3
The Moss Physcomitrella patens
A Novel Model System for Plant Development
and Genomic Studies
David J. Cove, Pierre-François Perroud, Audra J. Charron,
Stuart F. McDaniel, Abha Khandelwal, and Ralph S. Quatrano
Department of Biology, Washington University, St. Louis, Missouri 63130
ABSTRACT
The moss Physcomitrella patens has been used
PROTOCOLS
as an experimental organism for more than
80 years. Within the last 15 years, its use as a 1 Culturing the Moss Physcomitrella patens, 75
model to explore plant functions has 2 Isolation and Regeneration of Protoplasts, 80
3 Somatic Hybridization in P. patens Using PEGincreased enormously. The ability to use gene
induced Protoplast Fusion, 82
targeting and RNA interference methods to 4 Chemical and UV Mutagenesis of Spores and
study gene function, the availability of many
Protonemal Tissue, 84
tools for comparative and functional 5 Transformation Using Direct DNA Uptake by
genomics (including a sequenced and assemProtoplasts, 87
bled genome, physical and genetic maps, and 6 Transformation Using T-DNA Mutagenesis, 89
>250,000 expressed sequence tags), and a 7 Transformation of Gametophytes Using a
dominant haploid phase that allows direct
Biolistic Projectile Delivery System, 91
forward genetic analysis have all led to a surge 8 Isolation of DNA, RNA, and Protein from P.
patens Gametophytes, 93
of new activity. P. patens can be easily cultured and 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. Although mosses lack vascular tissue, true
roots/stems/leaves, and flowers and seeds, 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, about 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. The methods outlined in this chapter will enhance the use of the P. patens model system in many laboratories throughout the world.
This chapter, with full-color images, can be found online at www.cshprotocols.org/emo.
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Chapter 3
BACKGROUND INFORMATION
The moss P. patens (Hedw.) Bruch & Schimp was first established as a laboratory experimental system in the 1920s by Fritz von Wettstein (1924), who studied the effects of ploidy variation and
inheritance patterns in interspecific and intergeneric crosses within the moss family Funariaceae.
The modern era of Physcomitrella research dates to the work of Paulinus Engel (1968), who generated the first biochemical and morphological mutants in the species.
Like all land plants, the moss life cycle consists of a multicellular haploid gametophyte generation that alternates with a morphologically distinct diploid sporophyte generation. But unlike
vascular plants, the gametophyte (Fig. 1C) is the dominant portion of the moss life cycle. Haploid
spores germinate to produce a filamentous protonemal stage (Fig. 1D). Protonemata are initially
AU: Changed “at 8°C”
to “at 10°C” because
that’s the temp used in
Protocol 1. OK?
FIGURE 1. P. patens cultures. (A) Six-day-old P. patens protonemata grown on cellophane over solid BCD medium
supplemented with diammonium tartrate. (B) Easy harvesting, with a spatula, of protonemata grown on solid media
overlaid with cellophane. (C) Four-week-old inoculum grown on BCD supplemented with diammonium tartrate.
Note the presence of the two major tissues that are characteristic of the haploid growth phase of P. patens development: the filamentous protonemata and the leafy gametophore. (D) P. patens protonemata displaying the characteristic branching pattern. (E) Six-week-old P. patens gametophores. (F) Three 3-day-old filaments regenerating
from protoplast on PRMB medium. (G) Transformation plate after 2 weeks 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 weeks’ growth. Up to 32 independent isolates can be grown and stored this way.
(I) Multiple plates can be easily stored after growth in an incubator with 2 hours of light per day at 10°C.
The Moss Physcomitrella patens
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71
composed of chloronemal cells that are full of large chloroplasts. Chloronemal cells extend by
serial division of the apical cell, and subapical cells branch to form new apices. Some apical
chloronemal cells develop into a second cell type, caulonemata. Caulonemal filaments contain
fewer and less-well-developed chloroplasts. But they extend more rapidly than chloronema; the
division times of the apical cells of caulonema and chloronema are about 6 and 24 hours, respectively. The subapical cells of caulonemal filaments branch to form more filaments and leafy stems,
called gametophores (Fig. 1E), on which gametes are produced. Moss is monoecious: Both male
and female gametes are produced on the same gametophore. Although self-fertilization is common, cross-fertilization can occur when two strains are grown adjacent to each other. Fertilized
zygotes develop into sporophytes that remain attached 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. It
is mostly found on wet soil and, in particular, on sites that are exposed to seasonal flooding, such
as the banks of lakes, ponds, rivers, and drainage ditches (Crum and Anderson 1981). Although it
is distributed widely in the northern hemisphere, it is uncommon throughout its range. Natural
populations produce spores from September to March, depending on the locality. Although P.
patens itself is restricted to North America 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 the degree to which the morphological features that distinguish these taxa have a genetic basis has not been established experimentally. Natural hybrids
between closely related species in the family Funariaceae, similar to those produced in culture by
von Wettstein, have been documented in several localities (Pettet 1964).
SOURCES AND HUSBANDRY
The strain of P. patens used by Engel was generated from a single spore that was obtained in 1962
from a plant in Gransden Wood, Huntingdonshire, England by Dr. H.W.K. Whitehouse. This
strain has since been used by many laboratories, but it is routinely taken through its sexual cycle
about every year, during which time cultures are reestablished from individual spores. Therefore,
Gransden strains will often be identified by the laboratory and the year in which a spore was used
to start a new culture (e.g., the material used to sequence the P. patens genome came from the
Gransden St. Louis 2004 strain). Because of gametophytic haploidy, all such strains differ only as
a result of mutation or epigenetic variation. More recently, additional collections of P. patens have
been made from Europe and North America, and these are now being genetically and morphologically characterized (von Stackelberg 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 Protocol
1). Temperatures between 24°C and 26°C are 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 temperature when temperature-sensitive mutants are sought. For
routine culture, continuous light from fluorescent tubes at an intensity of between 5 and 20 Wm–2
is generally satisfactory, although the exact quality of light is not critical. Many laboratories use
intermittent light, most commonly a 16-hour light/8-hour dark cycle. This regime entrains the cell
cycle of chloronemata. Development is slower under intermittent light regimes; developmental
landmarks are achieved in response to the total hours of illumination experienced.
RELATED SPECIES
Two additional moss species are currently used for experimental research: Ceratodon purpureus and
Tortula ruralis. C. purpureus is one of the most common mosses in exposed rock and soil in temperate regions of the northern and southern hemispheres. In the spring, it is easily recognized by
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Chapter 3
the purple seta that elevates the diploid sporophyte. This species also has a long history in experimental biology; the term “heterochromatin” was coined for the dark-staining sex chromosomes of
this and other moss species (Heitz 1928). A genetic map of C. purpureus has been constructed, and
several natural isolates have been extensively characterized (for review, see McDaniel et al. 2008).
Cultures of C. purpureus, isolated by E. Hartmann in Germany and by D.J. Cove in Austria, are used
by several labs worldwide, principally to study phototropism and gravitropism. C. purpureus is so
abundant that the isolation of additional cultures is fairly straightforward; isolates currently in use
are available from D.J. Cove (Washington University, St. Louis). 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 complete desiccation, similar to seeds. Although a modest collection of expressed sequence tags (ESTs)
is available for T. ruralis (Oliver et al. 2004), it is less amenable to growth in culture than either C.
purpureus or P. patens and has not been shown to be easily transformable or to undergo efficient
gene targeting.
USES OF THE P. PATENS MODEL SYSTEM
AU: Verify ref.
The common ancestor of mosses, such as P. patens, and seed plants, such as Arabidopsis thaliana,
pines, and other species, lived approximately 480 million years ago (Mya). Comparative studies
including members of both of these lineages allow us to infer biological properties of this common ancestor, giving us a richer understanding of the diversity of plant life. This may have practical value to the extent that understanding diverse 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
of cell, developmental, and evolutionary plant biology. Its development is 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 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
(Quatrano et al. 2007; Rensing et al. 2008).
RNA interference (RNAi) methods (Bezanilla et al. 2005) have been used to analyze the role of
ARPC1 (Harries et al. 2005), a member of the Arp2/3 complex, and profilin (Vidali et al. 2007) in
tip growth. 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 intermediate in the NOTCH signaling pathway of animal cells; however, unlike animal cells, P. patens
and other plants do not contain this pathway although the protein is present. The observed mutant
phenotype indicates a possible role for presenilin that is independent of γ-secretase activity and
the NOTCH pathway, thus raising the possibility of using P. patens as a novel system for studying
the off-target effects of AD therapy and drug discovery.
Targeted gene deletion and replacement methods have been used to study the role of another
member of the Arp2/3 complex, ARPC4 (Perroud and Quatrano 2006), and BRICK1, a member of
the Scar/Wave family (Perroud and Quatrano 2008). Like the other papers referenced above, the
excellent 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 studies (Thelander et al.
2005; Kaewsuwan et al. 2006; Schulte et al. 2006), as well as detailed analyses of microRNAs (Axtell
et al. 2006, 2007), have now appeared using P. patens. Finally, comparative genomic studies have elucidated the role of the transcriptional regulators LEAFY (Maizel et al. 2005), ABI3 (Marella et al.
2006), and transcription factors involved in rooting function (Menand et al. 2007) in P. patens as
well as in A. thaliana.
