**2. Reducing the glycoalkaloid content in transgenic tubers**

α-Chaconine and α-solanine are the two main glycoalkaloids that are toxic secondary metabolites present in the tubers of cultivated potato (*Solanum tuberosum* L.) [37, 38]. Symptoms of glycoalkaloid poisoning include abdominal pain, vomiting, and diarrhea in humans. Due to their toxic properties, 200 mg/kg fresh weight is the safety limit for total glycoalkaloid content in the tubers of released commercial potato cultivars [39, 40] because glycoalkaloids cannot be destroyed during food-processing treatments, such as boiling, baking or frying and even at high temperatures [41]. The general pathway of glycoalkaloid metabolism and important candidate genes used in the modification of glycoalkaloid content are highlighted in **Figure 2** and **Table 1**).

The biosynthesis of γ-solanine is catalyzed by the enzyme UDPgalactose:solanidine galactosyltransferase (SGT) from galactose and solanidine [44]. Transgenic potato lines were produced using an antisense version of a cDNA encoding *SGT* under the control of Cauliflower Mosaic Virus 35S (CaMV35S) promoter or a tuber-specific granule bound starch synthase (GBSS) promoter. Transgenic lines produced from potato cv. 'Lenape' expressing antisense *SGT* exhibited significantly lower steroidal glycoalkaloids in the tubers. In another study [45], antisense suppression of the genes that encode the enzyme for the biosynthesis of γ-solanine from UDP-galactose and solanidine (SGT1), γ-chaconine from UDP-glucose and solanidine (SGT2), and α-solanine and α-chaconine from UDP-rhamnose, β-solanine and β-chaconine (*SGT3*) were down-regulated under the control of GBSS6 promoter. Down-regulation of *SGT1* reduced the concentration of α-solanine without affecting

#### **Figure 2.**

*Glycoalkaloid biosynthetic pathway. The glycoalkaloid biosynthetic pathway starts from Acetyl-Co-A. The representative enzymes are HMGR1, 3-Hydroxy-3-methylglutaryl coenzyme A reductase; PVS1, vetispiradiene sesquiterpene cyclase; PSS1, squalene synthase; SMT1, sterol C24-methyltransferase type1; CH, cholestrole hydroxylase; SGT1, solanidine galactosyltransferase; SGT2, solanidine glucosyltransferas; SGT3, glycosterol rhamnosyltransferase; SMO, C-4 sterol methyl oxidase; SD, sterol C-5(6) desaturase; SSR, sterol side chain reductase; and GAME, glycoalkaloid metabolism genes. The figure is adapted and modified from Arnqvist et al. [27], Khan et al. [42], Sonawane et al. [43].*

the levels of α-chaconine. In contrast, down-regulation of *SGT2* resulted in reduction of α-chaconine and increased levels of α-solanine. Down-regulation of *SGT3* reduced concentrations of both α-chaconine and α-solanine [45]. Antisense manipulation of the *SGT* caused reduced glycoalkaloids content thereby decreasing toxicity in potato tubers.

RNAi, TALENs and CRISPR/Cas9-based systems have been used to reduce glycoalkaloid content [46, 52]. Glycoalkaloid biosynthesis is carried out by *PGA1* and *PGA2* encoding cytochrome P450 monooxygenases (CYP72A208 and CYP72A188) respectively. Transgenic lines using RNAi expressing either *PGA1* or *PGA2* showed very little steroidal glycoalkaloids accumulation and no effect on vegetative growth and tuber production [46]. Cholesterol and sterol side chain reductase 2 (SSR2) is a key enzyme in the biosynthesis of cholesterol and related steroidal glycoalkaloids [47, 56]. TALEN induced mutations in the *SSR2* gene have lower levels of cholesterol and steroids without affecting the plant growth [48–50]. CRISPR/Cas9-edited *StSSR2* resulted in a significant reduction of steroidal glycoalkaloids content [57]. CRISPR-Cas9-based knockout of *CYP88B1*, resulted in reduced levels of α-solanine and α-chaconine [51]. Likewise, knockout of dioxygenase *St16DOX*, responsible for


#### **Table 1.**

*Genes used in the modification of glycoalkaloid contents in transgenic potatoes.*

steroid 16α-hydroxylation abolished *St16DOX* expression and prevented glycoalkaloids production [52]. These studies indicate that suppression or knockout of genes involved in glycoalkaloid biosynthesis is a viable strategy to manipulate the steroidal glycoalkaloid levels in potato.

