Multiple Transgenic Strategies Positively Regulate Cold-Induced Sweetening in Low Temperature Stored Potato Tubers

Abstract

Cold-induced sweetening (CIS) is a common phenomenon in many plants including potatoes that help in osmoregulation and cryoprotection. However, CIS is associated with quality deterioration in potato tubers due to accumulation of reducing sugars at low temperatures. We investigated two different strategies to modulate CIS in potato, overexpression of RING finger (SbRFP1) as anti-sweetening gene and by double-stranded RNA-mediated gene silencing of the vacuolar invertase gene. In silico analysis predicted that the ubiquitination activity of the RING finger protein was responsible for regulating the expression of invertase during cold-induced stress. Moreover, the in silico predicted binding stability of siRNA-mRNA duplex suggested efficient gene silencing of the invertase gene. We successfully generated four single and three dual transgenic potato lines that were positive for transgene insertion and integration as revealed in PCR and Southern blot. The amount of reducing sugars found in tubers obtained from single transgenics showed maximum decrease of 1.67 folds while tubers obtained from dual transgenic line depicted 4.86 folds reduced accumulation of reducing sugars compared to non-transgenic control when analyzed through HPLC analysis post 60-day storage at low temperature (4°C). Further, the invertase activity was 1.46 folds reduced in single transgenics while this reduction was 2.13 folds in dual transgenics. The downregulation of the invertase gene was up to 3.36 folds in dual transgenic potato lines, 2.26 folds in single transgenic compared to control, non-transgenic post 60-day cold storage at low temperature. Conclusively, the utilization of multiple strategies to regulate CIS in low-temperature stored potato tubers positively regulate CIS in transgenic potatoes and can be employed to generate CIS resistant potato varieties.