The Moss Physcomitrella patens
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73
GENETICS, GENOMICS, AND ASSOCIATED RESOURCES
P. patens is amenable to classical genetics studies, with the haploidy of the gametophyte allowing
straightforward analysis (Cove 2005). Efforts to identify polymorphisms among isolates of P. patens
are proceeding and will enable map-based cloning of both ethylmethanesulfonate (EMS)- and UVgenerated mutants (for a method to generate such mutants, see Protocol 4), as well as quantitative
trait locus (QTL) mapping of natural variants (von Stackelberg et al. 2006). A number of mutant
strains are available from different laboratories (http://www.cosmoss.org; http://biology4.wustl.
edu/moss), including those having requirements for the vitamins p-aminobenzoic acid, nicotinic
acid, and thiamine. Vitamin requirements have been exploited to increase the frequency of crossfertilization. When two complementary p-aminobenzoic acid or nicotinic acid auxotrophs are
grown together on a medium with only a limited level of supplementation, the sporophytes 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
general agreement that annotated genes will be identified by numbers. Trivial names can then be
added as synonyms. Originally, the system used for trivial names was similar to that used for many
bacteria and fungi: Each symbol was composed of a three-letter lowercase code to designate the
mutant gene family, 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. thaliana communities, 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 eight times coverage, has been
released by 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 Genome Consortium website (http://www.mossgenome.org). Various libraries and
vectors are available (see links at http://biology4.wustl.edu/moss/links.html), as is an Agilent
microarray (MO gene), which contains 44,000 features (~28,000 gene models) based on all of the
open 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 dexamethasone- (Chakhparonian 2001), heat-shock- (Saidi et al. 2005), and homoserine-lactone(You et al. 2006) inducible promoter systems have all been successfully used in this system.
Forward genetics 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 via polyethylene glycol (PEG)–mediated DNA uptake by isolated protoplasts (see Protocol 5), via Agrobacterium (see Protocol 6), or via a gene gun (see
Protocol 7), and somatic hybridization has been used to analyze mutants genetically (see Protocol
3) (Cove and Quatrano 2006). Reverse genetics using gene targeting is a tool of choice for manipulating individual genes in P. patens, and RNAi allows the down-regulation of gene families. An
RNAi system has been developed in 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
The following eight protocols outline techniques for manipulating P. patens in the laboratory and
include three transformation methods (Protocols 5–7). P. patens (and C. purpureus) have a high
frequency of gene targeting (Kamisugi et al. 2005, 2006); when a transforming construct contains
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Chapter 3
A
Array Layout
Array 1
Array 2
Array 3
Array 4
design : 41,382 features
~28,000 unique genes
60-mer oligos
B
C
FIGURE 2. P. patens microarray. (A) Layout of the P. patens 4 x 44K Agilent microarray. (B) Photograph of a scanned
microarray slide diagrammed in A. (C) Enlarged image of a single array showing differential gene expression.
a genomic sequence, the construct is targeted to the corresponding sequence in the genome. This
can be exploited to knock out or modify a gene.
For knockout, aim to replace the coding sequence with a selection cassette and to border this
on each side by about 1000 bp of genomic sequence. Linear DNA fragments generated by polymerase chain reaction (PCR) give the highest rates of targeting. It is convenient to perform a number of transformations at the same time (ten is not difficult). For each experiment, make sure to
include a minus DNA control to assess protoplast viability.
Figure 1G shows a plate of transformants that have been growing for 2 weeks 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 transmitted through meiosis and is rapidly lost when selection is relaxed.
Stable: These grow on selective medium almost as fast as on nonselective medium. Resistance
is transmitted regularly through meiosis and is retained even when selection is absent.
AU: Fig 1G citation OK?
Protocol 1 = your protocols 1-6 + 12. Pls review carefully.
Protocol 1
Culturing the Moss Physcomitrella patens
AU: Please provide complete reference.
AU: Pls mention here (one sentence) why one might culture
moss in liquid media. (The liquid cultures are not used in any
subsequent protocols, so it is
not clear to me why one might
choose liquid media over solid
media.)
Here, we present a series of methods for culturing the moss P. patens at all stages of its life cycle.
Gametophytes are axenically cultured on solid agar-based media (Methods IA and IB, adapted
from Grimsley et al. 1979) and in shaken liquid cultures (Method IC). Growth rates in shaken liquid cultures are not as great as those obtained on solid media or in bioreactors (Boyd et al. 1988),
especially if these are supplied with CO2. AU: See query. For long-term storage of gametophytes,
cultures are maintained on solid medium at 10°C in a very short day (see Method IIA; Fig. 1H,I),
but cryopreservation (Method IIB, adapted from Grimsley and Withers 1983) may also be used.
Finally, sporophytes are generated by self-fertilization and sexual crossing (Method III, adapted
from Ashton and Cove 1977).
AU: Fig 1H,I citations OK?
MATERIALS
The recipes for items marked with <R> are on page XX.
CAUTION: See Appendix XX for appropriate handling of materials marked with <!>.
Reagents
BCD medium (liquid and solid) <R>, containing appropriate supplements
AU: Pls double-check
that references to supplements and antibiotics
in the media are correct
in ALL protocols in this
chapter, and that they
correspond to the
appropriate recipes
(indicated by <R>) at
the end of the chapter.
A common moss media supplement <R> is diammonium tartrate, which is added to BCD medium
<R> at a final concentration of 5 mM. For cryopreservation, also include mannitol (500 mM) in the
appropriately supplemented liquid BCD medium (see Step 1 of Method IIB).
Dimethylsulfoxide (DMSO)–glucose solution <R>
Ethanol (70%) <!>
Liquid nitrogen <!>
Nitrogen-free medium <R> containing 400 µM of KNO3 <!>
Somatic tissue or spores from P. patens
To establish a new culture, a fragment of tissue 1–2 mm in diameter is sufficient. For routine subculture, it is best to use protonemal tissue from a vigorously growing culture that is no more than 20 days
old, which will be composed mostly of chloronemal tissue. Chloronemal tissue, the growth of which is
enhanced when ammonium is provided as nitrogen source, is easiest to subculture. Tissue other than
chloronemata (e.g., leaf cells) may take a long time to regenerate. For sexual crosses, it is best that at
least one of the two strains to be crossed is self-sterile. Strains containing vitamin auxotrophies (e.g.,
for thiamine, p-aminobenzoic acid, or nicotinic acid) are normally self-sterile but cross-fertile
(Courtice and Cove 1978).
Equipment
Blender (e.g., Fisher Scientific PowerGen Model 125 Homogenizer)
AU: Pls check supplier
name for cellophane discs.
I cannot find Cannings
online; does the company
have a new name, or is the
item available elsewhere?
Different tissue blenders are used in different laboratories, and some have been made specifically for
blending moss tissue. Provided the blending assembly can be sterilized, it appears that there is little
difference among blenders in the end result—a tissue inoculum that will grow rapidly.
Cellophane discs (type 325P), sterile (Cannings)
These are most conveniently sterilized dry, by autoclaving interleaved with disks of filter paper.
Erlenmeyer flasks (1 liter, sterile)
Ethanol bath (controlled, low temperature) <!>
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Chapter 3
Forceps (fine)
Incubator with temperature control and white light at intensities between 5 and 20 Wm–2 (e.g.,
Percival Scientific Model CU-36L5)
Because of the radiant heat from light sources, it may be necessary to keep the air temperature below
the desired culture temperature. In this protocol, the temperatures given are those of the medium in
which the cultures are grown.
Liquid nitrogen storage facility
Magenta jars (optional; see Method III)
Microcentrifuge tubes (1.5 ml)
Micropore surgical tape (3M, 1530-0)
Parafilm (optional; see Step 4 of Method IIA)
Petri dishes (sterile)
Most work uses 90-mm-diameter, presterilized disposable plastic Petri dishes, but glass Petri dishes
are also suitable. Ideally, the dishes should be vent-free to slow evaporation and limit contamination.
Shaker (platform)
Spatula (sterile)
Test tubes (25 x 150 mm, sterile) with caps
Tubes (2 ml, plastic) containing 1.5 ml of agar medium (see Step 1 of Method IIA)
If space is not at a premium, 15-ml tubes containing 10 ml of medium provide a better resource for
long-term storage.
Vials (2 ml, plastic)
Water bath, set at 30°C
METHODS
I. Growth of Gametophytes
A. Using Petri Dishes Containing Solid Medium
1. Inoculate a Petri dish containing appropriately supplemented solid BCD medium as follows:
• For spores, use standard microbiological procedures to spread them on the agar. If individual plants are required, add about 100–200 spores per 90-mm-diameter Petri dish.
• For somatic tissue, place a clump of gametophyte tissue (usually 1–2 mm in diameter) on
the agar. The size of the clump is not critical. However, for growth tests, the clumps
should be as uniform as possible, and uniformity is best achieved by picking the tissue
while looking through a microscope.
2. Seal the Petri dishes with Micropore surgical tape.
This reduces the risk of contamination without affecting the growth or development of the cultures.
Do not seal cultures with Parafilm; this slows growth and prevents regeneration.
3. Incubate the cultures in an incubator at 25°C with constant white light at intensities between
5 and 20 Wm–2.
AU: Fig 1A cite OK?
Depending on the purpose of the procedure, growth under these conditions may range from a few
days (e.g., to score antibiotic resistance) to several weeks (e.g., to score the requirements of some vitamins). For an image of 6-day-old protonemata grown under these conditions, see Figure 1A.
B. Using Petri Dishes Containing Solid Medium Overlaid with Cellophane
1. Overlay a Petri dish containing appropriately supplemented solid BCD medium with a sterilized cellophane disc. Allow the dish to stand for at least 10 minutes to allow the cellophane to
hydrate and then, if necessary, straighten the disc while maintaining sterility.
