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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.

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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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Flow-scheme for the establishment of a Physcomitrella gene disruption mutant collection.
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Figure 1: Flow-scheme for the establishment of a Physcomitrella gene disruption mutant collection.

Mentions: The ease of its genetic manipulation, together with a high degree of conservation of morphological features, biochemical pathways and signal transduction mechanisms between Physcomitrella patens and higher plants [11-14] has made the moss an important model system for plant functional genomics. To facilitate a large-scale study of plant gene function using Physcomitrella patens as a model organism, we are developing a collection of Physcomitrella plants with insertion mutations that affect a wide variety of developmental, morphological and physiological characteristics. Transformation with constructs carrying sequences homologous to the genome typically results in 10-fold higher transformation frequencies then the use of non-homologous constructs, and among these transformants a high proportion shows integration of the construct at the homologous genomic locus [3,12]. We argued that – compared to a random mutagenesis strategy [15] – targeting insertion mutations towards expressed genes would increase the proportion of transformants displaying altered properties, and would decrease the total number of transformants to be screened to find a particular change in phenotype. We therefore developed an efficient transposon-based shuttle mutagenesis system for moss cDNA libraries, and have used pools of insertion-mutagenised cDNA clones tagged with a nos-regulated nptII selection cassette for the transformation of Physcomitrella plants (Fig. 1).


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)

Flow-scheme for the establishment of a Physcomitrella gene disruption mutant collection.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Flow-scheme for the establishment of a Physcomitrella gene disruption mutant collection.
Mentions: The ease of its genetic manipulation, together with a high degree of conservation of morphological features, biochemical pathways and signal transduction mechanisms between Physcomitrella patens and higher plants [11-14] has made the moss an important model system for plant functional genomics. To facilitate a large-scale study of plant gene function using Physcomitrella patens as a model organism, we are developing a collection of Physcomitrella plants with insertion mutations that affect a wide variety of developmental, morphological and physiological characteristics. Transformation with constructs carrying sequences homologous to the genome typically results in 10-fold higher transformation frequencies then the use of non-homologous constructs, and among these transformants a high proportion shows integration of the construct at the homologous genomic locus [3,12]. We argued that – compared to a random mutagenesis strategy [15] – targeting insertion mutations towards expressed genes would increase the proportion of transformants displaying altered properties, and would decrease the total number of transformants to be screened to find a particular change in phenotype. We therefore developed an efficient transposon-based shuttle mutagenesis system for moss cDNA libraries, and have used pools of insertion-mutagenised cDNA clones tagged with a nos-regulated nptII selection cassette for the transformation of Physcomitrella plants (Fig. 1).

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