Targeted gene mutation in tetraploid potato through transient TALEN expression in protoplasts

Nicolia, Alessandro; Proux‐Wéra, Estelle; Åhman, Inger; Onkokesung, Nawaporn; Andersson, Mariette; Andréasson, Erik; Zhu, Lu

doi:10.1016/j.jbiotec.2015.03.021

Cited by 113 publications

(82 citation statements)

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“…The protoplast isolation and transfection method used was essentially as described by Nicolia et al 2015, where a method to induce mutations in potato via TALEN was developed (Nicolia et al 2015). In this study, the protocol was further developed and adapted for CRISPR-Cas9.…”

Section: Resultsmentioning

confidence: 99%

“…Recently, the first studies using TALEN and CRISPR-Cas9 to induce mutations in potato were published (Butler et al 2015, 2016; Clasen et al 2016; Nicolia et al 2015; Sawai et al 2014; Wang et al 2015). In a TALEN study, stable transformation was used for studying the disruption of a sterol side chain reductase 2 (StSSR2).…”

Section: Introductionmentioning

confidence: 99%

“…In that study, two lines with confirmed indels were described in detail of which one line was concluded to have all four alleles mutated (Sawai et al 2014). Further, a transient method for TALEN expression was developed and induced mutations were verified in the target gene, ACETOLACTATE SYNTHASE (ALS), in 2 out of 20 regenerated shoots (Nicolia et al 2015). In that study, no shoots with multiple mutated alleles were described.…”

Section: Introductionmentioning

confidence: 99%

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Efficient targeted multiallelic mutagenesis in tetraploid potato (Solanum tuberosum) by transient CRISPR-Cas9 expression in protoplasts

et al. 2016

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Key message Altered starch quality with full knockout of GBSS gene function in potato was achieved using CRISPR-Cas9 technology, through transient transfection and regeneration from isolated protoplasts. AbstractSite-directed mutagenesis (SDM) has shown great progress in introducing precisely targeted mutations. Engineered CRISPR-Cas9 has received increased focus compared to other SDM techniques, since the method is easily adapted to different targets. Here, we demonstrate that transient application of CRISPR-Cas9-mediated genome editing in protoplasts of tetraploid potato (Solanum tuberosum) yielded mutations in all four alleles in a single transfection, in up to 2 % of regenerated lines. Three different regions of the gene encoding granule-bound starch synthase (GBSS) were targeted under different experimental setups, resulting in mutations in at least one allele in 2–12 % of regenerated shoots, with multiple alleles mutated in up to 67 % of confirmed mutated lines. Most mutations resulted in small indels of 1–10 bp, but also vector DNA inserts of 34–236 bp were found in 10 % of analysed lines. No mutations were found in an allele diverging one bp from a used guide sequence, verifying similar results found in other plants that high homology between guide sequence and target region near the protospacer adjacent motif (PAM) site is essential. To meet the challenge of screening large numbers of lines, a PCR-based high-resolution fragment analysis method (HRFA) was used, enabling identification of multiple mutated alleles with a resolution limit of 1 bp. Full knockout of GBSS enzyme activity was confirmed in four-allele mutated lines by phenotypic studies of starch. One remaining wild-type (WT) allele was shown sufficient to maintain enough GBSS enzyme activity to produce significant amounts of amylose.Electronic supplementary materialThe online version of this article (doi:10.1007/s00299-016-2062-3) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Efficient targeted multiallelic mutagenesis in tetraploid potato (Solanum tuberosum) by transient CRISPR-Cas9 expression in protoplasts

et al. 2016

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“…Using recombinant gene techniques, the PGA1-and PGA2-knockdown plants contained significantly less SGA. As an alternative to transgenic technology, functionless mutants can also be obtained from mutation breeding, using such techniques as TILLING (targeting induced local lesions in genome; Elias et al, 2009) and genome editing techniques (Sawai et al, 2014;Nicolia et al, 2015).…”

