High-efficiency induction of soybean hairy roots and propagation of the soybean cyst nematode

Cotyledon explants of 10 soybean [Glycine max (L.) Merr.] cultivars were in oculated with Agrobacterium rhizogenes strain K599 with and without binary vectors pBI121 or pBINm-gfp5-ER possessing both neomycin phosphotransferase II (nptII) and beta-glucuronidase (gus) or nptII and green fluorescent pro tein (gfp) genes, respectively. Hairy roots were produced from the wounded surface of 54-95% of the cotyledon explants on MXB selective medium contain ing 200 mu g ml(-1) kanamycin and 500 mu g ml(-1) carbenicillin. Putative i ndividual transformed hairy roots were identified by cucumopine analysis an d were screened for transgene incorporation using polymerase chain reaction . All of the roots tested were found to be co-transformed with T-DNA from t he Ri-plasmid and the transgene from the binary vectors, Southern blot anal ysis confirmed the presence of the 35S-gfp5 gene in the plant genomes, Tran sgene expression was also confirmed by histochemical GUS assay and Western blot analysis for the GFP. Attempts to induce shoot formation from the hair y roots failed. Infection of hairy roots of the soybean cyst nematode (Hete rodera glycines Ichinohe)-susceptible cultivar, Williams 82, with eggs of H . glycines race 1, resulted in the development of mature cysts about 4-5 we eks after inoculation. Thus the soybean cyst nematode could complete its en tire life cycle in transformed soybean hairy-root cultures expressing GFP. This system should be ideal for testing genes that might impart resistance to soybean cyst nematode.