Co-transformation Efficiency Research Articles

Stable co-transformation of maize protoplasts with gusA and neo genes

An efficient co-transformation protocol using polyethylene glycol was developed for Zea mays L. (cv. A188 x BMS) protoplasts isolated from suspension culture cells. Co-transformation was accomplished by using plasmid constructions containing beta-glucuronidase (gusA) or neomycin phosphotransferase (neo) gene coding sequences; both were under control of the CaMV 35S promoter. Protoplast culture and transformation conditions were optimized to assure efficient recovery of transformed cells. The overall efficiency of transformation was 1 x 10(-4) (calculated per viable protoplast plated). Among kanamycin-resistant lines, 50% showed a high level of GUS activity (above one unit). Southern blot hybridization confirmed the presence of numerous gusA and neo coding sequences in the maize genome. In two analyzed lines, integrated sequences appeared to be organized in tandem head-to-tail repeats. Results also indicated that the integrated sequences were partially methylated.

Transformation of Aspergillus oryzae using the A. niger pyrG gene.

A transformation system for Aspergillus oryzae based on the orotidine-5'-phosphate decarboxylase gene (pyrG) was developed. Transformation frequencies of up to 16 transformants per microgram of DNA were obtained with the vector pAB4-1, which carries the pyrG gene of A. niger. Southern blotting analysis showed that vector DNA sequences were integrated into the chromosomal DNA, in various copy numbers and presumably at different sites. Efficient cotransformation of an unselectable gene was also shown. Under the conditions used no transformants were obtained with the equivalent pyr4 gene of Neurospora crassa.

A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei

An efficient transformation system for the cellulolytic filamentous fungus Trichoderma reesei has been developed. Transformation was obtained with plasmid carrying the dominant selectable marker amdS or the argB gene of Aspergillus nidulans, which was found to complement the respective argB mutation of T. reesei. The transformation frequency can be up to 600 transformants per μg of transforming DNA. The efficiency of co-transformation with unselected DNA was high (approx. 80%). The transforming DNA was found to be integrated at several different locations, often in multiple tandem copies in the T. reesei genome. In addition, the Escherichia coli β-galactosidase was expressed in T. reesei in enzymatically active form from the A. nidulans gpd promoter.

Assessment of the efficiency of cotransformation of the T-DNA of disarmed binary vectors derived from Agrobacterium tumefaciens and the T-DNA of A. rhizogenes

Co-transfer of Agrobacterium rhizogenes T-DNA and T-DNA from the A. tumefaciens binary vector pBin19 (Bevan, 1984) was studied in detail using Nicotiana rustica. High frequencies of co-transfer of T-DNA's were observed, even when no selection pressure was exerted. Increased levels of pBin19 T-DNA were found in hairy root cultures with selection at higher levels of kanamycin sulphate (50-200 μg ml(-1)). Several other species were also transformed by A. rhizogenes carrying pBin19 and A. rhizogenes harbouring a different binary factor, pAGS125 (Van den Elzen et al., 1985), was used to transform N. rustica hairy roots to confer hygromycin B resistance.