivated recombinant CP4-EPSPS. Therefore, in everyday life, the risk associated with consuming GM foods containing recombinant CP4-EPSPS is substantially reduced after heat treatment. [ABSTRACT FROM AUTHOR]

Generating CRISPR/Cas9-mediated mutants in tomato (Solanum lycopersicum L.) involves screening shoots regenerated fromcultured cells transformedwith a T-DNA harboring sequences encoding Cas9 and single guide RNAs (sgRNAs). Production of transformants can be inconsistent and obtaining transformants in large numbersmay be difficult, resulting in a limited variety of mutations. Here, I report a method for generating various types of mutations from one transgenic plant harboring the CRISPR/Cas9 system. In this method, a wild-type plant was crossed with a T0 biallelic mutant expressing two sgRNAs targeting the RIPENING INHIBITOR (RIN) gene, and the resulting F1 seedlings were classified using a kanamycin resistancemarker on the T-DNA. Genotyping of the RIN locus revealed that kanamycin-sensitive F1 seedlings, which carried no T-DNA, always harbored the wild-type allele and a mutant allele from the transgenic parent. Kanamycin-resistant F1 seedlings, which do carry the TDNA, harbored a variety of novel mutant alleles, but not the wild-type allele, suggesting that it was mutated during crossing. The novel mutations included one-base insertions or short deletions at each target site, or large deletions across the two target sites. Thismethod was also successfully applied to producemutations in Geranylgeranyl pyrophosphate synthase 2 (GGPS2). Because thismethod involves crossing rather than transformation, it can be readily scaled up to produce numerous novel mutations, even in plant species or cultivars for which transformation is inefficient. Therefore, when initial transgene experiments fail to induce the desired mutation, this method provides additional opportunities for generating mutants. [ABSTRACT FROM AUTHOR]

Promoters are one of the most important components for many gene-based research as they can fine-tune precise gene expression. Many unique plant promoters have been characterized, but strong promoters with dual expression in both monocot and dicot systems are still lacking. In this study, we attempted to make such a promoter by combining specific domains from monocot-infecting pararetroviral-based promoters sugarcane bacilliform virus (SCBV) and banana streak virus (BSV) to a strong dicot-infecting pararetroviral-based promoter mirabilis mosaic virus (MMV). The generated chimeric promoters, MS, SM, MB, and BM, were tested in monocot and dicot systems and further validated in transgenic tobacco plants. We found that the developed chimeric promoters were species-specific (monocot or dicot), which depended on their respective core promoter (CP) region. Furthermore, with this knowledge, deletion-hybrid promoters were developed and evaluated, which led to the development of a unique dual-expressing promoter, MSD3, with high gene expression efficiency (GUS and GFP reporter genes) in rice, pearl millet, and tobacco plants. We conclude that the MSD3 promoter can be an important genetic tool and will be valuable in plant biology research and application. [ABSTRACT FROM AUTHOR]

Summary: Posttranslational modification of multiple ABA signaling components is an essential process for the adaptation and survival of plants under stress conditions. In our previous study, we established that the pepper group A PP2C protein CaAITP1, one of the core components of ABA signaling, undergoes ubiquitination mediated by the RING‐type E3 ligase CaAIRE1.In this study, we discovered an additional form of regulation mediated via the SUMOylation of CaAITP1. Pepper plants subjected to drought stress were characterized by reductions in both the stability and SUMOylation of CaAITP1 protein.Moreover, we identified a SUMO protease, Capsicum annuum DeSUMOylating Isopeptidase 2 (CaDeSI2), as a new interacting partner of CaAITP1. In vitro and in vivo analyses revealed that CaAITP1 is deSUMOylated by CaDeSI2. Silencing of CaDeSI2 in pepper plants led to drought‐hypersensitive and ABA‐hyposensitive phenotypes, whereas overexpression of CaDeSI2 in transgenic Arabidopsis plants resulted in the opposite phenotypes. Importantly, we found that the CaAITP1 protein was stabilized in response to the silencing of CaDeSI2, and CaDeSI2 and CaAITP1 co‐silenced pepper plants were characterized by drought‐tolerant phenotypes similar to those observed in CaAITP1‐silenced pepper.Collectively, our findings indicate that CaDeSI2 reduces the stability of CaAITP1 via deSUMOylation, thereby positively regulating drought tolerance. [ABSTRACT FROM AUTHOR]

As a typical bulb flower, lily is widely cultivated worldwide because of its high ornamental, medicinal and edible value. Although breeding efforts evolved over the last 10000 years, there are still many problems in the face of increasing consumer demand. The approach of biotechnological methods would help to solve this problem and incorporate traits impossible by conventional breeding. Target traits are dormancy, development, color, floral fragrance and resistances against various biotic and abiotic stresses, so as to improve the quality of bulbs and cut flowers in planting, cultivation, postharvest, plant protection and marketing. Genetic transformation technology is an important method for varietal improvement and has become the foundation and core of plant functional genomics research, greatly assisting various plant improvement programs. However, achieving stable and efficient genetic transformation of lily has been difficult worldwide. Many gene function verification studies depend on the use of model plants, which greatly limits the pace of directed breeding and germplasm improvement in lily. Although significant progress has been made in the development and optimization of genetic transformation systems, shortcomings remain. Agrobacterium-mediated genetic transformation has been widely used in lily. However, severe genotypic dependence is the main bottleneck limiting the genetic transformation of lily. This review will summarizes the research progress in the genetic transformation of lily over the past 30 years to generate the material including a section how genome engineering using stable genetic transformation system, and give an overview about recent and future applications of lily transformation. The information provided in this paper includes ideas for optimizing and improving the efficiency of existing genetic transformation methods and for innovation, provides technical support for mining and identifying regulatory genes for key traits, and lays a foundation for genetic improvement and innovative germplasm development in lily. [ABSTRACT FROM AUTHOR]

