OPEN ACCESS ARTICLE

This Article Free via Open Access: OA OA Full Text Full Text (PDF) Supplemental Data OA All Versions of this Article: 151/3/1077    most recent pp.109.142919v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via CrossRef Google Scholar Articles by Rogers, C. Articles by Oldroyd, G. PubMed PubMed Citation Articles by Rogers, C. Articles by Oldroyd, G. Agricola Articles by Rogers, C. Articles by Oldroyd, G.

BREAKTHROUGH TECHNOLOGIES - FOCUS ISSUE

Deletion-Based Reverse Genetics in Medicago truncatula^1,[W],[OA]

Department of Disease and Stress Biology, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom (C.R., G.O.); and Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73401 (J.W., R.C.)

The primary goal of reverse genetics, the identification of null mutations in targeted genes, is achieved through screening large populations of randomly mutagenized plants. T-DNA and transposon-based mutagenesis has been widely employed but is limited to species in which transformation and tissue culture are efficient. In other species, TILLING (for Targeting Induced Local Lesions IN Genomes), based on chemical mutagenesis, has provided an efficient method for the identification of single base pair mutations, only 5% of which will be null mutations. Furthermore, the efficiency of inducing point mutations, like insertion-based mutations, is dependent on target size. Here, we describe an alternative reverse genetic strategy based on physically induced genomic deletions that, independent of target size, exclusively recovers knockout mutants. Deletion TILLING (De-TILLING) employs fast neutron mutagenesis and a sensitive polymerase chain reaction-based detection. A population of 156,000 Medicago truncatula plants has been structured as 13 towers each representing 12,000 M2 plants. The De-TILLING strategy allows a single tower to be screened using just four polymerase chain reaction reactions. Dual screening and three-dimensional pooling allows efficient location of mutants from within the towers. With this method, we have demonstrated the detection of mutants from this population at a rate of 29% using five targets per gene. This De-TILLING reverse genetic strategy is independent of tissue culture and efficient plant transformation and therefore applicable to any plant species. De-TILLING mutants offer advantages for crop improvement as they possess relatively few background mutations and no exogenous DNA.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Christian Rogers (christian.rogers{at}bbsrc.ac.uk).

^[W] The online version of this article contains Web-only data.

^[OA] Open Access articles can be viewed online without a subscription.

www.plantphysiol.org/cgi/doi/10.1104/pp.109.142919

^* Corresponding author; e-mail christian.rogers{at}bbsrc.ac.uk.

Received June 19, 2009; accepted September 15, 2009; published September 16, 2009.