Non-transgenic Approach Research Articles

RNAi-based biocontrol for crops: a revised expectation for a non-recent technology.

The exogenous application of RNAi technology offers new promises for crops improvement. Cell-based or synthetically produced strands are economical, non-transgenic and could induce the same responses. The substantial population growth demands novel strategies to produce crops without further damaging the environment. RNA interference mechanism is one of the promising technologies to biologically control pests and pathogens in crops, suppressing them by cancelling protein synthesis related to parasitism/pathogenesis. The transgenic approach to generate host-induced gene silencing demonstrated high efficacy in controlling pests or pathogens by RNAi mechanism. However, transgenic technology is tightly regulated and still negatively perceived by global consumers. This review presents the basic biology of small RNA, the main actor of the RNAi mechanism, and tested non-transgenic approaches to induce RNAi exogenously. Novel avenues are offered by the discovery of cross-kingdom RNAi, that naturally, plants also deliver small RNA to suppress the growth of their threats. Future applications of non-transgenic RNAi-based biocontrol will involve the production of dsRNA on an industrial scale. Here, the attempts to provide dsRNA for routine application in farms are also discussed, emphasizing that the technology must be accessible by the countries with the greatest population which mostly are poorer ones.

Fungal effector genes involved in the suppression of chitin signaling as novel targets for the control of powdery mildew disease via a nontransgenic RNA interference approach.

Chitin is a crucial component of fungal cell walls and an effective elicitor of plant immunity; however, phytopathogenic fungi have developed virulence mechanisms to counteract the activation of this plant defensive response. In this study, the molecular mechanism of chitin-induced suppression through effectors involved in chitin deacetylases (CDAs) and their degradation (EWCAs) was investigated with the idea of developing novel dsRNA-biofungicides to control the cucurbit powdery mildew caused by Podosphaera xanthii. The molecular mechanisms associated with the silencing effect of the PxCDA and PxEWCAs genes were first studied through dsRNA cotyledon infiltration assays, which revealed a ≈80% reduction in fungal biomass and a 50% decrease in gene expression. To assess the impact on powdery mildew disease control, in vitro and in planta assays were carried out in growth chamber and glasshouse experiments, with ≈50% reduction in disease symptoms after 8 days postinoculation (dpi) in leaf discs and 12 dpi in plants' leaves, respectively. This control was extended for 21 days when the dsRNAs were protected on the carbon dot nanocarriers. Additionally, the uptake of the dsRNAs by fungal spores was observed 12 h postapplication via confocal microscopy, and efficient processing of dsRNAs into siRNAs by the melon RNAi machinery was observed 24 h postspraying through sRNA-seq. This study highlights notable advancements in environmentally friendly disease management, and features the technological potential of RNA-based fungicides together with nanotechnology for cucurbit powdery mildew control. © 2025 Society of Chemical Industry.

Identification of pathogenicity determinants in ToLCNDV and their RNAi-based knockdown for disease management in Nicotiana benthamiana and tomato plants.

Begomovirus solanumdelhiense (tomato leaf curl New Delhi virus, ToLCNDV), is member of the genus Begomovirus, family Geminiviridae, is a prolific bipartite whitefly transmitted begomovirus in the Indian sub-continent has a wide host range, including solanaceous, cucurbitaceous and other plants. Recently, dsRNA-mediated non-transgenic approaches have been promising in managing plant viruses. Such an approach could be effective if the pathogenicity determinants of a virus are targeted. In the case of ToLCNDV, viral pathogenicity has been demonstrated with coat protein (AV1), pre-coat protein (AV2), transcription activator protein (AC2) and nuclear shuttle protein (NSP). In the present study, we investigated the involvement of the three RNA silencing suppressor proteins (AV2, AC2, AC4) encoded by ToLCNDV in pathogenicity determinants through transient overexpression and hairpin RNAi-based knockdown assays in Nicotiana benthamiana plants. Further, we showed that the transcripts of AV2, AC2, and AC4 genes can systemically move and express their proteins. Hairpin RNAi constructs targeting each pathogenicity determinant could effectively reduce symptom development and virus titer upon inoculation of ToLCNDV in N. benthamiana plants. Exogenous application of dsRNA individually (dsAV2/dsAC2/dsAC4) or together (cocktail dsRNA: dsCk) against the pathogenicity determinants showed a significant reduction of viral load and reduced severity of disease in plants treated with dsCk followed by dsAC4. The present report reconfirms that the RNA silencing suppressor proteins encoded by DNA-A genomic component of ToLCNDV, can also act as pathogenicity determinants. Further, we demonstrated for the first time that exogenous application of dsRNA targeting those pathogenicity determinants reduces ToLCNDV load and limits symptom development in tomato plants.

