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.

Advantages:

• Is a model to explore plant functions.
• Has a sequenced and assembled genome, physical and genetic maps, and more than 250,000 expressed sequence tags [ESTs].
• Has a 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.
• Its development is relatively simple, and it generates only a few tissues that contain a limited number of cell types
• Has many signaling pathways found in angiosperms are intact in moss (phytohormones auxin, cytokinin, and abscisic acid, etc)
• 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 3mm 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: 10 ml water + 200mg moss 'trees' (plants), blend with hand blender in a beaker (cuisine art). May need to use more water in order to blend, in which case centrifuging moss particles and re-suspending them in water to get the desired concentration is recommended.

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

Storing on solid medium can keep cultures in a healthy state for a considerable period of time (at least 3 years). Grow cultures in an incubator at 25 C for 3 weeks then transfer to an incubator at 10 C with a 2-hour light/22-hour dark cycle with white light intensities between 5 and 20 Wm^-2.

• Cryopreservation

1. For each strain to be preserved, transfer 100 mg of tissue onto a Petri dish overlaid with fresh cellophane. Pipette 1 mL of appropriately supplemented liquid BCD containing 500 mM of mannitol onto the surface of the tissue.

2. Incubate the culture for an additional 7 d at 25°C under a 16-h light/8-h dark cycle (with white light at intensities between 5 and 20 W/m2).

3. Add 1.5 mL of DMSO-glucose solution to 2 mL sterile plastic vials. Then add one-tenth of the tissue on the plate to each vial. Incubate the vials for 1 h at 20°C.

4. Freeze the vials at a rate of 1°C per minute to –35°C using a controlled low-temperature ethanol bath.

5. Place the vials in liquid nitrogen for storage. It is advisable to freeze multiple aliquots of tissue.

Recovery from cryopreservation is usually good for vigorous strains, but some mutant strains recover poorly. To thaw the cultures, proceed with Steps 23-25. Successful recovery has been achieved after 10 yr of storage; longer storage periods may be possible.

To thaw Samples: 1. Retrieve the vials from the liquid nitrogen, and place them in a water bath at 30°C until thawed.

2.. After thawing, add tissue from one vial to 10 mL of H2O. Allow the mixture to stand for 30 min at room temperature.

3. Inoculate the tissue suspension onto appropriate solid medium (use 1-2 mL of the tissue suspension per plate).

Transforming Phycomitrella patens

Important references

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.

Decker, EL. "Moss – An Innovative Tool for Protein Production." Bioforum 2003 (2003): 2-3. Web. 18 May 2010. <http://www.plant-biotech.net/paper/Bioforum_2003.pdf>.

Decker, EL and Reski, R. (2007): Moss bioreactors producing improved biopharmaceuticals. Current Opinion in Biotechnology 18, 393-398.

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.

Murén E., Nilsson A., Ulfstedt M., Johansson M., Ronne H. (2009). Rescue and characterization of episomally replicating DNA from the moss Physcomitrella. Proc. Natl. Acad. Sci. USA 106, 19444-19449.

Reski, R. and Frank, W. (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.