Bachelor project in the Horticultural Science programme 2007:6, 10 p (15 ECTS) Evaluation of the dao1 gene as a selectable marker in transformation of apple rootstock M26 as well as transformation of a vector containing GA20 oxidase gene into Agrobacterium Utvärdering av dao1 genen som en selektionsmarkör vid transformering av äpplegrundstammen M26 samt transformering av en vektor innehållande GA20 oxidasgen till Agrobacterium By Sara Larsson Fakulteten för landskapsplanering, trädgårds- och jordbruksvetenskap SLU-Alnarp Evaluation of the dao1 gene as a selectable marker in transformation of apple rootstock M26 as well as transformation of a vector containing GA20 oxidase gene into Agrobacterium Utvärdering av dao1 genen som en selektionsmarkör vid transformering av äpplegrundstammen M26 samt transformering av en vektor innehållande GA20 oxidasgen till Agrobacterium By Sara Larsson Handledare: Docent Li-Hua Zhu Växtförädling och bioteknik, LTJ, SLU 2 SAMMANFATTING ................................................................................................................................... 4 ABSTRACT .................................................................................................................................................. 5 1. INTRODUCTION .................................................................................................................................... 6 1.1 BACKGROUND ...................................................................................................................................... 6 1.1.1 Tissue culture ............................................................................................................................... 6 1.1.2 Gene transfer strategies for plants ............................................................................................... 7 1.1.2.1 Indirect gene transfer through Agrobacterium ......................................................................... 7 1.1.2.1.1 Binary vectors ........................................................................................................................ 8 1.1.2.2 Direct gene transfer .................................................................................................................. 8 1.1.2.2.1 Microinjection ........................................................................................................................ 8 1.1.2.2.2 Particle Bombardment ........................................................................................................... 9 1.1.2.2.3 Electroporation ...................................................................................................................... 9 1.1.3 Selectable markers ....................................................................................................................... 9 1.1.3.1 Selection with antibiotics .......................................................................................................... 9 1.1.3.1.1 Aminoglycoside-modifying enzymes ..................................................................................... 10 1.1.3.1.2 Aminoglyside-O-phosphotransferases ................................................................................. 10 1.1.3.1.3 Aminoglycoside-N-acetyltransferases .................................................................................. 10 1.1.3.1.4 Aminoglycoside-O-nucleotidyltransferases.......................................................................... 10 1.1.3.2 Selection with herbicides ........................................................................................................ 11 1.1.3.2.1 Phosphinothicin ................................................................................................................... 11 1.1.3.2.2 Glyphosate ........................................................................................................................... 11 1.1.3.2.3 Oxynil herbicides ................................................................................................................. 11 1.1.3.2.4 Gabaculine ........................................................................................................................... 12 1.1.3.2.5 Cyanamide ........................................................................................................................... 12 1.1.3.3 Selection using inhibitors in metabolic pathways ................................................................... 12 1.1.3.3.1 2-Deoxyglucose .................................................................................................................... 12 1.1.3.3.2 Inhibitors of the aspartate pathway ..................................................................................... 12 1.1.3.3.3 Methotrexate and trimethoprim ........................................................................................... 13 1.1.3.3.4 4-methyltryptophan (4-mT) .................................................................................................. 13 1.1.3.4 Selection using non-toxic metabolic intermediates ................................................................. 13 1.1.3.4.1 Xylose and mannose ............................................................................................................. 13 1.1.3.4.2 Glucuronide derivate of benzyladenine ................................................................................ 14 1.1.3.5 Selection using gene expression for enhanced cytokinin levels .............................................. 14 1.1.3.6 Selection using D-amino acids ................................................................................................ 14 1.2 OBJECTIVE .......................................................................................................................................... 15 2. MATERIAL AND METHODS ............................................................................................................. 15 2.1 TRANSFORMATION OF M26 WITH DAO1 GENE .................................................................................... 15 2.1.1 Plant material ............................................................................................................................ 15 2.1.2 Agrobacterium strain and binary vector .................................................................................... 15 2.1.3 Transformation .......................................................................................................................... 15 2.1.4 DNA isolation and PCR analysis ............................................................................................... 16 2.2 TRANSFORMATION OF GA20 OXIDASE TO AGROBACTERIUM ............................................................. 16 2.2.1 Competent cell ........................................................................................................................... 16 2.2.2 Transformation .......................................................................................................................... 17 2.2.3 PCR analysis and DNA digestion .............................................................................................. 17 3. RESULTS ................................................................................................................................................ 17 3.1 TRANSFORMATION OF M26 WITH THE DAO1 GENE ............................................................................. 17 3.2 TRANSFORMATION OF GA20 OXIDASE TO AGROBACTERIUM ............................................................. 17 4. DISCUSSION .......................................................................................................................................... 18 3 Sammanfatting Syftet med detta projekt var att undersöka möjligheten att vid transformering av äpplegrundstammen M26 genomföra selektion med hjälp av D-aminosyra (D-alanin) istället för andra vanligt förekommande selektionsämnen, så som antibiotika och herbicider. Transformeringar med D-alanin som selektionsämne genomfördes, med Agrobacterium tumefaciens stammen C58C1(pGV3101) innehållande vektorn pPCV70:dao1. Vektorn innehöll även genen nptII, som ger de transformerade explantaten resistans mot antibiotika, kanamycin. dao1 genen kodar för enzymet D-amino-acid oxidase (DAAO). Enzymet bryter ner det giftiga ämnet D-alanin till ofarliga ämnen för växten. De transformerade explantaten odlades på medium innehållande ökande koncentration av D-alanin, med utgångskoncentrationen 1.2 mM. Åtta putativa transformerade kloner selekterades på selektionsmediet. Ytterligare molekylära analyser måste dock genomföras för att bekräfta resultaten. Försök genomfördes även för att transformera en vektor innehållande GA20 oxidas genen i A. tumefaciens, för att få fram en vektor färdig för användning vid transformering av växter. Vektorn innehållande GA20 oxidas genen och nptII genen transformerades in i A. tumefaciens stammen C58C1(pGV3850). DNA från A. tumefaciens var sedan isolerad och fragmenterad. Resultatet visualiserades på en agarose gel under UV ljus. Slutsatsen av försöket är att vektorn transformerats in i A. tumefaciens stammen C58C1. 4 Abstract The aim of this project was to evaluate the possibility to use D-alanine as a selective marker instead of commonly used antibiotics and herbicides in transformation of apple rootstock M26. The transformation was carried out using Agrobacterium tumefaciens strain C58C1(pGV3101) containing the vector