2009; doi: 10.1101/pdb.emo115Cold Spring Harb Protoc  Ralph S. QuatranoDavid J. Cove, Pierre-François Perroud, Audra J. Charron, Stuart F. McDaniel, Abha Khandelwal and Development and Genomic Studies

: A Novel Model System for PlantPhyscomitrella patensThe Moss

ServiceEmail Alerting click here.Receive free email alerts when new articles cite this article -

CategoriesSubject Cold Spring Harbor Protocols.Browse articles on similar topics from

(118 articles)Plant Biology, general (98 articles)Plant

(873 articles)Laboratory Organisms, general (316 articles)Genetics, general

(283 articles)Emerging Model Organisms (558 articles)Developmental Biology

http://cshprotocols.cshlp.org/subscriptions go to: Cold Spring Harbor Protocols To subscribe to

Cold Spring Harbor Laboratory Press at MASSACHUSETTS on October 5, 2012 - Published by http://cshprotocols.cshlp.org/Downloaded from

The Moss Physcomitrella patens: A Novel Model System for PlantDevelopment and Genomic Studies

David J. Cove, Pierre-François Perroud, Audra J. Charron, Stuart F. McDaniel,Abha Khandelwal, and Ralph S. Quatrano1

Department of Biology, Washington University, St. Louis, MO 63130, USA

INTRODUCTION

The moss Physcomitrella patens has been used as an experimental organism for more than 80 years.Within the last 15 years, its use as a model to explore plant functions has increased enormously. Theability to use gene targeting and RNA interference methods to study gene function, the availability ofmany tools for comparative and functional genomics (including a sequenced and assembled genome,physical and genetic maps, and more than 250,000 expressed sequence tags [ESTs]), and a dominanthaploid phase that allows direct forward genetic analysis have all led to a surge of new activity. P.patens can be easily cultured and spends the majority of its life cycle in the haploid state, allowing theapplication of experimental techniques similar to those used in microbes and yeast. Its developmentis relatively simple, and it generates only a few tissues that contain a limited number of cell types.Although mosses lack vascular tissue, true roots/stems/leaves, and flowers and seeds, many signalingpathways found in angiosperms are intact in moss. For example, the phytohormones auxin, cytokinin,and abscisic acid, as well as the photomorphogenic pigments phytochrome and cryptochrome, are allinterwoven into distinct but overlapping pathways and linked to clear developmental phenotypes. Inaddition, about one-quarter of the moss genome contains genes with no known function based onsequence motifs, raising the likelihood of successful discovery efforts to identify new and novel genefunctions.

BACKGROUND INFORMATION

The moss P. patens (Hedw.) Bruch & Schimp was first established as a laboratory experimental systemin the 1920s by Fritz von Wettstein (1924), who studied the effects of ploidy variation and inheritancepatterns in interspecific and intergeneric crosses within the moss family Funariaceae. The modern eraof Physcomitrella research dates to the work of Paulinus Engel (1968), who generated the first bio-chemical and morphological mutants in the species.

Like all land plants, the moss life cycle consists of a multicellular haploid gametophyte genera-tion that alternates with a morphologically distinct diploid sporophyte generation. But unlike vascu-lar plants, the gametophyte (Fig. 1C) is the dominant portion of the moss life cycle. Haploid sporesgerminate to produce a filamentous protonemal stage (Fig. 1D). Protonemata are initially composedof chloronemal cells that are full of large chloroplasts. Chloronemal cells extend by serial division ofthe apical cell, and subapical cells branch to form new apices. Some apical chloronemal cells developinto a second cell type, caulonemata. Caulonemal filaments contain fewer and less-well-developedchloroplasts. But they extend more rapidly than chloronema; the division times of the apical cells ofcaulonema and chloronema are ~6 and 24 h, respectively. The subapical cells of caulonemal fila-ments branch to form more filaments and leafy stems, called “gametophores” (Fig. 1E), on whichgametes are produced. Moss is monoecious: Both male and female gametes are produced on the