Stomata play a crucial role in plants by controlling water status and responding to drought stress. However, simultaneously improving stomatal opening and drought tolerance has proven to be a significant challenge. To address this issue, we employed the OnGuard quantitative model, which accurately represents the mechanics and coordination of ion transporters in guard cells. With the guidance of OnGuard, we successfully engineered plants that overexpressed the main tonoplast Ca2+‐ATPase gene, ACA11, which promotes stomatal opening and enhances plant growth. Surprisingly, these transgenic plants also exhibited improved drought tolerance due to reduced water loss through their stomata. Again, OnGuard assisted us in understanding the mechanism behind the unexpected stomatal behaviors observed in the ACA11 overexpressing plants. Our study revealed that the overexpression of ACA11 facilitated the accumulation of Ca2+ in the vacuole, thereby influencing Ca2+ storage and leading to an enhanced Ca2+ elevation in response to abscisic acid. This regulatory cascade finely tunes stomatal responses, ultimately leading to enhanced drought tolerance. Our findings underscore the importance of tonoplast Ca2+‐ATPase in manipulating stomatal behavior and improving drought tolerance. Furthermore, these results highlight the diverse functions of tonoplast‐localized ACA11 in response to different conditions, emphasizing its potential for future applications in plant enhancement. [ABSTRACT FROM AUTHOR]

Petunia hybrida, widely grown as a bedding plant, has reduced growth and flower quality at temperatures above 30 °C (heat stress), primarily due to heat stress‐induced ethylene (ET) production. The gene acdS encodes the 1‐aminocyclopropane‐1‐carboxylate (ACC) deaminase (ACCD) enzyme, which is known for its role in reducing ET production by breaking down the ET precursor, ACC, in plant tissues.This study investigated the impact of heat stress on both 'Mirage Rose' WT petunia and its acdS‐overexpressing transgenic lines.Heat stress‐induced growth inhibition was observed in WT plants but not in transgenic plants. The increased stress tolerance of transgenic plants over WT plants was associated with lower ET production, ROS accumulation, higher SPAD values, water content, and relative water content. Furthermore, higher sensitivity of the WT to heat stress than the transgenic plants was confirmed by analysing ET signalling genes, heat shock transcription factor genes, and antioxidant‐ and proline‐related genes, more strongly induced in WT than in transgenic plants.Overall, this study suggests the potential application of acdS overexpression in other floriculture plants as a viable strategy for developing heat stress‐tolerant varieties. This approach holds promise for advancing the floricultural industry by overcoming challenges related to heat‐induced growth inhibition and loss of flower quality. [ABSTRACT FROM AUTHOR]

Summary: Salinity, as one of the most challenging environmental factors restraining crop growth and yield, poses a severe threat to global food security. To address the rising food demand, it is urgent to develop crop varieties with enhanced yield and greater salt tolerance by delving into genes associated with salt tolerance and high‐yield traits. MiR396b/GRF6 module has previously been demonstrated to increase rice yield by shaping the inflorescence architecture. In this study, we revealed that miR396b/GRF6 module can significantly improve salt tolerance of rice. In comparison with the wild type, the survival rate of MIM396 and OE‐GRF6 transgenic lines increased by 48.0% and 74.4%, respectively. Concurrent with the increased salt tolerance, the transgenic plants exhibited reduced H2O2 accumulation and elevated activities of ROS‐scavenging enzymes (CAT, SOD and POD). Furthermore, we identified ZNF9, a negative regulator of rice salt tolerance, as directly binding to the promoter of miR396b to modulate the expression of miR396b/GRF6. Combined transcriptome and ChIP‐seq analysis showed that MYB3R serves as the downstream target of miR396b/GRF6 in response to salt tolerance, and overexpression of MYB3R significantly enhanced salt tolerance. In conclusion, this study elucidated the potential mechanism underlying the response of the miR396b/GRF6 network to salt stress in rice. These findings offer a valuable genetic resource for the molecular breeding of high‐yield rice varieties endowed with stronger salt tolerance. [ABSTRACT FROM AUTHOR]

The flowering period of rice significantly impacts variety adaptability and yield formation. Properly shortening the reproductive period of rice varieties can expand their ecological range without significant yield reduction. Targeted genome editing, like CRISPR/Cas9, is an ideal tool to fine-tune rice growth stages and boost yield synergistically. In this study, we developed a CRISPR/Cas9-mediated multiplex genome-editing vector containing five genes related to three traits, Hd2, Ghd7, and DTH8 (flowering-stage genes), along with the recessive rice blast resistance gene Pi21 and the aromatic gene BADH2. This vector was introduced into the high-quality japonica rice variety in Zhejiang province, Jiahe212 (JH212), resulting in 34 T0 plants with various effective mutations. Among the 17 mutant T1 lines, several displayed diverse flowering dates, but most exhibited undesirable agronomic traits. Notably, three homozygous mutant lines (JH-C15, JH-C18, and JH-C31) showed slightly earlier flowering dates without significant differences in yield-related traits compared to JH212. Through special Hyg and Cas marker selection of T2 plants, we identified seven, six, and two fragrant glutinous plants devoid of transgenic components. These single plants will serve as sib lines of JH212 and potential resources for breeding applications, including maintenance lines for indica–japonica interspecific three-line hybrid rice. In summary, our research lays the foundation for the creation of short-growth-period CMS (cytoplasmic male sterility, CMS) lines, and also provides materials and a theoretical basis for indica–japonica interspecific hybrid rice breeding with wider adaptability. [ABSTRACT FROM AUTHOR]

Bananas are one of the most important cash crops in the tropics and subtropics. Drought and low-temperature stress affect the growth of banana. The DREB (dehydration responsive element binding protein) gene family, as one of the major transcription factor families, plays crucial roles in defense against abiotic stress. Currently, systematic analyses of the banana DREB (MaDREB) gene family have not yet been reported. In this study, 103 members of the MaDREB gene family were identified in the banana genome. In addition, transcriptomic analysis results revealed that MaDREBs responded to drought and cold stress. The expression of MaDREB14/22/51 was induced by drought and cold stress; these geneswere selected for further analysis. The qRT-PCR validation results confirmed the transcriptome results. Additionally, transgenic Arabidopsis plants overexpressing MaDREB14/22/51 exhibited enhanced resistance to drought and cold stress by reducing MDA content and increasing PRO and soluble sugar content. This study enhances our understanding of the function of the MaDREB gene family, provides new insights into their regulatory role under abiotic stress, and lays a good foundation for improving drought and cold stress-tolerant banana verities. [ABSTRACT FROM AUTHOR]

