Early Identification of Stable Transformation Events by Combined Use of Antibiotic Selection and Vital Detection of Green Fluorescent Protein (GFP) in Carrot (Daucus carota L.) Callus

作     者：Yuan-Yeu Yau Seth J Davis Ahmet Ipek Philipp W Simon 

作者机构：U.S. Department of Agriculture Agricultural Research Service Department of Horticulture University of Wisconsin-Madison Laboratory of Genetics University of Wisconsin-Madison Madison WI 53706 USA U.S. Department of Agriculture Agricultural Research Service Department of Horticulture University of Wisconsin-Madison Madison WI 53706 USA 

出 版 物：《Agricultural Sciences in China》 (中国农业科学（英文版）)

年 卷 期：2008年第7卷第6期

页      面：664-671页

学科分类：09[农学] 0902[农学-园艺学] 090202[农学-蔬菜学] 

基　　金：Acknowledgements The research was supported by the Fresh Carrot Board (N328) 

主　　题：Agrobacterium tumefaciens antibiotic selection Daucus carota genetic transformation reporter gene,stable transformed transgene 

摘      要：Genetic transformation is a useful technique to complement conventional breeding in crop improvement. Although carrot has been a model organism for in vitro embryogenesis study, genetic transformation of carrot is still lengthy and labor intensive. An efficient transformation and detection system is desirable. Direct infection of Agrobacterium to carrot calli has provided an easy way for carrot genetic transformation. To improve the efficiency of antibiotic selection in this method, we report the combined use of an improved green-fluorescent protein, referred to as smGFP, to establish a versatile selection method for carrot callus transformation system. By combining antibiotic selection with the bright fluorescence observed in the callus tissue, we were able to easily identify stable transformants in early stage of the transformation process. In addition to the GFP expression of the callus cells, the transgenic nature of callus cells was confirmed with Southern and Western analysis. We found we can link the simplicity of carrot-callus-cell transformation, early detection of stable transformants with antibiotic selection, visualization of GFP fluorescence, and molecular analysis （Southern and Western） of callus tissue （non-photosynthetic tissue） to provide a more efficient way in identifying stable transformants at early stage of carrot transformation.