A Chimeric Peptide for Shielding Plant Photosynthetic Systems against Excess Light Stress via Chloroplast-Targeted ROS Quenching.

The ability to quench reactive oxygen species (ROS) overproduced in plant chloroplasts under light stress conditions is essential for securing plant photosynthetic performance and agricultural yield. Although genetic engineering can enhance plant stress resistance, its widespread application faces limitations due to challenges in successful transformation across plant species and public acceptance concerns. This study proposes a nontransgenic chemical approach using a designed chimeric peptide that scavenges ROS within plant chloroplasts for managing light stress. The chimeric peptide was strategically designed by combining cell-penetrating and chloroplast-targeting sequences, each with antioxidant ability against destructive ROS such as hydroxyl radical (•OH) and singlet oxygen (1O2). Our analyses involving various cell-penetrating peptides and a chloroplast-targeting peptide revealed that the •OH-scavenging ability predominantly relied on side chain oxidation in tryptophan residues, while the 1O2-quenching capacity was attributed to the oxidation of cysteine and methionine side chains. We further demonstrated that the chimeric peptide could traverse the cell wall and membranes to reach chloroplasts, where it scavenged •OH and 1O2 and alleviated light-stress-induced chlorophyll degradation in leaves. Foliar spraying of the peptide successfully protected photosynthetic activity in leaves exposed to excessive light, highlighting its potential for practical agricultural applications. This work can offer a promising approach for managing abiotic stress without genetic modifications and provide valuable insights into the design of effective peptide-based ROS quenchers specifically targeting plant chloroplasts.

Molecular approaches for the management of papaya ringspot virus infecting papaya: a comprehensive review.

Papaya ringspot virus (PRSV) is a catastrophic disease that causes huge yield losses in papaya cultivation around the world. Yield losses in severely infected plants can be upto 100%. Because of this disease, papaya cultivation has been shifted to other crops in some areas of the world. Many conventional methods and breeding approaches are used against this disease, which turns out to be less effective. Considering the yield loss caused by PRSV in papaya, it is high time to focus on alternative control methods. To implement effective management strategies, molecular approaches such as Marker Assisted Breeding (MAS) or transgenic methods involving post-transcriptional gene silencing targeting the genome viz., coat protein, replicase gene, or HC Pro can be pursued. However, the public's reluctance to widely accept the transgenic approach due to health and environmental concerns necessitates a consideration of non-transgenic alternatives. Prioritizing safety and ensuring efficient virus control, non-transgenic approaches which encompass cross-protection, genome editing, and topical applications of dsRNA to induce gene silencing within the host, can be adopted. This review aims to provide comprehensive insights of various molecular tools used in managing PRSV which in turn will help in sustainable agriculture.

CRISPR/Cas9 mediated validation of spermatogenesis-related gene, tssk2 as a component of genetic pest management of fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae).