The Moss Physcomitrella patens
AU: Fig 1B citation OK?
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77
2. Obtain one dish of protonemal tissue from plants that have developed for about 10 days from
tissue-clump inocula growing on appropriately supplemented BCD medium (see Method
IA). Harvest the tissue with a spatula as shown in Figure 1B.
3. Add the tissue to 10 ml of H2O, and blend it for about 2 minutes.
The exact procedure will depend on the type of blender used. Blending should result in an easily
pipettable suspension, but it should still consist of tissue clumps containing 20–50 cells.
Overblending leads to poor regeneration.
4. Pipette 1–2 ml of the protonemal suspension from Step 3 onto each Petri dish from Step 1.
Spread the suspension evenly.
There should be sufficient tissue to inoculate 5–10 Petri dishes.
5. Incubate the culture for 7 days at 25°C under a 16-hour light/8-hour dark cycle (with white
light at intensities between 5 and 20 Wm–2).
For a wild-type culture, each 90-mm-diameter Petri dish will yield about 200 mg (fresh weight) of
vigorously growing protonemal tissue, consisting mainly of chloronemata.
6. Harvest the tissue by scraping it from the cellophane using a sterile spatula.
Once tissue is growing on cellophane, it is convenient to use this tissue (instead of the tissue
described in Step 2) for further inocula by repeating Steps 3–6. However, do not repeat this cycle
more than three times.
C. Using Liquid Medium
1. Add tissue from one cellophane-overlay plate (see Method IB) to 10 ml of H2O and blend as
described in Step 3 of Method IB.
AU: Step 3. At what
temp, for how long,
and under what light
conditions? What is
the typical yield here?
2. Inoculate 200 ml of appropriately supplemented liquid BCD medium (in a 1-liter Erlenmeyer
flask) with 2 ml of the tissue suspension from Step 1.
Other quantities of the liquid BCD medium and/or the tissue suspension may be used instead.
3. Shake the cultures on a platform shaker.
Vigorous agitation is not necessary for growth.
II. Long-term Storage of Gametophyte Tissue
A. Storage on Solid Medium
1. Place a clump of gametophyte tissue (usually 1–2 mm in diameter) in each plastic tube containing agar medium (usually, appropriately supplemented solid BCD medium).
AU: Culturing conditions in Step 3 correct?
2. Make sure that the lids are not tightly sealed. For screw-cap lids, tighten the cap and then
release it about one quarter of a turn.
3. Grow the cultures for about 3 weeks in an incubator at 25°C with continuous white light at
intensities between 5 and 20 Wm–2.
4. After 3 weeks, tightly seal each tube. If the tube has an air-tight seal, this is sufficient; if it does
not, seal the tube with Parafilm.
5. Transfer the tubes to an incubator at 10°C with a 2-hour light/22-hour dark cycle (with white
light at intensities between 5 and 20 Wm–2) for long-term storage.
Cultures can be kept in a healthy state for a considerable period of time (at least 3 years) under these
conditions.
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Chapter 3
B. Cryopreservation
1. For each strain to be preserved, grow gametophyte tissue as described in Steps 1–5 of Method
IB. After 7 days of incubation (Method IB, Step 5), transfer 100 mg of tissue onto a Petri dish
AU: For Step 2, changed
overlaid
with fresh cellophane as described in Step 1 of Method IB. Pipette 1 ml of appropri“standard conditions” to “at
25°C under...” because that
ately supplemented liquid BCD containing 500 mM of mannitol onto the surface of the tisis what’s used in Method IB.
sue.
Correct?
AU: Step 3. Assume you’re
using the 2-ml tubes listed
in the Equipment list so I
added here. But please
check (are they too small?
will the solution spill over
once you add 2 ml of solution plus tissue to a 2-ml
tube?).
AU: Step 5. How long can
the cultures be stored at
this temp? Pls add as note
here.
AU: Step 8. Added “add tissue from one vial to 10 ml”
(Step 7) and “(use 1-2 ml of
the tissue suspension per
plate)” for clarity. Pls check.
2. Incubate the culture for an additional 7 days at 25°C under a 16-hour light/8-hour dark cycle
(with white light at intensities between 5 and 20 Wm–2).
3. To each of ten 2-ml sterile plastic vials, add 2 ml of DMSO–glucose solution. Then add one
tenth of the tissue on the plate to each vial. Incubate the vials for 1 hour 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 6–8.
6. Retrieve the vials from the liquid nitrogen and place them in a water bath at 30°C until
thawed.
7. After thawing, add tissue from one vial to 10 ml of H2O. Allow the mixture to stand for 30
minutes at room temperature.
8. Inoculate the tissue suspension from Step 7 onto appropriate solid medium as described in
Method IA (use 1–2 ml of the tissue suspension per plate).
III. Production of Sporophytes and Isolation of Spores
1. Prepare test tubes (25 x 150 mm) with between 15 and 20 ml of solid nitrogen-free medium
containing 400 µM of KNO3.
Alternatively, fill Magenta jars two-thirds full with the same medium.
2. Inoculate the medium with protonemata by placing a clump of tissue (usually 1–2 mm in
diameter) on the agar.
• If sporophytes resulting from self-fertilization are required, place a single inoculum into
a test tube. Alternatively, place four inocula into a Magenta jar.
• To establish a sexual cross, place one or more inocula of each strain into a tube (or a
Magenta jar).
• Harvest and test the sporophytes produced from sexual crosses individually because they
may result from either self- or cross-fertilization. Strains that have been kept for prolonged periods in vegetative culture may loose fertility.
3. Place the cap on the tube, but do not completely tighten it.
AU: Step 4. Changed
the second “sporophyte production” to
“sporophyte yield”. Pls
check.
4. Culture for about 4 weeks at 25°C in continuous white light at intensities between 5 and 20 Wm–2.
Continuous white light accelerates sporophyte production somewhat, but sporophyte yield may be
reduced.
5. Transfer the culture to 15°C for an additional 3 weeks under an 8-hour light/16-hour dark
cycle.
6. Irrigate the culture with H2O to facilitate fertilization. Make sure to thoroughly wet but not
submerge the culture. Allow it to stand for 24 hours.
The Moss Physcomitrella patens
AU: Added “under
an 8-hour light/16hour dark cycle” in
Steps 7 and 9 – correct?
/
79
7. After 24 hours, decant excess H2O. Place the culture for 1 week at 15°C under an 8-hour
light/16-hour dark cycle.
8. Repeat the irrigation procedure as described in Step 6.
9. After 24 hours, decant excess H2O. Place the culture for 2–5 weeks at 15°C under an 8-hour
light/16-hour dark cycle.
After this incubation period, sporophytes will have developed.
10. Use fine forceps to harvest pale brown sporophytes by separating them from the gametophytic
material. Do not take the green or yellow sporophytes (these are immature) or the darkbrown sporophytes (these burst easily).
11. Pinch the base of the seta, which can be identified by a zone of pigmentation, to release the
spore capsule.
Each capsule of a mature sporophyte contains 1 x 103 to 4 x 103 viable, haploid, uninucleate spores.
AU: Step 12. “sporophyte capsules” OK?
AU: Step 14. For
how long? Pls add
details to Step 14
(i.e., exactly how do
you dry the spores?)
or delete it altogether.
AU: Step 15. Using
what piece of equipment? pls add to
Equip list, too.
12. Place one or more sporophyte capsules in a sterile 1.5-ml microcentrifuge tube. Sterilize the
capsules by adding 1 ml of 70% ethanol and incubating for 4 minutes at room temperature.
13. Remove the ethanol. Gently rinse the sporophyte capsules three times with 1 ml of H2O at
room temperature.
14. Add 1 ml of H2O and transfer the tube to 4°C.
This step increases spore germination, but some spores will germinate if it is omitted.
15. Crush the sporophyte capsules and mix to produce a spore suspension.
Any residual sporophytic tissue will not regenerate and can be ignored. The spore suspension may
be kept for several weeks at 4°C.
16. If desired, dry the spores.
Dry spores may be stored for several years.
Protocol 2
Isolation and Regeneration of Protoplasts
This protocol describes how to isolate individual protoplasts from young gametophyte tissue
(Method I) and how to regenerate them into plants (Method II) if desired. It is adapted from a
protocol described by Grimsley et al. (1977).
MATERIALS
The recipes for items marked with <R> are on page XX.
Reagents
AU: In the protocol, you
said “0.5% driselase
solution”; however, the
recipe produces a 2%
solution. So I deleted
“0.5%” from the protocol. Please change the
concentration in the
recipe (end of chapter),
if necessary.
Driselase solution (sterile) <R>
Protonemal tissue from P. patens
This tissue is grown on cellophane-overlay plates as described in Method IB of Protocol 1.
Protoplast regeneration medium for the bottom layer (PRMB) <R>
Prepare Petri dishes (50 mm or 90 mm) containing PRMB and overlay with sterile cellophane.
Protoplast regeneration medium for the top layer (PRMT) <R>
This medium should be melted and kept at 45°C in a water bath.
Protoplast wash (PW) solution <R>
Equipment
Centrifuge
Filters (pore sizes of 50 µm and 100 µm, sterile)
Two filters are needed for each protoplast isolation. Filters can be made of stainless steel or nylon.
