Beyond Overexpression: A Critical Appraisal of RNAi, VIGS and CRISPR/Cas Approaches for Abiotic-Stress Resilience in Upland Cotton

Abstract

Drought, salinity and temperature extremes constrain the productivity of upland cotton (Gossypium hirsutum L.), and four molecular toolkits are now used to address them: gain-of-function overexpression, stable RNA interference, virus-induced gene silencing and CRISPR/Cas genome editing. This review re-examines what each toolkit has actually established in cotton, rather than what it is expected to deliver. Published evidence was classified by host species, experimental scale and validation method, from heterologous model systems through controlled-environment assays to multi-location field trials, and the scheme was applied to every example discussed. On that basis, overexpression emerges as an efficient discovery tool whose reported yield penalties are conditional on gene identity, promoter, expression level, developmental stage and genetic background rather than intrinsic to the approach; RNA interference provides stable, heritable knockdown and has produced the clearest field-validated cotton example of simultaneous gains in fibre quality and yield; virus-induced gene silencing is a rapid prioritization tool whose transient, partial and tissue-dependent silencing does not predict the phenotype of a stable knockout; and CRISPR/Cas multiplexing permits dissection of duplicated homologues but does not by itself deliver complete functional knockouts, negligible off-target risk or transgene-free germplasm. We then set out an explicit decision framework that specifies which tool suits which gene class, regulatory sign and stress type, together with validation checkpoints, known bottlenecks and falsifiable predictions. The sequential pipeline it describes remains a proposal: no published study has yet executed the full progression within one genetic background, and multi-environment field testing remains the decisive missing evidence.