Genetic and Phenotypic Characterization of Oriental Lilies Transformed with Genes of Herbicide Tolerance and Flavonoid Biosynthesis

Oriental lilies 'Marco Polo' and 'Sorbonne' were transformed either biolistically or Agrobacterium mediated transformation method with a herbicide tolerance gene, bar, and pCAMBIA-3p, harboring Flavonoid 3’, 5’ Hydroxylase gene (f3,5h) genes. Fifty plates of 0.5 g suspension cells of ‘Marco Polo’ were bombarded with gold particle coated with pDM302 DNA and transformants were selected with 2 mg/L phosphinothricin and regenerated. Bar gene transgenic plants were grown repeatedly with new bulbs every year and challenged with 1% gluphosinate herbicide.  For the Agrobacterium transformation, 2 g of embryogenic callus of 'Sorbonne' were transformed with the plasmid and selection-cultured with subsequent exposure to 10 mg/L Hygromycin so to produce 167 plantlets.  PCR analysis indicated that 60% of these plantlets contained the transgene. And, from the putatively-transformed plantlets, mGFP5 gene, a reporter gene in the transgene cassettes, was confirmed to express, which indicates that the protocols were very efficient and reliable for the transformation of lilies. One hundred twenty six plants of an Oriental lily 'Marco Polo' transformed with bar gene have been propagated vegetatively and those transgenic lily lines have been resistant to 1,000 mg/L of phosphinothricin over years.  Those transgenic lines were used as parents in hybridization with other cultivars and their progenies expressed the inherited herbicide tolerance trait.  To investigate the copy number and sequences of flanking regions of the transgene insertion, inverse PCRs were conducted for the herbicide and f3,5h transgenic lines.  Majority of the lines were found out to contain more than two copies, but some events were confirmed to have a single copy and their flaking region sequences were not matched with any reported coding sequences so far.