Hemocytometer
Incubator with temperature control and white light at intensities between 5 and 20 Wm–2 (e.g.,
Percival Scientific Model CU-36L5)
METHODS
I. Protoplast Isolation
1. After culturing protonemal tissue for 7 days (Method IB, Steps 1–5), harvest the tissue as
AU: Step 1. For clarity,
changed “5 ml” to “2 ml”
described in Step 6 of Method IB. Weigh the tissue and add 1 ml of sterile driselase solution
and added “per 90-mmfor every 100 mg of tissue (fresh weight) (i.e., use 2 ml of driselase solution per 90-mm-diamdiameter plate” because in
eter plate of tissue).
Step 5 of Method 1B, you
say that a typical 90-mm
It is difficult to obtain protoplasts from tissue other than gametophytes growing on cellophane-overplate contains 200 mg of
lay plates. Use young protonemal tissue; older tissue may leave clumps of undigested tissue, which
tissue. OK?
can act like glue and stick protoplasts together.
AU: Step 2. How can we
2. Incubate the tissue-driselase mixture for 30–60 minutes at room temperature with occasional
determine when adequate
gentle shaking. Monitor the breakdown of the tissue (no microscope is necessary).
breakdown has occurred –
For some strains, the incubation time may need to be longer. Protoplast viability is not greatly affected
what do we look for? Pls
by exposure to driselase for up to 2 hours, but it will decrease as the incubation time increases.
add here.
80
The Moss Physcomitrella patens
/
81
3. To isolate protoplasts from the digested tissue, sterilely filter the tissue-driselase mixture
through mesh with a pore size of 100 µm.
4. Sterilely refilter the filtrate from Step 3 through mesh with a pore size of 50 µm.
5. Sediment the protoplasts by centrifuging the filtrate from Step 4 at 100–200g for 4 minutes at
room temperature with no braking.
6. Discard the supernatant.
7. Resuspend the protoplast pellet in PW. Use about the same volume as the volume of driselase
used in Step 1.
AU: Pls check step
nos; believe these
were incorrect in
the original ms.
8. Repeat Steps 5–7.
This should yield about 106 protoplasts per 90-mm-diameter plate of tissue cultured for 7 days as
described in Method IB. Protoplasts can be used in a number of techniques (e.g., Protocols 3, 5, and
6). If protoplasts are to be plated without further manipulation, proceed to Method II.
II. Protoplast Regeneration
AU: Added the last sentence in Step 1. Please
fill in details.
1. Estimate the density of protoplasts using a hemocytometer. Adjust the volume of the protoplast suspension with XXXX so that the density of protoplasts is approximately XXXXX.
2. Determine the quantity of molten PRMT needed (use 1 volume of protoplast suspension for
every 2 volumes of molten PRMT). Gently add the protoplast suspension from Step 1 to the
appropriate volume of molten PRMT.
3. Pipette 1 ml of the protoplast-PRMT mixture gently but quickly onto a 90-mm PRMB plate
overlaid with cellophane.
Alternatively, use 400 µl of the protoplast-PRMT mixture to cover a 50-mm dish.
AU: Step 4 note. Fig.
1F citation OK?
4. Incubate the plate for 3 or 4 days at 25°C with strong, constant light (intensities >5 Wm–2).
AU: Step 5. Pls fill in
XX.
5. After about 4 days, transfer the protoplasts to appropriately supplemented, fresh BCD
medium as described in Method XX of Protocol 1.
For images of 3-day-old filaments regenerating from protoplasts, see Figure 1F.
Protocol 3
Somatic Hybridization in P. patens Using PEG-induced Protoplast Fusion
As an alternative to sexual crossing (see Protocol 1), protoplasts from two strains of moss (P.
patens) can be hybridized using polyethylene glycol (PEG). This protocol for PEG-induced protoplast fusion was adapted from the protocol described by Grimsley et al. (1977). Although the efficiency is low, it requires no sophisticated apparatus. Hybrids are readily obtained using
complementary auxotrophic mutants or strains with transgenic antibiotic resistance markers. It is
now routine to obtain hybrids using transgenic strains that are hygromycin- or G418-resistant by
selecting hybrids on medium containing both antibiotics.
MATERIALS
The recipes for items marked with <R> are on page XX.
Reagents
BCD medium (solid) <R>, containing common moss media supplements <R> and antibiotics
<R> as necessary
PEG solution for protoplast fusion (PEG/F) <R>
For each hybridization, 750 µl of PEG/F is required.
Protoplast regeneration medium for the bottom layer (PRMB) <R>
For each hybridization, prepare eight plates containing PRMB and overlay with sterile cellophane.
Protoplast regeneration medium for the top layer (PRMT) <R>
This medium should be melted and kept at 45°C in a water bath.
Protoplast wash (PW) solution <R>
For each hybridization, prepare 30 ml of PW solution.
Protoplasts from two different P. patens strains
These protoplasts are isolated as described in Protocol 2, Method I.
Equipment
Centrifuge
Hemocytometer (optional; see Step 2)
Incubator with temperature control and white light at intensities between 5 and 20 Wm–2 (e.g.,
Percival Scientific Model CU-36L5)
Petri dishes (90 mm), sterile
METHOD
1. For each strain to be hybridized, resuspend 106 protoplasts (approximately the number produced on one 90-mm plate in Protocol 2, Method I) in 2.5 ml of PW.
82
The Moss Physcomitrella patens
/
83
2. Combine 2.5 ml of each of the two strains to be hybridized.
Judge the density of protoplasts by eye (if preferred, density can be determined using a hemocytometer). If the yield of protoplasts from one strain is poor, the yield of hybrids involving that strain
can be maximized by mixing with an excess of the other strain.
3. Sediment the mixed protoplasts by centrifugation at 100–200g for 4 minutes at room temperature with no braking.
4. Discard the supernatant. Resuspend the protoplasts in 250 µl of PW.
5. Hybridize the two strains of protoplasts by performing the following steps on schedule:
i. At 0 minutes, add 750 µl of PEG/F and mix gently.
ii. At 40 minutes, add 1.5 ml of PW and mix gently.
iii. At 50 minutes, add 10 ml of PW and mix gently.
iv. At 60 minutes, add 10 ml of PW and mix gently.
v. At 70 minutes, sediment the protoplasts by centrifugation at 100–200g for 4 minutes at
room temperature with no braking. Discard the supernatant and resuspend the pellet in
1 ml of PW.
Multiple hybridizations can be performed in parallel. It should be possible to do these at 30second intervals.
AU: Steps 6 and 7.
Incubation conditions
OK?
6. Add the protoplast suspension to 7 ml of molten PRMT and plate 2 ml onto each of four
plates of PRMB overlaid with cellophane. In addition, to estimate the survival of the individual component strains, add 50 µl of the protoplast suspension to 8 ml of molten PRMT and
plate 2 ml onto each of four plates of PRMB overlaid with cellophane. Incubate all plates at
25°C with strong, constant light (intensities >5 Wm–2).
7. After 5–6 days (when protoplasts have regenerated, but growth is still not great), transfer the
protoplasts from Step 6 to Petri dishes containing the appropriate BCD selective medium to
select for hybrids. In addition, to estimate the survival of the individual component strains,
transfer the cellophane overlays from Step 6 onto the appropriate BCD media to select for one
or the other component strain. Incubate all plates under the conditions described in Step 6.
Selection of hybrids using vitamin auxotrophies usually takes about 3 weeks; using transgenic
antibiotic resistances, hybrids can usually be identified after only 7 days.
AU: Protocol 4 = your protocols 8-10. Pls review.
Protocol 4
Chemical and UV Mutagenesis of Spores and Protonemal Tissue
This protocol describes how to mutagenize spores and protonemal tissue from moss (P. patens) using
chemicals or UV light. Spores are mutagenized using the alkylating agents N-methyl-N′-nitro-Nnitrosoguanidine (NTG) and ethylmethanesulfonate (EMS) (Methods IA and IB, respectively), and
protonemal tissue is mutagenized with NTG and UV light (Methods IIA and IIB, respectively). The
chemical mutagenesis protocols were adapted from those described by Ashton and Cove (1977) and
Boyd et al. (1988). Compared to alkylating agents, UV is less effective as a mutagen, but it may be
advantageous because it is less hazardous and may not lead to clustered lesions.
MATERIALS
The recipes for items marked with <R> are on page XX.
CAUTION: See Appendix XX for appropriate handling of materials marked with <!>.
Reagents
BCD medium (solid) <R>, containing common moss media supplements <R> as necessary
Ethylmethanesulfonate (EMS) <!>
N-methyl-N′-nitro-N-nitrosoguanidine (NTG) <!>
Moss (P. patens) spores (see Protocol 1, Method III) or protonemal tissue on a cellophane-overlay plate (see Protocol 1, Method IB)
Orthophosphoric acid (H3PO4; 100 mM, pH 7.0; sterile) <!>
Sodium thiosulfate (Na2S2O3; 400 mM, sterile )
Tris-maleate buffer (sterile) <R>
Equipment
Centrifuge
Filter (pore size 100 µm, sterile)
Petri dishes (90 mm)
UV light source <!>
Any standard mutagenic UV source is likely to be effective, but this will need to be calibrated.
METHODS
Perform all chemical mutagenesis procedures in safe handling facilities for mutagens.
I. Mutagenesis of Spores
AU: In Step 1 of Method
IIA below, you say
“Incubate the mixture for
30 minutes at 25°C.” Is
that necessary here, too?
Please check.
84
A. Using NTG
1. Prepare 10 ml of a spore suspension containing about 106 spores in sterile Tris-maleate buffer.
2. Observing strict safety procedures, carefully add 1.2 mg of NTG to a separate tube containing
10 ml of Tris-maleate buffer. Incubate the tube for 30 minutes at 25°C.
