GBSS mutations in an SBE mutated background restore the potato starch granule morphology and produce ordered granules despite differences to native molecular structure.

Potato starch with mutations in starch branching enzyme genes (SBEI, SBEII) and granule-bound starch synthase gene (GBSS) was characterized for molecular and thermal properties. Mutations in GBSS were here stacked to a previously developed SBEI and SBEII mutation line. Additionally, mutations in the GBSS gene alone were induced in the wild-type variety for comparison. The parental line with mutations in the SBE genes showed a âˆ¼ 40 % increase in amylose content compared with the wild-type. Mutations in GBSS-SBEI-SBEII produced non-waxy, low-amylose lines compared with the wild-type. An exception was a line with one remaining GBSS wild-type allele, which displayed ∼80 % higher amylose content than wild-type. Stacked mutations in GBSS in the SBEI-SBEII parental line caused alterations in amylopectin chain length distribution and building block size categories of whole starch. Correlations between size categories of building blocks and unit chains of amylopectin were observed. Starch in GBSS-SBEI-SBEII mutational lines had elevated peak temperature of gelatinization, which was positively correlated with large building blocks.

Spatio-temporal transcriptome and storage compound profiles of developing faba bean (Vicia faba) seed tissues.

Faba bean (Vicia faba) is a legume grown in diverse climate zones with a high potential for increased cultivation and use in food due to its nutritional seeds. In this study, we characterized seed tissue development in faba bean to identify key developmental processes; from embryo expansion at the expense of the endosperm to the maturing storage stages of the bean seed. A spatio-temporal transcriptome profiling analysis, combined with chemical nutrient analysis of protein, starch, and lipid, of endosperm and embryo tissues at different developmental stages, revealed gene expression patterns, transcriptional networks, and biochemical pathways in faba bean. We identified key players in the LAFL (LEC1, ABI3, FUS3, and LEC2) transcription factor network as well as their major repressors VAL1 and ASIL1. Our results showed that proteins accumulated not only in the embryo but also in the endosperm. Starch accumulated throughout seed development and oil content increased during seed development but at very low levels. The patterns of differentially expressed transcripts encoding proteins with functions in the corresponding metabolic pathways for the synthesis of these storage compounds, to a high extent, aligned with these findings. However, the early expression of transcripts encoding WRI1 combined with the late expression of oil body proteins indicated a not manifested high potential for lipid biosynthesis and oil storage. Altogether, this study contributes to increased knowledge regarding seed developmental processes applicable to future breeding methods and seed quality improvement for faba bean.

Pho1a (plastid starch phosphorylase) is duplicated and essential for normal starch granule phenotype in tubers of Solanum tuberosum L.

Reserve starch from seeds and tubers is a crucial plant product for human survival. Much research has been devoted to quantitative and qualitative aspects of starch synthesis and its relation to abiotic factors of importance in agriculture. Certain aspects of genetic factors and enzymes influencing carbon assimilation into starch granules remain elusive after many decades of research. Starch phosphorylase (Pho) can operate, depending on metabolic conditions, in a synthetic and degradative pathway. The plastidial form of the enzyme is one of the most highly expressed genes in potato tubers, and the encoded product is imported into starch-synthesizing amyloplasts. We identified that the genomic locus of a Pho1a-type starch phosphorylase is duplicated in potato. Our study further shows that the enzyme is of importance for a normal starch granule phenotype in tubers. Null mutants created by genome editing display rounded starch granules in an increased number that contained a reduced ratio of apparent amylose in the starch.

Improved bioenergy value of residual rice straw by increased lipid levels from upregulation of fatty acid biosynthesis.

BACKGROUND: Rice (Oryza sativa) straw is a common waste product that represents a considerable amount of bound energy. This energy can be used for biogas production, but the rate and level of methane produced from rice straw is still low. To investigate the potential for an increased biogas production from rice straw, we have here utilized WRINKLED1 (WRI1), a plant AP2/ERF transcription factor, to increase triacylglycerol (TAG) biosynthesis in rice plants. Two forms of Arabidopsis thaliana WRI1 were evaluated by transient expression and stable transformation of rice plants, and transgenic plants were analyzed both for TAG levels and biogas production from straw. RESULTS: Both full-length AtWRI1, and a truncated form lacking the initial 141 amino acids (including the N-terminal AP2 domain), increased fatty acid and TAG levels in vegetative and reproductive tissues of Indica rice. The stimulatory effect of the truncated AtWRI1 was significantly lower than that of the full-length protein, suggesting a role for the deleted AP2 domain in WRI1 activity. Full-length AtWRI1 increased TAG levels also in Japonica rice, indicating a conserved effect of WRI1 in rice lipid biosynthesis. The bio-methane production from rice straw was 20% higher in transformants than in the wild type. Moreover, a higher producing rate and final yield of methane was obtained for rice straw compared with rice husks, suggesting positive links between methane production and a high amount of fatty acids. CONCLUSIONS: Our results suggest that heterologous WRI1 expression in transgenic plants can be used to improve the metabolic potential for bioenergy purposes, in particular methane production.