© 2009 Cold Spring Harbor Laboratory Press 1 Vol. 4, Issue 2, February 2009

1Corresponding author (rsq@wustl.edu)This article is also available in Emerging Model Organisms: A LaboratoryManual, Vol. 1. CSHL Press, Cold Spring Harbor, NY, USA, 2009.Cite as: Cold Spring Harb. Protoc.; 2009; doi:10.1101/pdb.emo115 www.cshprotocols.org

Emerging Model Organisms

Cold Spring Harbor Laboratory Press at MASSACHUSETTS on October 5, 2012 - Published by http://cshprotocols.cshlp.org/Downloaded from

www.cshprotocols.org 2 Cold Spring Harbor Protocols

same gametophore. Although self-fertilization is common, cross-fertilization can occur when twostrains are grown adjacent to each other. Fertilized zygotes develop into sporophytes that remainattached to the gametophore. Within the sporophyte, spore mother cells give rise to spores meiotically.

P. patens is small, and in nature, the gametophores seldom reach more than 5 mm in height. Itis mostly found on wet soil and, in particular, on sites that are exposed to seasonal flooding, such asthe banks of lakes, ponds, rivers, and drainage ditches. (For a general guide to the mosses of NorthAmerica, see Crum and Anderson 1981.) Although it is distributed widely in the NorthernHemisphere, it is uncommon throughout its range. Natural populations produce spores fromSeptember to March, depending on the locality. Although P. patens itself is restricted to NorthAmerica and Europe, other morphologically similar species are found in Africa, Asia, South America,and Australia. Many variants of P. patens have been given species or subspecies rank, although thedegree to which the morphological features that distinguish these taxa have a genetic basis has not

FIGURE 1. P. patens cultures. (A) Six-day-old P. patens protonemata grown on cellophane over solid BCD medium sup-plemented with diammonium tartrate (for details, see Culturing the Moss Physcomitrella patens [Cove et al. 2008a]).(B) Easy harvesting, with a spatula, of protonemata grown on solid media overlaid with cellophane. (C) Four-week-oldinoculum grown on BCD supplemented with diammonium tartrate. Note the presence of the two major tissues that arecharacteristic of the haploid growth phase of P. patens development: the filamentous protonemata and the leafy game-tophore. (D) P. patens protonemata displaying the characteristic branching pattern. (E) Six-week-old P. patens game-tophores. (F) Three 3-d-old filaments regenerating from protoplast on PRMB medium. (G) Transformation plate after 2wk on antibiotic selection. Transformants surviving selection are easily identified as individual growing plants. (H)Individual spot-inoculums of P. patens strains on a 9-cm Petri dish after 2 wk of growth. Up to 32 independent isolatescan be grown and stored this way. (I) Multiple plates can be easily stored after growth in an incubator with 2 h of lightper day at 10°C.

Cold Spring Harbor Laboratory Press at MASSACHUSETTS on October 5, 2012 - Published by http://cshprotocols.cshlp.org/Downloaded from

been established experimentally. Natural hybrids between closely related species in the familyFunariaceae, similar to those produced in culture by von Wettstein, have been documented in severallocalities (Pettet 1964).

SOURCES AND HUSBANDRY

The strain of P. patens used by Engel was generated from a single spore that was obtained in 1962from a plant in Gransden Wood, Huntingdonshire, England by Dr. H.W.K. Whitehouse. This strain hassince been used by many laboratories, but it is routinely taken through its sexual cycle about everyyear, during which time cultures are re-established from individual spores. Therefore, Gransden strainswill often be identified by the laboratory and the year in which a spore was used to start a new cul-ture (e.g., the material used to sequence the P. patens genome came from the Gransden St. Louis 2004strain). Because of gametophytic haploidy, all such strains differ only as a result of mutation or epige-netic variation. More recently, additional collections of P. patens have been made from Europe andNorth America, and these are now being characterized genetically and morphologically (vonStackelberg et al. 2006). This new collection is curated and distributed from the University of Freiburg,Germany (see http://www.cosmoss.org).