Invasive insect pests, currently, pose a serious economic threat to several staple crops all over the world, one such being the fall armyworm, Spodoptera frugiperda. It was first observed in Africa since 2016, outside of its natural habitat in the Americas. Subsequently, it invaded several countries in South and South East Asia and also very recently in Australia. In all the newly invaded regions, maize is the principal crop attacked causing a serious economic concern to the poor farmers, particularly in the developing countries. Owing to the innate genetic ability, it defies many of the management options that include insecticides, Bt transgenics, and so forth. This is due to its high mobility, polyphagy and ability for quick development of resistance to several classes of insecticides. At this critical juncture, CRISPR/Cas9 mediated genome editing has shown a lot of promise in developing a novel area-wide pest management strategy called precision-guided sterile insect technique (pgSIT). pgSIT was initially demonstrated in Drosophila melanogaster which holds a greater promise for the environmentally friendly management of several globally significant agricultural pests such as S. frugiperda. Therefore, before developing both sgRNA and Cas9 transgenic lines, we have validated the target gene such as tssk2 through a non-transgenic approach by microinjecting ribo nucleo protein complex (Cas9 protein and tssk2 sgRNA) into G0 eggs of S. frugiperda. In the current investigation, we have obtained five edited males with distinct mutations which were further used for crossing studies to ascertain the effect of tssk2 editing affecting egg hatchability.

Potato Biofortification: A Systematic Literature Review on Biotechnological Innovations of Potato for Enhanced Nutrition

Potato biofortification is a comprehensive approach aimed at enhancing the nutritional content of potatoes, addressing widespread nutrient deficiencies and contributing to global food security. This systematic review examines the existing literature on various aspects of potato biofortification, encompassing genetic, agronomic, and biotechnological strategies. The review highlights the nutritional significance of potatoes, emphasizing their role as a staple food in many regions. Genetic approaches to biofortification involve the identification and use of natural variations in potato germplasm to develop varieties with elevated levels of essential nutrients. This includes targeting key micronutrients, such as iron, zinc, and vitamins, through traditional breeding methods. The review explores the genetic diversity within potato germplasm and the potential for breeding programs to develop nutrient-rich varieties. Agronomic practices play a crucial role in potato biofortification, with studies demonstrating the impact of tuber priming and the application of mineral fertilizers on nutrient concentrations in potatoes. The review delves into the intricacies of agronomic biofortification, emphasizing the importance of precise dosages and timing for optimal results. Biotechnological tools, including transgenic and non-transgenic approaches, are discussed in the context of potato biofortification. The review evaluates the efficiency and ethical considerations associated with the development of biofortified transgenic potatoes and emphasizes the significance of non-transgenic approaches in addressing consumer concerns and regulatory barriers. Overall, this systematic review provides a comprehensive overview of the current state of potato biofortification research. It synthesizes findings from diverse studies, offering insights into the potential of biofortified potatoes to address hidden hunger and contribute to improved nutritional outcomes. This review also identifies knowledge gaps and areas for future research, guiding the direction of efforts to harness the full potential of potato biofortification for global food and nutrition security.

Exogenous Application of dsRNA for Protection against Tomato Leaf Curl New Delhi Virus.

Tomato leaf curl New Delhi virus (ToLCNDV) is an emerging plant pathogen, fast spreading in Asian and Mediterranean regions, and is considered the most harmful geminivirus of cucurbits in the Mediterranean. ToLCNDV infects several plant and crop species from a range of families, including Solanaceae, Cucurbitaceae, Fabaceae, Malvaceae and Euphorbiaceae. Up to now, protection from ToLCNDV infection has been achieved mainly by RNAi-mediated transgenic resistance, and non-transgenic fast-developing approaches are an urgent need. Plant protection by the delivery of dsRNAs homologous to a pathogen target sequence is an RNA interference-based biotechnological approach that avoids cultivating transgenic plants and has been already shown effective against RNA viruses and viroids. However, the efficacy of this approach against DNA viruses, particularly Geminiviridae family, is still under study. Here, the protection induced by exogenous application of a chimeric dsRNA targeting all the coding regions of the ToLCNDV DNA-A was evaluated in zucchini, an important crop strongly affected by this virus. A reduction in the number of infected plants and a delay in symptoms appearance, associated with a tendency of reduction in the viral titer, was observed in the plants treated with the chimeric dsRNA, indicating that the treatment is effective against geminiviruses but requires further optimization. Limits of RNAi-based vaccinations against geminiviruses and possible causes are discussed.

