Investigation of a genetic transformation system in onion
A genetic transformation system via the pollen tube pathway was investigated. Plasmid pD0432 (Ow et al., 1986), containing the cauliflower mosaic virus 35S promoter fused to the firefly(Photinus pyralis Linnaeus) luciferase coding sequence and a nopaline synthase terminator sequence, was used as the vector. Plasmid DNA prepared with each of 4 different methods was applied to the surface of the remaining style of florets decapitated 18 or 24 hours following pollination. There were no significant differences in percentage of fruit set between exogenous DNA application time (18 or 24 hours following pollination), among treatments (plasmid DNA solution, control solution, and no solution applied), or interaction between application time and treatments. According to the observation of the movement of EB-labeled plasmid DNA, plasmid DNA reached the ovules of decapitated florets within 24 hours after its application to the surface of remaining styles at 18 hours after pollination. Plasmid DNA mixed with 1/2 liquid MS medium (Musrashige & Skoog, 1964) containing 4 mg/l NAA and 4 mg/l GA3, plus 0.25% DMSO may have facilitated the entrance of plasmid DNA into onion ovules via the pollen tube pathway. Based on the result of PCR analysis of genomic DNA, 12 to 15% of the plants tested had the 282 bp fragment, the specific portion of the luciferase gene construct. Exogenous DNA should be applied to the decapitated onion florets no later than 18 hour following pollination. Southern blotting of genomic DNA from PCR positive plants indicated that the firefly luciferase gene construct may have been incorporated into the genomic DNA of onion. However, insertion of the foreign DNA in some of the PCR positive plants may have been chimeric since the 282 bp PCR amplified fragment could not be detected at their maturing stages and thereafter (in Allium, cepa cv. DG133), and in some sexually produced progeny (in A fistulosum cv. Heshiko). In addition, expression of the firefly luciferase gene construct was not detected in any of the putative transgenic plants. Some of the PCR positive Heshiko plants, derived from seeds harvested from florets treated with a DNA preparation containing 1/2 liquid MS medium supplied with 4 mg/l NAA, 4 mg/l GA3, still had the 282 bp fragment of the luciferase gene construct in their sexually produced progenies. These results indicated that transformation via the pollen tube pathway has the potential to produce stably transformed onion plants.