A selection procedure for identifying transgenic cells and embryos of cotton without the use of antibiotics

Abstract

Transgenic cells containing inserted antibiotic resistance genes and linked genes of interest are routinely selected by exposure to antibiotics. Concerns over the widespread use of antibiotic resistance genes as selectable markers for genetic transformation have motivated researchers to find alternative selection procedures. This study describes the evaluation of an alternative protocol using temperature as the selection tool. In this method, a population of host cells is transformed with a foreign DNA construct that includes at least one gene of interest and an additional sequence encoding a protein that enhances cellular high temperature tolerance. Following transformation, the population of cells is transiently cultured under temperature conditions wherein growth of non-transformed cells is suppressed or prevented, while growth of cells containing the DNA construct continues. Thus, survival and/or significant additional growth is an indication that a cell has been successfully transformed with the DNA construct and can be subsequently recovered for further growth and development. The present study used a heat shock protein (hsp101) gene from Arabidopsis thaliana under the control of a constitutive promoter as a selectable marker; however, alternative potentially suitable genes include: other heat shock proteins; heat shock transcription factors; cold regulated proteins (COR); or protein transcription factors associated with the induction of cold tolerance.