**Background:** Wheat (Triticum aestivum L.) is a staple food for over 35% of the world's population, but its production is constrained by biotic stresses, particularly the grain aphid Sitobion avenae, which causes severe yield losses through direct feeding and virus transmission. Chemical control is deleterious to the environment, and transgenic insecticidal approaches face tolerance issues. RNA interference (RNAi) offers an alternative by silencing essential insect genes. Laccase (lac), a multi-copper oxidase found in aphid salivary glands, is involved in plant defense breakdown, cuticle hardening, and insecticide resistance, making it a promising RNAi target. This study aimed to evaluate the efficacy of the S. avenae laccase (Salac1) gene as an RNAi target and to develop transgenic wheat cultivars resistant to grain aphids.
**Methods:** S. avenae aphids were collected from wheat fields in Gujrat, Pakistan, and reared on T. aestivum cv. Faisalabad 2008 under controlled conditions (16h light/8h dark at 22°C, 70% relative humidity). Total RNA was extracted from 3–5-day-old adult aphids, and cDNA was synthesized. The lac gene was amplified by RT-PCR using primers designed with Primer-BLAST, producing a 615 bp fragment. The PCR product was cloned into pTZ57R/T plasmid and transformed into E. coli DH5α. The plasmid was sequenced (GenBank accession ON703252), and phylogenetic analysis was performed using phylogeny.fr. The ERNAi tool (version 3.2) was used to design 143 siRNAs and one dsRNA target against the lac1 mRNA sequence with Acyrthosiphon pisum as reference. For dsRNA feeding assays, dsRNA was produced using the MEGAscript RNAi kit, and aphids were fed an artificial diet of 20% sucrose (pH 7.2) containing 20 ng μL⁻¹ dsRNA. Mortality was recorded from 1 to 8 days post-feeding (n=25 per treatment, p<0.05). qRT-PCR was performed using SYBR Green to measure lac expression relative to actin (primer efficiency 109.5%). For spray application, the second leaves of 3-week-old wheat plants were detached and sprayed with 20 ng μL⁻¹ dsRNA. Systemic movement was assessed by measuring lac expression in the first leaf, second leaf, shoot, and root at 9, 11, and 13 days post-spray. The ihpRNA construct was assembled using the Golden Gate (GG) cloning strategy with the pRNAi-GG vector (15,796 bp), which uses BsaI restriction and T4 DNA ligase in a single reaction. The construct was transformed into A. tumefaciens LBA4404. Both in vitro (callus-based) and in planta transformation methods were used for three wheat cultivars: Anaj 2017, Galaxy 2012, and Punjab. Acetosyringone concentrations (50–300 μM) and co-cultivation time (3h) were optimized. Transgenic plants were selected on hygromycin (50 mg L⁻¹) and confirmed by PCR and qRT-PCR. Insect bioassays were conducted by rearing aphids on transgenic and non-transgenic plants, including resistant cv. Zincol 2016 as positive control.
**Key Results:** The lac gene sequence (ON703252) showed 90% homology with Myzus persicae, A. pisum, and Diuraphis noxia. The lac protein domain was similar to copper oxidase copper-binding domains found in Aphis craccivora, Tribolium castaneum, and other species. In the dsRNA artificial feeding assay, 69% aphid mortality was observed 8 days post-feeding with lac dsRNA compared to controls (p<0.05). qRT-PCR revealed a 61% reduction in lac expression 8 days post-feeding relative to the dsGFP control. In the spray application experiment, systemic movement of dsRNA was confirmed: relative lac expression was highest in roots (60%), followed by shoots (40%), and lowest in the second leaf (20%) at 9–13 days post-spray compared to controls. For in vitro transformation, maximum transformation efficiency was achieved with 250 μM acetosyringone and 3h co-cultivation. Galaxy 2012 showed the highest in vitro efficiency (1.5%), followed by Anaj 2017 (0.8%) and Punjab (0.2%). In planta transformation yielded substantially higher efficiencies: Galaxy 2012 (16%), Anaj 2017 (10%), and Punjab (5%). Insect bioassays on transgenic T1 plants showed the lowest aphid survival on Galaxy 2012 (22.6%), followed by Punjab (37.6%) and Anaj 2017 (52%), compared to the resistant control Zincol 2016 (20%). qRT-PCR confirmed lac expression in transgenic plants: Galaxy 2012 (70%), Punjab (50%), and Anaj 2017 (30%), with no expression in non-transgenic controls. Actin (internal control) showed 100% expression across all cultivars.
**Clinical Implications:** This study demonstrates that the laccase gene is an effective RNAi target for controlling S. avenae in wheat, with dsRNA feeding achieving 69% mortality and 61% gene silencing. The systemic movement of sprayed dsRNA from leaves to roots supports its potential as a bio-pesticide for field application. The Golden Gate cloning strategy simplified the construction of ihpRNA vectors, and in planta transformation proved more efficient (5–16%) than in vitro methods (0.2–1.5%). Transgenic wheat cv. Galaxy 2012 showed aphid mortality comparable to the resistant check Zincol 2016, indicating that Salac1 silencing confers substantial resistance. These findings provide a practical RNAi-based approach for developing aphid-resistant wheat cultivars, reducing reliance on chemical insecticides. However, the mechanism of dsRNA movement across the apoplastic-symplastic barrier and the full role of laccase in insect immunity require further investigation.