BACKGROUND: Transgenic crops producing insecticidal toxins from the bacterium Bacillus thuringiensis (Bt) have been used to manage insect pests for nearly 30 years. Dose of a Bt crop is key to assessing the risk of resistance evolution because it affects the heritability of resistance traits. Western corn rootworm (Diabrotica virgifera virgifera, LeConte), a major pest of maize, has evolved resistance to all commercially available Bt traits targeting it, and threatens resistance to future transgenic traits. Past research shows the dose of Bt maize targeting western corn rootworm can be confounded by larval density‐dependent mortality. We conducted a 2‐year field study at two locations to quantify larval density‐dependent mortality in Bt and non‐Bt maize. We used these results to calculate dose for our method and compared it to three previously published methods. Additionally, adult emergence and root injury were analyzed for predicting initial egg density. RESULTS: Increased pest density caused greater proportions of larvae to die in Bt maize than in non‐Bt maize. All methods for calculating dose produced values less than high‐dose, and stochastic variation had the greatest impact on dose at high and low pest densities. Our method for calculating dose did not produce values positively correlated with pest density while the three other methods did. CONCLUSION: To achieve the most accurate calculation of dose for transgenic maize targeting western corn rootworm, density‐dependent mortality should be taken into account for both transgenic and non‐transgenic maize and assessed at moderate pest densities. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Griffithsin (GRFT) is a highly effective, broad-spectrum, safe, and stable viral inhibitor used to suppress a variety of viruses. However, little information is available on whether GRFT can prevent plant viral diseases. In this study, we constructed a GRFT overexpression vector containing the sweetpotato storage cell signal peptide and generated exogenous GRFT overexpression lines through genetic transformation. The transgenic plants showed notable resistance to sweetpotato virus disease in the virus nursery. To verify the antiplant virus function of GRFT, transient expression in tobacco leaves showed that GRFT inhibited the sweetpotato leaf curl virus (SPLCV). The replication of SPLCV was entirely inhibited when the concentration of GRFT reached a certain level. The results of pulldown and BIFC assays showed that GRFT did not interact with the six components of SPLCV. In addition, the mutated GRFTD/A without the binding ability of carbohydrate and anticoronavirus function, in which three aspartate residues at carbohydrate binding sites were all mutated to alanine, also inhibited SPLCV. Quantitative reverse-transcription PCR analyses showed that the tobacco antiviral-related genes HIN1, ICS1, WRKY40, and PR10 were overexpressed after GRFT/GRFTD/A injection. Furthermore, HIN1, ICS1, and PR10 were more highly expressed in the leaves injected with GRFTD/A. The results suggest that sweetpotato is able to express GRFT exogenously as a bioreactor. Moreover, exogenous GRFT expression inhibits plant viruses by promoting the expression of plant antiviral genes. [ABSTRACT FROM AUTHOR]

Molecular farming, the practice of engineering plants to produce recombinant proteins, presents novel challenges and opportunities for domestic markets and international trade. This article explores the multifaceted risks associated with these biotechnological advancements, including public health concerns related to recombinant animal proteins produced in plants, cross-contamination and unintended allergens, and the necessity for stringent identity preservation systems to avoid past failures. On the global stage, the trade of such genetically engineered crops brings about unique regulatory concerns, underscoring the need for internationally harmonized policies and reevaluating existing low-level presence (LLP) thresholds to address unexpected allergens. Moreover, molecular farming ventures into complex religious and ethical territories, particularly affecting communities with strict dietary laws, such as Islamic, Jewish, and those following vegan or vegetarian lifestyles. Addressing these concerns requires a collaborative approach among scientists, regulatory bodies, industry leaders, and religious figures, aiming to foster an inclusive dialogue that navigates the ethical, religious, and environmental implications of integrating animal proteins into plant-based systems. Such efforts are essential for ensuring the responsible development of molecular farming technologies, contributing to a future of sustainable, secure, and inclusive food systems that respect diverse cultural and ethical values. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) have started to play an important role in pest control, and novel miRNA‐based transgenic insect‐resistant plants are now emerging. However, an environmental risk assessment of these novel transgenic plants expressing insect miRNAs must be undertaken before promoting their application. Here, transgenic miR‐14 rice, which has high resistance to the rice stem borer Chilo suppressalis, was used as an example for evaluation in this study. Taking the tier 1 risk assessment method in Bacillus thuringiensis (Bt) crops as a reference, the effects of the direct exposure of a non‐target parasitoid, Cotesia chilonis, to a high concentration of miRNA were evaluated. The results showed that direct feeding with miR‐14 at high concentration had no significant effects on the biological parameters of Co. chilonis, whereas when miR‐14 was injected into Ch. suppressalis‐parasitized larvae, the development duration of Co. chilonis was significantly affected. In combination with the real conditions of the rice paddy field, it could be inferred that transgenic miR‐14 rice has no significant negative effects on the important non‐target parasitoid, Co. chilonis. These results will provide a foundation for the establishment of a new safety evaluation system for novel RNAi‐based transgenic plants. [ABSTRACT FROM AUTHOR]

Summary: During arbuscular mycorrhizal (AM) symbiosis, plant innate immunity is modulated to a prime state to allow for fungal colonization. The underlying mechanisms remain to be further explored. In this study, two rice genes encoding LysM extracellular (LysMe) proteins were investigated.By obtaining OsLysMepro:GUS transgenic plants and generating oslysme1, oslysme2 and oslysme1oslysme2 mutants via CRISPR/Cas9 technique, OsLysMe genes were revealed to be specifically induced in the arbusculated cells and mutations in either gene caused significantly reduced root colonization rate by AM fungus Rhizophagus irregularis. Overexpression of OsLysMe1 or OsLysMe2 dramatically increased the colonization rates in rice and Medicago truncatula.The electrophoretic mobility shift assay and dual‐luciferase reporter assay supported that OsLysMe genes are regulated by OsWRI5a. Either OsLysMe1 or OsLysMe2 can efficiently rescue the impaired AM phenotype of the mtlysme2 mutant, supporting a conserved function of LysMe across monocotyledonous and dicotyledonous plants.The co‐localization of OsLysMe proteins with the apoplast marker SP‐OsRAmy3A implies their probable localization to the periarbuscular space (PAS) during symbiosis. Relative to the fungal biomass marker RiTEF, some defense‐related genes showed disproportionately high expression levels in the oslysme mutants. These data support that rice plants deploy two OsLysMe proteins to facilitate AM symbiosis, likely by diminishing plant defense responses. [ABSTRACT FROM AUTHOR]

