Agrobacterium tumefaciens-mediated transformation of Phellodendron amurense Rupr. using mature-seed explants.
An efficient transformation protocol was developed for Agrobacterium-mediated transformation of Phellodendron amurense Rupr. for using explants from mature seeds. The binary vector pCAMBIA1303, which contained hygromycin phosphotransferase (hptII) as a selectable marker gene and β-glucuronidase (GUS) as a reporter gene, was used for transformation studies. Different factors that affect survival of transformed buds, namely Agrobacterium infection method, bacterial strain, pre-culture duration, acetosyringone concentration, co-culture duration, and co-culture temperature were examined and optimized for transformation efficiency on the basis of GUS staining of hygromycin-resistant buds. Polymerase chain reaction (PCR), Southern blot and reverse transcription PCR confirmed the presence of the GUS gene. A transformation frequency of 13.1 % was achieved under optimized conditions for transformation (A. tumefaciens strain EHA105, 4 days co-cultivation at 4 °C, and infection of the pre-cultured mature-seed explants for 2 days). This is the first report of a successful genetic transformation protocol for P. amurense.
State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin 150040, China.
Jingli
J
Yang
Bo
B
Zhao
Yeon Bok
YB
Kim
Chenguang
C
Zhou
Chunyan
C
Li
Yunlin
Y
Chen
Haizhen
H
Zhang
Cheng Hao
CH
Li
D016960:Agrobacterium tumefaciens / Q000502:physiology*; D002934:Cinnamates / Q000494:pharmacology; D018920:Coculture Techniques; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D006921:Hygromycin B / Q000031:analogs & derivatives / Q000494:pharmacology; D032102:Phellodendron / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D018520:Plant Shoots / Q000187:drug effects / Q000235:genetics; D030821:Plants, Genetically Modified; D012639:Seeds / Q000235:genetics*; D014170:Transformation, Genetic
D002934:Cinnamates; D010940:Plant Proteins; D006921:Hygromycin B; C026273:hygromycin A; D005966:Glucuronidase