CRISPR/Cas9 Technology for Potato Functional Genomics and Breeding.

Cultivated potato (Solanum tuberosum L.) is one of the most important staple food crops worldwide. Its tetraploid and highly heterozygous nature poses a great challenge to its basic research and trait improvement through traditional mutagenesis and/or crossbreeding. The establishment of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) as a gene editing tool has allowed the alteration of specific gene sequences and their concomitant gene function, providing powerful technology for potato gene functional analysis and improvement of elite cultivars. This technology relies on a short RNA molecule called single guide RNA (sgRNA) that directs the Cas9 nuclease to induce a site-specific double-stranded break (DSB). Further, repair of the DSB by the error-prone non-homologous end joining (NHEJ) mechanism leads to the introduction of targeted mutations, which can be used to produce the loss of function of specific gene(s). In this chapter, we describe experimental procedures to apply the CRISPR/Cas9 technology for potato genome editing. First, we provide strategies for target selection and sgRNA design and describe a Golden Gate-based cloning system to obtain a sgRNA/Cas9-encoding binary vector. We also describe an optimized protocol for ribonucleoprotein (RNP) complex assembly. The binary vector can be used for both Agrobacterium-mediated transformation and transient expression in potato protoplasts, while the RNP complexes are intended to obtain edited potato lines through protoplast transfection and plant regeneration. Finally, we describe procedures to identify the gene-edited potato lines. The methods described here are suitable for potato gene functional analysis and breeding.

An alternative pathway to plant cold tolerance in the absence of vacuolar invertase activity.

To cope with cold stress, plants have developed antioxidation strategies combined with osmoprotection by sugars. In potato (Solanum tuberosum) tubers, which are swollen stems, exposure to cold stress induces starch degradation and sucrose synthesis. Vacuolar acid invertase (VInv) activity is a significant part of the cold-induced sweetening (CIS) response, by rapidly cleaving sucrose into hexoses and increasing osmoprotection. To discover alternative plant tissue pathways for coping with cold stress, we produced VInv-knockout lines in two cultivars. Genome editing of VInv in 'Désirée' and 'Brooke' was done using stable and transient expression of CRISPR/Cas9 components, respectively. After storage at 4°C, sugar analysis indicated that the knockout lines showed low levels of CIS and maintained low acid invertase activity in storage. Surprisingly, the tuber parenchyma of vinv lines exhibited significantly reduced lipid peroxidation and reduced H2 O2 levels. Furthermore, whole plants of vinv lines exposed to cold stress without irrigation showed normal vigor, in contrast to WT plants, which wilted. Transcriptome analysis of vinv lines revealed upregulation of an osmoprotectant pathway and ethylene-related genes during cold temperature exposure. Accordingly, higher expression of antioxidant-related genes was detected after exposure to short and long cold storage. Sugar measurements showed an elevation of an alternative pathway in the absence of VInv activity, raising the raffinose pathway with increasing levels of myo-inositol content as a cold tolerance response.

A seed-like proteome in oil-rich tubers.

There are numerous examples of plant organs or developmental stages that are desiccation-tolerant and can withstand extended periods of severe water loss. One prime example are seeds and pollen of many spermatophytes. However, in some plants, also vegetative organs can be desiccation-tolerant. One example are the tubers of yellow nutsedge (Cyperus esculentus), which also store large amounts of lipids similar to seeds. Interestingly, the closest known relative, purple nutsedge (Cyperus rotundus), generates tubers that do not accumulate oil and are not desiccation-tolerant. We generated nanoLC-MS/MS-based proteomes of yellow nutsedge in five replicates of four stages of tuber development and compared them to the proteomes of roots and leaves, yielding 2257 distinct protein groups. Our data reveal a striking upregulation of hallmark proteins of seeds in the tubers. A deeper comparison to the tuber proteome of the close relative purple nutsedge (C. rotundus) and a previously published proteome of Arabidopsis seeds and seedlings indicates that indeed a seed-like proteome was found in yellow but not purple nutsedge. This was further supported by an analysis of the proteome of a lipid droplet-enriched fraction of yellow nutsedge, which also displayed seed-like characteristics. One reason for the differences between the two nutsedge species might be the expression of certain transcription factors homologous to ABSCISIC ACID INSENSITIVE3, WRINKLED1, and LEAFY COTYLEDON1 that drive gene expression in Arabidopsis seed embryos.

