New Plant Breeding Techniques Research Articles

Knockout of phytoene desaturase gene using CRISPR/Cas9 in highbush blueberry.

Among the New Plant Breeding Techniques (NPBTs), the CRISPR/Cas9 system represents a useful tool for target gene editing, improving the traits of the plants rapidly. This technology allows targeting one or more sequences simultaneously, as well as introducing new genetic variations by homology-directed recombination. However, the technology of CRISPR/Cas9 remains a challenge for some polyploid woody species, since all the different alleles for which the mutation is required must be simultaneously targeted. In this work we describe improved protocols adapting the CRISPR/Cas9 system to highbush blueberry (Vaccinium corymbosum L.), using Agrobacterium-mediated transformation. As a proof of concept, we targeted the gene encoding for phytoene desaturase, whose mutation disrupts chlorophyll biosynthesis allowing for the visual assessment of knockout efficiency. Leaf explants of in vitro-cultured blueberry cv. Berkeley has been transformed with a CRISPR/Cas9 construct containing two guide RNAs (gRNA1 and gRNA2) targeting two conserved gene regions of pds and subsequently maintained on a selection medium enriched with kanamycin. After 4 weeks in culture on the selection medium, the kanamycin-resistant lines were isolated, and the genotyping of these lines through Sanger sequencing revealed successful gene editing. Some of mutant shoot lines included albino phenotypes, even if the editing efficiencies were quite low for both gRNAs, ranging between 2.1 and 9.6% for gRNA1 and 3.0 and 23.8 for gRNA2. Here we showed a very effective adventitious shoot regeneration protocol for the commercial cultivar of highbush blueberry "Berkeley", and a further improvement in the use of CRISPR/Cas9 system in Vaccinium corymbosum L., opening the way to the breeding mediated by biotechnological approaches.

European consumer and societal stakeholders' response to crop improvements and new plant breeding techniques

AbstractThe global demand for providing nutritious, sustainable, and safe diets for a 10 billion population by 2050 while preserving affordability, reducing environmental impacts, and adapting to climate change will require accelerating the transition to sustainable agri‐food systems. A plausible way to help tackle these challenges is by developing new plant varieties that have improved crop yield, plant nutritional quality, and sustainability (or resilience) traits. However, stakeholders, consumers, and citizens' concerns and appreciation of future‐proofing crops and the acceptability of new plant breeding strategies are not well‐established. These groups are actors in the agri‐food systems, and their views, values, needs, and expectations are crucial in helping to co‐design fair, ethical, acceptable, sustainable, and socially desirable policies on new plant breeding techniques (NPBTs) and the transition to sustainable agri‐food systems. In this study, we engaged with consumer experts and societal stakeholders to consider their perceptions, expectations, and acceptability of improving crops and NPBTs for future‐proofing the agri‐food systems. Our analysis points to a need for governments to take a proactive role in regulating NPBTs, ensure openness and transparency in breeding new crop varieties, and inform consumers about the effects of these breeding programmes and the risks and benefits of the new crop varieties developed. Consumer experts and societal stakeholders considered these strategies necessary to instil confidence in society about NPBTs and accelerate the transition to sustainable agri‐food systems.

Freedom of Choice—Organic Consumers’ Discourses on New Plant Breeding Techniques

In recent years, there have been significant developments in biotechnology, specifically regarding New Plant Breeding Techniques (NPBTs). Such advancements have been driven by the need to develop improved and more sustainable crops while reducing pesticides and fertilisers. NPBTs include a heterogeneous group of methods that allow performing plant mutations more precisely than in genetically modified (GM) technologies, saving time and effort. Although some experts consider NPBTs an opportunity for organic farming expansion, the European Court of Justice in 2018 pronounced against their use in organic farming since all plants obtained by NPBTs should follow the same regulations as Genetically Modified Organisms (GMOs). This study aims to understand consumers’ attitudes and viewpoints towards new breeding techniques. Focus groups and Q methodological approach were used to uncover consensus and divergence among organic consumers in seven selected European countries (Germany, Italy, Latvia, the Netherlands, Spain, Switzerland, United Kingdom). Results of qualitative studies suggest that organic consumers are generally hostile towards NPBTs in organic farming. Using Q methodology, three distinct factors were identified: the “Risk Averse”, the “Technological Optimists”, and the “Socially Concerned”. The results highlight that consumers’ subjective knowledge and understanding of NPBTs diverge from the discourse of NPBTs lobbyists and proponents.

