Submitted to: International Rhizoctonia Symposium
Publication Type: Abstract Only
Publication Acceptance Date: August 15, 2008
Publication Date: January 11, 2010
Citation: Takeshi, T., Upchurch, R.G., and Marc, G. 2010. Development of an Agrobacterium-based transformation system for Rhizoctonia solani. Proc. 4th International Rhizoctonia Symposium, S1028 Berlin, Germany.
A 8.7 kb binary vector containing the 1.9 kb hygromycin B phosphortransferase (hyg) gene was constructed with promoter and terminator regions from the glyceraldehyde-3-phosphate- dehydrogenase (gpd) gene of Rhizoctonia solani anastomosis group 3 (AG-3) at the 5'- and 3'- gene termini of hyg. Promoter (TATA and CAAT box, GATA factor) and terminator (poly-A-tail) regions were confirmed by sequencing the up- and downstream regions of the gpd gene. The hyg construct was inserted into the plasmid pCAMBIA0380 and subsequently transformed into Agrobacterium tumefaciens strains AGL1 and EHA101 by electroporation. For transformation, isolates of R. solani AG-1 IA and AG-3 were incubated on cellophane membranes placed on 1/4 strength potato dextrose agar (PDA) for 24 h. After incubation, the cellophane membranes with mycelium were cut into 3 x 3 mm2 segments and placed onto 2% water agar induction medium with A. tumefaciens carrying the recombinant pCAMBIA0380 with the hyg cassette, 200 mM acetosyringone and 50mg/L kanamycin and incubated at 20-22 C for 2-4 d. Cellophane membranes with mycelium of R. solani were transferred to selection medium containing 1/4 PDA with ampicillin, tetracycline and hygromycin to kill cells of A. tumefaciens and recover transformed colonies of R. solani. Isolates of both R. solani AGs were recovered from selection medium but had slower growth rates than their respective parental strains and were not stable. Research is currently in progress to determine if genomic integration of the hyg construct occurred and the relative stability of the new construct in transformed cells of R. solani.