Transient and stable transformation in mango by particle bombardment
1999
Botella, J.R. | Cavallaro, A.S. | Town, L. | Cruz-Hernandez, A.(University of Queensland, Brisbane (Australia). Dept. of Botany. Plant Genetic Engineering Laboratory)
Genetic manipulation of plants requires a system for the introduction of foreign DNA into the cells. Particle bombardment offers a rapid and efficient method for the delivery of DNA for transient and stable transformation studies. In this work we describe a system for the transfer of DNA using mango proembryogenic masses as a target tissue for particle bombarment. The kanamycin resistance gene (npt II) was used as a selectable marker and the B-glucuronidase (gus A) and the green fluorescent protein (gfp) as reporter genes. The parameters associated with particle bombardment were optimized using transient expression assays of the B-glucuronidase gene in proembryogenic masses. Osmotical treatment, distance and particle acceleration pressure had a major effect on GUS transitory expression. Optimally, more than 1000 GUS loci were observed per microgram of bombarded DNA. Stable transformation assays of proembryogenic tissue were initiated using the green fluorescent protein as the reporter gene. The bombarded proembryogenic masses were transferred to selection media containing 400 mg/l kanamycin and the progress of selection was monitored using an inverted fluorescence microscope. The implications of the system for transformation and regeneration of embryogenic tissue will be discussed.
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