A Non-Canonical Pathway Induced by Externally Applied Virus-Specific dsRNA in Potato Plants.

The external application of double-stranded RNA (dsRNA) has recently been developed as a non-transgenic approach for crop protection against pests and pathogens. This novel and emerging approach has come to prominence due to its safety and environmental benefits. It is generally assumed that the mechanism of dsRNA-mediated antivirus RNA silencing is similar to that of natural RNA interference (RNAi)-based defence against RNA-containing viruses. There is, however, no direct evidence to support this idea. Here, we provide data on the high-throughput sequencing (HTS) analysis of small non-coding RNAs (sRNA) as hallmarks of RNAi induced by infection with the RNA-containing potato virus Y (PVY) and also by exogenous application of dsRNA which corresponds to a fragment of the PVY genome. Intriguingly, in contrast to PVY-induced production of discrete 21 and 22 nt sRNA species, the externally administered PVY dsRNA fragment led to generation of a non-canonical pool of sRNAs, which were present as ladders of ~18-30 nt in length; suggestive of an unexpected sRNA biogenesis pathway. Interestingly, these non-canonical sRNAs are unable to move systemically and also do not induce transitive amplification. These findings may have significant implications for further developments in dsRNA-mediated crop protection.

RNAi Technology: A New Path for the Research and Management of Obligate Biotrophic Phytopathogenic Fungi.

Powdery mildew and rust fungi are major agricultural problems affecting many economically important crops and causing significant yield losses. These fungi are obligate biotrophic parasites that are completely dependent on their hosts for growth and reproduction. Biotrophy in these fungi is determined by the presence of haustoria, specialized fungal cells that are responsible for nutrient uptake and molecular dialogue with the host, a fact that undoubtedly complicates their study under laboratory conditions, especially in terms of genetic manipulation. RNA interference (RNAi) is the biological process of suppressing the expression of a target gene through double-stranded RNA that induces mRNA degradation. RNAi technology has revolutionized the study of these obligate biotrophic fungi by enabling the analysis of gene function in these fungal. More importantly, RNAi technology has opened new perspectives for the management of powdery mildew and rust diseases, first through the stable expression of RNAi constructs in transgenic plants and, more recently, through the non-transgenic approach called spray-induced gene silencing (SIGS). In this review, the impact of RNAi technology on the research and management of powdery mildew and rust fungi will be addressed.

Nanocarrier spray: a nontransgenic approach for crop engineering.

Genetic modification allows engineering of important traits in crops through expensive and tedious procedures to alter their genetic background. Recently, Thagun et al. developed a nanocarrier-based foliar spray method to translocate bioactive molecules of interest into plant cells to engineer important traits without introducing a transgene.

Scion–Rootstock Relationship: Molecular Mechanism and Quality Fruit Production

Most tree fruits are commercially grown on different root systems, hence called composite plants. The section provides the root system as the rootstock, and the atop ground portion is called the scion. The combination is selected based on different traits of scion varieties, rootstock, and prevailing edaphic situations. The dated back plant propagation technique of joining two plants (grafting/budding) that directly communicates new physiological traits to the desirable scion variety from the rootstock remains unclear. In spite of this, this propagation technique continues widely applied in the multiplication of several fruit plant species. In a grafted plant, rootstocks impacted the scion variety’s growth, yield and quality attributes, physiology, nutrient accumulation as well as biotic and abiotic stress tolerance in many ways. Modern research in plant science for next-generation sequencing providing new vital information about the molecular interactions in composite plants multiplied using grafting. Now it was confirmed that genetic exchange is occurring between rootstock and scion variety through grafting joints. In this aspect, we discuss the process and the molecular mechanism of rootstock scion interactions. This review finally explains the dynamics of rootstock–scion interactions as well as their effect on physiology in terms of production, environmental stresses, and fruit quality. The morphological, physiochemical, and molecular mechanisms have been reviewed to develop an integrated understanding of this unknowable process that questions existing genetic paradigms. The present review summarizes the reported molecular mechanism between scion and rootstock and the impact of rootstocks on the production biology of scion varieties of economically important fruit crops and identifies numerous key points to consider when conducting rootstock scion interaction experiments. Rootstocks may offer a non-transgenic approach to rapidly respond to the changing environment and expand agricultural production of perennial fruit crops where grafting is possible in order to meet the global demand for fruit, food, and demands of the future.

