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High frequency of phenotypic deviations in Physcomitrella patens plants transformed with a gene-disruption library.

Egener T, Granado J, Guitton MC, Hohe A, Holtorf H, Lucht JM, Rensing SA, Schlink K, Schulte J, Schween G, Zimmermann S, Duwenig E, Rak B, Reski R - BMC Plant Biol. (2002)

Bottom Line: The resulting gene-disruption library was then used to transform Physcomitrella.An immediate phenotypic analysis of transformants is made possible by the predominance of the haploid gametophytic state in the life cycle of the moss.Among the first 16,203 transformants analysed so far, we observed 2636 plants (= 16.2%) that differed from the wild-type in a variety of developmental, morphological and physiological characteristics.

View Article: PubMed Central - HTML - PubMed

Affiliation: Plant Biotechnology, Freiburg University, Sonnenstrasse 5, D-79104 Freiburg/Br, Germany. tanja.egener@biologie.uni-freiburg.de

ABSTRACT

Background: The moss Physcomitrella patens is an attractive model system for plant biology and functional genome analysis. It shares many biological features with higher plants but has the unique advantage of an efficient homologous recombination system for its nuclear DNA. This allows precise genetic manipulations and targeted knockouts to study gene function, an approach that due to the very low frequency of targeted recombination events is not routinely possible in any higher plant.

Results: As an important prerequisite for a large-scale gene/function correlation study in this plant, we are establishing a collection of Physcomitrella patens transformants with insertion mutations in most expressed genes. A low-redundancy moss cDNA library was mutagenised in E. coli using a derivative of the transposon Tn1000. The resulting gene-disruption library was then used to transform Physcomitrella. Homologous recombination of the mutagenised cDNA with genomic coding sequences is expected to target insertion events preferentially to expressed genes. An immediate phenotypic analysis of transformants is made possible by the predominance of the haploid gametophytic state in the life cycle of the moss. Among the first 16,203 transformants analysed so far, we observed 2636 plants (= 16.2%) that differed from the wild-type in a variety of developmental, morphological and physiological characteristics.

Conclusions: The high proportion of phenotypic deviations and the wide range of abnormalities observed among the transformants suggests that mutagenesis by gene-disruption library transformation is a useful strategy to establish a highly diverse population of Physcomitrella patens mutants for functional genome analysis.

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Isolation of metabolic mutants. Physcomitrella plants derived from transformation with the gene-disruption library and regenerated on supplemented medium were split in two parts, which were transferred to minimal Knop medium with and without supplements (see Materials) and cultured for 8 weeks. The picture shows four independent Physcomitrella transformants cultured on minimal medium, one of which (bottom right) displays a clear growth defect. All four plants grew equally well on supplemented medium (data not shown). The scale bar at the top indicates size in centimetres.
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Figure 3: Isolation of metabolic mutants. Physcomitrella plants derived from transformation with the gene-disruption library and regenerated on supplemented medium were split in two parts, which were transferred to minimal Knop medium with and without supplements (see Materials) and cultured for 8 weeks. The picture shows four independent Physcomitrella transformants cultured on minimal medium, one of which (bottom right) displays a clear growth defect. All four plants grew equally well on supplemented medium (data not shown). The scale bar at the top indicates size in centimetres.

Mentions: To test for physiological alterations induced in moss plants after transformation with the gene-disruption library, we looked for changed nutritional requirements induced in the transformants (Fig. 3). 7.2% (1,163 of 16,203) of the regenerated plants showed a retarded growth during the culture on minimal medium but a normal growth during the culture on the same medium containing a combination of supplements (see Materials), whereas wild-type plants grew well on both media. This suggests a metabolic defect in transformants with a slow-growth phenotype on minimal medium that could be rescued by supplying metabolites in the medium. 3.1% (508 of 16,203) of the transformants showed a retarded growth during the culture on minimal medium as well as on supplemented medium. Those plants might require substances that are not present in our supplemented medium, or they might be defective in another way. Different nutritional mutants of Physcomitrella patens, e.g. resulting from mutagenised spores [22] have been described previously.