P. patens can be grown on either solid (agar-based) or liquid media (for details, see Culturing theMoss Physcomitrella patens [Cove et al. 2009a] and Fig. 1). Temperatures between 24°C and 26°Care used for routine culture, although little difference in growth rate is observed from 20°C to 26°C.Growth is slower but still satisfactory at 15°C, and this has been used as the permissive temperaturewhen temperature-sensitive mutants are sought. For routine culture, continuous light from fluorescenttubes at an intensity of between 5 and 20 W/m2 is generally satisfactory, although the exact qualityof light is not critical. Many laboratories use intermittent light, most commonly a 16-h light/8-h darkcycle. This regime entrains the cell cycle of chloronemata. Development is slower under intermittentlight regimes; developmental landmarks are achieved in response to the total hours of illuminationexperienced.

RELATED SPECIES

Two additional moss species are currently used for experimental research: Ceratodon purpureus andTortula ruralis. C. purpureus is one of the most common mosses in exposed rock and soil in temperateregions of the Northern and Southern Hemispheres. In the Spring, it is easily recognized by the pur-ple seta that elevates the diploid sporophyte. This species also has a long history in experimental biol-ogy; the term “heterochromatin” was coined for the dark-staining sex chromosomes of this and othermoss species (Heitz 1928). A genetic map of C. purpureus has been constructed, and several naturalisolates have been extensively characterized (McDaniel et al. 2008). Cultures of C. purpureus, isolatedby E. Hartmann in Germany and by D.J. Cove in Austria, are used by several laboratories worldwide,principally to study phototropism and gravitropism. C. purpureus is so abundant that the isolation ofadditional cultures is fairly straightforward; isolates currently in use are available from D.J. Cove(Washington University, St. Louis, MO). All of the experimental procedures that are described for P.patens are also used for C. purpureus with few modifications.

T. ruralis has been used principally to study water stress because it is able to withstand completedesiccation, similar to seeds. Although a modest collection of ESTs is available for T. ruralis (Oliver etal. 2004), it is less amenable to growth in culture than either C. purpureus or P. patens and has notbeen shown to be easily transformable or to undergo efficient gene targeting.

USES OF THE P. PATENS MODEL SYSTEM

The common ancestor of mosses, such as P. patens, and seed plants, such as Arabidopsis thaliana andpines, lived ~480 million years ago (Mya). Comparative studies including members of both of theselineages allow us to infer biological properties of this common ancestor, giving us a richer under-standing of the diversity of plant life. This may have practical value to the extent that understandingdiverse plant systems yields novel solutions to problems in crop breeding, for example.

During the last several years, P. patens has been used as a model to study various components ofcell, developmental, and evolutionary plant biology. Its development is relatively simple, and it gen-erates only a few tissues that contain a limited number of cell types. Although mosses lack vascular

www.cshprotocols.org 3 Cold Spring Harbor Protocols

Cold Spring Harbor Laboratory Press at MASSACHUSETTS on October 5, 2012 - Published by http://cshprotocols.cshlp.org/Downloaded from

www.cshprotocols.org 4 Cold Spring Harbor Protocols

tissue, true roots/stems/leaves, and flowers and seeds, many signaling pathways found inangiosperms are intact in moss. For example, the phytohormones auxin, cytokinin, and abscisic acid,as well as the photomorphogenic pigments phytochrome and cryptochrome, are all interwoven intodistinct but overlapping pathways and linked to clear developmental phenotypes (Quatrano et al.2007; Rensing et al. 2008).

RNA interference (RNAi) methods (Bezanilla et al. 2005) have been used to analyze the role ofARPC1 (Harries et al. 2005), a member of the Arp2/3 complex, and profilin (Vidali et al. 2007) in tipgrowth. Khandelwal et al. (2007) studied the role of the P. patens presenilin protein using RNAi.Presenilin possesses γ-secretase activity, is involved in Alzheimer’s disease (AD), and is an intermediatein the NOTCH signaling pathway of animal cells; however, unlike animal cells, P. patens and otherplants do not possess this pathway, although the protein is present. The observed mutant phenotypeindicated a possible role for presenilin that is independent of γ-secretase activity and the NOTCH path-way, thus raising the possibility of using P. patens as a novel system for studying the off-target effectsof AD therapy and drug discovery.

