Improving cold storage and processing traits in potato through targeted gene knockout

Abstract

Cold storage of potato tubers is commonly used to reduce sprouting and extend postharvest shelf life. However, cold temperature stimulates the accumulation of reducing sugars in potato tubers. Upon high-temperature processing, these reducing sugars react with free amino acids, resulting in brown, bitter-tasting products and elevated levels of acrylamide--a potential carcinogen. To minimize the accumulation of reducing sugars, RNA interference (RNAi) technology was used to silence the vacuolar invertase gene (VInv), which encodes a protein that breaks down sucrose to glucose and fructose. Because RNAi often results in incomplete gene silencing and requires the plant to be transgenic, here we used transcription activator-like effector nucleases (TALENs) to knockout VInv within the commercial potato variety, Ranger Russet. We isolated 18 plants containing mutations in at least one VInv allele, and five of these plants had mutations in all VInv alleles. Tubers from full VInv-knockout plants had undetectable levels of reducing sugars, and processed chips contained reduced levels of acrylamide and were lightly coloured. Furthermore, seven of the 18 modified plant lines appeared to contain no TALEN DNA insertions in the potato genome. These results provide a framework for using TALENs to quickly improve traits in commercially relevant autotetraploid potato lines.

Keywords: acrylamide reduction; cold-induced sweetening; gene editing; potato; transcription activator-like effector nucleases; vacuolar invertase.

Figure 1

Targeting the Solanum tuberosum cv Ranger Russet VInv gene with transcription activator‐like effector nucleases (TALENs). (a) During cold storage, potato tubers accumulate acrylamide through a nonenzymatic Maillard reaction, which uses reducing sugars (primarily glucose and fructose) and free amino acids (asparagine [Asn]). Reducing sugars accumulate through hydrolysis of sucrose by vacuolar acid invertase (VInv). In addition to accumulating acrylamide, cold‐stored potatoes also produce brown‐ to black‐pigmented products. (b) Schematic of the VInv gene. TALEN target sites are indicated with black, grey and white triangles (VInv_T1, VInv_T2, VInv_T3, respectively). (c) TALEN target sites within exon 1. Single nucleotide polymorphisms are indicated by lowercase bold letters. Underlined letters indicate TALEN‐binding sites. A1, allele 1; A2(1), copy 1 of allele 2; A2(2) copy 2 of allele 2; A(3), allele 3.

Figure 2

Assessment of transcription activator‐like effector nuclease (TALEN) activity in Solanum tuberosum cv Ranger Russet protoplasts. (a) Protoplasts from leaves on 3‐week‐old potato plants (Ranger Russet) were isolated and transformed with plasmids encoding TALEN pairs. Following transformation, exon 1 was amplified by PCR and mutations were assessed by 454 pyro‐sequencing. (b) Percentage of vacuolar invertase sequences containing TALEN‐induced mutations. Total refers to the combined percentage of mutations in all four alleles. Else refers to sequences that could not be assigned an allele type due to TALEN‐induced mutations that removed the allele‐defining single nucleotide polymorphisms.

Figure 3

Recovery of potato lines carrying mutations within vacuolar invertase (VInv). (a) Approach and timeline to regenerate plants with mutations in VInv. Protoplasts were transformed with plasmids encoding the VInv_T2 transcription activator‐like effector nucleases pair and were cultured in nonselective regeneration medium. Following shoot and root formation, potato plantlets were transferred to soil. (b) Examples of plant lines carrying mutations in one or more of the VInv alleles. WT, wild type.

Figure 4

Quality assessment of mutant potato lines. (a) Analysis of sugar content within potato tubers stored at 4 °C for 14 days. Error bars represent standard deviation. (b) Analysis of acrylamide content in potato chips that were processed from tubers that were stored at 4 °C for 14 days. (c) Images of potato chips after being processed from tubers stored at 4 °C for 14 days. The colorimetric score is listed to the right of the image.