Establishment of a DNA-free genome editing and protoplast regeneration method in cultivated tomato (Solanum lycopersicum).

KEY MESSAGE: We have established a DNA-free genome editing method via ribonucleoprotein-based CRISPR/Cas9 in cultivated tomato and obtained mutant plants regenerated from transfected protoplasts with a high mutation rate. The application of genome editing as a research and breeding method has provided many possibilities to improve traits in many crops in recent years. In cultivated tomato (Solanum lycopersicum), so far only stable Agrobacterium-mediated transformation carrying CRISPR/Cas9 reagents has been established. Shoot regeneration from transfected protoplasts is the major bottleneck in the application of DNA-free genome editing via ribonucleoprotein-based CRISPR/Cas9 method in cultivated tomato. In this study, we report the implementation of a transgene-free breeding method for cultivated tomato by CRISPR/Cas9 technology, including the optimization of protoplast isolation and overcoming the obstacle in shoot regeneration from transfected protoplasts. We have identified that the shoot regeneration medium containing 0.1 mg/L IAA and 0.75 mg/L zeatin was the best hormone combination with a regeneration rate of up to 21.3%. We have successfully obtained regenerated plants with a high mutation rate four months after protoplast isolation and transfection. Out of 110 regenerated M0 plants obtained, 35 (31.8%) were mutated targeting both SP and SP5G genes simultaneously and the editing efficiency was up to 60% in at least one allele in either SP or SP5G genes.

Manufacturing specialized wax esters in plants.

Biologically produced wax esters can fulfil different industrial purposes. These functionalities almost drove the sperm whale to extinction from hunting. After the ban on hunting, there is a niche in the global market for biolubricants with properties similar to spermaceti. Wax esters can also serve as a mechanism for producing insect sex pheromone fatty alcohols. Pheromone-based mating disruption strategies are in high demand to replace the toxic pesticides in agriculture and manage insect plagues threatening our food and fiber reserves. In this study we set out to investigate the possibilities of in planta assembly of wax esters, for specific applications, through transient expression of various mix-and-match combinations of genes in Nicotiana benthamiana leaves. Our synthetic biology designs were outlined in order to pivot plant lipid metabolism into producing wax esters with targeted fatty acyl and fatty alcohols moieties. Through this approach we managed to obtain industrially important spermaceti-like wax esters enriched in medium-chain fatty acyl and/or fatty alcohol moieties of wax esters. Via employment of plant codon-optimized moth acyl-CoA desaturases we also managed to capture unusual, unsaturated fatty alcohol and fatty acyl moieties, structurally similar to moth pheromone compounds, in plant-accumulated wax esters. Comparison between outcomes of different experimental designs identified targets for stable transformation to accumulate specialized wax esters and helped us to recognize possible bottlenecks of such accumulation.

Characterisation of Grains and Flour Fractions from Field Grown Transgenic Oil-Accumulating Wheat Expressing Oat WRI1.

Wheat (Triticum aestivum L.) is one of the major staple crops in the world and is used to prepare a range of foods. The development of new varieties with wider variation in grain composition could broaden their use. We characterized grains and flours from oil-accumulating transgenic wheat expressing the oat (Avena sativa L.) endosperm WRINKLED1 (AsWRI1) grown under field conditions. Lipid and starch accumulation was determined in developing caryopses of AsWRI1-wheat and X-ray microtomography was used to study grain morphology. The developing caryopses of AsWRI1-wheat grains had increased triacylglycerol content and decreased starch content compared to the control. Mature AsWRI1-wheat grains also had reduced weight, were wrinkled and had a shrunken endosperm and X-ray tomography revealed that the proportion of endosperm was decreased while that of the aleurone was increased. Grains were milled to produce two white flours and one bran fraction. Mineral and lipid analyses showed that the flour fractions from the AsWRI1-wheat were contaminated with bran, due to the effects of the changed morphology on milling. This study gives a detailed analysis of grains from field grown transgenic wheat that expresses a gene that plays a central regulatory role in carbon allocation and significantly affects grain composition.