Targeted gene deletion and replacement methods have been used to study the role of anothermember of the Arp2/3 complex, ARPC4 (Perroud and Quatrano 2006), and BRICK1, a member of theScar/Wave family (Perroud and Quatrano 2008). As in the other papers referenced above, the excel-lent cell biology of P. patens was used to localize ARPC4 and BRICK1 in growing tip filaments.Transcriptome (Nishiyama et al. 2003; Cuming et al. 2007) and metabolic (Thelander et al. 2005;Kaewsuwan et al. 2006; Schulte et al. 2006) studies, as well as detailed analyses of microRNAs (Axtellet al. 2006, 2007), have now appeared using P. patens. Finally, comparative genomics studies haveelucidated the role of the transcriptional regulators LEAFY (Maizel et al. 2005) and ABI3 (Marella et al.2006) and transcription factors involved in rooting function (Menand et al. 2007) in P. patens as wellas in A. thaliana.

GENETICS, GENOMICS, AND ASSOCIATED RESOURCES

P. patens is amenable to classical genetics studies, with the haploidy of the gametophyte allowingstraightforward analysis (Cove 2005). Efforts to identify polymorphisms among isolates of P. patens areproceeding and will enable map-based cloning of both ethyl methanesulfonate (EMS)- and ultravio-let (UV)-generated mutants (for a method to generate such mutants, see Chemical and UVMutagenesis of Spores and Protonemal Tissue from the Moss Physcomitrella patens [Cove et al.2009b]), as well as quantitative trait locus (QTL) mapping of natural variants (von Stackelberg et al.2006). Several mutant strains are available from different laboratories (http://www.cosmoss.org;http://biology4.wustl.edu/moss), including those having requirements for the vitamins p-aminoben-zoic acid, nicotinic acid, and thiamine. Vitamin requirements have been exploited to increase the fre-quency of cross-fertilization. When two complementary p-aminobenzoic acid or nicotinic acidauxotrophs are grown together on a medium with only a limited level of supplementation, the sporo-phytes produced are the result of cross-fertilization (Courtice and Cove 1978).

No universally accepted system of gene nomenclature has been adopted, but there has been gen-eral agreement that annotated genes will be identified by numbers. Trivial names can then be addedas synonyms. Originally, the system used for trivial names was similar to that used for many bacteriaand fungi: Each symbol was composed of a three-letter lowercase code to designate the mutant genefamily, an uppercase letter to designate the family member, and a number to designate the allele (e.g.,pabA4). More recently, some laboratories have adopted the system used by the yeast and A. thalianacommunities, designating the family member by a number rather than by an uppercase letter (e.g.,pab1-4). But no general agreement has yet been reached as to which system should be adopted.

The assembled P. patens genome (~487 Mb), representing eightfold coverage, has been releasedby the Joint Genome Institute (http://shake.jgi-psf.org/Phypa1/Phypa1.home.html; Rensing et al.2008). In parallel, sequences of full-length cDNAs, additional ESTs, and bacterial artificial chromosome(BAC) ends are being developed, and updates can be accessed through the Physcomitrella GenomeConsortium website (http://www.mossgenome.org). Various libraries and vectors are available (seelinks at http://biology4.wustl.edu/moss/links.html), as is an Agilent microarray (MO gene;http://www.mogene.com), which contains 41,382 features (~28,000 gene models) based on all of theopen reading frames (ORFs) in the draft genome (Fig. 2).

Several tools are available for the functional analysis of genes in P. patens. For example, the dex-amethasone (Chakhparonian 2001), heat-shock (Saidi et al. 2005), and homoserine-lactone (You etal. 2006) inducible promoter systems have all been successfully used in this system. Forward genetics

