Methylation Pattern Analysis of Transgenic Tobacco Plants

Tobacco (Nicotiana tabacum cv. Havana SR1) was transfomed with Agrobacterium tumefaciens LBA4404 containing a rescue cloning vector (pRCV2). Southern hybridization data showed that the copy number of integrated genes varied from one copy to multiple copies. Transgenic plants confirmed with Southern hybridization were selfed and the T₁ seeds from the transgenic lines with various copy numbers were tested in hygromycin medium to examine the progeny segregation ratio. The result showed that T-DNA was stably transferred to eight T₁ lines. Polymerase chain reaction and Southern analyses were used to reconfirm the presence of hpt gene. Rescue cloning was performed to analyze methylation pattern of inserted T-DNA region. As a result, rescued plasmids were obtained from six lines and digested with AvaI and SeaI. Cleaved-pattern appeared to methylated-pattern band or not. Particularly, the cleaved-pattern in line 14 revealed bands that were expected to methylated up to 355 promoter and hygromycin phosphotransferase coding region.