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Efficient screening of transgenic plant lines for ecological research.

Abstract：

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

journal_name

Mol Ecol Resour

journal_title

Molecular ecology resources

authors

Gase K,Weinhold A,Bozorov T,Schuck S,Baldwin IT

doi

10.1111/j.1755-0998.2011.03017.x

subject

Has Abstract

pub_date

2011-09-01 00:00:00

pages

890-902

issue

5

eissn

1755-098X

issn

1755-0998

journal_volume

11

pub_type

杂志文章

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