The Moss Physcomitrella patens
/
85
3. Mix the spore suspension with the NTG solution and shake it gently for an additional 30 minutes at 25°C.
AU: In Step 4, pls fill
in “XXXg for XX
minutes”, and room
temp OK?
AU: Added more specific
instructions for the
washes in Step 5. Please
review and fill in “XXXg
for XX minutes” if centrifugation OK.
4. Terminate the treatment by sedimenting the spores by centrifugation (XXXg for XX minutes
at room temperature) and dispose of the supernatant safely.
Usually about 10% of the originally viable spores survive this treatment.
5. Wash the spores three times as follows:
i. Add 10 ml of H2O to the tube.
ii. Centrifuge the tube at XXXg for XX minutes at room temperature.
iii. Discard the supernatant.
6. After the final wash, resuspend the spores in XXX. Spread about 100 surviving spores onto the
AU: Pls add details to
surface of appropriately supplemented BCD medium in 90-mm Petri dishes.
Step 6. Added “After the
7. Incubate the plate under the desired conditions. For standard cultivating procedures and confinal wash...” but can you
ditions, see Protocol 1.
explain what/how much
you add to the spore pelThe mutagenic treatment results in an initial delay in development, but once germinated, the
let? (Add any new
sporelings grow as usual.
reagents to Reagents list.)
How can you tell which
spores are “surviving” vs. B. Using EMS
non-viable? Or do you
simply plate out 1,000 of
1. Prepare 9.5 ml of a spore suspension containing about 106 spores in sterile 100 mM orthothe resulting spores, and
phosphoric acid (pH 7.0).
assume that 10% of those
(100) are “surviving”? If
2. Observing strict safety procedures, add 500 µl of EMS.
you start with 106 spores,
you will need quite a few
3. Incubate the mixture for 45 minutes at 25°C.
plates!
4. Terminate the treatment by adding 10 ml of sterile 400 mM sodium thiosulfate.
AU: In Step 5, pls fill in
5. Sediment the spores by centrifugation (XXXg for XX minutes at room temperature) and dis“XXXg for XX minutes”,
pose of the supernatant safely.
and room temp OK?
Usually 35–40% of the originally viable spores survive this treatment.
AU: Added more specific
instructions for the
washes in Step 6. Please
review and fill in “XXXg
for XX minutes” if centrifugation OK.
AU: Pls add details to
Step 7. Added “After the
final wash...” but can you
explain what/how much
you add to the spore pellet? (Add any new
reagents to Reagents list.)
How can you tell which
spores are “surviving” vs.
nonviable?
6. Wash the spores three times as follows:
i. Add 10 ml of H2O to the tube.
ii. Centrifuge the tube at XXXg for XX minutes at room temperature.
iii. Discard the supernatant.
7. After the final wash, resuspend the spores in XXX. Spread about 100 surviving spores onto the
surface of appropriately supplemented BCD medium in 90-mm Petri dishes.
8. Incubate the plate under the desired conditions. For standard cultivating procedures and conditions, see Protocol 1.
The mutagenic treatment results in an initial delay in development, but once germinated, the
sporelings grow as usual.
II. Mutagenesis of Protonemal Tissue
A. Using NTG
1. Add 1 g (fresh weight) of young protonemal tissue from a cellophane-overlay plate to 10 ml
of sterile Tris-maleate buffer. Incubate the mixture for 30 minutes at 25°C.
2. Observing strict safety procedures, carefully add 1.2 mg of NTG to a separate tube containing
10 ml of Tris-maleate buffer. Incubate the tube for 30 minutes at 25°C.
86
/
Chapter 3
3. Mix the tissue with the NTG solution and shake it gently for an additional 30 minutes at 25°C.
4. Terminate the treatment by filtering the mixture through a sterile filter.
The filter retains the protonemal tissue. This procedure results in a cell survival rate of about 10%.
AU: Step 5 note. Are
there alternatives
here? If protoplasting
is not necessary, then
what?
5. Wash the tissue thoroughly, with at least 100 ml of H2O.
If individual cells are required, these may be obtained by protoplasting the tissue as described in
Method I of Protocol 2.
B. Using UV Light
1. Remove the lid from a cellophane-overlay plate and irradiate the tissue with UV light. The kill
level is difficult to determine, but a dose of 2000 Jm–2 should give about 10% survival. It is best
to perform a number of experiments to calibrate the UV source in order to get a feel for the
correct exposure. Petri dishes are opaque to UV light, so make sure to remove the lid.
2. Immediately after irradiation, transfer the plate to darkness for at least 24 hours to prevent
photoreactivation.
AU: Step 2 note.
Pls fill in XX.
Following incubation in the dark, the tissue can be protoplasted (see Protocol 2, Method I), but very
few protoplasts regenerate following UV irradiation. Yields are better if the tissue is allowed to grow
following incubation (see Protocol 1, Method XX), but this may yield mutant clones.
Protocol 5
Transformation Using Direct DNA Uptake by Protoplasts
AU: Please check
last sentence.
This protocol describes how to transform moss (P. patens) protoplasts using PEG-mediated DNA
uptake. The transformation rates for direct uptake by protoplasts of DNA with and without
genomic sequence (a targeting construct) are typically 10–5 and 10–3, respectively (these are the frequencies of stable transformants among regenerants surviving the transformation procedure).
MATERIALS
The recipes for items marked with <R> are on page XX.
CAUTION: See Appendix XX for appropriate handling of materials marked with <!>.
Reagents
BCD medium (solid) <R>, containing common moss media supplements <R> and antibiotics
<R> as necessary (optional; see Step 15)
DNA to be used in transformation
Use 15–30 µg of DNA in a volume no greater than 30 µl.
D-Mannitol (8.5%, w/v) <R>
MMM solution <R>
Prepare 10 ml of MMM solution; this is sufficient for 10–15 transformations.
PEG solution for transformation (PEG/T)
To prepare sufficient PEG/T for 15 transformations, melt 2 g of PEG (MW 6000) in a microwave on
the day of transformation. Add 5 ml of MCT solution <R>, mix well, and allow the mixture to stand
for 2–3 hours at room temperature before use.
Protoplast regeneration medium for the bottom layer (PRMB) <R>
For each transformation, prepare four plates containing PRMB and overlay with sterile cellophane.
Protoplast regeneration medium for the top layer (PRMT) <R>
This medium should be melted and kept at 45°C in a water bath.
Protoplasts, isolated as described in Method I of Protocol 2
Equipment
Centrifuge
Hemocytometer
Tube (10 ml, sterile)
Water bath, set at 45°C
METHOD
Except where stated otherwise, perform all procedures at room temperature, providing this is between
20°C and 25°C. Otherwise, use a water bath. The ten-step dilution described in Steps 8–11 maximizes
the recovery rate; however, the dilution can be made in fewer steps if maximum recovery is not
required.
87
88
/
Chapter 3
1. Resuspend the protoplasts in 8.5% (w/v) D-mannitol solution. Use 2.5 ml for each plate of tissue digested.
2. Use a hemocytometer to estimate protoplast density.
AU: Step 3. At what
speed (in g), for how
long, and at what
temp?
AU: Step 5. Invert
OK?
3. Centrifuge Speed (in g), for how long, and at what temp? and discard the supernatant.
Resuspend the protoplasts in MMM solution at a concentration of 1.6 x 106 protoplasts/ml.
4. Prepare the DNA to be used in transformation by dispensing 15–30 µg of DNA into a sterile
10-ml tube. Centrifuge briefly to bring the DNA to the bottom of the tube.
5. Add 300 µl of protoplast suspension from Step 3 and 300 µl of PEG/T solution to the DNA
from Step 4. Gently invert the tube to mix.
6. Heat the mixture in a water bath for 5 minutes at 45°C.
7. Transfer the tube to room temperature (20°C) and leave it for 5 minutes.
8. Add 300 µl of 8.5% D-mannitol solution. Gently invert the tube to mix. Wait at least 1 minute.
AU: Pls check Step
numbers in Steps 9
and 11 – I think they
were incorrectly numbered in the original.
AU: Step 14. Do the
PRMT or PRMB need
to have antibiotics for
selection? If so, please
say so here and in
Materials list above.
AU: Step 15 note.
When is “appropriate”? Pls explain
briefly here. Also,
Added BCD medium
to Reagents list –OK?
9. Repeat Step 8 four times.
10. Add 1 ml of 8.5% D-mannitol solution. Invert gently to mix. Wait at least 1 minute.
11. Repeat Step 10 four times.
12. Centrifuge at 100–200g for 5 minutes at room temperature.
13. Discard the supernatant and resuspend the pellet in 500 µl of 8.5% D-mannitol solution.
14. Add 2.5 ml of molten PRMT. Dispense 1 ml of the mixture to each of three 90-mm plates containing PRMB and overlaid with cellophane.
15. Incubate the plates in continuous white light for 5 days at 25°C.
If appropriate, transfer the protoplasts on the top layer to the appropriate BCD selective medium.
Protocol 6
Transformation Using T-DNA Mutagenesis
In this protocol, the transformation of moss (P. patens) protoplasts is performed via Agrobacteriummediated transfer of T-DNA. The transformation rate for this protocol is typically 10–4 (expressed
as the frequency of stable transformants among regenerants surviving the transformation procedure).
MATERIALS
The recipes for items marked with <R> are on page XX.
CAUTION: See Appendix XX for appropriate handling of materials marked with <!>.
