Studies on plant gene transfer systems

Abstract

A number of methods for the transfer of genes to plants are assessed in this work. The potential of Agrobacterium tumefaciens infection of germinating pollen tubes in vitro as a method of gene transfer was investigated and evidence presented for an essential pre-requisite of infection, that of attachment of Agrobacteria to the pollen tube wall, with both a dicot and a monocot species. In addition, the possibility of direct uptake of DNA molecules by germinating pollen tubes was suggested by in vitro uptake studies. Microinjection of DNA molecules into the loculus of ovaries, with the aim of facilitating in vivo DNA uptake by the male and/or female gametes, was investigated with Salpiglossis sinuata ovaries. Evidence was presented for the persistence of DNA molecules in the ovary loculus and gene transfer using a non-oncogenic Agrobacterium plasmid vector was attempted and the resulting progeny were screened for transformation: The techniques of Agrobacterium-infection of leaf discs and direct DNA uptake by protoplasts were applied to Nicotiana tabacum. DNA transformation vectors containing a kanamycin resistance marker gene and a chimeric pea seed storage protein gene were constructed for use in this study. The seed-specific promoter of the pea legumin A gene was replaced with the nopaline synthase promoter that is expressed in a more constitutive manner in plant tissues. Kanamycin resistant transgenic plants were regenerated following both transformation techniques and the presence and structure of inserted foreign DNA was determined by Southern blot hybridizations. The transmission of kanamycin resistance to transformant progeny after self-fertilization demonstrated characteristic Mendelian inheritance. The expression of the inserted legumin gene in leaf tissue of a number of Agrobacterium-denved transformants was assessed by Northern blot hybridization; legumin transcripts were detected, although the protein immuno-detection procedures of Western blotting and ELISA did not detect legumin in the seeds or leaf tissue of transgenic plants derived from either transformation technique.