[Display omitted] • Steam gasification of a genetically modified (GMO) rice straw is investigated. • GMO rice straw exhibits superior gasification performance compared to regular one. • Co-gasification of GMO rice straw with Giant Miscanthus is investigated. • Seaweed biochar addition can greatly enhance co-gasification with reduced tar. • Promoting synergistic effect by low-cost catalyst for co-gasification is clarified. Future sustainability visions include clean, renewable energy from hydrogen, which can be produced, among other ways, by biomass steam gasification. This study explores strategies addressing the limitations in steam co-gasification of herbaceous biomass, using Monster-TUAT1 rice straw, a genetically modified rice plant with a taller and bigger stalk developed by Tokyo University of Agriculture and Technology (TUAT), and Giant Miscanthus, a promising energy crop, as the feedstock. Firstly, compared with the typical rice straw, the Monster TUAT1 demonstrated superior steam gasification performance with a 1.75 times higher hydrogen gas yield and 27.0 % less tar generation. With a focus on overcoming the challenges posed by high silica content in the Monster TUAT1, co-gasification of it with an energy crop of Giant Miscanthus was performed. However, even under the optimum operation condition (750 °C, steam flowrate: 0.15 g/min), the hydrogen gas yield was only 29.3 mmol/g-C with a tar yield of 27.6 %wt. and a carbon conversion efficiency of 45.9 %, which is deemed unsatisfactory for hydrogen production. Thus, strategies for enhancement were proposed, including the incorporation seaweed biochar with high alkali and alkaline earth species, calcined scallop shell powder, and alkali metal salt into the gasifier. Consequently, the introduction of 10 %wt. of calcined scallop shell resulted in an increase in H 2 yield to 37.0 mmol/g-C and 24.3 % CO 2 reduction. The addition of alkali metal salt led to 43.9 % increase of H 2 product with a 15 %wt. tar yield. The most significant improvement occurred with the introduction of seaweed biochar at 50 %wt., increasing of the hydrogen gas yield to 62.0 mmol/g-C with 86 % of carbon conversion efficiency and tar reduction to 5.5 %. These findings demonstrate the viability of utilizing herbaceous biomass such as rice straw in conjunction with the strategic solutions of co-gasification to overcome constraints in improving hydrogen production. [ABSTRACT FROM AUTHOR]

The amino acid proline accumulates in plants during abiotic stresses such as drought and salinity and is considered a reliable marker of environmental stress. While its accumulation is well established, its precise role in stress tolerance and its underlying molecular mechanism remain less clear. To address these issues, we performed a meta-analysis—a robust statistical technique that synthesizes results from multiple independent studies while accounting for experimental differences. We focused on 16 physiological and morphological parameters affected by drought and salt stress in transgenic plants expressing proline metabolic genes. For each parameter, we calculated the effect size as the response ratio ( R R ), which represents the logarithm of the mean value in the transgenic group over the mean value of the control group ( ln R R ). Under stress, most parameters exhibited significantly higher response ratios in the transgenic group, confirming the beneficial effects of proline during drought and salt stress. Surprisingly, under non-stressed conditions, most stress markers showed no significant differences between transgenic and non-transgenic plants, despite elevated proline levels in the former. These results suggest that the benefits of proline may be related to proline catabolism or may only become apparent during stress, possibly due to interactions with reactive oxygen species (ROS), which accumulate predominantly under stress conditions. [ABSTRACT FROM AUTHOR]

ABSTRACT Prime editing is a versatile CRISPR/Cas‐based precise genome‐editing technique for crop breeding. Four new types of prime editors (PEs) named PE6a–d were recently generated using evolved and engineered reverse transcriptase (RT) variants from three different sources. In this study, we tested the editing efficiencies of four PE6 variants and two additional PE6 constructs with double‐RT modules in transgenic rice (Oryza sativa) plants. PE6c, with an evolved and engineered RT variant from the yeast Tf1 retrotransposon, yielded the highest prime‐editing efficiency. The average fold change in the editing efficiency of PE6c compared with PEmax exceeded 3.5 across 18 agronomically important target sites from 15 genes. We also demonstrated the feasibility of using two RT modules to improve prime‐editing efficiency. Our results suggest that PE6c or its derivatives would be an excellent choice for prime editing in monocot plants. In addition, our findings have laid a foundation for prime‐editing‐based breeding of rice varieties with enhanced agronomically important traits. [ABSTRACT FROM AUTHOR]

Plant roots shape the rhizosphere microbiome, recruiting microbes with beneficial functions. While genetically engineered crops offer yield advantages, their impacts on rhizosphere microbial communities remain understudied. This study evaluated the effects of transgenic maize, alongside a non-transgenic counterpart, on rhizosphere bacterial and fungal community composition using 16S rRNA and ITS amplicon sequencing. Additionally, glyphosate was used to evaluate its impact on microbial assembly and the magnitude of its effect at various maize growth stages. The results showed that transgenic maize D105 line significantly increased bacterial alpha diversity but not fungal diversity. Beta diversity analysis showed clear separation between bacterial and fungal communities at higher glyphosate treatment. Specific bacterial taxa such as Pseudomonas and Sphingomonas were enriched, while fungal taxa such as Ascomycota, Lasiosphaeriaceae, Verticillium were differentially abundant in glyphosate treatments. LEfSe analysis identified distinct enrichment patterns of bacterial (Proteobacteria and Actinobacteria) and fungal taxa (Verticillium and Guehomyces) associated with the transgenic line and glyphosate levels. KEGG functional analysis suggested potential impacts on bacterial metabolic pathways and shifts in fungal trophic modes (saprotrophs, pathogens) within the rhizosphere microbiome. This research provides insights into the classification, functional relationships, and underlying mechanisms shaping microbial communities carrying insect resistance and glyphosate resistance traits. [ABSTRACT FROM AUTHOR]

Abstract\nNOVELTY STATEMENTVesicular sequestration is a potential strategy for enhancing plant tolerance to cadmium (Cd) and arsenic (As). In this study, the ectopic overexpression of yeast-derived ScSMF2 in Arabidopsis thaliana was found to enhance the accumulation and tolerance of Cd and As in transgenic plants. ScSMF2 was localized on vacuole membranes and formed puncta structures in plant cells when agro-infiltrated for transient expression. Transgenic Arabidopsis showed less retardation on root elongation and shoot weight and more accumulation of Cd, As (III) and As (V) when cultured on medium containing Cd or As. Overexpression of ScSMF2 promoted accumulation of Cd and arsenic in transgenic Arabidopsis, which were over twice higher than in WT plants when cultured in soil. This study provides insights into the mechanisms involved in the vesicular sequestration of heavy metals in plant and presents a potential strategy for enhancing the phytoremediation capacity of plants toward heavy metals.Ectopic overexpression of the yeast Mn2+ transporter SMF2 in Arabidopsis thaliana substantially boosts the accumulation and tolerance to Cd and As in plants. This augmentation is attributed to the enhanced efficacy of intracellular vesicle sequestration, thereby bolstering the capacity of plants to sequester and detoxify these toxic heavy metals. This investigation introduces a potential approach for cultivating plants with improved phytoremediation capabilities, thereby advancing eco-friendly and sustainable remediation initiatives against heavy metal pollution. [ABSTRACT FROM AUTHOR]

