Articles
ANALYSIS OF TISSUE UNIFORMITY IN TRANSGENIC APPLE PLANTS
Article number
738_33
Pages
301 – 306
Language
English
Abstract
Putative transgenic plants are commonly analyzed for integration and expression of the transferred genes by molecular techniques.
Most of these techniques cannot distinguish between plants containing a mixture of transformed and non-transformed cells (chimera) and plants containing only transgenic cells (uniform). Putative transgenics with detectable DNA fragments of the expected size and acceptable level of transgenic proteins are often assumed to be uniform and chimeras remain undetected.
In addition, plants containing marker genes, such as nptII, are usually grown under selection pressure for marker gene expression, but there is no direct selection for the gene of interest.
Since both cell types with and without expression of the gene of interest are able to grow under selective conditions, we can not rely on current methods to confirm the homogeneity of gene integration and gene expression.
To study the uniformity of T-DNA insertion and gene expression in transgenic plants, and to mimic natural conditions for transgenic trees in the field, 26 transgenic apple lines containing the attacin E and the nptII genes were propagated in vitro on propagation medium without antibiotics.
After four years of subculture, ten individual shoots of each line were characterized using PCR, RT-PCR, Southern blot, ELISA and histochemical GUS staining.
Individual shoots with partially or completely silenced transgenes were identified as well as shoots with no detectable T-DNA. Several lines appeared chimeric based upon histochemical GUS staining.
Although most molecular techniques can reliably detect the presence of transgenic cells, they often fail to detect mixtures of transformed and non-transformed cells, or mixtures of cells expressing transgenes and cells with silenced transgenes.
Evaluation of transgenic apple plants using standard molecular techniques provides no information about the uniformity of transgenic tissue.
Most of these techniques cannot distinguish between plants containing a mixture of transformed and non-transformed cells (chimera) and plants containing only transgenic cells (uniform). Putative transgenics with detectable DNA fragments of the expected size and acceptable level of transgenic proteins are often assumed to be uniform and chimeras remain undetected.
In addition, plants containing marker genes, such as nptII, are usually grown under selection pressure for marker gene expression, but there is no direct selection for the gene of interest.
Since both cell types with and without expression of the gene of interest are able to grow under selective conditions, we can not rely on current methods to confirm the homogeneity of gene integration and gene expression.
To study the uniformity of T-DNA insertion and gene expression in transgenic plants, and to mimic natural conditions for transgenic trees in the field, 26 transgenic apple lines containing the attacin E and the nptII genes were propagated in vitro on propagation medium without antibiotics.
After four years of subculture, ten individual shoots of each line were characterized using PCR, RT-PCR, Southern blot, ELISA and histochemical GUS staining.
Individual shoots with partially or completely silenced transgenes were identified as well as shoots with no detectable T-DNA. Several lines appeared chimeric based upon histochemical GUS staining.
Although most molecular techniques can reliably detect the presence of transgenic cells, they often fail to detect mixtures of transformed and non-transformed cells, or mixtures of cells expressing transgenes and cells with silenced transgenes.
Evaluation of transgenic apple plants using standard molecular techniques provides no information about the uniformity of transgenic tissue.
Authors
H. Flachowsky, M. Riedel, S. Reim, M.V. Hanke
Keywords
apple, Malus × domestica Borkh., T-DNA leakage, silencing
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