In contrast to gene suppression strategies, other studies have shown that the overexpression of genes involved in steroidal glycoalkaloid biosynthesis pathway are also an effective strategy to reduce cholesterol and glycoalkaloid levels [27]. Steroidal glycoalkaloids are thought to be synthesized from cholesterol that is converted to solanidine, and then by two separate pathways to α-solanine and α-chaconine. Altered glycoalkaloid content has been associated with overexpression of genes such as sterol *24-C-methyltransferase* (*SMT1*), *sterol desaturase* (*SD*) and *C-4 sterol methyl oxidase* (*SMO*) [53]. *SMTs* are involved in the biosynthesis of sterols and other products [58]. Overexpressing soybean *SMT* (*GmSMT1*) increased total sterols accompanied by a decrease in cholesterol and glycoalkaloids in leaves and tubers [27]. *Glycoalkaloid metabolism*9 (GAME9) is an APETALA2/Ethylene Response Factor associated with a major quantitative trait for steroidal glycoalkaloid content in tubers [54, 59]. Overexpression of *GAME9* altered the levels of steroidal glycoalkaloids leaves and tuber skin [54, 55].

### **3. Genetic modification for vitamin C content**

A whole, baked potato is an excellent source of vitamin C, vitamin B6, niacin and folate [60–62]. High consumption of potato tubers has been correlated with increased antioxidant level in blood and tissues and increased protection against oxidative stress [60]. However, the level of vitamin C is reduced if the potato is frozen, stored under refrigerated conditions, boiled or fried [63–65]. Thus far, there has been limited success in increasing vitamin C content in transgenic potato tubers [66, 67].

#### **Figure 3.**

*L-ascorbic acid biosynthesis pathway. The representative enzymes are GGP, GDP-L-galactose phosphorylase; GalUR, D-galacturonic acid reductase; DHAR, dehydroascorbate reductase; GALDH, L-galactono-1,4-lactone dehydrogenase; and AL, aldono lactonase. The figure is adapted and modified from Hemavathi et al. [68], Venkatesh and Park [69].*

The pathway of ascorbic acid (vitamin C) production (**Figure 3**) includes the reduction of D-galacturonic acid to L-galactonic acid by D- galacturonic acid reductase (GalUR), followed by conversion to L-galactono-1,4-lactone by aldonolactonase. The L-galactono-1,4-lactone is then oxidized to ascorbic acid by L-galactono-1,4 lactone dehydrogenase (GALDH) [70]. **Table 2** outlines the key genes used in the modification of ascorbic acid content. Overexpression of the strawberry *GalUR* resulted in increased ascorbic acid levels in potato [3]. Dehydroascorbate reductase (DHAR) plays an important role in maintaining the normal level of ascorbic acid by recycling oxidized ascorbic acid. Transgenic potato over-expressing the cytosolic *DHAR* significantly increased DHAR activity and ascorbic acid content in potato leaves and tubers, whereas chloroplastic *DHAR* overexpression only increased DHAR activity and ascorbic acid content in leaves [29]. Overexpression of the *Arabidopsis GDP-*L*-galactose phosphorylase* (*GGP*) resulted in significantly enhanced ascorbic acid content. Overall studies suggest that genetic alteration of specific vitamin-related genes could be excellent targets for improving the nutritional content of potato.


#### **Table 2.**

*Genes used in the modification of ascorbic acid content in transgenic potatoes.*
