Location: Temperate Tree Fruit and Vegetable Research
Project Number: 2092-21220-003-048-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 30, 2026
End Date: Feb 28, 2028
Objective:
Identify target genes involved in sprouting regulation, construct trans-encapsulated RNA replicons, and evaluate their ability to deliver genetic material to potato tuber eyes in order to inhibit sprouting. Evaluate effect of trans-cleaving ribozymes (TcRz) on sprout development, tuber physiology, and storage longevity.
Approach:
Candidate genes regulating dormancy release and sprouting will be identified through literature and bioinformatics analyses. Tissue-specific expression across developmental stages and dormancy phases will determine which key isoforms are expressed. Two isoforms each of StTCP15, StGID1, and stu-miR319c will be prioritized, and five top-ranking, functionally distinct isoforms will be selected as gene-silencing targets most likely to prolong tuber dormancy. Potato virus X–derived RNA replicons lacking the coat protein will be assembled with GFP as a visual cargo. Replicon and coat protein constructs (including CPP fusions) will be assembled using Golden Gate assembly into T-DNA vectors and verified by whole-plasmid sequencing. Replicon T-DNAs will be transiently expressed in N. benthamiana, either alone or co-infiltrated with coat protein variants. Lysates containing packaged virions will be applied to potato eyes via carborundum abrasion or pressurized spray. Twenty tubers per treatment will be analyzed for GFP fluorescence to quantify delivery efficiency and identify the optimal coat protein–CPP combination. Using the most efficient system, treated tubers (n = 40) will be monitored weekly for GFP expression over eight weeks at room temperature and storage temperatures (4 °C, 10 °C). Fading GFP signals will trigger RNA extraction and qPCR for replicon detection, to evaluate the duration of persistence across storage conditions. Replicon constructs replacing GFP with TcRzs or 300 bp antisense RNAi sequences for stu-miR319c, TCP, and GID will be built and delivered to tubers at 4 °C. After half the maximal persistence time, eye tissues will be harvested for RNA extraction and qPCR. Relative expression will be compared with GFP controls to determine which method achieves greater knockdown. Layered double hydroxide (LDH) nanomaterials will be synthesized via co-precipitation and loaded with dsRNA to protect against degradation and ensure sustained release. Application methods (dip, spray, fogging) and adjuvants will be optimized. Delivery efficiency, gene silencing, phenotypic outcomes, and cost-effectiveness will identify whether LDH or TcRzs offers the superior large-scale delivery approach. TcRz-treated and control tubers (targeting stu-miR319c, StTCP15, StGID1) will be cured, then stored at 4 °C, 10 °C, and room temperature. Days to sprouting (DTS), defined as 80 % of tubers showing = 2 mm sprouts, will be recorded. Weekly measurements of sprout number and length will quantify inhibition kinetics and allow genotype comparisons across treatments.
Weight loss, respiration rate, starch degradation, and reducing sugar (glucose, fructose) accumulation will be tracked to assess metabolic health during storage. Treated and control tubers will be evaluated for dry matter content, fry color, texture (TPA), oil uptake*, and the accumulation of reducing sugars and glycoalkaloids at 3 and 6 months of storage.
2.5 Efficacy and Longevity: At 3- and 6-month intervals, sprout incidence, average sprout length, appearance, spoilage, and weight loss will be recorded to establish storage longevity.