case_images: PMC11817997_01
case_images: a Schematic representation of RNA interference target regions in TST1, TST2b-1A, and TST2b-1C marked with diagonal hatching. TST-RNAi and NPTII are marker gene constructs used for biolistic transformation. TST-S, sense strands of TST1, TST2b-1A, and TST2b-1C; TST-AS, anti-sense strands TST1, TST2b-1A, and TST2b-1C; intron, 4CL intron from bahiagrass. b Relative gene expression levels of TST1, TST2a, and TST2b in internode 5 were determined by qPCR. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) served as an internal control for normalization at maturity of plants (mean +- standard error, n = 3). c Height of stems from 7-month-old plants showed no significant differences between the TC and RNAi lines (n = 12, each background). d Diameter of internode 15 of TC and RNAi events at 7 months old (n = 3, each background). The boxes are drawn from the 25th percentile to the 75th percentile and the medians are shown. Whiskers extend to 1.5 times the interquartile range from the 25th and 75th percentiles. Student's t-test was used for the significance test. Plants were grown under controlled temperature (28 C/22 C, day/night) with a 14-h light (~300-950 mumol m-2 s-1)/10-h dark regimen via a mix of natural photoperiod and light conditions. e Total soluble sugar (sucrose + glucose + fructose) contents using high-performance liquid chromatography (HPLC) equipped with a refractive index detector (RID) (mean +- standard error, n = 3). Statistically significant differences among samples were determined using one-way ANOVA followed by multiple comparison tests (Fisher's LSD method) with different letters indicating significant differences (P < 0.05).
case_images: uhae312f1.jpg
case_images: PMC11817997_01_uhae312f1.jpg
case_images: f1
case_images: TST1 and TST2b-1A and TST2b-1C are responsible for sugar accumulation in the vacuoles of sugarcane stems. To test this hypothesis, we generated simultaneous knockdown events for these genes using the RNA interference (RNAi) strategy in sugarcane cultivar UFCP84-1047 (Fig. 1a), a high-fiber sugarcane cultivar for cellulosic ethanol production. We selected four sugarcane events that exhibited a >2-fold reduction in the expression levels of all tested genes by quantitative PCR (qPCR) from the tissue of internode 5 (counting from the top of the plant) of 7-month-old sugarcane plants (Fig. 1b). These RNAi events did not show significant phenotypic differences from the transformation control (TC) at maturity under our greenhouse conditions, including height (Fig. 1c) and diameter (Fig. 1d). TC (Fig. 1e). The total sugar level of RNAi lines was, on average, reduced by 80% relative to the total sugar levels in the TC.