Abstract
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.
Highlights
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
Based on the number and combination of alleles mutated, the lines were divided into three groups: five lines had mutations in all four alleles of Sbe1, 82007, 82050, 82079, 104011 and 104032 (Group 1), six lines had four-allele mutations in Sbe[1] combined with two to three alleles mutated in Sbe2, 104001, 104005, 104006, 104016, 104018 and 104034 (Group 2) and two lines, 104010 and 104023, had all eight alleles mutated (Group 3)
To develop a starch with a significant increase in chain lengths corresponding to the amylose fraction, mutations in all alleles of both genes were needed (Group 3)
Summary
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 increasing the average chain length of potato starch, a starch with health benefits can be developed. The development of potatoes with a high ratio of amylose starch and/or altered starch chain length distribution has been achieved by targeting two starch branching enzymes (SBEs) using traditional gene silencing technologies and recently genome editing[12,13,14]. A very high amylose starch content was found synthesised at the expense of total starch content and plant d evelopment[15] Based on those results and the fact that, so far, no potato studies have resulted in pure amylose starch, it could be speculated that the presence of a fraction of amylopectin in potato starch is essential for plant development. The high amylose content only had a minor impact on grain yield and starch content
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