First Report on Genome Editing via Ribonucleoprotein (RNP) in Castanea sativa Mill.

Castanea sativa is an important tree nut species worldwide, highly appreciated for its multifunctional role, in particular for timber and nut production. Nowadays, new strategies are needed to achieve plant resilience to diseases, climate change, higher yields, and nutritional quality. Among the new plant breeding techniques (NPBTs), the CRISPR/Cas9 system represents a powerful tool to improve plant breeding in a short time and inexpensive way. In addition, the CRISPR/Cas9 construct can be delivered into the cells in the form of ribonucleoproteins (RNPs), avoiding the integration of exogenous DNA (GMO-free) through protoplast technology that represents an interesting material for gene editing thanks to the highly permeable membrane to DNA. In the present study, we developed the first protoplast isolation protocol starting from European chestnut somatic embryos. The enzyme solution optimized for cell wall digestion contained 1% cellulase Onozuka R-10 and 0.5% macerozyme R-10. After incubation for 4 h at 25 °C in dark conditions, a yield of 4,500,000 protoplasts/mL was obtained (91% viable). The transfection capacity was evaluated using the GFP marker gene, and the percentage of transfected protoplasts was 51%, 72 h after the transfection event. The direct delivery of the purified RNP was then performed targeting the phytoene desaturase gene. Results revealed the expected target modification by the CRISPR/Cas9 RNP and the efficient protoplast editing.

Exploring Consumers’ Attitudes towards Food Products Derived by New Plant Breeding Techniques

New plant breeding techniques (NPBTs) are seen as promising and innovative tools to achieve food security and food safety. Biotechnological innovations have great potential to address sustainable food development, and they are expected in the near future to play a critical role in feeding a growing population without exerting added pressure on the environment. There is, however, a considerable debate as to how these new techniques should be regulated and whether some or all of them should fall within the scope of EU legislation on genetically modified organisms (GMOs), despite the product obtained being free from genes foreign to the species. In the EU, the adoption of these methods does not rely only on the scientific community but requires social acceptance and a political process that leads to an improved regulatory framework. In this paper, we present the results of an online survey carried out in Italy with 700 randomly selected participants on consumer attitudes towards food obtained by NPBTs. By applying the decision tree machine learning algorithm J48 to our dataset, we identified significant attributes to predict the main drivers of purchasing such products. A classification model accuracy assessment has also been developed to evaluate the overall performance of the classifier. The result of the model highlighted the role of consumers’ self-perceived knowledge and their trust in the European approval process for NPBT, as well as the need for a detailed label. Our findings may support decision makers and underpin the development of NPBT products in the market.

Metabolomic approaches for the characterization of carotenoid metabolic engineering in planta.

Carotenoid biosynthesis has now been subjected to metabolic engineering for over two decades. The outputs clearly show that carotenoid formation is an integral component of metabolism. Perturbations can affect intermediary metabolism and other isoprenoids. The advances in omic technologies have enabled the quantitative assessment of changes in the transcriptome, proteome and metabolome in response to altered carotenoid biosynthesis. In the present article, the approaches and procedures relating to the capture of the metabolome in response to modulation of the carotenoid biosynthetic pathway are described. These data will contribute to the fundamental understanding of metabolic biology, underpinning future rationale design of New Plant Breeding Techniques (NPBTs) and associated regulatory affairs.