The hepatitis B virus is a dangerous infectious agent, with an estimated 300 million carriers worldwide. Therefore, developing effective vaccine production methods is essential. One such method involves the use of transgenic plants. Several factors influence foreign gene expression in transgenic plants, including the type of genes, their chromosomal location, and the promoter used. Based on these factors, various expression cassettes can be designed for transgenic plants. In this study, we designed an expression cassette for transgenic potato plants. Promoters play a crucial role, as some are tissue-specific while others are inducible. Additionally, certain elements can enhance or silence gene expression in transgenic plants. Enhancer sequences derived from plant viruses are also utilized. To increase the expression level of HBsAg in transgenic potato plants, the tobacco etch virus (TEV) 5' untranslated region and the Patatin promoter were employed. The results demonstrated that in the highest-producing transgenic plant, the expression level of HBsAg increased threefold when both patatin and TEV were used together, compared to plants containing only the patatin promoter without TEV. Furthermore, a comparison with pCaMV combined with TEV revealed that the use of patatin and TEV resulted in a 4-5 times higher expression level. [ABSTRACT FROM AUTHOR]

EFSA was asked by the European Parliament to provide a scientific opinion on the analysis by the French Agency for Food, Environmental and Occupational Health & Safety (ANSES) of Annex I of the European Commission proposal for a regulation 'on plants obtained by certain new genomic techniques (NGTs) and their food and feed, and amending regulation (EU) 2017/625'. The Panel on genetically modified organisms (GMO) assessed the opinion published by ANSES, which focuses on (i) the need to clarify the definitions and scope, (ii) the scientific basis for the equivalence criteria and (iii) the need to take potential risks from category 1 NGT plants into account. The EFSA GMO Panel considered the ANSES analysis and comments on various terms used in the criteria in Annex I of the European Commission proposal and discussed definitions based on previous EFSA GMO Panel opinions. The EFSA GMO Panel concluded that the available scientific literature shows that plants containing the types and numbers of genetic modifications used as criteria to identify category 1 NGT plants in the European Commission proposal do exist as the result of spontaneous mutations or random mutagenesis. Therefore, it is scientifically justified to consider category 1 NGT plants as equivalent to conventionally bred plants with respect to the similarity of genetic modifications and the similarity of potential risks. The EFSA GMO Panel did not identify any additional hazards and risks associated with the use of NGTs compared to conventional breeding techniques in its previous Opinions. [ABSTRACT FROM AUTHOR]

The structure and expression of exogenous genes in transgenic crops are critical for the target traits. R7569 has the same exogenous insertion structure as the transgenic insect-resistant cotton MON531 but with a deletion in the 3′ end of the cry1Ac gene and the terminator region. Thus, in the present study, transcription, expression, and insecticidal activity assays were conducted to determine the function of the truncated cry1Ac gene. R7569 has a truncated cry1Ac gene with a length of 2554 bp encoding 881 amino acids, and the transcription termination site was mainly concentrated downstream of the truncated position and extended 160–270 bp from the truncated position using rapid amplification of cDNA ends (RACE). The transcript levels of the cry1Ac gene in R7569 were significantly higher than those of MON531 implants, except for during the boll stage. The content of the Cry1Ac protein in R7569 was higher than that of MON531 in the cotton leaf in all three periods. The corrected mortality rates of R7569 and MON531 against bollworms were 93.09% and 88.83%, respectively. The LC50 value of R7569 was 0.732 ng/g (dw), indicating a high level of resistance to bollworm. In this study, for the first time, we found a partial deletion of the target gene in commercially applied transgenic crops, and the partial deletion of the 3′ end of the cry1Ac gene retained a better transcription, expression level, and insecticidal activity, which can provide a specific case for the safety evaluation of transgenic crops. [ABSTRACT FROM AUTHOR]

Water deficit is the main limiting factor constraining sugarcane productivity, and its impact is expected to increase due to climate changes. During prolonged drought periods, most plants become extremely vulnerable to ROS accumulation, which can severely damage their photosynthetic apparatus. The PetC gene, encoding a Rieske FeS protein (ISP), has been shown to regulate the electron transport chain and protect photosystems (PSs) under drought conditions in some plant species. In sugarcane, transcriptome analysis revealed that ScPetC is repressed during drought stress in the field. In this study, we have overexpressed ScPetC in tobacco plants and evaluated its role in water-deficit tolerance. Our results indicate that the ScPetC protein structure is conserved when compared to other species. ScPetC overexpression reduced the negative impact of water deficit on plant development. This effect was associated with a positive impact on ScPetC quantum efficiency and the electron transport rate of PSII, the photosynthetic rate, and water use efficiency. The total chlorophyll content under water deficit was higher in plants overexpressing ScPetC, and this was correlated with less chlorophyll degradation from oxidative damage. Together, these results demonstrate that ScPetC confers tolerance to water deficit and oxidative stresses, making it a candidate gene for crop improvement. [ABSTRACT FROM AUTHOR]

Bacillus thuringiensis (Bt) toxins are potential alternatives to synthetic insecticides for the control of lepidopteran pests. However, the evolution of resistance in some insect pest populations is a threat and can reduce the effectiveness of Bt toxins. In this review, we summarize the results of 161 studies from 20 countries reporting field and laboratory-evolved resistance, cross-resistance, and inheritance, mechanisms, and fitness costs of resistance to different Bt toxins. The studies refer mainly to insects from the United States of America (70), followed by China (31), Brazil (19), India (12), Malaysia (9), Spain (3), and Australia (3). The majority of the studies revealed that most of the pest populations showed susceptibility and a lack of cross-resistance to Bt toxins. Factors that delay resistance include recessive inheritance of resistance, the low initial frequency of resistant alleles, increased fitness costs, abundant refuges of non-Bt, and pyramided Bt crops. The results of field and laboratory resistance, cross-resistance, and inheritance, mechanisms, and fitness cost of resistance are advantageous for predicting the threat of future resistance and making effective strategies to sustain the effectiveness of Bt crops. [ABSTRACT FROM AUTHOR]

