Working group session:
Functional Genomics
Presentation type:
oral
Authors:
Mansoor, Shahid; Mansoor, Shahid
Presenter:
Mansoor, Shahid; Mansoor, Shahid
Correspondent:
Abstract:
Cotton (Gossypium spp.) is one of the most economically important crops of the world which has unique qualities. It is used in the production of textile fiber and also serves as food and feed stock. There are more than 50 species of cotton, of which four are cultivated for commercial purposes; G. hirsutum (more than 90% of world production), G. barbadense (3-4%), G. herbaceumand G. arboreum (collectively 2%). The study described here reports the optimization and use of an Agrobacterium-mediated, virus-induced gene silencing (VIGS) system in three cultivated cotton species (G. hirsutum, G. arboreum and G. herbaceum) using a Tobacco rattle virus (TRV) vector for transient silencing of an endogenous gene. GrCLA1 is a homolog of the Arabidopsis thaliana chloroplast alterados 1 (AtCLA1) gene, and is involved in chloroplast development.The GrCLA1 gene serves as a marker in this study; silencing of this gene produced a foliar albino or white phenotype within 10 to 15 day after inoculation. Three independent biological replicates were used in case of G. hirsutum (cultivarsIR3701, NIBGE2, 777). Each replicate consisted of 20 plants inoculated with control (TRV without insert) and 20 plants with TRV-GrCLA1. Plants were kept at (28+ 2 oC). All plants inoculated with TRV-GrCLA1 showed typical the albino phenotype indicating 100% efficiency of silencing. Two independent replicate were done for G. herbeceum and G. arboreum with same results and efficiencies. RT-PCR was conducted using specific primers for the GrCLA1 gene while the 18S RNA gene was used as an internal control. RT-PCR analyses showed presence of GrCLA1 transcripts in control and healthy cotton plants. No amplification was achieved from plants expressing the albino phenotype, indicating that the GrCLA1 gene had been silenced by TRV-based VIGS. The study optimized the Agrobacterium-mediated VIGS in three cultivated species of cotton with different ploidy levels; tetraploid (G. hirsutum) and diploid (G. arboreum and G. herbaceum). With the availability of the genome sequence of cotton several species, this rapid and efficient Agrobacterium-mediated VIGS assay has the potential to provide a powerful tool for robust large-scale analysis of gene function in cotton.