Somatic Embryogenesis in Vitis for Genome Editing: Optimization of Protocols for Recalcitrant Genotypes

New Plant Breeding Techniques (NPBTs) protocols have been developed to produce new grape varieties with improved quantitative and qualitative characteristics. Reliable transformation protocols for grapes are based on the generation/induction of embryogenic callus cells that are then transformed. Varieties such as Italia have proven to be very recalcitrant to regeneration via somatic embryogenesis. In this work, the development of a protocol for improved production of embryogenic calluses is described. Two sterilization protocols were tested: (a) a lower active chlorine concentration for a longer time (LS); and (b) a higher chlorine concentration for a shorter time (HS), in combination with the absence or presence of citric acid in the growing substrate in the first growth media. The embryogenic calluses formation in Chardonnay, a cv. with a high embryogenic response, was significantly higher in presence of citric acid in the initial growing substrate regardless of the sterilization protocol. In Aglianico, a cv. with a lower embryogenic response, no significant differences were observed. Instead, in a recalcitrant cv. as Italia, we obtained a 13-fold increase in embryogenic calluses formation performing sterilization of flowers with the HS protocol compared to LS.

CRISPR/Cas systems: opportunities and challenges for crop breeding

Increasing crop production to meet the demands of a growing population depends largely on crop improvement through new plant-breeding techniques (NPBT) such as genome editing. CRISPR/Cas systems are NPBTs that enable efficient target-specific gene editing in crops, which is supposed to accelerate crop breeding in a way that is different from genetically modified (GM) technology. Herein, we review the applications of CRISPR/Cas systems in crop breeding focusing on crop domestication, heterosis, haploid induction, and synthetic biology, and summarize the screening methods of CRISPR/Cas-induced mutations in crops. We highlight the importance of molecular characterization of CRISPR/Cas-edited crops, and pay special attentions to emerging highly specific genome-editing tools such as base editors and prime editors. We also discuss future improvements of CRISPR/Cas systems for crop improvement.

Precision Genome Engineering for the Breeding of Tomatoes: Recent Progress and Future Perspectives.

Currently, poor biodiversity has raised challenges in the breeding and cultivation of tomatoes, which originated from the Andean region of Central America, under global climate change. Meanwhile, the wild relatives of cultivated tomatoes possess a rich source of genetic diversity but have not been extensively used for the genetic improvement of cultivated tomatoes due to the possible linkage drag of unwanted traits from their genetic backgrounds. With the advent of new plant breeding techniques (NPBTs), especially CRISPR/Cas-based genome engineering tools, the high-precision molecular breeding of tomato has become possible. Further, accelerated introgression or de novo domestication of novel and elite traits from/to the wild tomato relatives to/from the cultivated tomatoes, respectively, has emerged and has been enhanced with high-precision tools. In this review, we summarize recent progress in tomato precision genome editing and its applications for breeding, with a special focus on CRISPR/Cas-based approaches. Future insights and precision tomato breeding scenarios in the CRISPR/Cas era are also discussed.

Challenges and Prospects of New Plant Breeding Techniques for GABA Improvement in Crops: Tomato as an Example.

Over the last seven decades, γ-aminobutyric acid (GABA) has attracted great attention from scientists for its ubiquity in plants, animals and microorganisms and for its physiological implications as a signaling molecule involved in multiple pathways and processes. Recently, the food and pharmaceutical industries have also shown significantly increased interest in GABA, because of its great potential benefits for human health and the consumer demand for health-promoting functional compounds, resulting in the release of a plethora of GABA-enriched products. Nevertheless, many crop species accumulate appreciable GABA levels in their edible parts and could help to meet the daily recommended intake of GABA for promoting positive health effects. Therefore, plant breeders are devoting much effort into breeding elite varieties with improved GABA contents. In this regard, tomato (Solanum lycopersicum), the most produced and consumed vegetable worldwide and a fruit-bearing model crop, has received much consideration for its accumulation of remarkable GABA levels. Although many different strategies have been implemented, from classical crossbreeding to induced mutagenesis, new plant breeding techniques (NPBTs) have achieved the best GABA accumulation results in red ripe tomato fruits along with shedding light on GABA metabolism and gene functions. In this review, we summarize, analyze and compare all the studies that have substantially contributed to tomato GABA breeding with further discussion and proposals regarding the most recent NPBTs that could bring this process to the next level of precision and efficiency. This document also provides guidelines with which researchers of other crops might take advantage of the progress achieved in tomato for more efficient GABA breeding programs.

