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Agrobacterium-mediated transformation of Blakeslea

A transformation protocol allowing the generation of hygromycin-resistant mycelia of Blakeslea trispora has been developed based in the use of Agrobacterium tumefaciens containing a binary vector. Among five different Agrobacterium strains checked, best growth was exhibited by SK502 and GV2260. The first one was chosen for the transformation experiments.

Samples of 10e5, 10e6 and 10e7 fresh spores from Blakeslea in 100 µl were mixed with 100µl of the Agrobacterium culture previously treated with 4'-hydroxy-3,5-dimethoxyacetophenone

(acetosyringone). The strains SK561, SK562 and SK563 were obtained from SK502 upon introduction of the plasmid pBHT2, containing the HygR gene (strain SK561), or with derivatives of this plasmid containing the gene pyrG from either Phycomyces blakesleeanus (plasmid pIP1, strain S562) or from Mucor circinelloides (plasmid pIM1, strain SK563). Different Blakeslea wild type strains were used. Samples of the Blakeslea/Agrobacterium mixtures, previously incubated up to three days at temperatures from 22°C to 28°C on a medium containing acetosyringone, were transferred to selection plates supplemented with 0.5mM cefotaxime as an Agrobacterium-specific antibiotic, and 200mg/l hygromycin to select transformation.

The medium also contained 2% DMSO to increase hygromycin sensitivity. Incubations were done on cellophane or filter paper laying on the agar medium.
Putative transformants were obtained from strains F986 and CBS198.90, but no mycelia growing under selective conditions were obtained with other strains. Primary transformants were able to grow when transferred to the hygromycin/DMSO supplemented medium.

These putative transformants were able to grow upon a new transfer to fresh selective medium, although some of them exhibited a slower growth rate. The borders of the colonies were more irregular than control plates without selection, indicating either a lack of stability or segregation derived from heterokaryosis. Those with the best growth were selected for DNA extraction and demonstration of the presence of the hygR gene. These putative transformants retained the ability to grow in the presence of hygromycin and southern blot analysis with the hygR probe showed the presence in at least two of them of a low size band in undigested samples, suggesting the presence of free DNA carrying the hygR gene. This result demonstrates that at least some of the strains are authentic transformants.

However, the signals were lost upon subculturing, indicating lack of stability of the hygR DNA. Consequently, the transformants are not stably maintained.

Reported by

University Department
Departamento de Genetica, Apartado 1095
E-41080 Sevilla