Direct Gene Transfer to Plants

Summary

Gene transfer by biological vectors is limited by the restricted host range of a given vector. We have developed and exploited a vector-independent method for gene transfer. Incubation of protoplasts with genes under the control of plant gene expression signals leads to high frequencies of stable integrative transformation. The foreign gene is transmitted to sexual offspring and is inherited according to Mendelian laws. The integration of the foreign gene occurs at random sites; in more than 77% of primary transgenic plants studied this was at one locus, but integration at two, three and more independent or linked loci has also been found, as well as cases indicating maternal inheritance. In the majority of transgenic plants analysed, the foreign gene is absolutely stable; so far over eight sexual generations and for nearly three years without any selective pressure. However, transgenic plants expressing various degrees of instability or loss of the foreign gene have also been found. Transformation frequency can reach values of up to 10% of non-selected protoplast-derived clones, independent of which of two optimized methods is used. One is a combination of electroporation and polyethylene glycol treatment, the other a sequential treatment with magnesium ions and polyethylene glycol. The transformation frequency is, however, also species and genotype dependent: the high levels achieved with Nicotiana tabacum SR1 were never reached with N. plumbaginifolia or Petunia hybrida. Direct gene transfer is, apparently, possible with protoplasts from any plant species. Treatment with mixtures of selectable and non-selectable genes led to co-transformation rates of up to 88%. Treatment of protoplasts with sheared or partially digested total genomic DNA from a plant carrying one copy of a dominant, selectable marker gene led to the transfer, integration and expression of this gene. In situ hybridization of radioactively labelled probes of the foreign gene to metaphase chromosomes could be used to visualize the location of the gene. 5′ and 3′ deletions of a selectable gene with overlapping stretches of homology have been used to study homologous recombination within plant cells. Stable integration of non-functional 5′ deletions of the same gene into the host genome and subsequent transformation with complementing 3′ deletions were used to demonstrate gene targeting in plants. Microinjection of a marker gene into microspore-derived proembryos produced transgenic plants in Brassica napus.