SUMMARY Cassava is a crucial staple crop for smallholder farmers in tropical Asia and Sub‐Saharan Africa. Although high yield remains the top priority for farmers, the significance of nutritional values has increased in cassava breeding programs. A notable negative correlation between provitamin A and starch accumulation poses a significant challenge for breeding efforts. The negative correlation between starch and carotenoid levels in conventional and genetically modified cassava plants implies the absence of a direct genomic connection between the two traits. The competition among various carbon pathways seems to account for this relationship. In this study, we conducted a thorough analysis of 49 African cassava genotypes with varying levels of starch and provitamin A. Our goal was to identify factors contributing to differential starch accumulation. Considering carotenoid levels as a confounding factor in starch production, we found that yellow‐ and white‐fleshed storage roots did not differ significantly in most measured components of starch or de novo fatty acid biosynthesis. However, genes and metabolites associated with myo‐inositol synthesis and cell wall polymer production were substantially enriched in high provitamin A genotypes. These results indicate that yellow‐fleshed cultivars, in comparison to their white‐fleshed counterparts, direct more carbon toward the synthesis of raffinose and cell wall components. This finding is underlined by a significant rise in cell wall components measured within the 20 most contrasting genotypes for carotenoid levels. Our findings enhance the comprehension of the biosynthesis of starch and carotenoids in the storage roots of cassava. [ABSTRACT FROM AUTHOR]

Cereal grains are nutrient-dense food for a large section population in the world in general and developing and underdeveloped countries in Asia and Africa in particular along with their status as a staple food. Cereal crops such as rice and wheat are forerunners in achieving food security in the modern world of population explosion. Other cereals crops such as maize, sorghum, and pearl millets are proposed and promoted for various reasons such as the indirect promoter for agricultural diversification, promoter of diet diversification, capacity to contribute higher minerals (micronutrients) and other nutritional components in diet and their capacity to tolerate environmental odd situations. Besides these major cereal crops, the significance of millet has been highlighted in recent times. The millets are known for their tolerance to adverse environmental situations, nutritional superiority over major staple cereals, and multifarious uses. The availability in large quantities at an affordable price, policy support in production and distribution as well as strong institutional development activities (such as exploring genetic materials for high-yielding varieties development and success in tolerating biotic and abiotic stress) helps the cereal crops to remain a staple food. Besides staple and nutrient-dense food, cereals are also known for their very high crop residue production which is by default in most of the developing and underdeveloped countries used as fodder and feed to cattle, small ruminants, and also for poultry birds. Considering this status of cereals in human and animal nutrition, attempts have been made and still going on to enhance the nutritional status of these cereal crops in most of the developing and underdeveloped countries in Africa and Asia. Development of biofortified varieties, different methods of agronomic biofortification, success in developing genetically engineered nutrient-dense crops, different new and modified processing, and values addition in cereal grain-based nutrient-rich products and policies at the global and national level for the development of nutrient-dense food explain these attempts. On the other hand, lack of availability of seeds and low seed replacement ratio with nutrient-dense crops/varieties, socioeconomic constraints in the adoption of genetically engineered crops, lack of market incentives for biofortified cereals crops/varieties, lack of awareness and low adoption rate, constraints of bioavailability of nutrients in cereal grains and availability of a competitive source of nutrition other than cereals are other constraints in development and adoption of nutrient-dense cereal crops. Considering the significant contribution of cereal grains to the energy and protein supply to the majority of the population in developing and underdeveloped countries, all attempts to increase the nutritional status of cereal crops play a major role in supporting the attempts to achieve food and nutritional security in most of the Asian and African countries. The main objective of this chapter is to overview the status of cereal crops in Africa and Asia and the need for the development of nutrient-dense cereal crops. The chapter discussed the major nutrients designated for enriching the cereal grains and valid options for these enrichment. The development of nutrient-dense cereal grain varieties by exploring landraces and improved breeding techniques is the most important and largely explored option. This option will remain its highest significance in developing nutrient-dense cereal crops due to well-established institutional support and policy orientation and being a self-sustained avenue. The soil and plant-based approaches including bio-fortification, ferti-fortification, and diversification of cereal crops with millets stand second. This approach is gaining and will again intensify its impact on developing nutrient-dense crops. The harvesting complimentary and supplementary interactions of soil and plant-based approaches with newly developed nutrient-dense crop varieties will be forerunners in achieving the reach for the nutrient-dense crop to designated end beneficiaries. The legislation (mandatory or voluntary) for fortification of food products, supplementation of micronutrients through medication, the establishment of standards for food fortification, awareness creation in the market chain about nutrient-dense crops and their significance, and increasing attempts for incorporation of genetically modified crops in the food chain are the other avenues of significant role at present and future too. Considering the single major source of nutrition, the cereal grain will remain an affordable nutrient-dense grain for a large section of people from Africa and Asia. Therefore, the wider implications of the above-mentioned avenues are going on at present and will acquire more attention in the near future. [ABSTRACT FROM AUTHOR]

Mucilage is a gelatinous and sticky hydrophilic polysaccharide released from epidermal cells of seed coat after the hydration of mature seeds and is composed primarily of unbranched rhamnogalacturonan I (RG-I). In this study, we produced a recombinant endo-RG-I hydrolase from Aspergillus aculeatus (Aa RhgA) in the fission yeast Schizosaccharomyces pombe and examined its substrate preference for pyridylaminated (PA) RG-I with the various degrees of polymerization (DP). Recombinant Aa RhgA requires PA-RG-I with a DP of 10 or higher for its hydrolase activity. We heterologously expressed the AarhgA gene under the strong constitutive promoter, cauliflower mosaic virus 35S promoter, in Arabidopsis thaliana. In a series of biochemical analyses of each mucilage fraction released from the water-imbibed seeds of the transgenic plants, we found the enhanced deposition of the transparent mucilage layer that existed in the peripheral regions of the adherent mucilage and was not stained with ruthenium red. This study demonstrated the feasibility of manipulating the mucilage organization by heterologous expression of the endo-RG-I hydrolase. • A production system for recombinant Aa RhgA enzyme in S. pombe was established. • Substrate preference for PA-RG-I with various DP was demonstrated. • AarhgA was heterologously expressed under the CaMV promoter in A. thaliana. • Enhanced deposition of the transparent mucilage layer was revealed in transgenic plants. [ABSTRACT FROM AUTHOR]

