Production of transgenic soybean plants with two anti-fungal protein genes via Agrobacterium and particle bombardment
Li, H.Y.(Agriculture and Agri-Food Canada (Canada). Lethbridge Research Centre) | Zhu, Y.M.(Northeast Agricultural Univ., Harbin (China). Coll. of Life Science) | Chen, Q.(Agriculture and Agri-Food Canada (Canada). Lethbridge Research Centre) | Conner, R,L,(Agriculture and Agri-Food Canada (Canada). Morden Research Station) | Ding, X.D.(Northeast Agricultural Univ., Harbin (China). Coll. of Life Science) | Li, J.(Northeast Agricultural Univ., Harbin (China). Coll. of Life Science) | Zhang, B.B.(Northeast Agricultural Univ., Harbin (China). Coll. of Life Science)
Utilizing either Agrobacterium-mediated transformation or particle bombardment we obtained transgenic soybean plants expressing the chitinase gene (chi) and the barley ribosome-inactivating protein gene (rip). Six regenerated plants were grown to maturity and set seed. The identification of transgenic soybean plants that co-integrated the two anti-fungal protein genes was determined by PCR and Southern blot analysis. Protein detection from the soybean leaves demonstrated the expression of chitinase and the ribosome-inactivating protein (RIP) in the six Ro transformants. Soybean cotyledonary nodes were transformed using the bivalent plant expression vector pBRC containing chi and rip both driven by the CaMV 35S double promoter. Following vacuum (0.06 MPa) infiltration treatment of the tissue for 5 min, Agrobacterium was co-cultivated with the cotyledonary nodes for 3 d on MSB medium (MS salts and B5 vitamins, pH 5.2), the transformation frequency reached a maximum of 1.33%. The chi and rip genes were present in all the transgenic plants. Co-bombardment of immature cotyledons with plasmids pBchE (encoding chi) and pARIP (encoding rip) resulted in a maximum transformation frequency of 0.52% with a 50% co-integration rate. Our results demonstrate efficient co-transformation of multiple genes in soybean.
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