Reagents
Acetoseringone (3′5′-dimethoxy-4′-hydroxyacetophenone) (100 mM) <!>, prepared in ethanol <!>
Agrobacterium tumefaciens culture (strain Agl-1)
AU: Deleted “for every 5 x
About 1 ml of a mid-log-phase culture (OD600 = 0.4–0.6) is needed.
105 protoplasts to be transBCD medium (modified)
formed”. Is this OK? See
query in Step 5.
Prepare Petri dishes with solid BCD medium <R> containing 5 mM diammonium tartrate (920
mg/liter of BCD) and 0.05 mg/ml vancomycin <!> (1 ml of 1000 stock per liter of BCD).
Protoplast regeneration medium for the bottom layer (PRMB, modified)
AU: “After autoclaving...”
note OK?
After autoclaving 1 liter of PRMB <R>, add 1 ml of 1000x cefotaxime <!> and 1 ml of 1000x vancomycin <!> (for final concentrations of 0.2 mg/ml and 0.05 mg/ml, respectively). Pour the solution
into Petri dishes and overlay each with a sterile cellophane disc. Four dishes are required per transformation.
Protoplast regeneration medium for the top layer (PRMT, modified)
AU: “Melt 2.5 ml...” note
OK?
Melt 2.5 ml of PRMT <R> for each transformation and keep it at 45°C in a water bath. Add 1 µl of
1000x cefotaxime <!> and 1 µl of 1000x vancomycin <!> per milliliter of PRMT (for final concentrations of 0.2 mg/ml and 0.05 mg/ml, respectively).
Protoplast regeneration medium (PRML, liquid) <R>
Protoplast wash solution for Agrobacterium transformation (APW), maintained at 25°C <R>
Prepare 35 ml of APW for each transformation.
Protoplasts, isolated as described in Method I of Protocol 2
Selection medium
For the selection medium, prepare Petri dishes with solid BCD medium <R> containing 5 mM
diammonium tartrate (920 mg/liter of BCD), 0.2 mg/ml cefotaxime (1 ml of 1000x stock per liter of
BCD), 0.05 mg/ml vancomycin (1 ml of 1000x stock per liter of BCD), and an appropriate antibiotic
<R> used to select for T-DNA insertion.
Equipment
Centrifuge
Hemocytometer
Incubator with temperature control and white light at intensities between 5 and 20 Wm–2 (e.g.,
Percival Scientific Model CU-36L5)
89
90
/
Chapter 3
AU: What size?
Micropore surgical tape (3M, 1530-0)
Petri dishes (SIZE?) (sterile)
Spectrophotometer
METHOD
1. Use a hemocytometer to estimate the density of protoplasts.
2. Centrifuge the protoplasts at 100–200g for 4 minutes at room temperature with no braking
and discard the supernatant. Resuspend the protoplasts in PRML at a concentration of 5 x 105
protoplasts/ml.
3. For each milliliter of protoplast suspension, prepare a Petri dish containing 9 ml of PRML.
4. Add 1 ml of protoplast suspension to each prepared Petri dish.
AU: You originally
wrote “add
(110/OD600) µl”,
which I interpreted
as written here.
However, in the
reagents list above,
you originally said
that we will need 1
ml of a culture growing at 0.4–0.6 OD600
for every 5 x 105 protoplasts. If the culture is growing at an
OD600 of 0.5, then we
would add only 220
µl (not even close to
1 ml) of culture to
the Petri dish in Step
5. So I deleted “for
every 5 x 105 protoplasts to be transformed” from the
Reagents list. Is all
OK now?
5. Calculate the quantity of Agrobacterium culture to add by determining the OD600 with a spectrophotometer. Add 110 µl of culture per 1.0 OD600 to each Petri dish.
6. Add 20 µl of 100 mM acetoseringone to each Petri dish.
7. Wrap each dish with Micropore surgical tape and incubate for 48 hours at 25°C under a 16hour light/8-hour dark cycle.
The protoplasts will tend to adhere to the Petri dish surface during incubation.
8. Use a pipette to carefully resuspend the protoplasts.
9. Centrifuge the protoplasts at 100–200g for 4 minutes at room temperature with no braking
and discard the supernatant. Resuspend the pellet in 10 ml of APW.
10. Repeat Step 9 twice, but after the last centrifugation step, resuspend the pellet in 2 ml of APW
and add 2.4 ml of modified PRMT.
11. Dispense 1.1 ml of the protoplast suspension into each of four Petri dishes containing modified PRMB and overlaid with sterile cellophane discs. Incubate the dishes for 7 days at 25°C
under continuous white light.
12. Transfer the cellophane overlays onto plates containing selection medium and incubate for 7
days at 25°C under a 16-hour light/8-hour dark cycle.
13. Transfer the cellophane overlays to plates containing modified BCD medium and incubate for
7 days at 25°C under continuous white light. AU: incubation conditions OK in Steps 13, 15?
14. Transfer the cellophane overlays to plates containing selection medium and incubate for 7–10
days at 25°C under a 16-hour light/8-hour dark cycle.
15. Pick small clumps of tissue from each vigorously growing plant and place them on plates containing modified BCD medium. Incubate the plates for 7 days at 25°C under continuous white
light.
AU: Added details to this
note. Pls check.
These should be stable transformants. However, to confirm that these are transformants, test them
once more for resistance by growing small clumps of tissue on selection medium for 7–10 days under
a 16-hour light/8-hour dark cycle.
Protocol 7
Transformation of Gametophytes Using a
Biolistic Projectile Delivery System
This method is especially suitable for transient gene expression studies, but it can be used to obtain
stable transformants.
MATERIALS
CAUTION: See Appendix XX for appropriate handling of materials marked with <!>.
Reagents
CaCl2 (2.5 M) <!>
Ethanol (70% and 100%) <!>
Gold beads (1 µM)
Prepare in 100% ethanol and resuspend by vigorous mixing for 5 minutes.
Macrocarriers
Wash in 100% ethanol and allow to dry on sterile filter paper.
Plasmid DNA
Use 2.5 µg of plasmid DNA contained in less than 4 µl.
Protonemal tissue from P. patens
This tissue is grown as described in Protocol 1, Method IB.
Spermidine (0.1 M)
Equipment
Biolistic particle delivery system (Bio-Rad, PDS-1000/He)
Before use, clean the chamber with H2O followed by 100% ethanol and allow it to dry. The disruption disks (900 psi) should be immersed in 100% isopropanol <!> until bombardment.
Centrifuge
Incubator with temperature control and white light at intensities between 5 and 20 Wm–2 (e.g.,
Percival Scientific Model CU-36L5)
METHOD
1. While mixing vigorously, add the following solutions to 2.5 µg of plasmid DNA:
Gold beads (1 µM)
CaCl2 (2.5 M)
Spermidine (0.1 M)
AU: Step 2. At what
speed (in g), and at
what temp?
25 µl
25 µl
10 µl
2. Mix for an additional minute. Allow the beads to settle and then centrifuge the tube for 2 minutes.
91
92
/
Chapter 3
AU: Step 3. How
many µl? At what
speed (in g), for how
long, and at what
temp?
3. Discard the supernatant. Wash the pellet with 70% ethanol by adding XX µl of 70% ethanol
and centrifuging at XXX.
Do not resuspend the gold particles during this step.
4. Discard the supernatant. Resuspend the gold particles in 24 µl of 100% ethanol and apply 6
µl to each macrocarrier. Allow the ethanol to evaporate.
5. Place the protonemal tissue so that it is about 150 mm below the stopper plate of the Bio-Rad
PDS-1000/He biolistic particle delivery system. Make two discharges into each dish, moving
the position of the dish between discharges.
AU: Step 6. Incubation
conditions OK?
6. Following bombardment, incubate the tissue for 48 hours at 25°C under continuous white
light at intensities between 5 and 20 Wm_2.
7. After the 48-hour incubation, harvest the tissue and blend and plate it onto selective medium
as described in Method IB of Protocol 1.
Alternatively, if clones derived from single cells are required, isolate protoplasts from the tissue as
described in Method I of Protocol 2.
AU: Protocol 8 = Your protocols 16-19. Please review carefully.
Protocol 8
Isolation of DNA, RNA, and Protein from P. patens Gametophytes
AU: There were a lot of
similarities among these
methods, and each
method was relatively
short, so I combined them
and created a common
Introduction. Also reworded some of the
Methods for completeness, clarity, and consistency. Pls check all
carefully.
This protocol describes a series of procedures for isolating nucleic acids (DNA and RNA) and proteins from moss (P. patens) gametophyte tissue. Method IA, a rapid small-scale procedure for isolating DNA, results in genomic DNA that is suitable for PCR only. However, larger amounts of genomic
DNA suitable for Southern analysis may be obtained using Method IB (adapted from Luo and Wing
2003) together with the Nucleon PhytoPure Genomic DNA Extraction Kit (GE Healthcare). The
RNA isolation procedure (Method II) uses the Plant RNA Isolation Mini Kit (Agilent) with some
modifications. Proteins can be obtained from moss gametophytes using Method III.
MATERIALS
The recipes for items marked with <R> are on page XX.
CAUTION: See Appendix XX for appropriate handling of materials marked with <!>.
Reagents
AU: Cold? See query in Isopropanol <!> cold?
Step 6 of Method 1A.
Liquid nitrogen <!>
AU: Prechilled to 4C?
see query in Step 4 of
Method 1A.
AU: Tris-Cl/ OK?
Desired pH?
