Agrobacterium-mediated transgene delivery and integration into barley under a range of in vitro culture conditions

An efficient Agrobacterium-mediated transformation procedure for barley was developed for immature embryos. Factors considered crucial for the delivery and integration of transgenes, such as type of growth regulator, duration of preculture period, plasmolysis and micro wounding were evaluated for their effect on frequencies of transient expression and stable integration. Dicamba in the callus induction and maintenance media was generally superior to 2,4-D in promoting transient expression and subsequent stable transformation. Plasmolysis generally reduced the level of transient expression although transgenic plants were recovered when 1-day precultured embryos were plasmolysed and grown on 2,4-D containing media. Micro wounding by particle bombardment did not change the level or localisation of transient expression events after Agrobacterium infection. Transgenic plants were generated with five out of 12 different procedures with transformation efficiencies ranging from 1.7 to 6.3%. Integration of bar and uidA coding sequences was confirmed by PCR for 75 plants representing 42 independent transgenic lines. Expression of the uidA gene was detected in 74% of primary transformants while 91% of them expressed the bar gene. Southern analysis proved integration of the transgenes with copy numbers ranging from one to six. Most transgenic lines that expressed both marker and reporter genes showed simple integration patterns and Mendelian inheritance of the transgenes in TI progeny. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.