Cold Spring Harbor Laboratory Press at MASSACHUSETTS on October 5, 2012 - Published by http://cshprotocols.cshlp.org/Downloaded from

can be used to dissect gene function using a shuttle-mutagenesis library (Nishiyama et al. 2000;Hayashida et al. 2005). A targeted deletion library that was created using ESTs (Schween et al. 2005)has also been used for functional analysis (Schulte et al. 2006). Transformation can be performed viapolyethylene glycol (PEG)-mediated DNA uptake by isolated protoplasts (see Transformation of theMoss Physcomitrella patens Using Direct DNA Uptake by Protoplasts [Cove et al. 2009c]), viaAgrobacterium (see Transformation of the Moss Physcomitrella patens Using T-DNA Mutagenesis[Cove et al. 2009d]), or via a gene gun (see Transformation of Moss Physcomitrella patensGametophytes Using a Biolistic Projectile Delivery System [Cove et al. 2009e]), and somatichybridization has been used to analyze mutants genetically (see Somatic Hybridization in the MossPhyscomitrella patens Using PEG-Induced Protoplast Fusion [Cove et al. 2009f; Cove and Quatrano2006]). Reverse genetics using gene targeting is a tool of choice for manipulating individual genes inP. patens, and RNAi allows the down-regulation of gene families. An RNAi system has been developedin P. patens that silences the nucleus-localized green fluorescent protein:: β-glucuronidase (GFP::GUS)fusion protein at the same time that it silences the gene(s) of interest (Bezanilla et al. 2005).

TECHNICAL APPROACHES

P. patens can be easily cultured as described in Culturing the Moss Physcomitrella patens (Cove et al.2009a). A method for isolating protoplasts is given in Isolation and Regeneration of Protoplasts ofthe Moss Physcomitrella patens (Cove et al. 2009g). Other techniques for manipulating P. patens inthe laboratory include Somatic Hybridization in the Moss Physcomitrella patens Using PEG-InducedProtoplast Fusion (Cove et al. 2009f) and Chemical and UV Mutagenesis of Spores andProtonemal Tissue from the Moss Physcomitrella patens (Cove et al. 2009b). For isolating nucleicacids and protein from P. patens tissue, see Isolation of DNA, RNA, and Protein from the MossPhyscomitrella patens Gametophytes (Cove et al. 2009h).

www.cshprotocols.org 5 Cold Spring Harbor Protocols

FIGURE 2. P. patens microarray. (A) Layout of the P. patens 4 × 44K Agilent microarray. (B) Photograph of a scanned microarray slidediagrammed in A. (C) Enlarged image of a single array showing differential gene expression. (D) Differential expression of P. patens tran-scriptome showing those genes that are up-regulated (red) and those that are down-regulated (green). The genes within the diagonalred lines are not significantly changed (blue). (For color figure, see doi: 10.1101/pdb.emo115 online at www.cshprotocols.org.)

Cold Spring Harbor Laboratory Press at MASSACHUSETTS on October 5, 2012 - Published by http://cshprotocols.cshlp.org/Downloaded from

Axtell, M., Jan, C., Rajagopalan, R., and Bartel, D. 2006. A two-hittrigger for siRNA biogenesis in plants. Cell 127: 565–577.

Axtell, M.J., Snyder, J.A., and Bartel, D.P. 2007. Common functionsfor diverse small RNAs of land plants. Plant Cell 19: 1750–1769.

Bezanilla, M., Perroud, P.-F., Pan, A., Klueh, P., and Quatrano, R.S.2005. An RNAi system in Physcomitrella patens with an internalmarker for silencing allows for rapid identification of loss of func-tion phenotypes. Plant Biol. 7: 251–257.

Chakhparonian, M. 2001. Développement d’outils de la mutagenèseciblée par recombinaison homologue chez Physcomitrella patens.Ph.D. Thesis, Université de Lausanne, Lausanne, Switzerland.

Courtice, G.R.M. and Cove, D.J. 1978. Evidence for the restrictedpassage of metabolites into the sporophyte of the moss,Physcomitrella patens. J. Bryol. 10: 191–198.

Cove, D.J. 2005. The moss Physcomitrella patens. Annu. Rev. Genet.39: 339–358.

Cove, D.J. and Quatrano, R.S. 2006. Agravitropic mutants of themoss Ceratodon purpureus do not complement mutants having areversed gravitropic response. Plant Cell Environ. 29: 1379–1387.