Section: The Phenotype Of Pga1-and Pga2-knockdown Transgenic Potato Pmentioning

confidence: 99%

Two Cytochrome P450 Monooxygenases Catalyze Early Hydroxylation Steps in the Potato Steroid Glycoalkaloid Biosynthetic Pathway

et al. 2016

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Solanine and a-chaconine, steroidal glycoalkaloids (SGAs) found in potato (Solanum tuberosum), are among the best-known secondary metabolites in food crops. At low concentrations in potato tubers, SGAs are distasteful; however, at high concentrations, SGAs are harmful to humans and animals. Here, we show that POTATO GLYCOALKALOID BIOSYNTHESIS1 (PGA1) and PGA2, two genes that encode cytochrome P450 monooxygenases (CYP72A208 and CYP72A188), are involved in the SGA biosynthetic pathway, respectively. The knockdown plants of either PGA1 or PGA2 contained very little SGA, yet vegetative growth and tuber production were not affected. Analyzing metabolites that accumulated in the plants and produced by in vitro enzyme assays revealed that PGA1 and PGA2 catalyzed the 26-and 22-hydroxylation steps, respectively, in the SGA biosynthetic pathway. The PGA-knockdown plants had two unique phenotypic characteristics: The plants were sterile and tubers of these knockdown plants did not sprout during storage. Functional analyses of PGA1 and PGA2 have provided clues for controlling both potato glycoalkaloid biosynthesis and tuber sprouting, two traits that can significantly impact potato breeding and the industry.

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“…Sawai applied TALEN through a transgenic approach with stable transformation to disrupt the sterol side chain reductase 2 (St SSR2) gene, involved in glycoalkaloid synthesis, obtaining potatoes with a significant reduction in these toxic compounds [39]. Nicolia proposed a pipeline for tetraploid potato genome editing based on transient expression of TALEN in protoplasts, potentially usable also for other SDNs (e.g., Cas), assuring no T-DNA insertion in the plant genome [51]. Meanwhile, a similar approach was applied at the biotech Company Calyxt, to improve cold storage and processing traits of the variety Ranger Russet, through the knockout of potato vacuolar invertase gene (St Vlnv), involved in the production of reducing sugars in cold-stored tubers, resulting in a significant reduction of acrylamide levels in potato chips [38].…”

Section: Not All Gm Potatoes Are Equalmentioning

confidence: 99%

Effect of Organic Potato Farming on Human and Environmental Health and Benefits from New Plant Breeding Techniques. Is It Only a Matter of Public Acceptance?

Pacifico

Paris

2016

Sustainability

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Organic farming practices are commonly thought to reduce the environmental impact of agriculture and to preserve the naturalness of the products. Herein, we report the effect of crop management practices on nutritional and toxicological value of potato tubers. Comparative studies are often controversial and the results are dependent on genotype and methodological approach. Targeted analysis and "omics" strategies are discussed, pointing at the nutritional aspects and the corresponding biological and molecular processes involved. Organic farming supporters still do not accept the use of genetic modification to produce new varieties suited for organic agriculture and crop improvement by genetic engineering still sparks hot debate among various scientific and social factions whose major concern is the possible existence of unintended effects both on human and world health. In this context, the advent of "new plant breeding techniques" has reignited the discussion on genetic engineering and on the compatibility of the new technologies with an eco-friendly agriculture. Could cisgenic and genome-edited potatoes be new good options for organic agriculture? We discuss how these approaches can be used to address food security challenges and to overcome specific problems based on the biological characteristics of potato tubers, producing new varieties that can improve farmers' profit with a lower impact on public opinion. However, political, ethical, and social fears will probably persist much longer, mainly in Italy, historically a fiercely anti-GM country with a European leadership in organic food production and export.

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Targeted gene mutation in tetraploid potato through transient TALEN expression in protoplasts

Cited by 113 publications

References 53 publications

Efficient targeted multiallelic mutagenesis in tetraploid potato (Solanum tuberosum) by transient CRISPR-Cas9 expression in protoplasts

Efficient targeted multiallelic mutagenesis in tetraploid potato (Solanum tuberosum) by transient CRISPR-Cas9 expression in protoplasts

Two Cytochrome P450 Monooxygenases Catalyze Early Hydroxylation Steps in the Potato Steroid Glycoalkaloid Biosynthetic Pathway

Effect of Organic Potato Farming on Human and Environmental Health and Benefits from New Plant Breeding Techniques. Is It Only a Matter of Public Acceptance?

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