Development of genome editing in potato and tobacco

Potato (Solanum tuberosum) is the third most important food crop in the world (Dongyu, 2022). Fifty to sixty percent of global potato production is intended for human consumption (Raji, 2020) and is shifting from fresh potatoes to processed food products (Devaux et al., 2020). However, the potato industry faces a problem known as cold-induced sweetening, which occurs when tubers are stored at low temperatures. Under these conditions, phosphorylated starch breaks down into reducing sugars which, during frying, react with amino acids to form acrylamide, a potential carcinogen (Mottram et al., 2002; Stadler et al., 2002; Bhaskar et al., 2010). Acrylamide formation in potato chips and French fries has raised global food safety concerns (Grob, 2007; Medeiros Vinci et al., 2012). As a result, the potato processing industry is seeking methods to reduce acrylamide levels, with one effective way being to decrease starch degradation in cold‐stored tubers (Matsuura-Endo et al., 2006; Bhaskar et al., 2010). Studies on Arabidopsis and potato plants have shown that Glucan Water Dikinase 1 (GWD1) plays a crucial role in the first step of starch degradation (Kozlov et al., 2007; Wickramasinghe et al., 2009; Hejazi et al., 2014; Pirone et al., 2017; Ohnuma et al., 2023). Silencing of the GWD1 gene reduces GWD1 protein levels, thereby blocking starch breakdown and preventing the accumulation of reducing sugars (Lorberth et al., 1998; Viksø-Nielsen et al., 2001). Gene silencing can be achieved through gene editing, a method that allows precise alterations to an organism’s DNA (Zhang et al., 2017). Over the past decade, genome editing tools have been developed which enables targeted genetic modifications (Alamillo et al., 2023). One of these tools, CRISPR/Cas9, can be delivered using conventional transgenic methods, or non-transgenic methods, such as transfection of CRISPR/Cas9 ribonucleoproteins (RNPs) in protoplasts followed by plant regeneration (Chen et al., 2019). The non-transgenic approach is preferred in many cases as it avoids the introduction of trans DNA, helping to ease regulatory requirements in some jurisdictions (Gong et al., 2021). In this study, two strategies were used to try to mutate GWD1. The first involved gene editing of GWD1 in a transgene-free manner through the effective isolation of potato protoplasts and their regeneration into plants. Although protoplasts were successfully isolated it proved impossible to regenerate them into calli which is as essential step in plant regeneration. A transgenic approach was then used to target GWD1 and PDS for gene editing in tobacco. The use of tobacco was due to its faster regeneration time compared to potato (35-40 days for tobacco; Pathi et al., 2013 vs. 77 days for potato; Esna-Ashari and Villiers, 1998). CRISPR/Cas9 was introduced into tobacco using Agrobacterium-mediated transformation, where leaf explants were transformed to introduce the desired genetic changes. This resulted in transgenic plantlets which were screened for editing. Sequencing reactions and Tracking of Indels by Decomposition (TIDE) analysis revealed one plantlet had editing in the PDS gene, however, when the same sequencing reaction was analysed using Inference of CRISPR Edits (ICE), it was rejected due to low sequencing quality.

Grantor dc:publisher

Stellenbosch : Stellenbosch University

Year dc:date.issued

2026

Author dc:creator

• Keyte, Janet

Advisors dc:contributor.advisor

• Lloyd, James
• Campa, Manuela

Language dc:language.iso

en

Repository record dc:identifier.uri

https://scholar.sun.ac.za/handle/10019.1/136178

OAI identifier oai:identifier

oai:scholar.sun.ac.za:10019.1/136178