Summary: RNA interference (RNAi) has emerged as an efficient technology for pest control by silencing the essential genes of targeted insects. Owing to its nucleotide sequence‐guided working mechanism, RNAi has a high degree of species‐specificity without impacts on non‐target organisms. However, as plants are inevitably under threat by two or more insect pests in nature, the species‐specific mode of RNAi‐based technology restricts its wide application for pest control. In this study, we artificially designed an intermediate dsRNA (iACT) targeting two β‐Actin (ACT) genes of sap‐sucking pests Bemisia tabaci and Myzus persicae by mutual correction of their mismatches. When expressing hairpin iACT (hpiACT) from tobacco nuclear genome, transgenic plants are well protected from both B. tabaci and M. persicae, either individually or simultaneously, as evidenced by reduced fecundity and suppressed ACT gene expression, whereas expression of hpRNA targeting BtACT or MpACT in transgenic tobacco plants could only confer specific resistance to either B. tabaci or M. persicae, respectively. In sum, our data provide a novel proof‐of‐concept that two different insect species could be simultaneously controlled by artificial synthesis of dsRNA with sequence optimization, which expands the range of transgenic RNAi methods for crop protection. [ABSTRACT FROM AUTHOR]

The latest CRISPR-Cas9-mediated genome editing technology is expected to bring about revolution in rice yield and quality improvement, and thus validation of rice transformation protocols using CRISPR-Cas9-gRNA constructs is the need of the hour. Moreover, regeneration of more number of transgenic rice plants is prerequisite for developing genome-edited rice lines, as recalcitrant rice varieties were shown to have lower editing efficiencies which necessities screening of large number of transgenic plants to find the suitable edits. In the present study, we have simplified the Agrobacterium-mediated rice transformation protocol for both Indica and Japonica rice cultivars using CRISPR/Cas9 empty vector construct, and the protocols have been suitably optimized for getting large numbers of the regenerated plantlets within the shortest possible time. The Japonica transgenic lines were obtained within 65 days and for the Indica cultivars, it took about 76–78 days. We also obtained about 90% regeneration efficiency for both Japonica and Indica cultivars. The transformation efficiency was about 97% in the case of Japonica and 69–83% in the case of Indica rice cultivars. Furthermore, we screened the OsWRKY24 gene editing efficiency by transforming rice cultivars with CRISPR/Cas9 construct harbouring sgRNA against OsWRKY24 gene and found about 90% editing efficiency in Japonica rice cultivars, while 30% of the transformed Indica cultivars were found to be edited. This implicated the presence of a robust repair mechanism in the Indica rice cultivars. [ABSTRACT FROM AUTHOR]

Saline–alkali stress is a significant abiotic stress that restricts plant growth globally. Basic region leucine zipper (bZIP) transcription factor proteins are widely involved in plants in response to abiotic stress such as saline–alkali stress. Based on transcriptome and quantitative real-time PCR (qRT-PCR), we found that the MhbZIP23 gene could respond to saline–alkali stress. Despite this discovery, the underlying mechanism by which the MhbZIP23 transcription factor responds to saline–alkaline stress remains unexplored. To address this gap in knowledge, we successfully cloned the MhbZIP23 (MD05G1121500) gene from Malus halliana for heterologous expression in Arabidopsis thaliana, facilitating the investigation of its functional role in stress response. Compared to the wild type (WT), Arabidopsis plants demonstrated enhanced growth and a lower degree of wilting when subjected to saline–alkali stress. Furthermore, several physiological indices of the plants altered under such stress conditions. The transgenic Arabidopsis plants (OE-5, 6, and 8), which grew normally, exhibited a higher chlorophyll content and had greater root length in comparison to the control check (CK). MhbZIP23 effectively regulated the levels of the osmoregulatory substance proline (Pro), enhanced the activities of antioxidant enzymes such as peroxidase (POD) and superoxide dismutase (SOD), and reduced the levels of malondialdehyde (MDA) and relative conductivity (REC). These actions improved the ability of plant cells in transgenic Arabidopsis to counteract ROS, as evidenced by the decreased accumulation of O2− and hydrogen peroxide (H2O2). In summary, the MhbZIP23 gene demonstrated effectiveness in alleviating saline–alkali stress in M. halliana, presenting itself as an outstanding resistance gene for apples to combat saline–alkali stress. [ABSTRACT FROM AUTHOR]

Idesia polycarpa is a promising woody oilseed species because of its high oil yield. However, its use is greatly limited due to the lack of varieties with good qualities; additionally, gene function has been less studied in this plant because an efficient transformation method has not been established yet. In this study, we established a rapid and efficient hairy root transformation method by infecting the whole seedling, the rootless seedling, and the leaf petiole with Agrobacterium rhizogenes using different infection methods. Among these transformation methods, a higher transformation efficiency was obtained using the whole seedling, which could reach up to 71.91%. Furthermore, we found that the seedling age significantly affected the transformation efficiency, either using whole or rootless seedlings. Additionally, we found that the transgenic roots could regenerate transgenic shoots. Taken together, our study lays the foundation for future study and for genetically modifying wood traits in the future. [ABSTRACT FROM AUTHOR]

In order to screen candidate aquaporin genes involved in resisting osmotic stress, we analyzed the physiological responses and the expression levels of aquaporin genes in garlic under drought and salt stress with 'Er Shuizao' as plant material. Different physiological indicators were detected under drought and salt stress treatments. RT-qPCR was used to detect the expression levels of the candidate aquaporin genes in specific tissues. Finally, we screened AsPIP1-3 as a candidate gene and analyzed its function. The results showed that the relative water content and chlorophyll content of leaves decreased, the O2− production rate increased, and H2O2 accumulated in garlic under drought and salt stress. The activities of SOD, POD, and CAT enzymes first increased and then decreased in garlic. The content of soluble sugar and proline increased to maintain cell osmotic balance, and the content of MDA and relative conductivity continued to increase. Most aquaporin gene expression first increased and then decreased in garlic under drought and salt stress. AsPIP1-3 gene expression is up-regulated under drought and salt stress in garlic. The relative expression was the highest on the 6th day of stress, being related to antioxidant enzyme activity and osmotic regulation. The consistent changes in gene expressions and physiological responses indicated that AsPIP1-3 played a role in resisting garlic osmotic stress. AsPIP1-3 was located on the cell membrane, being consistent with the predicted results of subcellular localization. The germination rate and root length of transgenic Arabidopsis under drought stress were significantly different from the wild type. Drought stress reduced the ROS accumulation of transgenic Arabidopsis, and the antioxidant enzyme activity was significantly higher than the wild type. The relative conductivity and MDA content significantly decreased, and the proline content increased under drought stress. The expression level of the genes related to drought stress response (AtRD22, AtP5CS, AtABF3, and AtLEA) significantly increased. The overexpression of AsPIP1-3 genes improved the drought tolerance of transgenic Arabidopsis plants, showing that AsPIP1-3 proteins enhanced drought tolerance. Our study laid a foundation for exploring the regulatory mechanism of garlic to abiotic stress. [ABSTRACT FROM AUTHOR]

