Abstract
BackgroundGenetically 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.ResultsWe 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.ConclusionsThe 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.
Highlights
Modified cotton accounts for 64% of the world’s cotton growing area (22.3 million hec‐ tares)
We describe the development of a transient expression system for cotton, and used it to analyze the localization and expression patterns of two previously characterized members of the glutathione peroxidase (GPX) gene family, GhGPX1 and GhGPX8 [21]
Plasmid construction To study the subcellular localization of GhGPX1 and GhGPX8, their coding sequences (CDS) were ligated in frame with the green fluorescent protein (GFP) coding region in the p35S-GFP vector, to construct the recombinant plasmid p35S-GhGPX1/8-GFP
Summary
Modified cotton accounts for 64% of the world’s cotton growing area (22.3 million hec‐ tares). 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. Cotton (Gossypium spp.) is a very important economic crop, providing the basic resource for thousands of consumer and industrial products worldwide and its contribution to the fiber and food industries continues to grow in importance. It is important to improve the agronomic performance of cotton, and especially to enhance the insect and disease resistance of cotton plants as well as cotton fiber quality and yield. The development of transgenic techniques for cotton in the last decade has contributed to improvements in insect resistance, fiber quality and yield [3, 4]. Transient expression systems have inherent limitations, they can have important applications for gene functional studies as well as the rapid and scalable production of recombinant proteins in plants
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