Release of moth pheromone compounds from Nicotiana benthamiana upon transient expression of heterologous biosynthetic genes.

BACKGROUND: Using genetically modified plants as natural dispensers of insect pheromones may eventually become part of a novel strategy for integrated pest management. RESULTS: In the present study, we first characterized essential functional genes for sex pheromone biosynthesis in the rice stem borer Chilo suppressalis (Walker) by heterologous expression in Saccharomyces cerevisiae and Nicotiana benthamiana, including two desaturase genes CsupYPAQ and CsupKPSE and a reductase gene CsupFAR2. Subsequently, we co-expressed CsupYPAQ and CsupFAR2 together with the previously characterized moth desaturase Atr∆11 in N. benthamiana. This resulted in the production of (Z)-11-hexadecenol together with (Z)-11-hexadecenal, the major pheromone component of C. suppressalis. Both compounds were collected from the transformed N. benthamiana headspace volatiles using solid-phase microextraction. We finally added the expression of a yeast acetyltransferase gene ATF1 and could then confirm also (Z)-11-hexadecenyl acetate release from the plant. CONCLUSIONS: Our results pave the way for stable transformation of plants to be used as biological pheromone sources in different pest control strategies.

Potato trait development going fast-forward with genome editing.

Implementations and improvements of genome editing techniques used in plant science have increased exponentially. For some crops, such as potato, the use of transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR) has moved to the next step of trait development and field trials, and should soon be applied to commercial cultivation.

Green Chemistry Production of Codlemone, the Sex Pheromone of the Codling Moth (Cydia pomonella), by Metabolic Engineering of the Oilseed Crop Camelina (Camelina sativa).

Synthetic pheromones have been used for pest control over several decades. The conventional synthesis of di-unsaturated pheromone compounds is usually complex and costly. Camelina (Camelina sativa) has emerged as an ideal, non-food biotech oilseed platform for production of oils with modified fatty acid compositions. We used Camelina as a plant factory to produce mono- and di-unsaturated C12 chain length moth sex pheromone precursors, (E)-9-dodecenoic acid and (E,E)-8,10-dodecadienoic acid, by introducing a fatty acyl-ACP thioesterase FatB gene UcTE from California bay laurel (Umbellularia californica) and a bifunctional ∆9 desaturase gene Cpo_CPRQ from the codling moth, Cydia pomonella. Different transgene combinations were investigated for increasing pheromone precursor yield. The most productive Camelina line was engineered with a vector that contained one copy of UcTE and the viral suppressor protein encoding P19 transgenes and three copies of Cpo_CPRQ transgene. The T2 generation of this line produced 9.4% of (E)-9-dodecenoic acid and 5.5% of (E,E)-8,10-dodecadienoic acid of the total fatty acids, and seeds were selected to advance top-performing lines to homozygosity. In the T4 generation, production levels of (E)-9-dodecenoic acid and (E,E)-8,10-dodecadienoic acid remained stable. The diene acid together with other seed fatty acids were converted into corresponding alcohols, and the bioactivity of the plant-derived codlemone was confirmed by GC-EAD and a flight tunnel assay. Trapping in orchards and home gardens confirmed significant and specific attraction of C. pomonella males to the plant-derived codlemone.

Amylose starch with no detectable branching developed through DNA-free CRISPR-Cas9 mediated mutagenesis of two starch branching enzymes in potato.

DNA-free genome editing was used to induce mutations in one or two branching enzyme genes (Sbe) in tetraploid potato to develop starch with an increased amylose ratio and elongated amylopectin chains. By using ribonucleoprotein (RNP) transfection of potato protoplasts, a mutation frequency up to 72% was achieved. The large variation of mutations was grouped as follows: Group 1 lines with all alleles of Sbe1 mutated, Group 2 lines with all alleles of Sbe1 as well as two to three alleles of Sbe2 mutated and Group 3 lines having all alleles of both genes mutated. Starch from lines in Group 3 was found to be essentially free of amylopectin with no detectable branching and a chain length (CL) distribution where not only the major amylopectin fraction but also the shortest amylose chains were lost. Surprisingly, the starch still formed granules in a low-ordered crystalline structure. Starch from lines of Group 2 had an increased CL with a higher proportion of intermediate-sized chains, an altered granule phenotype but a crystalline structure in the granules similar to wild-type starch. Minor changes in CL could also be detected for the Group 1 starches when studied at a higher resolution.

