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KMID : 0578319950050060658
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Molecules and Cells
1995 Volume.5 No. 6 p.658 ~ p.667
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Transformation of Plant Pathogenic Fungus, fusarium Ooxysporum f. sp. Niverun, to Hygromycin B Resistance and Altered Pathogenicity
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Kim, Dae-Hyuk
Magil, Clint W./Martyn, Raymond D.
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Abstract
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Fusarium oxysporum f. sp. niveum (FON) race2 isolate was transformed to a hygromycin B (Hyg B)-resistant using two heterologous vectors, pDH25 and CosHyg1. Five and 15 transformed protoplasts, respectively, were obtained per μg of pDH25 and CosHyg1. However, 1-10% of them lost resistance during alternation transfers on Hyg B^(+) and Hyg B^(-) media. Five pDH25-mediated transformants and twelve CosHyg1-mediated transformants were assayed for stability of Hyg B resistance through conidiation. All but two produced Hyg B resistant-microconidia consistently. Of four transformants examined by Southern hybridization, none contained the autonomous replicating vectors. Southern blot analysis showed that multiple integration of vectors occurred. However, no change in pathogenicity was observed among four transformants tested. A genomic library of FL-60-3A race 0 isolate was constructed in CosHyg1 with the average size of 40 kb insert. 960 recombinant clones were hybridized to [^(32) P]-labeled total DNA probe from FL-60-3A race 0 and the TX-XID race 2 isolates. Nineteen FL-60-3A isolatespecific clones and nine repetitive clones were selected by differential hybridization with total DNA probe and used to transform the TX-XID race 2 isolate. Transformation efficiency was 49.4/㎍ DNA. Nineteen transformants representing 11 specific clones and 7 repetitive clones were bioassayed on watermelon differential cultivars and three transformants showed the difference in pathogenicity. Probe and in situ digestion analysis of recombinant clone-mediated transformants indicated that all of them were integrative transformants and had multiple integration of the vectors. They severe rearrangement of vectors after integration due to a large homologous area represented in the insert.
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