TRANSFORMATION OF BRASSICA-NAPUS BY USING THE AADA GENE AS SELECTABLEMARKER AND INHERITANCE STUDIES OF THE MARKER GENES

An Agrobacterium tumefaciens-mediated transformation system for Brassi ca napus has been improved. We investigated several marker genes for t ransformation of Brassica napus, and the aadA gene, which confers resi stance to streptomycin and spectinomycin, was found to be the most sui table. Forty-three out of 193 putative transformants in the T-1 genera tion were investigated by Southern blot analysis. Transformants contai ning a range of 1 to 10 integrated T-DNA copies per genome were found. Total DNA from 35 plants showed hybridisation to both the aadA and th e nptII marker gene probes, from 5 plants only to one marker gene prob e and from 3 plants DNA did not hybridise to any of the gene probes. F urthermore, more complex integration patterns such as direct repeated copies of the T-DNA, both as tandem and inverted copies, were observed . Inheritance of the marker genes in the T-2 generation was studied in 37 of the plants. This revealed that 22% of the plants that contained both marker genes, segregated as one single locus (3:1) for both gene s, while 46% of the plants gave a segregation pattern corresponding to one T-DNA locus for at least one of the marker genes. Moreover, these inheritance patterns appeared to be more or less independent of the n umber of genes seen in the Southern blot analysis of the T-1 generatio n. In this study we show that the introduced marker genes are inherite d by the T-2 generation in a less predictable way than was earlier rep orted for B. napus.