Genetic transformation of Brassica campestris var. rapa protoplasts with an engineered cauliflower mosaic virus genome

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Abstract
A hybrid Cauliflower Mosaic Virus (CaMV) genome containing a selectable marker gene was constructed by replacing the gene VI coding region with the aminoglycoside (neomycin) phosphotransferase type II [APH(3′)II] gene from Tn5. This modified viral genome was tested for its infectivity both in planta and in a protoplast transformation system of Brassica campestris var. rapa. Stable, genetically transformed cell lines of B. campestris var. rapa were obtained after transformation. DNA of the hybrid CaMV genome was found to be integrated into high molecular weight plant genomic DNA. Transformation was achieved only when the hybrid genome was supplied together with wild type viral DNA. A possible complementation of the modified CaMV genome with the wild type viral DNA as a helper molecule in planta and in the protoplast system is discussed.