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Exploitable results
a. Somatic hybrid production, characterization and propagation.
A large number of somatic hybrids between Brassica napus and Lesquerella fendleri have been produced. Since the two species represent different tribes within the family of Brassicaceae they cannot be crossed sexually and thus the hybrids produced are unique results in respect of the possibility to combine the two genomes.
Furthermore, the hybrids were fertile, at least male fertile, which means that progeny could be produced from the hybrids and the traits of interest could be followed and investigated more in detail. A detailed presentation of the results and the material is presented in the publication by Skarzinskaya et aL. Theor. Appl. Genet.93:1242-1250, 1996.
b. Cytological characterization of the somatic hybrids including karyotyping and in situ hybridization, GISH.
ln order to analyse and characterize the somatic hybrids cytological construction of the karyotypes of Brassica napus and Lesquerella fendleri were made. By analysing the karyotypes and performing genomic in situ hybridization (GlSH) the chromosome complements of a certain number of selected somatic hybrids were made. Both symmetric and asymmetric hybrids were analysed. From fusions without any pretreatment resulting in symmetric hybrids, hybrid plants containing L. fendleri chromosome in number varying from 2 chromosomes to 2 chromosome complements. While in the asymmetric fusion experiments in which the L.fendleri protoplasts were irradiated before fusion plants with 38 to more than 76 chromosomes were obtained. ln the hybrids with 38 chromosomes, presence of L.fendleri chromosomes was not detected by GISH analysis even though L.fendleri DNA ,was revealed by Southern blotting. Intra- and intergenomic recombinations were more prevalent in plants resulting from asymmetric fusions. Modifications of the phenotype and reduced fertility of the hybrid plants were correlated with increased dosages of the L. fendleri genome. At least one genome complement of L. fendleri was required to express its morphological features. For further details see the Ms of Skarzinskaya et al. 1997 (submitted to Genome).
c. Fatty acid analysis.
Fatty acid analysis have been performed from all hybrid plants that produced seeds after selfing and back-crossing. The seeds have been analysed with gas chromatography and HPLC and the total fatty acid pattern has been modified in some of the progeny in which an increase in the content of erucic acid has been found. Since the rapeseed cultivar used in the sommatic hybridization experiments is a double low variety of rapeseed containing no erucic acid the hypothesis is that the gene(s) are derived from Lesquerella fendleri. Further crosses to rapeseed with high content of erucic acid has been performed to investigate whether the content could be increased. So far, the level has reached about 60% which most probably is the optimal level possible to obtain in rapeseed. Genetic and molecular analysis are performed to determine the number of loci involved regulating the production of erucic acid. By this investigation it will be possible to establish whether the genes are derived from L.fendleri and whether the loci are non-alellic. From one of the hybrid lines a progeny was obtained exhibiting a low amount of the hydroxy-fatty acid. Further analysis of this material and the progeny obtained from that line has been performed, but the seeds tested so far have not revealed presence of the hydroxy-fatty acid. Thus, the modification does not seem to be stably inherited. Molecular investigations to follow presence of the hydroxy-fatty acid coding gene will be performed to confirm whether this is present in the material or not. These analysis are performed in the laboratory by one of our PhD students Mia Schröder-Pontoppidan. Results of the analysis of the fatty acid composition in the hybrid plants and their progeny will be reported later (manuscript in preparation).
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