High frequency of phenotypic deviations in Physcomitrella patens plants transformed with a gene-disruption library.

Egener T, Granado J, Guitton MC, Hohe A, Holtorf H, Lucht JM, Rensing SA, Schlink K, Schulte J, Schween G, Zimmermann S, Duwenig E, Rak B, Reski R - BMC Plant Biol. (2002)

Isolation of metabolic mutants. Physcomitrella plants derived from transformation with the gene-disruption library and regenerated on supplemented medium were split in two parts, which were transferred to minimal Knop medium with and without supplements (see Materials) and cultured for 8 weeks. The picture shows four independent Physcomitrella transformants cultured on minimal medium, one of which (bottom right) displays a clear growth defect. All four plants grew equally well on supplemented medium (data not shown). The scale bar at the top indicates size in centimetres.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC117800&req=5

Figure 3: Isolation of metabolic mutants. Physcomitrella plants derived from transformation with the gene-disruption library and regenerated on supplemented medium were split in two parts, which were transferred to minimal Knop medium with and without supplements (see Materials) and cultured for 8 weeks. The picture shows four independent Physcomitrella transformants cultured on minimal medium, one of which (bottom right) displays a clear growth defect. All four plants grew equally well on supplemented medium (data not shown). The scale bar at the top indicates size in centimetres.
Mentions: To test for physiological alterations induced in moss plants after transformation with the gene-disruption library, we looked for changed nutritional requirements induced in the transformants (Fig. 3). 7.2% (1,163 of 16,203) of the regenerated plants showed a retarded growth during the culture on minimal medium but a normal growth during the culture on the same medium containing a combination of supplements (see Materials), whereas wild-type plants grew well on both media. This suggests a metabolic defect in transformants with a slow-growth phenotype on minimal medium that could be rescued by supplying metabolites in the medium. 3.1% (508 of 16,203) of the transformants showed a retarded growth during the culture on minimal medium as well as on supplemented medium. Those plants might require substances that are not present in our supplemented medium, or they might be defective in another way. Different nutritional mutants of Physcomitrella patens, e.g. resulting from mutagenised spores [22] have been described previously.

Bottom Line: The resulting gene-disruption library was then used to transform Physcomitrella.An immediate phenotypic analysis of transformants is made possible by the predominance of the haploid gametophytic state in the life cycle of the moss.Among the first 16,203 transformants analysed so far, we observed 2636 plants (= 16.2%) that differed from the wild-type in a variety of developmental, morphological and physiological characteristics.

View Article: PubMed Central - HTML - PubMed

Affiliation: Plant Biotechnology, Freiburg University, Sonnenstrasse 5, D-79104 Freiburg/Br, Germany. tanja.egener@biologie.uni-freiburg.de

ABSTRACT

Background: The moss Physcomitrella patens is an attractive model system for plant biology and functional genome analysis. It shares many biological features with higher plants but has the unique advantage of an efficient homologous recombination system for its nuclear DNA. This allows precise genetic manipulations and targeted knockouts to study gene function, an approach that due to the very low frequency of targeted recombination events is not routinely possible in any higher plant.

Results: As an important prerequisite for a large-scale gene/function correlation study in this plant, we are establishing a collection of Physcomitrella patens transformants with insertion mutations in most expressed genes. A low-redundancy moss cDNA library was mutagenised in E. coli using a derivative of the transposon Tn1000. The resulting gene-disruption library was then used to transform Physcomitrella. Homologous recombination of the mutagenised cDNA with genomic coding sequences is expected to target insertion events preferentially to expressed genes. An immediate phenotypic analysis of transformants is made possible by the predominance of the haploid gametophytic state in the life cycle of the moss. Among the first 16,203 transformants analysed so far, we observed 2636 plants (= 16.2%) that differed from the wild-type in a variety of developmental, morphological and physiological characteristics.

Conclusions: The high proportion of phenotypic deviations and the wide range of abnormalities observed among the transformants suggests that mutagenesis by gene-disruption library transformation is a useful strategy to establish a highly diverse population of Physcomitrella patens mutants for functional genome analysis.

Show MeSH
Related in: MedlinePlus