A tale of two metals: Biofortification of rice grains with iron and zinc.

Iron (Fe) and zinc (Zn) are essential micronutrients needed by virtually all living organisms, including plants and humans, for proper growth and development. Due to its capacity to easily exchange electrons, Fe is important for electron transport in mitochondria and chloroplasts. Fe is also necessary for chlorophyll synthesis. Zn is a cofactor for several proteins, including Zn-finger transcription factors and redox metabolism enzymes such as copper/Zn superoxide dismutases. In humans, Fe participates in oxygen transport, electron transport, and cell division whereas Zn is involved in nucleic acid metabolism, apoptosis, immunity, and reproduction. Rice (Oryza sativa L.) is one of the major staple food crops, feeding over half of the world's population. However, Fe and Zn concentrations are low in rice grains, especially in the endosperm, which is consumed as white rice. Populations relying heavily on rice and other cereals are prone to Fe and Zn deficiency. One of the most cost-effective solutions to this problem is biofortification, which increases the nutritional value of crops, mainly in their edible organs, without yield reductions. In recent years, several approaches were applied to enhance the accumulation of Fe and Zn in rice seeds, especially in the endosperm. Here, we summarize these attempts involving transgenics and mutant lines, which resulted in Fe and/or Zn biofortification in rice grains. We review rice plant manipulations using ferritin genes, metal transporters, changes in the nicotianamine/phytosiderophore pathway (including biosynthetic genes and transporters), regulators of Fe deficiency responses, and other mutants/overexpressing lines used in gene characterization that resulted in Fe/Zn concentration changes in seeds. This review also discusses research gaps and proposes possible future directions that could be important to increase the concentration and bioavailability of Fe and Zn in rice seeds without the accumulation of deleterious elements. We also emphasize the need for a better understanding of metal homeostasis in rice, the importance of evaluating yield components of plants containing transgenes/mutations under field conditions, and the potential of identifying genes that can be manipulated by gene editing and other nontransgenic approaches.

Increasing the Grain Yield and Grain Protein Content of Common Wheat (Triticum aestivum) by Introducing Missense Mutations in the Q Gene.

Grain yield (GY) and grain protein content (GPC) are important traits for wheat breeding and production; however, they are usually negatively correlated. The Q gene is the most important domestication gene in cultivated wheat because it influences many traits, including GY and GPC. Allelic variations in the Q gene may positively affect both GY and GPC. Accordingly, we characterized two new Q alleles (Qs1 and Qc1-N8) obtained through ethyl methanesulfonate-induced mutagenesis. Compared with the wild-type Q allele, Qs1 contains a missense mutation in the sequence encoding the first AP2 domain, whereas Qc1-N8 has two missense mutations: one in the sequence encoding the second AP2 domain and the other in the microRNA172-binding site. The Qs1 allele did not significantly affect GPC or other processing quality parameters, but it adversely affected GY by decreasing the thousand kernel weight and grain number per spike. In contrast, Qc1-N8 positively affected GPC and GY by increasing the thousand kernel weight and grain number per spike. Thus, we generated novel germplasm relevant for wheat breeding. A specific molecular marker was developed to facilitate the use of the Qc1-N8 allele in breeding. Furthermore, our findings provide useful new information for enhancing cereal crops via non-transgenic approaches.

