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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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Deviating phenotypes induced in gene-disruption library transformants. Physcomitrella wild-type and transformed plants were grown on minimal Knop medium to induce differentiation and development of gametophores. For each plant, an overview (upper row, scale bar corresponds to 1 mm) and a close-up (bottom row, scale bar equals 0.5 mm) is shown. A, Haploid wild-type moss plant completely covered with leafy gametophores and close-up of wild-type leaf. B, Transformant BC22189 affected in differentiation, mostly comprising of filamentous protonema with reduced number of gametophores, but normal leaf morphology (haploid). C, Transformant BC11280 showing retarded growth, a reduced number of gametophores per moss plant and altered leaf morphology ("drehzipfel" phenotype; twisted tips of leaves, haploid). D, Transformant BC1015 displaying altered growth habitus ("wasserpest" phenotype, reminiscent of the waterweed Elodea) and altered leaf morphology (polyploid). E, Transformant BC22288 showing retarded growth and elongated, narrow leafs (polyploid).
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Figure 4: Deviating phenotypes induced in gene-disruption library transformants. Physcomitrella wild-type and transformed plants were grown on minimal Knop medium to induce differentiation and development of gametophores. For each plant, an overview (upper row, scale bar corresponds to 1 mm) and a close-up (bottom row, scale bar equals 0.5 mm) is shown. A, Haploid wild-type moss plant completely covered with leafy gametophores and close-up of wild-type leaf. B, Transformant BC22189 affected in differentiation, mostly comprising of filamentous protonema with reduced number of gametophores, but normal leaf morphology (haploid). C, Transformant BC11280 showing retarded growth, a reduced number of gametophores per moss plant and altered leaf morphology ("drehzipfel" phenotype; twisted tips of leaves, haploid). D, Transformant BC1015 displaying altered growth habitus ("wasserpest" phenotype, reminiscent of the waterweed Elodea) and altered leaf morphology (polyploid). E, Transformant BC22288 showing retarded growth and elongated, narrow leafs (polyploid).

Mentions: To screen for morphological and developmental mutations, transformants were microscopically observed after regeneration and selection on supplemented Knop medium for 11 weeks followed by growth on minimal Knop medium for 8 weeks. Under these conditions, the filamentous structure (protonema) that is initially formed by regenerating wild-type protoplasts undergoes a developmental switch. It forms a three-faced apical meristem bud, and differentiates into leafy stems, the gametophores (Fig. 4A), that will eventually carry the sex organs. 16.2% (2,631 of 16,203) of the gene-disruption library transformants showed morphological deviations in one or more of the features observed: structure and colour of the moss plant, coverage of the plant by gametophores, shape and uniformity of leaves and arrangement of cells within the leaves (Table 1). Such deviations were not observed to occur spontaneously in 350 analysed wild-type plants and in less than 1% of 400 plants derived from protoplasts mock-transformed without DNA and regenerated in the absence of antibiotic. Examples for deviating phenotypes observed in gene-disruption library transformants are shown in Fig. 4b to 4E. In addition to the phenotypic classes described above, we observed other developmental abnormalities, like the formation of outgrowths on the leaf surface, the formation of thread-like appendages on leaf tips, and an increased number of dark-coloured sectors on leaves. Therefore, a wide spectrum of morphological and developmental alterations is observed in moss plants transformed with a gene-disruption library. In addition, between the three parameters we assayed for each transformant – growth requirements, morphology and ploidy level – there did not appear to be a strict correlation, and we found various combinations of characteristics (Fig. 5).


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)

Deviating phenotypes induced in gene-disruption library transformants. Physcomitrella wild-type and transformed plants were grown on minimal Knop medium to induce differentiation and development of gametophores. For each plant, an overview (upper row, scale bar corresponds to 1 mm) and a close-up (bottom row, scale bar equals 0.5 mm) is shown. A, Haploid wild-type moss plant completely covered with leafy gametophores and close-up of wild-type leaf. B, Transformant BC22189 affected in differentiation, mostly comprising of filamentous protonema with reduced number of gametophores, but normal leaf morphology (haploid). C, Transformant BC11280 showing retarded growth, a reduced number of gametophores per moss plant and altered leaf morphology ("drehzipfel" phenotype; twisted tips of leaves, haploid). D, Transformant BC1015 displaying altered growth habitus ("wasserpest" phenotype, reminiscent of the waterweed Elodea) and altered leaf morphology (polyploid). E, Transformant BC22288 showing retarded growth and elongated, narrow leafs (polyploid).
© Copyright Policy
Related In: Results  -  Collection

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Figure 4: Deviating phenotypes induced in gene-disruption library transformants. Physcomitrella wild-type and transformed plants were grown on minimal Knop medium to induce differentiation and development of gametophores. For each plant, an overview (upper row, scale bar corresponds to 1 mm) and a close-up (bottom row, scale bar equals 0.5 mm) is shown. A, Haploid wild-type moss plant completely covered with leafy gametophores and close-up of wild-type leaf. B, Transformant BC22189 affected in differentiation, mostly comprising of filamentous protonema with reduced number of gametophores, but normal leaf morphology (haploid). C, Transformant BC11280 showing retarded growth, a reduced number of gametophores per moss plant and altered leaf morphology ("drehzipfel" phenotype; twisted tips of leaves, haploid). D, Transformant BC1015 displaying altered growth habitus ("wasserpest" phenotype, reminiscent of the waterweed Elodea) and altered leaf morphology (polyploid). E, Transformant BC22288 showing retarded growth and elongated, narrow leafs (polyploid).
Mentions: To screen for morphological and developmental mutations, transformants were microscopically observed after regeneration and selection on supplemented Knop medium for 11 weeks followed by growth on minimal Knop medium for 8 weeks. Under these conditions, the filamentous structure (protonema) that is initially formed by regenerating wild-type protoplasts undergoes a developmental switch. It forms a three-faced apical meristem bud, and differentiates into leafy stems, the gametophores (Fig. 4A), that will eventually carry the sex organs. 16.2% (2,631 of 16,203) of the gene-disruption library transformants showed morphological deviations in one or more of the features observed: structure and colour of the moss plant, coverage of the plant by gametophores, shape and uniformity of leaves and arrangement of cells within the leaves (Table 1). Such deviations were not observed to occur spontaneously in 350 analysed wild-type plants and in less than 1% of 400 plants derived from protoplasts mock-transformed without DNA and regenerated in the absence of antibiotic. Examples for deviating phenotypes observed in gene-disruption library transformants are shown in Fig. 4b to 4E. In addition to the phenotypic classes described above, we observed other developmental abnormalities, like the formation of outgrowths on the leaf surface, the formation of thread-like appendages on leaf tips, and an increased number of dark-coloured sectors on leaves. Therefore, a wide spectrum of morphological and developmental alterations is observed in moss plants transformed with a gene-disruption library. In addition, between the three parameters we assayed for each transformant – growth requirements, morphology and ploidy level – there did not appear to be a strict correlation, and we found various combinations of characteristics (Fig. 5).

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