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Agrobacterium tumefaciens-mediated transformation of Phellodendron amurense Rupr. using mature-seed explants.

Molecular biology reports (2012-10-16)
Jingli Yang, Bo Zhao, Yeon Bok Kim, Chenguang Zhou, Chunyan Li, Yunlin Chen, Haizhen Zhang, Cheng Hao Li
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Hygromycin B solution from Streptomyces hygroscopicus, ≥60% (HPLC), 45-60 mg/mL in H2O
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Hygromycin B solution from Streptomyces hygroscopicus, ≥60% (HPLC), 45-60 mg/mL in H2O, γ-irradiated
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Hygromycin B from Streptomyces hygroscopicus, suitable for plant cell culture, BioReagent, ≥60% (HPLC), lyophilized powder
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Hygromycin B from Streptomyces hygroscopicus, powder, BioReagent, suitable for cell culture, suitable for insect cell culture
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Hygromycin B from Streptomyces hygroscopicus, lyophilized powder