AU: Note. Assume this
protocol is for gametophyte tissue only?
Nuclei isolation buffer containing β-mercaptoethanol (NIBM) <R> <!>
Nuclei isolation buffer containing Triton X-100 (NIBT) <R> <!>
Protein extraction solution, prechilled to 4°C <R>
Protein wash solution, prechilled to 4°C <R>
1x Shorty buffer <R> prechilled?
Tris-Cl (10 mM, pH XX) containing EDTA (1 mM, pH XX) (TE buffer)
Tissue of interest from P. patens gametophytes (see Protocol 1)
For DNA isolation (Method I), use protonemal tissue grown for 6–7 days on cellophane-overlay plates
as described in Method IB of Protocol 1.
Equipment
Indicated this here
and in the
Introduction; pls
check.
Centrifuge, at 4°C
Centrifuge tubes (50 ml)
Cheesecloth (pore size of 1.4 mm x 2.4 mm)
Erlenmeyer flask (1 liter)
Filter paper
Incubator, set at 65°C
Microcentrifuge
Microcentrifuge tube pestles (prechilled; Pellet Pestles, Kontes)
Microcentrifuge tubes (1.5 ml and 2 ml, prechilled)
Miracloth (pore size of 25 µm; Calbiochem)
Mortar and pestle (prechilled)
Nucleon PhytoPure Genomic DNA Extraction Kit (GE Healthcare)
The pellet that results from Step 15 of Method IB is the starting material for the large sample of the
kit (RPN8511).
93
94
/
Chapter 3
Paintbrush (small)
Paper towels
Plant RNA Isolation Mini Kit (Agilent)
Before performing the procedure, prepare the Extraction Solution from the kit by adding 10 µl of βmercaptoethanol <!> per milliliter of Extraction Buffer and store at 4°C.
Rotator, at 4°C
Spatula (prechilled)
METHODS
I. DNA Isolation
AU: Some of these methods instructed
to weigh tissue before freezing, other
methods instructed to weigh tissue
after freezing, and others were unclear.
Changed all methods so that you
weigh the tissue after blotting out
moisture but BEFORE freezing. OK?
A. Rapid Small-scale Preparation of DNA
1. Blot the tissue of moisture by sandwiching the tissue between sheets of filter paper and firmly
compressing the tissue with the thumb.
AU: Step 3. Is the
previous sentence
needed here?
2. Weigh 100 mg of tissue and freeze it in liquid nitrogen. Then, using a prechilled spatula, transfer the frozen tissue to a prechilled 2-ml microcentrifuge tube.
3. Grind the frozen tissue in liquid nitrogen using a prechilled microcentrifuge tube pestle. Make
sure the tissue remains frozen.
AU: Step 4. Prechilled?
4. Add 400 µl of prechilled? 1x Shorty buffer to the tube and mix well.
AU: Step 5. At what
temp?
6. Transfer 300 µl of the supernatant to a fresh 2-ml microcentrifuge tube containing 300 µl of
cold?? isopropanol. Mix by inverting the tube.
AU: Step 6. Cold?
7. Centrifuge the sample at 16,000g for 10 minutes temp??.
AU: Step 7. At what
temp.?
8. Discard the supernatant and dry the pellet by inverting the tube on a paper towel.
AU: Step 9. Added
“or” in previous sentence – correct?
9. Once the tube is dry, add 300 µl of TE. Resuspend the pellet by incubating for 30 minutes at
65°C or overnight at 4°C.
AU: Step 10. How
long? Sentence added
– OK?
5. Centrifuge the sample in a microcentrifuge at maximum speed for 5 minutes .
The pellet may not be obvious, but DNA is there—don’t worry!
10. Store the DNA at 4°C for up to ??.
For PCR, use 1 µl of DNA per reaction.
B. Isolation of High-molecular-weight DNA
1. Blot the tissue of moisture by sandwiching the tissue between sheets of filter paper and firmly
compressing the tissue with the thumb.
AU: Step 2. Do you
recommend an
approximate starting
quantity here? Pls
add as note.
2. Weigh the blotted tissue and record the mass in grams.
3. Freeze the tissue in liquid nitrogen. Then, using a liquid-nitrogen-chilled mortar and pestle,
grind the frozen tissue to a coarse powder. Transfer the ground tissue to a 1-liter Erlenmeyer
flask.
4. To the flask, add 13.3 ml of cold NIBM per gram of tissue.
5. Gently shake the flask by slowly rotating it for 15 minutes at 4°C.
6. Filter the ground tissue through four layers of cheesecloth plus one layer of Miracloth.
The filtrate will contain nuclei.
7. Squeeze the pellet to allow maximum recovery of nucleus-containing solution.
The Moss Physcomitrella patens
/
95
8. Filter the filtrate again through one layer of Miracloth.
9. To the filtrate, add 1 ml of NIBT for every 30 ml of filtrate. Gently shake the mixture by slowly
rotating it for 15 minutes at 4°C.
10. Transfer the mixture to 50-ml centrifuge tube and centrifuge at 2400g for 15 minutes at 4°C.
11. Discard the supernatant and gently resuspend the pellets in the residual buffer by tapping the
tubes or by mixing with a small paintbrush.
12. Dilute the nuclei suspensions with NIBM and combine them into two 50-ml centrifuge tubes.
Adjust the total volume in each tube to 50 ml with NIBM and centrifuge at 2400g for 15 minutes at 4°C.
13. Discard the supernatant and gently resuspend the pellets in the residual buffer by tapping the
tubes or by mixing with a small paintbrush.
14. Dilute the nuclei suspensions with NIBM and combine them into one 50-ml centrifuge tube.
Adjust the total volume to 50 ml with NIBM and centrifuge at 2400g for 15 minutes at 4°C.
15. Discard the supernatant and resuspended the pellet in the residual buffer (1 ml). Use the resuspended pellet, which is enriched in nuclei, as the starting material for genomic DNA
extraction with the Nucleon PhytoPure Genomic DNA Extraction Kit (GE Healthcare).
II. RNA Isolation
1. Blot the tissue of moisture by sandwiching the tissue between sheets of filter paper and firmly
compressing the tissue with the thumb.
2. Weigh 100 mg of the tissue and, without delay, freeze the tissue in liquid nitrogen. Use a
prechilled spatula to transfer the frozen tissue to a prechilled 1.5-ml microcentrifuge tube.
3. Grind the frozen tissue in liquid nitrogen using a prechilled microcentrifuge tube pestle. Make
sure that the tissue remains frozen.
AU: Step 4. Added this
note because the kit
protocol says that all
steps can be performed at RT – OK?
4. Add 500 µl of Extraction buffer (containing β-mercaptoethanol) from the Plant RNA
Isolation Mini Kit (Agilent) and grind continuously for at least 7 minutes to maximize yield.
This and all subsequent steps can be performed at room temperature.
5. Centrifuge the sample at 12,000g for 1.5 minutes and transfer the supernatant to the prefiltration column that is supplied with the Plant RNA Isolation Mini Kit (Agilent). Proceed
per the kit protocol. Store all RNA samples at –80°C.
III. Protein Isolation
1. Blot the tissue of moisture by sandwiching the tissue between sheets of filter paper and firmly
compressing the tissue with the thumb.
AU: Step 4. Changed
“aliquots as
required” to “100-µl
aliquots” for specificity. Also added
“1.5-ml microcentrifuge tubes based
on the volumes
needed below – OK?
And how many 100µl aliquots do you
typically obtain from
700 mg of tissue?
Add as note here.
2. Weigh about 700 mg of the blotted tissue and freeze it in liquid nitrogen.
3. Using a liquid-nitrogen-chilled mortar and pestle, grind the frozen tissue to a fine powder.
4. Divide the tissue powder into 100-µl aliquots in 1.5-ml microcentrifuge tube and store them
at –80°C until needed for protein extraction.
5. When required, suspend about 100 µl of powder in 1 ml of prechilled protein extraction solution. Incubate the mixture for 2 hours at –20°C.
6. Centrifuge the mixture at 20,000g for 30 minutes at 4°C and discard the supernatant.
7. To remove pigments, lipids, and nucleic acids, add 1 ml of prechilled protein wash solution,
mix, and incubate for 1 hour at –20°C.
96
/
Chapter 3
AU: Step 8. Added “at
8. Centrifuge the mixture at 20,000g for 30 minutes at 4°C and discard the supernatant.
4°C and discard the
supernatant” – correct? 9. Repeat Steps 7 and 8 until the supernatant is completely clear and uncolored.
AU: Step 9 note. Can
you give an example
here, please?
AU: Step 10. For
approx. how long?
Also, added “the pellets” to previous sentence and deleted the
note about dissolving
the pellet for downstream applications
because it was vague –
OK?
The number of washes depends on the tissue type and its age.
10. Dry the pellet by leaving the centrifuge tubes open at room temperature. Store the pellets at
–80°C.
Recipes
AU: Please doublecheck all recipes
for accuracy!
The recipes for items marked with <R> are also listed here.
CAUTION: See Appendix XX for appropriate handling of materials marked with <!>.
BCD MEDIUM, LIQUID OR SOLID
Reagent
Quantity (for 1 liter)
Final concentration
7g
111 mg
12.5 mg
10 ml
10 ml
10 ml
1 ml
to 1 liter
0.7% (w/v)
1 mM
45 µM
1 mM MgSO4
1.84 mM KH2PO4
10 mM KNO3
trace
Agar (Sigma-Aldrich A9799)
CaCl2 <!>
FeSO4 • 7H2O <!>
Solution B <R>
Solution C <R>
Solution D <R>
TES <R>
H2O
AU: At what temp
should the medium
be stored and for
how long will it last?
