Agrobacterim-mediated transformation (Kolitha Wijesekara)

Agrobacterium mediated transformation of Brassica napus is a routine method in many laboratories. However, the use of diploid explants (hypocotyls or cotyledonary segments) obtained from in vitro germinated seedlings has some disadvantages. The seeds used may not be completely homozygous. This may contribute to differences found among individual first generation transgenic plants (T1-plants). The regenerated transgenic T1-plants are hemizygous for all transgene loci. Transgene copy number determination by Southern Blot is not always giving reliable results and segregration in the first seed generation may be complex (T2-seeds). Phenotypical results observed in segregating T2-plants may be confounded by effects caused by using not completely homozygous seed. Surprisingly only few researchers have used seeds derived from doubled haploid lines.
The performed research work concentrates on the development of a Agrobacterium mediated transformation protocol using explants (leaf and petiole segments) taken from in vitro propagated haploid plants. The haploid plants used were obtained via microspore culture from the spring cultivar Drakkar and are maintained and propagated in vitro. Within the research work the following conditions are optimised: