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NAZV project QH71290 2007-2011 Cry3A transgenic, bark beetle resistant spruce Spruce bark beetle (Ips typographus) Bark beetle galleries Bacillus thuringiensis crystal protein (Cry, δ-endoTOXin) Agrobacterium interaction with the plant cell Major problems: Spruce is difficult to transform No known Bt toxin is toxic enough to bark beetle Spruce callus cultures Somatic embryogenesis induction conversion cultivation Modified Cry3A genes Cry3A orig Synt TOX-1 (602 aa)-codons Picea optimized Ndel/sTOX-1 Cdel/sTOX-1 (602 aa) Cdel/Ndel/sTOX-1 GUS Refined modification of domain II Expression vector PAGE analysis of modified Cry3A expression WESTERN detection of modified Cry3A proteins Toxicity tests on live beetles Konec presentace