Protoplast-Based Method for Genome Editing in Tetraploid Potato.

The cultivated potato is tetraploid with four probably equivalent loci for each gene. A potato variety is furthermore commonly genetically heterogeneous and selected based on a beneficial genetic context which is maintained by clonal propagation. When introducing genetic changes by genome editing it is then desirable to achieve edits in all four loci for a certain gene target. This is in order to avoid crosses to achieve homozygosity for edited gene loci and at the same time reduce risk of inbreeding depression. In such a context transient transfection of protoplasts for the introduction of mutations, avoiding stable insertion of foreign DNA, would be very attractive. The protocol of this chapter has been shown to be applicable for the introduction of mutations by DNA vectors containing expression cassettes of TALEN, Cas9, and Cas9 deaminase fusions together with sgRNA expression cassettes on either single or separate vectors. Furthermore, the protoplast-based system has been shown to work very efficiently for mutations introduced by in vitro-produced and transfected RNP (ribonucleoprotein) complexes.

Transitions in wheat endosperm metabolism upon transcriptional induction of oil accumulation by oat endosperm WRINKLED1.

BACKGROUND: Cereal grains, including wheat (Triticum aestivum L.), are major sources of food and feed, with wheat being dominant in temperate zones. These end uses exploit the storage reserves in the starchy endosperm of the grain, with starch being the major storage component in most cereal species. However, oats (Avena sativa L.) differs in that the starchy endosperm stores significant amounts of oil. Understanding the control of carbon allocation between groups of storage compounds, such as starch and oil, is therefore important for understanding the composition and hence end use quality of cereals. WRINKLED1 is a transcription factor known to induce triacylglycerol (TAG; oil) accumulation in several plant storage tissues. RESULTS: An oat endosperm homolog of WRI1 (AsWRI1) expressed from the endosperm-specific HMW1Dx5 promoter resulted in drastic changes in carbon allocation in wheat grains, with reduced seed weight and a wrinkled seed phenotype. The starch content of mature grain endosperms of AsWRI1-wheat was reduced compared to controls (from 62 to 22% by dry weight (dw)), TAG was increased by up to nine-fold (from 0.7 to 6.4% oil by dw) and sucrose from 1.5 to 10% by dw. Expression of AsWRI1 in wheat grains also resulted in multiple layers of elongated peripheral aleurone cells. RNA-sequencing, lipid analyses, and pulse-chase experiments using 14C-sucrose indicated that futile cycling of fatty acids could be a limitation for oil accumulation. CONCLUSIONS: Our data show that expression of oat endosperm WRI1 in the wheat endosperm results in changes in metabolism which could underpin the application of biotechnology to manipulate grain composition. In particular, the striking effect on starch synthesis in the wheat endosperm indicates that an important indirect role of WRI1 is to divert carbon allocation away from starch biosynthesis in plant storage tissues that accumulate oil.

Oil crops for the future.

Agriculture faces enormous challenges including the need to substantially increase productivity, reduce environmental footprint, and deliver renewable alternatives that are being addressed by developing new oil crops for the future. The efforts include domestication of Lepidium spp. using genomics-aided breeding as a cold hardy perennial high-yielding oil crop that provides substantial environmental benefits, expands the geography for oil crops, and improves farmers' economy. In addition, genetic engineering in Crambe abyssinica may lead to a dedicated industrial oil crop to replace fossil oil. Redirection of photosynthates from starch to oil in plant tubers and cereal endosperm also provides a path for enhancing oil production to meet the growing demands for food, fuel, and biomaterials. Insect pheromone components are produced in seed oil plants in a cost-effective and environmentally friendly pest management replacing synthetically produced pheromones. Autophagy is explored for increasing crop fitness and oil accumulation using genetic engineering in Arabidopsis.

Charting oat (Avena sativa) embryo and endosperm transcription factor expression reveals differential expression of potential importance for seed development.

