Transformation of E. coli and Agrobacterium and molecular analysis of transformed apple (Malus domestica) rootstock A2 :

Abstract: This project includes two parts. The first part was transformation of Escherichia coli and Agrobacterium tumefaciens with a vector containing the GA2ox gene. The second part was molecular analyses of the apple (Malus domestica) rootstock A2 transformed with the rolC gene. The rolC and GA2ox genes are two potential genes for dwarfing plants. E. coli was transformed with a binary vector, which was supposed to contain the GA2ox and nptII genes. Transformation events were confirmed by restriction enzyme digestion and by PCR analysis, which led to the discovery that the GA2ox gene was not present in the original vector but the gus gene. A. tumefaciens was later transformed with the vector, isolated from the above transformed E. coli. Transformation of A. tumefaciens could not be confirmed by PCR analysis, even though the colonies grew well on the selectable medium containing kanamycin. In the second part of the project Southern blot was conducted to further confirm integration of the rolC and nptII genes in the transgenic clones of the apple rootstock A2 and to determine the copy number of the transgenes. The Southern results confirmed that six clones contained the nptII and rolC genes. Two of the transformed clones have a single copy of the T-DNA integrated.