Agrobacterium tumefaciens-mediated plant transformation is the most dominant technique for the transformation of plants. It is used to transform monocotyledonous and dicotyledonous plants. A. tumefaciens apply for stable and transient transformation, random and targeted integration of foreign genes, as well as genome editing of plants. The Advantages of this method include cheapness, uncomplicated operation, high reproducibility, a low copy number of integrated transgenes, and the possibility of transferring larger DNA fragments. Engineered endonucleases such as CRISPR/Cas9 systems, TALENs, and ZFNs can be delivered with this method. Nowadays, Agrobacterium-mediated transformation is used for the Knock in, Knock down, and Knock out of genes. The transformation effectiveness of this method is not always desirable. Researchers applied various strategies to improve the effectiveness of this method. Here, a general overview of the characteristics and mechanism of gene transfer with Agrobacterium is presented. Advantages, updated data on the factors involved in optimizing this method, and other useful materials that lead to maximum exploitation as well as overcoming obstacles of this method are discussed. Moreover, the application of this method in the generation of genetically edited plants is stated. This review can help researchers to establish a rapid and highly effective Agrobacterium-mediated transformation protocol for any plant species. [ABSTRACT FROM AUTHOR]

Introduction: Melatonin (N-acetyl-5-methoxytryptamine) is a molecule implicated in multiple biological functions, but exerts contrasting effects on plants owing to concentration differences. Hydroxyindole O-methyltransferase (HIOMT), which catalyzes the last step of melatonin synthesis, plays a crucial role in this context. Methods: Transgenic switchgrass overexpressing oHIOMT with different melatonin levels displayed distinct morphological changes in a concentrationdependent manner. In this study, we divided the transgenic switchgrass into two groups: melatonin-moderate transgenic (MMT) plants and melatonin-rich transgenic (MRT) plants. To determine the concentration-dependent effect of melatonin on switchgrass growth and stress resistance, we conducted comparative morphological, physiological, omics and molecular analyses between MMT, MRT and wild-type (WT) plants. Results: We found that oHIOMT overexpression, with moderate melatonin levels, was crucial in regulating switchgrass growth through changes in cell size rather than cell number. Moderate levels of melatonin were vital in regulating carbon fixation, stomatal development and chlorophyll metabolism. Regarding salt tolerance, melatonin with moderate levels activated numerous defense (e.g. morphological characteristics, anatomical structure, antioxidant enzymatic properties, non-enzymatic capacity and Na+/K+ homeostasis). Additionally, moderate levels of oHIOMT overexpression were sufficient to increase lignin content and alter monolignol compositions with an increase in the S/G lignin ratio. Discussion: Taken together, oHIOMT overexpression in switchgrass with different melatonin levels resulted in morphological, anatomical, physiological and molecular changes in a concentration-dependent manner, which characterized by stimulation at low doses and inhibition at high doses. Our study presents new ideas and clues for further research on the mechanisms of the concentration-dependent effect of melatonin. [ABSTRACT FROM AUTHOR]

BACKGROUND: Bacillus thuringiensis (Bt) and its crystal toxin or δ‐endotoxins (Cry) offer great potential for the efficient control of crop pests. A vast number of pests can potentially infect the same host plant, either simultaneously or sequentially. However, no effective Bt‐Cry protein has been reported to control both aphids and plant parasitic nematodes due to its highly specific activity. RESULTS: Our study indicated that the Cry5Ba2 protein was toxic to the green peach aphid Myzus persicae, which had a median lethal concentration (LC50) of 9.7 ng μL−1 and fiducial limits of 3.1–34.6 ng μL−1. Immunohistochemical localization of Cry5Ba2 revealed that it could bind to the apical tip of microvilli in midgut regions. Moreover, transgenic tobacco plants expressing Cry5Ba2 exhibited significant resistance to Myzus persicae, as evidenced by reduced insect survival and impaired fecundity, and also intoxicated the Meloidogyne incognita as indicated by a decrease in galls and progeny reproduction. CONCLUSION: In sum, we identified a new aphicidal Bt toxin resource that could simultaneously control both aboveground and belowground pests, thus extending the application range of Bt‐based strategy for crop protection. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Abstract As a typical bulb flower, lily is widely cultivated worldwide because of its high ornamental, medicinal and edible value. Although breeding efforts evolved over the last 10000 years, there are still many problems in the face of increasing consumer demand. The approach of biotechnological methods would help to solve this problem and incorporate traits impossible by conventional breeding. Target traits are dormancy, development, color, floral fragrance and resistances against various biotic and abiotic stresses, so as to improve the quality of bulbs and cut flowers in planting, cultivation, postharvest, plant protection and marketing. Genetic transformation technology is an important method for varietal improvement and has become the foundation and core of plant functional genomics research, greatly assisting various plant improvement programs. However, achieving stable and efficient genetic transformation of lily has been difficult worldwide. Many gene function verification studies depend on the use of model plants, which greatly limits the pace of directed breeding and germplasm improvement in lily. Although significant progress has been made in the development and optimization of genetic transformation systems, shortcomings remain. Agrobacterium-mediated genetic transformation has been widely used in lily. However, severe genotypic dependence is the main bottleneck limiting the genetic transformation of lily. This review will summarizes the research progress in the genetic transformation of lily over the past 30 years to generate the material including a section how genome engineering using stable genetic transformation system, and give an overview about recent and future applications of lily transformation. The information provided in this paper includes ideas for optimizing and improving the efficiency of existing genetic transformation methods and for innovation, provides technical support for mining and identifying regulatory genes for key traits, and lays a foundation for genetic improvement and innovative germplasm development in lily.