Efficient screening of transgenic plant lines for ecological research

摘要: Plants stably transformed to manipulate the expression of genes mediating ecological performance have profoundly altered research in plant ecology. Agrobacterium-mediated transformation remains most effective method creating plants harbouring a limited number transgene integrations low complexity. For ⁄physiological research, following requirements must be met: (i) regenerated should same ploidy level as corresponding wildtype and (ii) contain single copy homozygous state; (iii) T-DNA completely inserted without vector backbone sequence all its elements functional; (iv) integration not change phenotype by interrupting chromosomal or mutations occurring during regeneration procedure. The screening process obtain that meet above criteria is costly time-consuming, an optimized procedure presented. We developed flow chart optimizes efficiently select for research. It consists segregational analyses, which transgenic T1 T2 generation with copies are homozygous. Indispensable molecular genetic tests (flow cytometry, diagnostic PCRs Southern blotting) performed at earliest times process. qPCR quantify changes transcript accumulation confirm gene silencing overexpression last step selection Because we routinely transform wild tobacco, Nicotiana attenuata, constructs silence ectopically overexpress ecologically relevant genes, proposed protocol supported examples from this system.