Cove, D.J., Perroud, P.-F., Charron, A.J., McDaniel, S.F., Khandelwal,A., and Quatrano, R.S. 2009a. Culturing the moss Physcomitrellapatens. Cold Spring Harb. Protoc. (this issue). doi: 10.1101/pdb.prot5136.

Cove, D.J., Perroud, P.-F., Charron, A.J., McDaniel, S.F., Khandelwal,A., and Quatrano, R.S. 2009b. Chemical and UV mutagenesis ofspores and protonemal tissue from the moss Physcomitrellapatens. Cold Spring Harb. Protoc. (this issue). doi: 10.1101/pdb.prot5142.

Cove, D.J., Perroud, P.-F., Charron, A.J., McDaniel, S.F., Khandelwal,A., and Quatrano, R.S. 2009c. Transformation of the mossPhyscomitrella patens using direct DNA uptake by protoplasts.Cold Spring Harb. Protoc. (this issue). doi: 10.1101/pdb.prot5143.

Cove, D.J., Perroud, P.-F., Charron, A.J., McDaniel, S.F., Khandelwal,A., and Quatrano, R.S. 2009d. Transformation of the mossPhyscomitrella patens using T-DNA mutagenesis. Cold Spring Harb.Protoc. (this issue). doi: 10.1101/pdb.prot5144.

Cove, D.J., Perroud, P.-F., Charron, A.J., McDaniel, S.F., Khandelwal,A., and Quatrano, R.S. 2009e. Transformation of mossPhyscomitrella patens gametophytes using a biolistic projectiledelivery system. Cold Spring Harb. Protoc. (this issue). doi:10.1101/pdb.prot5145.

Cove, D.J., Perroud, P.-F., Charron, A.J., McDaniel, S.F., Khandelwal,A., and Quatrano, R.S. 2009f. Somatic hybridization in the mossPhyscomitrella patens using PEG-induced protoplast fusion. ColdSpring Harb. Protoc. (this issue). doi: 10.1101/pdb.prot5141.

Cove, D.J., Perroud, P.-F., Charron, A.J., McDaniel, S.F., Khandelwal,A., and Quatrano, R.S. 2009g. Isolation and regeneration of pro-toplasts of the moss Physcomitrella patens. Cold Spring Harb.Protoc. (this issue). doi: 10.1101/pdb.prot5140.

Cove, D.J., Perroud, P.-F., Charron, A.J., McDaniel, S.F., Khandelwal,A., and Quatrano, R.S. 2009h. Isolation of DNA, RNA, and proteinfrom the moss Physcomitrella patens gametophytes. Cold SpringHarb. Protoc. (this issue). doi: 10.1101/pdb.prot5146.

Crum, H.A. and Anderson, L.E. 1981. Mosses of eastern NorthAmerica, Vol. 1 and 2. Columbia University Press, New York.

Cuming, A.C., Cho, S.H., Kamisugi, Y., Graham, H., and Quatrano,R.S. 2007. Microarray analysis of transcriptional responses toabscisic acid and osmotic, salt, and drought stress in the moss,Physcomitrella patens. New Phytol. 176: 275–287.

Engel, P.P. 1968. The induction of biochemical and morphologicalmutants in the moss, Physcomitrella patens. Am. J. Bot. 55: 438–446.

Harries, P., Pan, A., and Quatrano, R.S. 2005. Actin-related protein2/3 complex component ARPC1 is required for proper cell mor-phogenesis and polarized cell growth in Physcomitrella patens.Plant Cell 17: 2327–2339.

Hayashida, A., Takechi, K., Sugiyama, M., Kubo, M., Itoh, R.D., Takio,S., Fujita, T., Hiwatashi, Y., Hasebe, M., and Takano, H. 2005.Isolation of mutant lines with decreased numbers of chloroplastsper cell from a tagged mutant library of the moss Physcomitrellapatens. Plant Biol. 7: 300–306.

Heitz, E. 1928. Das Heterochromatin der Moose. I. Jahrb. Wiss. Bot.69: 762–818.

