ABSTRACT
Root chicory (Cichorium intybus L. var. sativum) is used to extract inulin, a fructose polymer used as a natural sweetener and prebiotic. However, bitter tasting sesquiterpene lactones, giving chicory its known flavour, need to be removed during inulin extraction. To avoid this extraction and associated costs, recently chicory variants with a lower sesquiterpene lactone content were created by inactivating the four copies of the germacrene A synthase gene (CiGAS-S1, -S2, -S3, -L) which encode the enzyme initiating bitter sesquiterpene lactone biosynthesis in chicory. In this study, different delivery methods for CRISPR/Cas9 reagents have been compared regarding their efficiency to induce mutations in the CiGAS genes, the frequency of off-target mutations as well as their environmental and economic impacts. CRISPR/Cas9 reagents were delivered by Agrobacterium-mediated stable transformation or transient delivery by plasmid or preassembled ribonucleic complexes (RNPs) using the same sgRNA. All methods used lead to a high number of INDEL mutations within the CiGAS-S1 and CiGAS-S2 genes, which match the used sgRNA perfectly; additionally, the CiGAS-S3 and CiGAS-L genes, which have a single mismatch with the sgRNA, were mutated but with a lower mutation efficiency. While using both RNPs and plasmids delivery resulted in biallelic, heterozygous or homozygous mutations, plasmid delivery resulted in 30% of unwanted integration of plasmid fragments in the genome. Plants transformed via Agrobacteria often showed chimerism and a mixture of CiGAS genotypes. This genetic mosaic becomes more diverse when plants were grown over a prolonged period. While the genotype of the on-targets varied between the transient and stable delivery methods, no off-target activity in six identified potential off-targets with two to four mismatches was found. The environmental impacts (greenhouse gas (GHG) emissions and primary energy demand) of the methods are highly dependent on their individual electricity demand. From an economic view - like for most research and development activities - employment and value-added multiplier effects are high; particularly when compared to industrial or manufacturing processes. Considering all aspects, we conclude that using RNPs is the most suitable method for genome editing in chicory since it led to a high efficiency of editing, no off-target mutations, non-transgenic plants with no risk of unwanted integration of plasmid DNA and without needed segregation of transgenes.
ABSTRACT
In Europe, root chicory and other plants are cultivated for their prebiotic food fiber, inulin, which boosts the growth of beneficial gut bacteria and stimulates the human immune system. CHIC, a H2020 project, develops new chicory variants which produce more and reported to be healthier inulin as well as medicinal terpenes. This paper presents an environmental and socio-economic assessment of the whole value chain of the new chicory variants and their derived products using a case study based in the Netherlands. Two scenarios based on new chicory variants using new plant breeding technologies (NPBT) are analyzed and impacts thereof are compared to the reference scenario; the current commercial inulin process from conventional chicory. Both scenarios show higher inulin content, but the inulin adsorption process differs. While one aims to optimize inulin yield, the other one explores the potential of a multipurpose use, yielding inulin and health beneficial terpenes. Methodologically, we employ multi-regional input-output (MRIO) analysis to estimate additional economic benefits, added value and job creation, while by means of life cycle assessment (LCA) effects on greenhouse gas (GHG) emissions and primary energy demand are derived. Both methods, MRIO and LCA, are well suited to analyze the raised issues and draw on the same data. Generally, the results highlight the importance of inulin production at a national and EU-level in the reference scenario. In case of the two scenarios, we find that the related socio-economic impacts are much higher than in the reference scenario and thus highlight their ability to boost economic activity and increase competiveness of the EU, i.e. over 80% of the generated value added stays in the EU. In terms of environmental impacts, the two scenarios show lower GHG emissions and primary energy demand due to the higher efficiencies of the process in the scenarios compared to the reference inulin process. Additionally, regarding the goal of climate neutral production, we find that the majority of GHG emissions stem from the electricity mix and natural gas demand. Replacing these sources of energy with more renewable ones will contribute to this goal.