Construction and analysis of a Noccaea caerulescens TILLING population

BackgroundMetals such as Zn or Cd are toxic to plant and humans when they are exposed in high quantities through contaminated soil or food. Noccaea caerulescens, an extraordinary Zn/Cd/Ni hyperaccumulating species, is used as a model plant for metal hyperaccumulation and phytoremediation studies. Current reverse genetic techniques to generate mutants based on transgenesis is cumbersome due to the low transformation efficiency of this species. We aimed to establish a mutant library for functional genomics by a non-transgenic approach, to identify mutants with an altered mineral profiling, and to screen for mutations in bZIP19, a regulator of Zn homeostasis in N. caerulescens.ResultsTo generate the N. caerulescens mutant library, 3000 and 5000 seeds from two sister plants of a single-seed recurrent inbred descendant of the southern French accession Saint-Félix-de-Pallières (SF) were mutagenized respectively by 0.3 or 0.4% ethyl methane sulfonate (EMS). Two subpopulations of 5000 and 7000 M2 plants were obtained after 0.3 or 0.4% EMS treatment. The 0.4% EMS treatment population had a higher mutant frequency and was used for TILLING. A High Resolution Melting curve analysis (HRM) mutation screening platform was optimized and successfully applied to detect mutations for NcbZIP19, encoding a transcription factor controlling Zn homeostasis. Of four identified point mutations in NcbZIP19, two caused non-synonymous substitutions, however, these two mutations did not alter the ionome profile compared to the wild type. Forward screening of the 0.4% EMS treatment population by mineral concentration analysis (ionomics) in leaf material of each M2 plant revealed putative mutants affected in the concentration of one or more of the 20 trace elements tested. Several of the low-Zn mutants identified in the ionomic screen did not give progeny, illustrating the importance of Zn for the species. The mutant frequency of the population was evaluated based on an average of 2.3 knockout mutants per tested monogenic locus.ConclusionsThe 0.4% EMS treatment population is effectively mutagenized suitable for forward mutant screens and TILLING. Difficulties in seed production in low Zn mutants, obtained by both forward and reverse genetic approach, hampered further analysis of the nature of the low Zn phenotypes.

CRISPR/Cas- and Topical RNAi-Based Technologies for Crop Management and Improvement: Reviewing the Risk Assessment and Challenges Towards a More Sustainable Agriculture.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated gene (Cas) system and RNA interference (RNAi)-based non-transgenic approaches are powerful technologies capable of revolutionizing plant research and breeding. In recent years, the use of these modern technologies has been explored in various sectors of agriculture, introducing or improving important agronomic traits in plant crops, such as increased yield, nutritional quality, abiotic- and, mostly, biotic-stress resistance. However, the limitations of each technique, public perception, and regulatory aspects are hindering its wide adoption for the development of new crop varieties or products. In an attempt to reverse these mishaps, scientists have been researching alternatives to increase the specificity, uptake, and stability of the CRISPR and RNAi system components in the target organism, as well as to reduce the chance of toxicity in nontarget organisms to minimize environmental risk, health problems, and regulatory issues. In this review, we discuss several aspects related to risk assessment, toxicity, and advances in the use of CRISPR/Cas and topical RNAi-based technologies in crop management and breeding. The present study also highlights the advantages and possible drawbacks of each technology, provides a brief overview of how to circumvent the off-target occurrence, the strategies to increase on-target specificity, the harm/benefits of association with nanotechnology, the public perception of the available techniques, worldwide regulatory frameworks regarding topical RNAi and CRISPR technologies, and, lastly, presents successful case studies of biotechnological solutions derived from both technologies, raising potential challenges to reach the market and being social and environmentally safe.

RNAi as a Foliar Spray: Efficiency and Challenges to Field Applications.

