Efficient transient expression and transformation of PEG-mediated gene uptake into mesophyll protoplasts of pepper (Capsicum annuum L.) |
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Authors: | Joo Mi Jeon Nam Young Ahn Bo Hwa Son Cha Young Kim Chang-deok Han Gun-Do Kim Sang Wan Gal Sung-Ho Lee |
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Affiliation: | (1) Division of Applied Life Science (BK21) and Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju, 660-701, Korea;(2) Department of Microbiology, College of Natural Science, Pukyong National University, Busan, 608-737, Korea;(3) Department of Microbiological Engineering, Jinju National University, Jinju, 660-758, Korea |
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Abstract: | PEG-mediated transformation was used for gene delivery and evaluation of various parameters affecting the transient expression of a gene for ß-glucuronidase (gus) in mesophyll protoplasts of Capsicum annuum. Transient expression was found to be dependent on PEG concentration and exposure time of plasmid DNA to protoplasts as well as the amount of plasmid DNA. Maximum GUS activity was obtained when protoplasts were applied to 40% concentration and molecular weight was 6,000 of PEG solution with 30 min of exposure time. Protoplasts of pepper were transformed with a vector, pCAMBIA::Ac, which contained a pCAMBIA1302 T-DNA vector carrying a maize transposable element, Ac (activator), a selection marker HPT (hygromycin phosphotransferase), and a GFP-coding region driven by the 35S promoter in the presence of PEG. Approximately 30% of the protoplasts expressed GFP. Visibly transformed colonies were obtained from protoplasts after 2 months of culture and GFP was expressed. Southern hybridization confirmed the presence of Ac in the pepper genome. |
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Keywords: | Ac Transposable element Capsicum annuum L. Protoplast transformation Transient expression |
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