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

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.

Figure 1

Amylose content of extracted starches measured using a. enzymatic assay and b. colorimetric assay. The results are a mean of two technical replicates, error bar represents standard deviation (s.d.). Values that differ from the parental variety Desiree by Dunnett’s test (P < 0.05) are marked with *.

Figure 2

Chain-length distribution of debranched starches from the potato lines after normalisation for the peak area, analysed with HPSEC. The averages of the potato lines from Groups 1, 2 and 3 are shown. The parental variety Desiree and the high-amylose line T-2012 were included for comparison. The arrows from left to right point out the three populations of amylose chains, i.e. long, intermediate, and short chain amylose fraction, respectively. Software used is ASTRA software version 4.70.07 (wyatt.com/products/software/astra.html, Wyatt Technology Corp., Santa Barbara, CA).

Figure 3

Chain-length distribution of debranched starches on a relative molar basis (M%) with degree of polymerization (DP) 6–50, based on HPAEC analysis with averages of potato lines from Groups 1 and 2. No peak was detected for the starches from Group 3. The parental variety Desiree and the high-amylose line T-2012 were included for comparison.

Figure 4

¹H NMR spectra of starch from the Group 3 lines (a) 104010 and (b) 104023, and from (c) the parental variety Desiree. Glucose H1 at the α(1 → 4)-linkage (H1) and at the α(1 → 6)-linkage (H1′) are assigned as well as H4 of non-reducing end terminal residues (H4-t). Traces of EDTA and Tris are highlighted with asterisks.

Figure 5

X-ray diffraction patterns and polarised light microscopy of potato lines from Groups 1, 2, and 3. (a) Average diffraction intensity for each group. The parental variety Desiree and the high-amylose line T-2012 were included for comparison. (b) Images of selected potato starches using polarised light microscopy. Scale bar = 40 µm.

Figure 6

Starch granules (left images) and thin sliced tuber tissue (right images) stained with iodine and visualised under light microscope. (a,b) 82079 (Group 1) (c,d) 104018 (Group 2) (e,f) 104010 (Group 3). (g,h) RNAi line T-2012. (i,j) parental variety Desiree. Scale bar = 100 µm.

Figure 7

Transitory starch and green biomass of plants grown in greenhouse. Lines analysed are; 82079 (Group 1), 104006 (Group 2), 104018 (Group 2), 104010 (Group 3), 104023 (Group 3), T-2012 and parental variety Desiree. (a) Green biomass measured with low-cost RGB imaging phenotyping lab using digiCamControl (digiCamControl v2,1,2, http://www.digicamcontrol.com) and Easy Leaf Area. All lines are normalised to the parental variety Desiree, which is set to 1. The results are a mean of three biological replicates. Note that the vertical axis has a logarithmic scale. (b) Leaf tissue of top shoots harvested after a light (top row) and dark period (bottom row) stained with iodine.