RNA interference (RNAi) is a powerful tool that is being increasingly utilized for crop protection against viruses, fungal pathogens, and insect pests. The non-transgenic approach of spray-induced gene silencing (SIGS), which relies on spray application of double-stranded RNA (dsRNA) to induce RNAi, has come to prominence due to its safety and environmental benefits in addition to its wide host range and high target specificity. However, along with promising results in recent studies, several factors limiting SIGS RNAi efficiency have been recognized in insects and plants. While sprayed dsRNA on the plant surface can produce a robust RNAi response in some chewing insects, plant uptake and systemic movement of dsRNA is required for delivery to many other target organisms. For example, pests such as sucking insects require the presence of dsRNA in vascular tissues, while many fungal pathogens are predominately located in internal plant tissues. Investigating the mechanisms by which sprayed dsRNA enters and moves through plant tissues and understanding the barriers that may hinder this process are essential for developing efficient ways to deliver dsRNA into plant systems. In this review, we assess current knowledge of the plant foliar and cellular uptake of dsRNA molecules. We will also identify major barriers to uptake, including leaf morphological features as well as environmental factors, and address methods to overcome these barriers.

Inhibiting tricin biosynthesis improves maize lignocellulose saccharification

Lignin is a technological bottleneck to convert polysaccharides into fermentable sugars, and different strategies of genetic-based metabolic engineering have been applied to improve biomass saccharification. Using maize seedlings grown hydroponically for 24 h, we conducted a quick non-transgenic approach with five enzyme inhibitors of the lignin and tricin pathways. Two compounds [3,4-(methylenedioxy)cinnamic acid: MDCA and 2,4-pyridinedicarboxylic acid: PDCA] revealed interesting findings on root growth, lignin composition, and saccharification. By inhibiting hydroxycinnamoyl-CoA ligase, a key enzyme of phenylpropanoid pathway, MDCA decreased the lignin content and improved saccharification, but it decreased root growth. By inhibiting flavone synthase, a key enzyme of tricin biosynthesis, PDCA decreased total lignin content and improved saccharification without affecting root growth. PDCA was three-fold more effective than MDCA, suggesting that controlling lignin biosynthesis with enzymatic inhibitors may be an attractive strategy to improve biomass saccharification.

Delivery of Double-Stranded RNAs (dsRNAs) Produced by Escherichia coli HT115(DE3) for Nontransgenic RNAi-Based Insect Pest Management.

RNA interference (RNAi) is a powerful mechanism that can be exploited not only for physiology research but also for designing insect pest management approaches. Some insects cause harm by vectoring diseases dangerous to humans, livestock, or plants or by damaging crops. For at least a decade now, different insect control strategies that induce RNAi by delivering double stranded RNA (dsRNA) targeting essential genes have been proposed. Here, we focus on nontransgenic RNAi-based approaches that use oral delivery of dsRNA through feeding of inactivated bacteria to produce RNAi in disease vectors and in a crop pest. This potential pest management method could be easily adapted to target different genes or similar organisms.

Enrichment of provitamin A content in durum wheat grain by suppressing β-carotene hydroxylase 1 genes with a TILLING approach.

The suppression of the HYD-1 gene by a TILLING approach increases the amount of β-carotene in durum wheat kernel. Vitamin A deficiency is a major public health problem that affects numerous countries in the world. As humans are not able to synthesize vitamin A, it must be daily assimilated along with other micro- and macronutrients through the diet. Durum wheat is an important crop for Mediterranean countries and provides a discrete amount of nutrients, such as carbohydrates and proteins, but it is deficient in some essential micronutrients, including provitamin A. In the present work, a targeting induced local lesions in genomes strategy has been undertaken to obtain durum wheat genotypes biofortified in provitamin A. In detail, we focused on the suppression of the β-carotene hydroxylase 1 (HYD1) genes, encoding enzymes involved in the redirection of β-carotene toward the synthesis of the downstream xanthophylls (neoxanthin, violaxanthin and zeaxanthin). Expression analysis of genes involved in carotenoid biosynthesis revealed a reduction of the abundance of HYD1 transcripts greater than 50% in mutant grain compared to the control. The biochemical profiling of carotenoid in the wheat mutant genotypes highlighted a significant increase of more than 70% of β-carotene compared to the wild-type sibling lines, with no change in lutein, α-carotene and zeaxanthin content. This study sheds new light on the molecular mechanism governing carotenoid biosynthesis in durum wheat and provides new genotypes that represent a good genetic resource for future breeding programs focused on the provitamin A biofortification through non-transgenic approaches.