New Plant Breeding Techniques in Citrus for the Improvement of Important Agronomic Traits. A Review

New plant breeding techniques (NPBTs) aim to overcome traditional breeding limits for fruit tree species, in order to obtain new varieties with improved organoleptic traits and resistance to biotic and abiotic stress, and to maintain fruit quality achieved over centuries by (clonal) selection. Knowledge on the gene(s) controlling a specific trait is essential for the use of NPBTs, such as genome editing and cisgenesis. In the framework of the international scientific community working on fruit tree species, including citrus, NPBTs have mainly been applied to address pathogen threats. Citrus could take advantage of NPBTs because of its complex species biology (seedlessness, apomixis, high heterozygosity, and long juvenility phase) and aptitude for in vitro manipulation. To our knowledge, genome editing in citrus via transgenesis has successful for induced resistance to Citrus bacterial canker in sweet orange and grapefruit using the resistance gene CsLOB1. In the future, NPBTs will also be used to improve fruit traits, making them healthier. The regeneration of plants following the application of NPBTs is a bottleneck, making it necessary to optimize the efficiency of current protocols. The strengths and weaknesses of using explants from young in vitro plantlets, and from mature plants, will be discussed. Other major issues addressed in this review are related to the requirement for marker-free systems and shortening the long juvenility phase. This review aims to summarize methods and approaches available in the literature that are suitable to citrus, focusing on the principles observed before the use of NPBTs.

New Hope for a "Cursed" Crop? Understanding Stakeholder Attitudes to Plant Molecular Farming With Modified Tobacco in Europe.

Plant molecular farming (PMF) with tobacco could provide a sustainable and cheap platform for the production of high-value proteins for medical use. It could also offer European tobacco farmers an alternative, healthful end use for their crop. New plant breeding techniques (NPBTs) offer a means of quickly and precisely optimizing molecular farming platforms for this purpose. However, there has been little empirical research focussing on the barriers and facilitators of these technologies in the agricultural sphere. Here, we explore key stakeholder perceptions toward this combination of technologies, exploring their understanding of risk and opportunity. We interviewed N = 24 key stakeholders – tobacco farmers, agronomists, policymakers, and researchers – in three tobacco-growing areas of Spain and Italy. Our findings demonstrate these stakeholders have a favorable attitude toward PMF with tobacco due to its beneficial medical purpose and the opportunity it provides farmers to continue growing tobacco in a declining European market. Tobacco producers also reported favorable views toward NPBTs, though for some this was contingent on their use for non-food crops like tobacco. Most stakeholders’ concerns are economic in nature, such as potential profitability and demands for new agronomic practices or infrastructure. Tobacco producer associations were thought to be important facilitators for future PMF scale-up. The attitude toward these technologies by smoking tobacco companies is, however, unknown and constitutes a potential risk to the development of PMF.

Shotguns vs Lasers: Identifying barriers and facilitators to scaling-up plant molecular farming for high-value health products.

Plant molecular farming (PMF) is a convenient and cost-effective way to produce high-value recombinant proteins that can be used in the production of a range of health products, from pharmaceutical therapeutics to cosmetic products. New plant breeding techniques (NPBTs) provide a means to enhance PMF systems more quickly and with greater precision than ever before. However, the feasibility, regulatory standing and social acceptability of both PMF and NPBTs are in question. This paper explores the perceptions of key stakeholders on two European Union (EU) Horizon 2020 programmes–Pharma-Factory and Newcotiana–towards the barriers and facilitators of PMF and NPBTs in Europe. One-on-one qualitative interviews were undertaken with N = 20 individuals involved in one or both of the two projects at 16 institutions in seven countries (Belgium, France, Germany, Italy, Israel, Spain and the UK). The findings indicate that the current EU regulatory environment and the perception of the public towards biotechnology are seen as the main barriers to scaling-up PMF and NPBTs. Competition from existing systems and the lack of plant-specific regulations likewise present challenges for PMF developing beyond its current niche. However, respondents felt that the communication of the benefits and purpose of NPBT PMF could provide a platform for improving the social acceptance of genetic modification. The importance of the media in this process was highlighted. This article also uses the multi-level perspective to explore the ways in which NPBTs are being legitimated by interested parties and the systemic factors that have shaped and are continuing to shape the development of PMF in Europe.

