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Background  Genetically modified cotton accounts for 64% of the world’s cotton growing area (22.3 million hectares). The genome sequencing of the diploid cotton progenitors  Gossypium raimondii  and  Gossypium arboreum  as well as the cultivated  Gossypium hirsutum  has provided a wealth of genetic information that could be exploited for crop improvement. Unfortunately, gene functional characterization in cotton is lagging behind other economically important crops due to the low efficiency, lengthiness and technical complexity of the available stable transformation methods. We present here a simple, fast and efficient method for the transient transformation of  G .  hirsutum  that can be used for gene characterization studies.    Results  We developed a transient transformation system for gene characterization in upland cotton. Using β-glucuronidase as a reporter for  Agrobacterium -mediated transformation assays, we evaluated multiple transformation parameters such as  Agrobacterium  strain, bacterial density, length of co-cultivation, chemicals and surfactants, which can affect transformation efficiency. After the initial characterization, the  Agrobacterium  EHA105 strain was selected and a number of binary constructs used to perform gene characterization studies. 7-days-old cotton seedlings were co-cultivated with  Agrobacterium  and transient gene expression was observed 5 days after infection of the plants. Transcript levels of two different transgenes under the control of the cauliflower mosaic virus (CaMV) 35S promoter were quantified by real-time reverse transcription PCR (qRT-PCR) showing a 3–10 times increase over the levels observed in non-infected controls. The expression patterns driven by the promoters of two  G .  hirsutum  genes as well as the subcellular localization of their corresponding proteins were studied using the new transient expression system and our observations were consistent with previously published results using  Arabidopsis  as a heterologous system.    Conclusions  The  Agrobacterium -mediated transient transformation method is a fast and easy transient expression system enabling high transient expression and transformation efficiency in upland cotton seedlings. Our method can be used for gene functional studies such as promoter characterization and protein subcellular localization in cotton, obviating the need to perform such studies in a heterologous system such as  Arabidopsis .    Electronic supplementary material  The online version of this article (10.1186/s13007-018-0319-2) contains supplementary material, which is available to authorized users.

A transient transformation system for gene characterization in upland cotton (Gossypium hirsutum)