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United States Department of Agriculture

Agricultural Research Service

Title: A Selection Procedure for Identifying Transgenic Cells and Embryos of Cotton Without the Use of Antibiotics

Authors
item Burke, John
item O'Mahony, Patrick - FSAI
item Oliver, Melvin
item Velten, Jeffrey

Submitted to: In Vitro Plant
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 17, 2008
Publication Date: August 1, 2008
Citation: Burke, J.J., O'Mahony, P., Oliver, M.J., Velten, J.P. 2008. A selection procedure for identifying transgenic cells and embryos of cotton without the use of antibiotics. In Vitro Cellular & Development Biology. 44(4):246-253.

Interpretive Summary: Concerns over the wide-spread use of antibiotic resistance genes as selectable markers for genetic transformation has motivated researchers to find alternative selection procedures. This study describes the evaluation of an alternative procedure that uses temperature as a selection tool. A gene coding for a protein of the naturally occurring heat protection system of plant cells was constitutively expressed in cells. Following transformation, the population of cells was 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 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 results of this study show that naturally occurring heat protection proteins can be used to select plants containing transgenes.

Technical Abstract: Transgenic cells containing inserted antibiotic resistance genes and linked genes of interest are routinely selected by exposure to antibiotics. Concerns over the wide-spread use of antibiotic resistance genes as selectable markers for genetic transformation has motivated researchers to find alternative selection procedures. This study describes the evaluation of an alternative procedure that uses temperature as a selection tool. In this method, a population of host cells are 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 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 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 cold regulated protein transcription factors.

Last Modified: 4/19/2014
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