No abstract

Cold-induced sweetening (CIS) is an unwanted physiological phenomenon in which reducing sugars (RS) get accumulated in potato (Solanum tuberosum) upon cold storage. High RS content makes potato commercially unsuitable for processing due to the unacceptable brown color in processed products like chips, fries, etc., and the production of a potential carcinogen, acrylamide. UDP-glucose pyrophosphorylase (UGPase) catalyzes the synthesis of UDP-glucose towards the synthesis of sucrose and is also involved in the regulation of CIS in potato. The objective of the present work was RNAi-mediated downregulation of the StUGPase expression level in potato for the development of CIS tolerant potato. Hairpin RNA (hpRNA) gene construct was developed by placing UGPase cDNA fragment in sense and antisense orientation intervened by GBSS intron. Internodal stem explants (cv. Kufri Chipsona-4) were transformed with hpRNA gene construct, and 22 transgenic lines were obtained by PCR screening of putative transformants. Four transgenic lines showed the highest level of RS content reduction following 30 days of cold storage, with reductions in sucrose and RS (glucose & fructose) levels of up to 46% and 57.5%, respectively. Cold stored transgenic potato of these four lines produced acceptable chip colour upon processing. The selected transgenic lines carried two to five copies of the transgene. Northern hybridization revealed an accumulation of siRNA with a concomitant decrease in the StUGPase transcript level in these selected transgenic lines. The present work demonstrates the efficacy of StUGPase silencing in controlling CIS in potato, and the strategy can be employed for the development of CIS tolerant potato varieties.

Genome editing through the alteration of nucleotide sequence has already revolutionized the field of site-directed mutagenesis for a decade. However, research in terms of precision and efficacy in targeting the loci and reduction in off-target mutation has always been a priority when DNA is involved. Therefore, recent research interest lies in utilizing the same precision technology but results in non-transgenic. In this review article, different technological advancements have been explained, which may provide a holistic concept of and need for transgene-free genome editing. The advantage and lacunas of each technology have been critically discussed to deliver a transparent view to the readers. A systematic analysis and evaluation of published research articles implied that researchers across the globe are putting continuous efforts in this direction to eliminate the hindrance of transgenic regulation. Nevertheless, this approach has severe implications legitimate for mitigating the conflict of acceptance, reliability, and generosity of gene-editing technology and sustainably retorting to the expanding global population feeding challenges.

Diurnal regulation of gene expression encoding the dominant isoform of UDP-Glucose Pyrophosphorylase, StUGPase2, was studied in potato leaves at different developmental stages (pre-tuberization, tuber initiation, and tuber bulking). Across the developmental stages, the StUGPase2 steady-state mRNA level increased gradually from morning (6 AM) to noon (12 PM), followed by a transient drop at 2 PM. The mRNA level increased thereafter, and the highest transcript abundance was recorded at 8:00 PM. A sharp decline in StUGPase2 transcript abundance at 2 PM in all the stages studied is believed to be associated with the previous reports of diurnal behaviour of sucrose concentration and light responsiveness of sucrose synthase enzyme. Moreover, in all three stages, a steady state increase of StUGPase2 transcript despite a consistent decrease in irradiance after 12 PM, suggests the presence of more complex transcriptional regulation. Comparatively higher StUGPase2 transcript abundance at the tuberization initiation stage as compared to the pre and post-tuberization stages indicates the prominent role of the gene during tuber development. Bioinformatics analysis of the StUGPase2 promoter region predicted the presence of various light-responsive cis-regulatory nucleotide sequences, which are considered to be associated with the light-mediated diurnal change in StUGPase2 gene expression. This research will aid in understanding the pattern of UGPase gene regulation, which is ultimately connected to potato tuber yield and bulking attributes.