The cotton bollworm (Helicoverpa armigera, Lepidoptera: Noctuidae) is a globally distributed agricultural pest. When conducting expression analysis of its functional genes, appropriate reference genes should be selected to ensure the reliability of the results. In this study, five algorithms including Delta Ct, GeNorm, Normfinder, BestKeeper, and RefFinder were used to evaluate the expression stability of eleven candidate reference genes under different developmental stages, larval tissues, adult sexes, plant secondary substance stresses, and insecticide treatments in H. armigera. The candidate genes included Actin, Tubulin, EF-1α, RPS3, RPS15, RPL27, RPL32, 28S, GAPDH, SOD, and TRX. The reliability of the recommended reference gene combinations was validated using the growth arrest and DNA-damage-inducible gene 45 (GADD45). The results showed that normalizing relative expression of the target gene with the combination of the two most stable reference genes is recommended. Specifically, the combination of RPS3 + RPL27 is recommended for developmental stage comparisons; RPL32 + RPL27 for larval tissues; RPS3 + RPL27 for adult sex comparisons; GAPDH + RPL32 under tannic acid stress; RPL32 + RPS3 under quercetin stress; RPS15 + RPL32 under 2-tridecanone stress; RPS3 + RPL32 under ZQ-8 stress; RPL27 + TRX following chlorantraniliprole treatment; and RPL27 + RPL32 following indoxacarb treatment. Moreover, larvae exposed to three concentrations of plant secondary substances and to sublethal doses of insecticides exhibited significant upregulation of GADD45: after 4 h of exposure to 1% tannic acid, 0.1% and 1% quercetin, 1% 2-tridecanone, and 0.05% ZQ-8; after 15 h of chlorantraniliprole treatment; and after 24 h of indoxacarb treatment. Thus, GADD45 was overexpressed in response to various plant secondary substances and insecticide treatments, indicating its involvement in the detoxification and metabolism of H. armigera. This study proves to be helpful for selecting reference genes in H. armigera under plant secondary substance and insecticide stress and lays the foundation for further research utilizing GADD45 as a molecular target for pest control.

Eucommia ulmoides, a tree species native to China, holds considerable medicinal, ecological, and industrial importance. However, the absence of an efficient and stable genetic transformation system poses significant challenges to gene function studies and molecular breeding in E. ulmoides. Protoplasts, which lack cell walls, serve as effective receptors for transient transformation and are thus ideal for genetic engineering research. In this study, the optimal conditions for callus induction were identified, and formation of the embryogenic callus was confirmed by histological analysis. Furthermore, we developed an efficient protoplast isolation and PEG-mediated transient transformation system using suitable embryogenic callus as the starting material. Our findings revealed that the optimal medium for inducing embryogenic callus was B5 + 1.5 mg/L 6-BA + 0.5 mg/L NAA + 30 g/L sucrose + 7 g/L agar (pH = 5.8). In this medium, the induction rate of callus achieved 97.50%, and the rate of embryogenic callus formation was 86.30%. For protoplast isolation, the best conditions involved enzymatic digestion with 1.5% cellulase R-10 and 1.0% macerozyme R-10 at an osmotic pressure of 0.6 M for 4 h, resulting in 1.82 × 10⁶ protoplasts/g FW with 91.13% viability. The highest transfection efficiency (53.23%) was attained when protoplasts were cultured with 10 µg of plasmid and 40% PEG4000 for 20 min. This study successfully established a stable and efficient system for protoplast isolation and transient transformation in E. ulmoides, offering technical support for exploring somatic hybridisation and transient gene expression in this species.

Eucommia ulmoides, a tree species native to China, holds considerable medicinal, ecological, and industrial importance. However, the absence of an efficient and stable genetic transformation system poses significant challenges to gene function studies and molecular breeding in E. ulmoides. Protoplasts, which lack cell walls, serve as effective receptors for transient transformation and are thus ideal for genetic engineering research. In this study, we identified the optimal conditions for callus induction using a full-factorial experimental design and accurately identified embryogenic callus through paraffin sectioning. Furthermore, we developed an efficient protoplast isolation and PEG-mediated transient transformation system using embryogenic callus as the starting material. Our findings revealed that the optimal medium for inducing embryogenic callus was B5, supplemented with 1.5 mg/L 6-BA, 0.5 mg/L NAA, 30 g/L sucrose, and 7 g/L agar. This medium achieved a callus induction rate of 97.50% and an embryogenic callus induction rate of 86.30%. For protoplast isolation, the best conditions involved enzymatic digestion with 1.5% cellulase R-10 and 1.0% macerozyme R-10 at an osmotic pressure of 0.6 M for 4 h, resulting in 1.82×10⁶ protoplasts/g FW with 91.13% viability. The highest transfection efficiency (53.23%) was attained when protoplasts were cultured with 10 µg of plasmid and 40% PEG4000 for 20 min. This study successfully established a stable and efficient system for protoplast isolation and transient transformation in E. ulmoides, offering technical support for exploring somatic hybridization and transient gene expression in this species.