Stanford/M4: Day 3 - Moss Datasheet

M4: Day 3 - Moss Datasheet

Moss-ay Group

Uses and Advantages of Physcomitrella Patens

Physcomitrella patens is a useful organism in research because of its high recombination efficiency. This feature allows researchers to easily introduce and knockout genes into the organism. Additionally, the facility at which Physcomitrella Patens is grown enables the organism to function as an efficient bioreactor, cheaply producing complex biopharmaceutical therapeutics.

• a model to explore plant functions
• sequenced and assembled genome, physical and genetic maps, and more than 250,000 expressed sequence tags [ESTs]
• dominant haploid phase that allows direct forward genetic analysis
• can be easily cultured
• spends the majority of its life cycle in the haploid state, allowing the application of experimental techniques similar to those used in microbes and yeast.
• development is relatively simple, and it generates only a few tissues that contain a limited number of cell types
• many signaling pathways 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 all interwoven into distinct but overlapping pathways and linked to clear developmental phenotypes. In addition, abou
• one-quarter of the moss genome contains genes with no known function based on sequence motifs, raising the likelihood of successful discovery efforts to identify new and novel gene functions.

Disadvantages

• Must observe extreme sterility when using it. Contamination can retard moss growth.
• Requires a long period of time for growth (7-14 days).
• State of transformation has not been tested by this lab. Yet.

Culturing Phycomitrella patens

Culturing Moss

Procedure I: Growth of Gametophytes

*Using Petri Dishes Containing Solid Medium

1. ethanol materials to maintain sterility

2. slice moss 'trees'into 3mm pieces, about 1 culture per plate

3. plate moss peices in a grid formation (about 16-25) on BCD medium

4. seal plates with porous tape (ex. surgical)

5. store plates on a 16 hour light/8 hour dark cycle

*Using Petri Dishes Containing Solid Medium Overlaid with Cellophane

1. ethanol everything - important to maintain sterility because moss contains no resistance 2. use BCD plates with autoclaved cellophane overlaid on top 3. make moss smoothie: water + moss 'trees' (plants), blend with hand blender in a beaker (cuisine art) 4. put 1 mL of moss smoothie per plate 5. seal plates with porous tape (ex. surgical) 6. store ~7 days on 16 hour light/8 hour dark cycle

*Using Liquid Medium

Storing Phycomitrella patens

Culturing Moss

Procedure II: Long-Term Storage of Gametophyte Tissue

• Storage on Solid Medium
• Cryopreservation

Transforming Phycomitrella patens

Important references

Moss Protocols: All Moss Protocols

Cove, David J. "The Moss Physcomitrella patens: A Novel Model System for Plant Development and Genomic Studies." Emerging Model Organisms: A Laboratory Manual. 1. (2009): 69-88. Print.

Hohe A, Egener T, Lucht JM, Holtorf H, Reinhard C, Schween G, and Reski R. An improved and highly standardised transformation procedure allows efficient production of single and multiple targeted gene-knockouts in a moss, Physcomitrella patens. Curr Genet 2004 Jan; 44(6) 339-47.

Ralf Reski and Wolfgang Frank (2005) "Moss (Physcomitrella patens) functional genomics – Gene discovery and tool development with implications for crop plants and human health." Briefings in Functional Genomics and Proteomics 4, 48-57.

Schaefer, D. (2002) A new moss genetics: Mutagenesis in Physcomitrella patens. Annu. Rev. Plant Biol. 53: 477–501.