ISHS Acta Horticulturae 508: XIX International Symposium on Improvement of Ornamental Plants
TRANSFORMATION OF GLADIOLI FOR FUSARIUM RESISTANCE |
| Authors: | H.J.M. Löffler, J.R. Mouris, M.J. van Harmelen, J.M. van Tuyl |
| Abstract:
Fusarium oxysporum f.sp. gladioli is a major pathogen of Gladiolus in many countries of the world among which The Netherlands. Resistance to this fungus is needed but is not present in commercial gladioli. Therefore breeding efforts are necessary, which are undertaken at CPRO-DLO. Two approaches were chooses: cross breeding and molecular breeding. With cross breeding, new varieties were developed with high resistance to the fungus (Löffler et al., 1997; Straathof et al., 1997; Straathof et al., 1998). The disadvantage of this method, however, is that by crossing all good parental characteristics segregate in the progeny. So, only new, resistant varieties can be developed. Molecular breeding will allow the further improvement of existing excellent varieties. Therefore research now focuses on molecular breeding. To do so, a transformation is needed. A method based on that that of Kamo (1995) was used. Friable callus was induced on slices of in-vitro grown corms and used to initiate an embryogenic callus culture (Remotti, 1995; Remotti and Löffler, 1995). Embryogenic cells were subjected to particle bombardment with gold particles coated with a construct harbouring the gus reporter gene and the pat selection gene. The latter confers resistance to the herbicide Basta. Several promoters and a the presence of maize-intron were tested for optimal transient expression of the gus gene. The dicotyledon promoter 35S performed better than the monocotyledon promoters ubi or act. These results confirm those of Kamo (1995), suggesting that in this respect gladiolus acts as a dicotyledon rather than a monocotyledon plant. For further research, the 35S promoter was used. One week after treatment, embryogenic cells were plated on a medium with 10 mg/l Basta. Surviving cells were transferred to callus induction medium and subsequently to regeneration medium. Most regenerated shoots were found to express Gus. PCR analysis showed that both genes were present in the 4 analysed shoots. Southern blots showed the integration of both genes in the genome of the plant. An exact copy number is not yet established and is topic of current analyses. In the future, transformed plants will be studied in the greenhouse and inheritance of the transgenic traits will be studied. Furthermore, target genes will be introduced. To this goal, various proteins were tested for in-vitro anti-fungal activity. Two proteins with high toxicity for the fungus were selected for further study. Both proteins are counteracted by bivalent cations, suggesting that the mode of action is based on inducing ion channels in the membrane of the fungus. This sensitivity of the protein for cations may interfere with the in-vivo activity of those proteins. De-facto introduction of the encoding genes in our gladioli will answer this question. | |
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