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 deliver y and integration of transgenes, such as type of growth regulator, duration of preculture period, plasmolysis and micro wounding were evaluated for th eir effect on frequencies of transient expression and stable integration. D icamba in the callus induction and maintenance media was generally superior to 2,4-D in promoting transient expression and subsequent stable transform ation. Plasmolysis generally reduced the level of transient expression alth ough transgenic plants were recovered when 1-day precultured embryos were p lasmolysed and grown on 2,4-D containing media. Micro wounding by particle bombardment did not change the level or localisation of transient expressio n events after Agrobacterium infection. Transgenic plants were generated wi th five out of 12 different procedures with transformation efficiencies ran ging from 1.7 to 6.3%. Integration of bar and uidA coding sequences was con firmed by PCR for 75 plants representing 42 independent transgenic lines. E xpression of the uidA gene was detected in 74% of primary transformants whi le 91% of them expressed the bar gene. Southern analysis proved integration of the transgenes with copy numbers ranging from one to six. Most transgen ic lines that expressed both marker and reporter genes showed simple integr ation patterns and Mendelian inheritance of the transgenes in TI progeny. ( C) 2001 Elsevier Science Ireland Ltd. All rights reserved.