Agrobacterium tumefaciens - mediated transformation of soybean [Glycine max (L.) Merrill.] using immature zygotic cotyledon explants

Agrobacterium tumefnciens-mediated transformation of soybean [Glycine max ( L.) Merrill. cv. Jack] using immature zygotic cotyledons was investigated t o identify important factors that affected transformation efficiency and re sulted in the production of transgenic soybean somatic embryos. The factors evaluated were initial immature zygotic cotyledon size, Agrobacterium conc entration during inoculation and co-culture and the selection regime. Our r esults showed that 8- to 10-mm zygotic cotyledons exhibited a higher transf ormation rate, as indicated by transient GUS gene expression, whereas the s maller zygotic cotyledons, at less than 5 mm, died shortly after co-cultiva tion. However, the smaller zygotic cotyledon explants were found to have a higher embryogenic potential. Analysis of Agrobacterium and immature cotyle don explant interactions involved two Agrobacterium concentrations for the inoculation phase and three co-culture regimes. No differences in explant s urvival or somatic embyogenic potential were observed between the two Agrob acterium concentrations tested. Analysis of co-culture regimes revealed tha t the shorter co-culture times resulted in higher explant survival and high er somatic embryo production on the explants, whereas the co-culture time o f 4 days severely reduced survival of the cotyledon explants and lowered th eir embryogenic potential. Analysis of selection regimes revealed that dire ct placement of cotyledon explants on hygromycin 25 mg/l was detrimental to explant survival, whereas 10 mg/l gave continued growth and subsequent som atic embryo development and plant regeneration. The overall transformation frequency in these experiments, from initial explant to whole plant, was 0. 03%. Three fertile soybean plants were obtained during the course of these experiments. Enzymatic GUS assays and Southern blot hybridizations confirme d the integration of T-DNA and expression of the GUS-intron gene in the thr ee primary transformants. Analysis of 48 progeny revealed that three copies of the transgene were inherited as a single Mendelian locus.