INVESTIGADORES
PIECKENSTAIN Fernando Luis
congresos y reuniones científicas
Título:
A time and space-saving protocol to identify arabidopsis T-DNA null mutants
Autor/es:
CAMPODÓNICO MD; UCHIYA PA; PIECKENSTAIN FL; ) GONZALEZ ME, F MARCO, EG MINGUET, P CARRASCO, J CARBONELL, MA BLÁZQUEZ, OA RUIZ, FL PIECKENSTAIN
Reunión:
Congreso; L Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2014
Resumen:
Arabidopsis is widely used as a model organism in plant biology. Its genome can be easily modified by transformation with Agrobacterium tumefaciens and a large number of mutant lines are thus available from Stock Centers. Working with mutant lines of interest requires the identification of homozygous individual plants from seed-pools that might also contain WT and heterozygous plants. Standard protocols use purified nucleic acids from four-week-old plants to confirm T-DNA insertion in both alleles and the absence of gene transcript. The aim of this work was to develop a fast and efficient protocol to identify T-DNA null mutants. In order to achieve this goal we performed an alkali treatment on a small piece of a leaf from a two-week-old plant to use it as template in tissue PCRs to identify homozygous plants for T-DNA insertion. In this way the screening time was shortened in two weeks and less space was required in growing chambers because only those plants carrying the T-DNA in both alleles were selected and continue to grow. Finally, in order to confirm the absence of the transcript of interest, four-week-old plants were analyzed. This novel protocol provides improved management of time, space; and even money; to work with genetically modified Arabidopsis lines. Identification and characterization of the Arabidopsis polyamineoxidase 3 T-DNA null mutant is presented as an example