Inside Lobbying on the Regulation of New Plant Breeding Techniques in the European Union: Determinants of Venue Choices

AbstractIn July 2018, the Court of Justice of the European Union decided that new plant breeding techniques (NPBTs) fall within the scope of the restrictive provisions on genetically modified organisms (GMOs). Previously, various actors had lobbied in order to influence the European Union’s (EU’s) regulatory decision on NPBTs. This study examines the venue choices taken by Cibus, a biotech company that promoted NPBT deregulation. It shows that the firm bypassed the EU level and that it lobbied competent authorities (CAs) in certain member states to gain support for the deregulation of NPBTs. Cibus chose the CAs because their institutional “closedness” reduced the risk of the debate over the deregulation of NPBTs becoming public. However, the CA’s specific competences and their influence on EU decision making were of likewise importance. The firm lobbied CAs based in Finland, Germany, Ireland, Sweden, Spain, and the United Kingdom. Two factors appear to have influenced Cibus’ choices for these countries: high‐level political support for agribiotech and the high relevance of biotech sectors. In contrast, public support for GMOs turned out to have hardly any influence, and virtually no association could be observed for the agricultural application of biotechnology in the past nor for the weakness of domestic anti‐GMO lobby groups. Finally, the in‐depth study on Germany affirms that “closedness” was important for Cibus’ choices and reveals that technical information served as a venue‐internal factor that influenced the firm’s choices.

Gene editing offers dietary benefits

Gene editing offers dietary benefits

European Court of Justice decision for genome editing: Consequences on food/feed risk assessment and detection

On 25 July 2018 the European Court of Justice (ECJ) clarified that organisms obtained by mutagenesis are genetically modified organisms (GMOs) as defined in the European Directive 18/2001/EC. However, the GMO directive shouldn't apply to organisms obtained by mutagenesis using techniques developed and applied before the respective Directive came into force (i.e. the application of chemical or physical mutageneous agents) and that have a long ‘history of safe’ use. Accordingly a safety assessment and labelling of products containing or consisting of GMOs obtained by mutagenesis developed after the Directive came into force will be needed. From a scientific point of view a similar basis for the risk assessment can be assumed when comparing ‘classical’ GMOs and GMOs obtained by mutagenesis using new plant breeding techniques (NPBTs). In both cases a conventional organism has been modified. Therefore the classical approach of comparing the GMO with the non-GM variety is the straight way to conduct the safety assessment. In contrast, for the identification of GMOs obtained by mutagenesis using NPBTs the development and validation of new tools are needed. However, scientifically it will not be possible to distinguish between GMOs bearing a single mutation, if it is either the result of the intended use of NPBTs, the application of chemical or physical mutageneous agents or the natural process of spontaneously occurring mutations.

Introgressiomics: a new approach for using crop wild relatives in breeding for adaptation to climate change

The need to boost agricultural production in the coming decades in a climate change scenario requires new approaches for the development of new crop varieties that are more resilient and more efficient in the use of resources. Crop wild relatives (CWRs) are a source of variation for many traits of interest in breeding, in particular tolerance to abiotic and biotic stresses. However, their potential in plant breeding has largely remained unexploited. CWRs can make an effective contribution to broadening the genetic base of crops and to introgressing traits of interest, but their direct use by breeders in breeding programs is usually not feasible due to the presence of undesirable traits in CWRs (linkage drag) and frequent breeding barriers with the crop. Here we call for a new approach, which we tentatively call ‘introgressiomics’, which consists of mass scale development of plant materials and populations with introgressions from CWRs into the genetic background of crops. Introgressiomics is a form of pre-emptive breeding and can be focused, when looking for specific phenotypes, or un-focused, when it is aimed at creating highly diverse introgressed populations. Exploring germplasm collections and identifying adequate species and accessions from different genepools encompassing a high diversity, using different strategies like the creation of germplasm diversity sets, Focused identification of germplasm strategy (FIGS) or gap analysis, is a first step in introgressiomics. Interspecific hybridization and backcrossing is often a major barrier for introgressiomics, but a number of techniques can be used to potentially overcome these and produce introgression populations. The generation of chromosome substitution lines (CSLs), introgression lines (ILs), or multi-parent advanced inter-cross (MAGIC) populations by means of marker-assisted selection allows not only the genetic analysis of traits present in CWRs, but also developing genetically characterized elite materials that can be easily incorporated in breeding programs. Genomic tools, in particular high-throughput molecular markers, facilitate the characterization and development of introgressiomics populations, while new plant breeding techniques (NPBTs) can enhance the introgression and use of genes from CWRs in the genetic background of crops. An efficient use of introgressiomics populations requires moving the materials into breeding pipelines. In this respect public–private partnerships (PPPs) can contribute to an increased use of introgressed materials by breeders. We hope that the introgressiomics approach will contribute to the development of a new generation of cultivars with dramatically improved yield and performance that may allow coping with the environmental changes caused by climate change while at the same time contributing to a more efficient and sustainable agriculture.

