Large‐Scale Analysis of 73 329 Physcomitrella Plants Transformed with Different Gene Disruption Libraries: Production Parameters and Mutant Phenotypes

Abstract

Gene targeting in the moss Physcomitrella patens has created a new platform for plant functional genomics. We produced a mutant collection of 73 329 Physcomitrella plants and evaluated the phenotype of each transformant in comparison to wild type Physcomitrella. Production parameters and morphological changes in 16 categories, such as plant structure, colour, coverage with gametophores, cell shape, etc., were listed and all data were compiled in a database (mossDB). Our mutant collection consists of at least 1804 auxotrophic mutants which showed growth defects on minimal Knop medium but were rescued on supplemented medium. 8129 haploid and 11 068 polyploid transformants had morphological alterations. 9 % of the haploid transformants had deviations in the leaf shape, 7 % developed less gametophores or had a different leaf cell shape. Other morphological deviations in plant structure, colour, and uniformity of leaves on a moss colony were less frequently observed. Preculture conditions of the plant material and the cDNA library (representing genes from either protonema, gametophore or sporophyte tissue) used to transform Physcomitrella had an effect on the number of transformants per transformation. We found correlations between ploidy level and plant morphology and growth rate on Knop medium. In haploid transformants correlations between the percentage of plants with specific phenotypes and the cDNA library used for transformation were detected. The number of different cDNAs present during transformation had no effect on the number of transformants per transformation, but it had an effect on the overall percentage of plants with phenotypic deviations. We conclude that by linking incoming molecular, proteome, and metabolome data of the transformants in the future, the database mossDB will be a valuable biological resource for systems biology.

Differential Requirements for RAD51 inPhyscomitrella patensandArabidopsis thalianaDevelopment and DNA Damage Repair

“…The data suggest that the mechanisms of gene targeting operating in P. patens produce unpredicted and unusual recombination products that cannot be sufficiently discriminated from the desired gene replacement by conventional PCR analysis. In addition, although the gene targeting efficiencies were as high (36% for Pp RAD51A and 75% for Pp RAD51B) as predicted (Kamisugi et al, 2005), such complex, unclear gene targeting events and polyploidization (Schween et al, 2005) had reduced the number of knockout lines useful for phenotypic analysis considerably.…”

The mechanism of gene targeting in Physcomitrella patens: homologous recombination, concatenation and multiple integration

“…Efficient simultaneous delivery of multiple transforming DNAs has previously been used to create a large population of mutant plants (>50 000) by transformation with cDNA clones disrupted by the random insertion of a transposon-borne selection cassette (18,24). For efficient generation of this collection either normalized cDNA libraries, or batches of 7 or 20 disrupted cDNA clones, were delivered simultaneously to protoplasts.…”

The moss Physcomitrella patens: methods and tools from cultivation to targeted analysis of gene function

“…The tagged insertion constructs derived from cDNA libraries were assumed to integrate preferably by HR into transcribed genes and those derived from gDNA will integrate across the whole genome. High-throughput transformation of random and defined pools of cDNA-derived tagged insertion constructs resulted in a mutant collection of 65,198 stably transformed plants that have been grouped into 16 categories according to deviations in development, morphology, physiology, and growth parameters (Schween et al, 2005). Phenotypically catalogued mutants were deposited into a searchable database (mossDB) and cryopreserved for long-term storage.…”