Optimization of soybean (Glycine max (L.) Merrill) in planta ovary transformation using a linear minimal gus gene cassette.
Soybean transformation by ovary-drip was improved by optimizing the length of the transformation pathway by cutting the styles. These modifications facilitated soybean transformation manipulation and improved transformation reproducibility and efficiency. Using a linear minimal gus gene cassette as the foreign DNA, a maximum transformation frequency of 11% was obtained in flowers of the soybean cultivar 'Liaodou 14' with their styles mostly removed, whereas removal of only the stigma, partial style cutting and partial ovary cutting gave transformation frequencies of 0%, 1%, and 2%, respectively. An average transformation frequency of 8.2% was obtained when 619 flowers from three soybean cultivars ('Liaodou 14', 'Liaodou 13', and 'Tiefeng 29') were transformed by this optimized method. Southern blotting analysis showed that the gus reporter gene (encoding beta-glucuronidase) was stably inherited with a simple pattern. Reverse transcription-polymerase chain reaction (RT-PCR) and GUS staining confirmed the expression of the gus gene in transgenic plants.
Liu
Ming
M
Department of Bioscience and Biotechnology, Dalian University of Technology, Dalian 116024, China.
Yang
Jun
J
Cheng
Yun-qing
YQ
An
Li-jia
LJ
D001709:Biotechnology / Q000379:methods*; D018744:DNA, Plant / Q000378:metabolism; D035264:Flowers / Q000378:metabolism*; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005838:Genotype; D005966:Glucuronidase / Q000378:metabolism; D008957:Models, Genetic; D030821:Plants, Genetically Modified; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013025:Glycine max / Q000235:genetics*
D018744:DNA, Plant; D005966:Glucuronidase