The Interaction Among the Regulation of New Plant Breeding Techniques, GMO Labeling, and Coexistence and Segregation Costs: The Case of Rapeseed in the EU

We analyze the market and welfare effects of regulating crops derived by New Plant Breeding Techniques (NPBTs) as genetically modified (GM) or conventional products. We consider the EU mandatory scheme for labeling GM products and a voluntary non-GM scheme for labeling livestock products derived from non-GM feed. We develop a partial equilbrium model that explicitly takes into account both the coexistence costs at farm-level and the segregation and identity preservation costs at downstream level. By applying the model to EU rapeseed, we find that regulating NPBTs as GM (as compared to non-GM) in combination with mandatory and voluntary labeling increases prices and makes consumers overall worse off and producers better off. We also show that higher coexistence costs make the price increasing effect even stronger. Voluntary non-GM labeling applied to feed makes consumers in this sector overall worse off but benefits farmers and rapeseed oil consumers overall as long as segregation costs are low. Consumers of biodiesel and industrial products such as lubricants produced from GM rapeseed benefit from high segregation costs. We show that the effects of farm-level coexistence costs largely differ from the effects of downstream market segregation costs.

I Have a Dream: Organic Movements Include Gene Manipulation to Improve Sustainable Farming

Several papers in a Special Issue of Sustainability have recently discussed various aspects to evaluate whether organic farming and gene manipulation are compatible. A special emphasis was given to new plant breeding techniques (NPBTs). These new approaches allow the most predictable genetic alterations of crop plants in ways that the genetically modified plant is identical to a plant generated by conventional breeding. The articles of the Special Issue present the arguments pro and contra the inclusion of the plants generated by NPBTs in organic farming. Organic movements have not yet made a final decision whether some of these techniques should be accepted or banned. In my view these novel genetically manipulated (GM) crops could be used in such a way as to respect the requirements for genetically manipulated organisms (GMOs) formulated by the International Federation of Organic Movements (IFOAM). Reviewing the potential benefits of disease-resistant potatoes and bananas, it seems possible that these crops support organic farming. To this end, I propose specific requirements that the organic movements should proactively formulate as their standards to accept specific GM crops.

Should Organic Agriculture Maintain Its Opposition to GM? New Techniques Writing the Same Old Story

Biotechnology is diversifying rapidly through the development and application of new approaches to genome editing and ongoing research into synthetic biology. Proponents of biotechnology are enthusiastic about these new developments and have recently begun calling for environmental movements to abandon their campaigns against Genetically Modified Organisms (GMOs) and for organic agriculture to reconsider its exclusion of Genetic Modification (GM). In this article, we begin by describing the diversity of practices that cluster under both the terms GM and organic and show that although there is a clash of different cultures of agriculture at stake, there is also a spectrum of practices existing between these two poles. Having established the terms of the debate, we then go on to analyse whether the organic movement should reconsider its position on GM in light of new plant breeding techniques (NPBTs), using the criteria highlighted as important by the International Federation of Organic Agriculture Movements (IFOAM) in their 2016 draft revised position on GMOs. Through this analysis, we suggest that given the in-context-trajectory of biotechnology development, the continued narrow framing of agricultural problems and the ongoing exclusion of important socio-economic, political and cultural dimensions, the organic movement is justified in maintaining its opposition to GM in the face of NPBTs.