**6.2 Postharvest tuber quality for enhanced cold-induced sweetening**

After harvest, storage of potato tubers under cold conditions (below 8–10°C) is required for year-round processing as well as to mitigate the possibility of sprouting and diseases. Stored tubers loose some of their starch content and accumulate high RS in a process known as cold-induced sweetening (CIS). Upon frying at high temperatures, these RS interact with asparagine to produce dark-colored fried products [166, 167]. Therefore, minimizing the accumulation of RS in tubers is of high importance to potato processing industry. The amount of RS in a tuber is regulated by a balance between the activity of invertases (cell wall invertase, vacuolar invertase and neutral invertase), which convert sucrose into RS and invertase inhibitors which limit the activity of invertase via protein-protein interactions [28, 136, 168]. **Figure 6** and **Table 5** highlights the information on important candidate genes used in the modification of sugar content in transgenic potatoes. Among various invertases, the acid invertase (AI) is the key enzymes involved in the conversion of sucrose into RS and transgenic approaches confirmed that overexpression of the vacuolar invertase inhibitor (*INH2*) gene reduces the expression of vacuolar invertase gene, AI activity and RS in transgenic potato tubers under the control of CaMV 35S or class I patatin promoter [136–138, 166]. In another study, overexpression of the vacuolar invertase inhibitor isoforms from potato resulted in decreased vacuolar invertase activity, low RS and low acrylamide content with improved chip quality in cold-stored transgenic potato [138, 151]. Suppression of the AI activity by silencing of the vacuolar invertase gene using RNAi or TALENS resulted in a very strong decrease in RS accumulation, light colored chips and low acrylamide in cold-stored transgenic tubers of different potato cultivars [147, 169]. Knockout of the vacuolar invertase was performed in 'Ranger Russet' potatoes using the TALENs technology. Five regenerated plants contained knockouts of all four invertase alleles with no detectable RS, light brown chip color and lower levels of acrylamide [148].

Other than, the vacuolar invertase and its inhibitor gene, Zhang et al. [170] hypothesized that *RING finger gene* (*SbRFP1*) could be a potential target for manipulation of the CIS in potato tubers. RING finger proteins constitute a large protein family in higher plants involved in cold response [170]. When a novel *SbRFP1* was overexpressed (CaMV35S promoter) in potato microtubers (cv. 'E-potato 3'), it resulted in inhibition of beta-amylase and invertase activity. As a result, starch and sucrose degradation slowed down and the accumulation of RS in cold stored tubers was prevented [170]. Study suggested that other potential genes could be involved in CIS in potato tubers and transgenic manipulation can be performed to overcome this persistent problem.

So far, much attention has been given to the manipulation of the acid invertase activity via suppression and overexpression of the vacuolar invertase and invertase inhibitor genes to control the RS content in transgenic potato tubers respectively. In contrast to the acid invertase, the potential involvement of neutral invertase which is involved in the conversion of sucrose into RS has not been demonstrated and wellstudied in potato tubers. Datir and Regan [150] identified 8 neutral invertase genes from potato. Based on their expression pattern and enzymatic pattern in cold-stored potato tubers, they concluded that neutral invertases also may play a role in sucrose degradation. Therefore, genetic manipulation of neutral invertase may result in decreased RS along with improved processing quality of potato tubers.

#### **6.3 Reducing the enzymatic browning**

Enzymatic browning or discoloration of potato tubers occurs when phenolic compounds are oxidized by the enzyme polyphenol oxidase (PPO). This results in negative effects on color, taste, flavor, and nutritional value and forms undesired dark pigments that result in considerable economic losses to the potato food and processing industry [171, 172]. To prevent the quality loss and increase consumers acceptance, sulfiting agents (sulfur dioxide, sodium etc.) can be used to prevent the enzymatic browning, however, there are concerns about the health risks of sulfites [173, 174]. For this reason, there is a need to develop the alternative technologies for developing potatoes that are resistant to enzymatic browning [175]. Silencing of the *PPO* gene resulted in significantly reduced enzymatic browning in the tubers of transgenic lines [175, 176]. Two potato cultivars 'Van Gogh' and 'Diamant' transformed with antisense *PPO* placed under patatin and GBSS promoters abolished the expression of PPO in transgenic tubers [176]. In another study, the PPO activity was inhibited by expression of a sense as well as antisense *PPO* RNAs from a tomato *PPO* cDNA under the control of the CaMV 35S promoter in cv. 'Russet Burbank'. Transgenic lines expressing *PPO* by sense and antisense approaches resulted in reduction in black spot susceptibility, decreased PPO activity and reduced enzymatic browning [175].

Recent studies have demonstrated that genome editing using CRISPR/Cas9 system can be successfully used to reduce the enzymatic browning in potato [177, 178]. The Cas9 nuclease guided by two RNA molecule/s (sgRNA/s) introduced a double stranded break in the PPO gene. The system that introduced the mutations in the *PPO* gene was delivered into the protoplasts of cv 'Desiree'. 24% of CRISPR/Cas9-edited lines carried mutations in all four alleles of *PPO* without any off-target mutations in other *PPO* genes. Mutations induced in the four alleles of *StPPO2* gene showed 69% reduction in tuber PPO activity and a 73% reduction in enzymatic browning, compared to the control [177]. These studies revealed that CRISPR/Cas9 system represents an important step towards the development of potato varieties that maintain the organoleptic, antioxidant and nutritional properties during harvest and post-harvest procedures, without the utilization of potentially harmful browning controlling agents.