Uniquely, oat, among cereals, accumulates an appreciable amount of oil in the endosperm together with starch. Oat is also recognized for its soluble fibers in the form of ß-glucans. Despite high and increasing interest in oat yield and quality, the genetic and molecular understanding of oat grain development is still very limited. Transcription factors (TFs) are important regulatory components for plant development, product quality and yield. This study aimed to develop a workflow to determine seed tissue specificity of transcripts encoding transcription factors to reveal differential expression of potential importance for storage compound deposition and quality characters in oat. We created a workflow through the de novo assembly of sequenced seed endosperm and embryo, and publicly available oat seed RNAseq dataset, later followed by TF identification. RNAseq data were assembled into 33,878 transcripts with approximately 90% completeness. A total of 3875 putative TF encoding transcripts were identified from the oat hybrid assemblies. Members of the B3, bHLH, bZIP, C3H, ERF, NAC, MYB and WRKY families were the most abundant TF transcripts. A total of 514 transcripts which were differentially expressed between embryo and endosperm were identified with a threshold of 16-fold expression difference. Among those, 36 TF transcript homologs, belonging to 7 TF families, could be identified through similarity search in wheat embryo and endosperm EST libraries of NCBI Unigene database, and almost all the closest homologs were specifically expressed in seed when explored in WheatExp database. We verified our findings by cloning, sequencing and finally confirming differential expression of two TF encoding transcripts in oat seed embryo and endosperm. The developed workflow for identifying tissue-specific transcription factors allows further functional characterization of specific genes to increase our understanding of grain filling and quality.

WRINKLED1 Is Subject to Evolutionary Conserved Negative Autoregulation.

High accumulation of storage compounds such as oil and starch are economically important traits of most agricultural crops. The genetic network determining storage compounds composition in crops has been the target of many biotechnological endeavors. Especially WRINKLED1 (WRI1), a well-known key transcription factor involved in the allocation of carbon into oil, has attracted much interest. Here we investigate the presence of an autoregulatory system involving WRI1 through transient expression in Nicotiana benthamiana leaves. Different lengths of the Arabidopsis WRI1 promotor region were coupled to a GUS reporter gene and the activity was measured when combined with constitutive expression of different WRI1 homologs from Arabidopsis thaliana, oat (Avena sativa L.), yellow nutsedge (Cyperus esculentus L.), and potato (Solanum tuberosum L.). We could show that increasing levels of each WRI1 homolog reduced the transcriptional activity of the Arabidopsis WRI1 upstream region. Through structural analysis and domain swapping between oat and Arabidopsis WRI1, we were able to determine that the negative autoregulation was clearly dependent on the DNA-binding AP2-domains. A DNA/protein interaction assay showed that AtWRI1 is unable to bind to its corresponding upstream region indicating non-direct interaction in vivo. Taken together, our results demonstrate a negative feedback loop of WRI1 expression and that it is an indirect interaction most likely caused by downstream targets of WRI1. We also show that it is possible to release WRI1 expression from this autoregulation by creating semi-synthetic WRI1 homologs increasing the potential use of WRI1 in biotechnological applications.

Reduced Enzymatic Browning in Potato Tubers by Specific Editing of a Polyphenol Oxidase Gene via Ribonucleoprotein Complexes Delivery of the CRISPR/Cas9 System.

Polyphenol Oxidases (PPOs) catalyze the conversion of phenolic substrates to quinones, leading to the formation of dark-colored precipitates in fruits and vegetables. This process, known as enzymatic browning, is the cause of undesirable changes in organoleptic properties and the loss of nutritional quality in plant-derived products. In potato (Solanum tubersoum L.), PPOs are encoded by a multi-gene family with different expression patterns. Here, we have studied the application of the CRISPR/Cas9 system to induce mutations in the StPPO2 gene in the tetraploid cultivar Desiree. We hypothesized that the specific editing of this target gene would result in a lower PPO activity in the tuber with the consequent reduction of the enzymatic browning. Ribonucleoprotein complexes (RNPs), formed by two sgRNAs and Cas9 nuclease, were transfected to potato protoplasts. Up to 68% of regenerated plants contained mutations in at least one allele of the target gene, while 24% of edited lines carried mutations in all four alleles. No off-target mutations were identified in other analyzed StPPO genes. Mutations induced in the four alleles of StPPO2 gene, led to lines with a reduction of up to 69% in tuber PPO activity and a reduction of 73% in enzymatic browning, compared to the control. Our results demonstrate that the CRISPR/Cas9 system can be applied to develop potato varieties with reduced enzymatic browning in tubers, by the specific editing of a single member of the StPPO gene family.