Kaewsuwan, S., Cahoon, E.B., Perroud, P.-F., Wiwat, C., Panvisavas,N., Quatrano, R.S., Cove, D.J., and Bunyapraphatsara, N. 2006.Identification and functional characterization of the mossPhyscomitrella patens ∆5-desaturase gene involved in arachidonicand eicosapentaenoic acids biosynthesis. J. Biol. Chem. 281:21988–21997.

Kamisugi, Y., Cuming, A.C., and Cove, D.J. 2005. Parameters deter-mining the efficiency of gene targeting in the moss Physcomitrellapatens. Nucleic Acids Res. 33: e173. doi: 10.1093/nar/gni172.

www.cshprotocols.org 6 Cold Spring Harbor Protocols

Among the methods for transformation of P. patens are Transformation of the MossPhyscomitrella patens Using Direct DNA Uptake by Protoplasts (Cove et al. 2009c), Transformationof the Moss Physcomitrella patens Using T-DNA Mutagenesis (Cove et al. 2009d), andTransformation of Moss Physcomitrella patens Gametophytes Using a Biolistic Projectile DeliverySystem (Cove et al. 2009e). P. patens and C. purpureus have a high frequency of gene targeting(Kamisugi et al. 2005, 2006); when a transforming construct contains a genomic sequence, the con-struct is targeted to the corresponding sequence in the genome. This can be exploited to knock outor modify a gene.

For deletion or disruption, aim to replace the coding sequence with a selection cassette and toborder this on each side by ~1000 bp of genomic sequence. Linear DNA fragments generated by poly-merase chain reaction (PCR) give the highest rates of targeting. It is convenient to perform severaltransformations at the same time (10 is not difficult). For each experiment, make sure to include aminus DNA control to assess protoplast viability.

Figure 1G shows a plate of transformants that have been growing for 2 wk on selective media.Following transformation, the regenerants are of three types:

• Transient: These do not retain resistance upon subculture.

• Unstable: These exhibit slow growth on selective medium. Resistance is probably not transmit-ted through meiosis and is rapidly lost when selection is relaxed.

• Stable: These grow on selective medium almost as fast as on nonselective medium. Resistance istransmitted regularly through meiosis and is retained even when selection is absent.

REFERENCES

Cold Spring Harbor Laboratory Press at MASSACHUSETTS on October 5, 2012 - Published by http://cshprotocols.cshlp.org/Downloaded from

Kamisugi, Y., Schlink, K., Rensing, S.A., Schween, G., vonStackelberg, M., Cuming, A.C., Reski, R., and Cove, D.J. 2006.The mechanism of gene targeting in Physcomitrella patens:Homologous recombination, concatenation and multiple integra-tion. Nucleic Acids Res. 34: 6205–6214.

Khandelwal, A., Chandu, D., Roe, C.M., Kopan, R., and Quatrano,R.S. 2007. Moonlighting activity of presenilin in plants is inde-pendent of γ-secretase and evolutionarily conserved. Proc. Natl.Acad. Sci. 104: 13337–13342.

Maizel, A., Busch, M.A., Tanahashi, T., Perkovic, J., Kato, M.,Hasebe, M., and Weigel, D. 2005. The floral regulator LEAFYevolves by substitutions in the DNA binding domain. Science308: 260–263.

Marella, H.H., Sakata, Y., and Quatrano, R.S. 2006. Characterizationand functional analysis of ABSCISIC ACID INSENSITIVE3-likegenes from Physcomitrella patens. Plant J. 46: 1032–1044.

McDaniel, S.F., Willis, J.H., and Shaw, A.J. 2008. The genetic basis ofabnormal development in interpopulation hybrids of the mossCeratodon purpureus. Genetics 179: 1425–1435.

Menand, B., Yi, K.K., Jouannic, S., Hoffmann, L., Ryan, E., Linstead, P.,Schaefer, D.G., and Dolan, L. 2007. An ancient mechanism con-trols the development of cells with a rooting function in landplants. Science 316: 1477–1480.

Nishiyama, T., Hiwatashi, Y., Sakakibara, I., Kato, M., and Hasebe, M.2000. Tagged mutagenesis and gene-trap in the moss,Physcomitrella patens, by shuttle mutagenesis. DNA Res. 7: 9–17.

