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Only a few transcription factors (TFs) regulating which cells of the ovule epidermis differentiate into lint fibres have been identified in cotton (Gossypium hirsutum L.). In this study, the effect on lint yield and fibre quality of over-expressing three TFs in cotton, GhHD-1, GhMYB25 and GhMYB25Like, and their double and triple combinations, were evaluated in field experiments over two seasons. The expression of single or stacked TFs were all driven either by an ovule-specific promoter, FBP 7, or a constitutive promoter, Stunt 7, in a Coker 315 background. TF type, either singly or in combination, was found to be the most significant factor affecting lint yield. Among 64 transgenic lines tested, seven were higher yielding than null segregant lines in one or both seasons and were all from the sets with single and double over-expressed TF combinations. A reduced yield was associated with the set of triple combinations. The two most stable high yielding lines across the seasons recorded 12–22% higher yields than the nulls, although were not competitive to locally adapted commercial controls. Over-expression of TFs singly or in combination did not significantly alter fibre length and strength, but sometimes increased fibre micronaire. There were positive relationships between lint yield and lint percentage and lint yield and fibre density amongst the transgenic lines. Our preliminary results suggest that manipulating TF expression, either singly or in pairs, can increase the density of fibres initiated on developing seeds and fibre yields under field conditions while maintaining overall fibre quality. [ABSTRACT FROM AUTHOR]