Nishiyama, T., Fujita, T., Shin-I, T., Seki, M., Nishide, H., Uchiyama, I.,Kamiya, A., Carninci, P., Hayashizaki, Y., Shinozaki, K., et al. 2003.Comparative genomics of Physcomitrella patens gametophytictranscriptome and Arabidopsis thaliana: Implication for land plantevolution. Proc. Natl. Acad. Sci. 100: 8007–8012.

Oliver, M.J., Velten, J., and Mishler, B.D. 2004. Desiccation tolerancein bryophytes: A reflection of the primitive strategy for plant sur-vival in dehydrating habitats? Integr. Comp. Biol. 45: 788–799.

Perroud, P.-F. and Quatrano, R.S. 2006. The role of ARPC4 in tipgrowth and alignment of the polar axis in filaments ofPhyscomitrella patens. Cell Motil. Cytoskelet. 63: 162–171.

Perroud, P.-F. and Quatrano, R.S. 2008. BRICK1 is required for apicalcell growth in filaments of the moss Physcomitrella patens but notfor gametophore morphology. Plant Cell 20: 411–422.

Pettet, A. 1964. Hybrid sporophytes in the Funariaceae. Trans. Br.Bryol. Soc. 4: 642–648.

Quatrano, R.S., McDaniel, S.F., Khandelwal, A., Perroud, P.-F., andCove, D.J. 2007. Physcomitrella patens: Mosses enter the genomicage. Curr. Opin. Plant Biol. 10: 182–189.

Rensing, S.A., Lang, D., Zimmer, A., Terry, A., Salamov, A., Shapiro,H., Nishiyama, T., Perroud, P.-F., Lindquist, E.A., Kamisugi, Y., etal. 2008. The Physcomitrella genome reveals insights into the con-quest of land by plants. Science 319: 64–69.

Saidi, Y., Finka, A., Chakhporanian, M., Zryd, J.P., Schaefer, D.G., andGoloubinoff, P. 2005. Controlled expression of recombinantproteins in Physcomitrella patens by a conditional heatshockpromoter: A tool for plant research and biotechnology. Plant Mol.Biol. 59: 697–711.

Schulte, J., Erxleben, A., Schween, G., and Reski, R. 2006. Highthroughput metabolic screen of Physcomitrella transformants.Bryologist 109: 247–256.

Schween, G., Egener, T., Fritzowsky, D., Granado, J., Guitton, M.C.,Hartmann, N., Hohe, A., Holtorf, H., Lang, D., Lucht, J.M., et al.2005. Large-scale analysis of 73,329 Physcomitrella plants trans-formed with different gene disruption libraries: Productionparameters and mutant phenotypes. Plant Biol. 7: 228–237.

Thelander, M., Olsson, T., and Ronne, H. 2005. Effect of the energysupply on filamentous growth and development in Physcomitrellapatens. J. Exp. Bot. 56: 653–662.

Vidali, L., Augustine, R.C., Kleinman, K.P., and Bezanilla, M. 2007.Profiling is essential for tip growth in the moss Physcomitrellapatens. Plant Cell 19: 3705–3722.

von Stackelberg, M., Rensing, S., and Reski, R. 2006. Identification ofgenic moss SSR markers and a comparative analysis of twenty-four algal and plant gene indices reveal species-specific ratherthan group-specific characteristics of microsatellites. BMC PlantBiol. 6: 9. doi: 10.1186/1471-2229-6-9.

von Wettstein, F. 1924. Morphologie und Physiologie desFormwechsels der Moose auf genetischler Grundlage I. Z. Indukt.Abstammungs-Vererbungesl. 33: 1–236.

You, Y.S., Marella, H.H., Zentella, R., Zhou, Y., Ulmasov, T., Ho, T.-H.D., and Quatrano, R.S. 2006. Use of bacterial quorum sensingcomponents to regulate gene expression in plants. Plant Physiol.140: 1205–1212.

www.cshprotocols.org 7 Cold Spring Harbor Protocols

Cold Spring Harbor Laboratory Press at MASSACHUSETTS on October 5, 2012 - Published by http://cshprotocols.cshlp.org/Downloaded from