Do not include CaCl2 if the medium is to be used to prepare protoplast regeneration media (PRMB, PRMT,
or PRML). Include agar for solid media only. Most high-grade agars can be used, but some may affect the
pH of the medium. If an agar other than Sigma-Aldrich A9799 is used, the quantity (7 g) may need to be
altered. Sterilize the medium by autoclaving for 40 minutes at 121°C. Add common moss media supplements
<R> as necessary. Store at XX°C for up to XXX.
COMMON ANTIBIOTICS INCLUDED WITH MOSS MEDIA
AU: Pls fill in table for
“Common antibiotics
included with moss
media” and make sure it
is cited appropriately
(with <R>) in the
reagents lists of the protocols above.
Supplement
Quantity (per
liter of H2O) for
stock solution
Concentration
in stock solution
G418 <!>
Hygromycin <!>
Sulfadiazine <!>
Concentration
in medium (1x)
50 µg/ml
30 µg/ml
150 µg/ml
COMMON MOSS MEDIA SUPPLEMENTS
Supplement
AU: At what temp do
you store these stock
solutions, and for
how long will they
last? Also, Pls confirm the quantities
and concentrations
in this table are correct.
1000x p-Aminobenzoic acid <!>
100x Diammonium
tartrate
1000x Nicotinic acid
100x D-Sucrose
1000x Thiamine-HCl
Quantity (per
liter of H2O) for
stock solution
Concentration
in stock solution
Concentration
in medium (1x)
247 mg
1.8 mM
1.8 µM
92 g
500 mM
5 mM
1g
500 g
500 mg
8 mM
1.5 M
1.5 mM
8 µM
15 mM
1.5 µM
Stock solutions may be either autoclaved or filter-sterilized and added to growth media as required to give
the concentrations listed above. For crossing thiA1 strains, add thiamine-HCl to a final concentration of
15 nM (0.01x), not 1.5 µM (1x).
97
98
/
Chapter 3
DMSO–GLUCOSE SOLUTION
AU: Do you need to prepare this solution fresh? If
so, please indicate here. If
not, please indicate at
what temp you should
store this solution and
how long it will last.
Reagent
Quantity (for X X m l )
Final concentration
XX g
XX g
to XX ml
5% (v/v)
10% (w/v)
Quantity (for 100 ml)
Final concentration
2g
to 100 ml
2%
Dimethylsulfoxide (DMSO) <!>
Glucose
H2O
DRISELASE SOLUTION
Reagent
Driselase
D-Mannitol (8.5% w/v)
Stir to mix (but do not shake vigorously) for at least 15 minutes. Centrifuge at 2500g for 5 minutes and filter-sterilize the clear supernatant. There may be variation among batches of driselase, so adjustment to the
driselase quantity (2 g) may be necessary.
HOAGLAND’S A-Z TRACE ELEMENT SOLUTION (TES)
Reagent
Quantity (for 1 liter)
Final concentration
55 mg
55 mg
55 mg
614 mg
28 mg
28 mg
28 mg
389 mg
28 mg
55 mg
to 1 liter
0.006% (w/v)
0.006% (w/v)
0.006% (w/v)
0.061% (w/v)
0.003% (w/v)
0.003% (w/v)
0.003% (w/v)
0.039% (w/v)
0.003% (w/v)
0.006% (w/v)
Al2(SO4)3 • K2SO4 • 24H2O <!>
CoCl2 • 6H2O <!>
CuSO4 • 5H2O <!>
H3BO3 <!>
KBr <!>
KI <!>
LiCl <!>
MnCl2 • 4H2O <!>
SnCl2 • 2H2O <!>
ZnSO4 • 7H2O <!>
H2O
The exact composition of this solution is probably not important.
MCT SOLUTION
Reagent
Quantity (for 10 ml)
Final concentration
1 ml
9 ml
100 µl
100 mM
7.65% (w/v)
10 mM
Ca(NO3)2 (1 M) <!>
D-Mannitol (8.5% w/v)
Tris-Cl (1 M, pH 8.0)
Prepare fresh on day of use and filter-sterilize.
The Moss Physcomitrella patens
/
99
MMM SOLUTION
Reagent
M-Mannitol
2-[N-morpholino] ethanesulfonic
acid (MES) (1% w/v, pH 5.6) <!>
MgCl2 (1 M)
H2O
Quantity (for 10 ml)
Final concentration
910 mg
1 ml
9.1%
10%
150 µl
8.85 ml
15 mM
Dissolve the D-mannitol in the H2O, sterilize by autoclaving, and store at room temperature if necessary. On
the day of use, add the MES and MgCl2 to the D-mannitol solution and filter-sterilize.
NITROGEN-FREE MEDIUM
AU: For the Nitrogen-free
medium, added CaCl2 to the
list of ingredients and
deleted the note about
“except for protoplast
regeneration media”
because Nitrogen-free
medium is not used to prepare any of the protoplast
regeneration media. OK?
Reagent
Agar (Sigma-Aldrich A9799)
CaCl2 <!>
FeSO4 • 7H2O <!>
Solution B <R>
Solution C <R>
TES <R>
H2O
Quantity (for 1 liter)
Final concentration
7g
111 mg
12.5 mg
10 ml
10 ml
1 ml
to 1 liter
0.7% (w/v)
1 mM
45 µM
1 mM MgSO4
1.84 mM KH2PO4
trace
Include the agar for solid media only. Most high-grade agars can be used, but some may affect the pH of the
medium. If an agar other than Sigma-Aldrich A9799 is used, the quantity (7 g) may need to be altered.
NUCLEI ISOLATION BUFFER CONTAINING β-MERCAPTOETHANOL (NIBM)
Reagent
EDTA (500 mM)
KCl (1 M)
β-Mercaptoethanol <!>
Spermidine (1 M) <!>
Spermine (1 M) <!>
D-Sucrose
H2O
AU: for up to how
long?
Quantity (for 1 liter)
Final concentration
20 ml
100 ml
20 µl
4 ml
1 ml
171 g
to 1 liter
10 mM
100 mM
2% (v/v)
4 mM
1 mM
17.1% (w/v)
Mix all ingredients except for the β-mercaptoethanol and filter-sterilize. Store at 4°C how long?. Add the βmercaptoethanol immediately before use.
NUCLEI ISOLATION BUFFER CONTAINING TRITON X-100 (NIBT)
AU: for up to how long?
For NIBT, assumed a 20%
stock concentration of
Triton X-100, for a final
concentration of 2% in
the solution. Are these values correct?
Reagent
EDTA (500 mM)
KCl (1 M)
Spermidine (1 M) <!>
Spermine (1 M) <!>
D-Sucrose
Triton X-100 (20%) <!>
H2O
Filter-sterilize. Store at 4°C how long?.
Quantity (for 1 liter)
Final concentration
20 ml
100 ml
4 ml
1 ml
171 g
100 ml
to 1 liter
10 mM
100 mM
4 mM
1 mM
17.1% (w/v)
2%
100
/
Chapter 3
PEG SOLUTION FOR PROTOPLAST FUSION (PEG/F)
Reagent
AU: Store at what temp
and for how long?
AU: For PEG/F, pls fill
in XXs in table.
CaCl2 • 6H2O
H2O
Polyethylene glycol (PEG) (MW 6000)
Quantity (for X X m l )
Final concentration
109 mg
10 ml
5g
XX
XX
XX
Melt the PEG in a microwave oven or water bath. Dissolve the CaCl2 • 6H2O in H2O and then mix the solution with the melted PEG. Dispense into appropriate aliquots (750 µl is needed for each fusion).
PROTEIN EXTRACTION SOLUTION
Reagent
Quantity (for 100 ml)
Final concentration
70 µl
10 ml
to 100 ml
0.07% (v/v)
1% (w/v)
β-Mercaptoethanol <!>
Trichloroacetic acid (TCA) (10% w/v) <!>
Ethanol <!>
AU: for up to how long? Filter-sterilize. Store at 4°C how long?.
PROTEIN WASH SOLUTION
Reagent
Quantity (for 100 ml)
Final concentration
400 µl
70 µl
100 µl
to 100 ml
2 mM
0.07% (v/v)
100 mM
EDTA (500 mM)
β-Mercaptoethanol <!>
Phenylmethylsulfonyl fluoride (PMSF) (1 M) <!>
Acetone <!>
AU: for up to how long?
Filter-sterilize. Store at 4°C how long?.
PROTOPLAST REGENERATION MEDIUM FOR THE BOTTOM LAYER (PRMB)
Reagent
AU: Added CaCl2 to
table – OK? At what
temp should the
PRMB be stored, and
for how long will it
last?
Agar (Sigma-Aldrich A9799)
CaCl2 <!>
Diammonium tartrate
D-Mannitol
BCD medium, liquid <R>
Quantity (for 1 liter)
Final concentration
7g
1.1 g
920 mg
60 g
to 1 liter
0.7% (w/v)
10 mM
5 mM
6% (w/v)
Most high-grade agars can be used, but some may affect the pH of the medium. If an agar other than SigmaAldrich A9799 is used, the quantity (7 g) may need to be altered. Sterilize the medium by autoclaving for 40
minutes at 121°C.
The Moss Physcomitrella patens
/
101
PROTOPLAST REGENERATION MEDIUM FOR THE TOP LAYER (PRMT)
Reagent
AU: Added CaCl2 to table
– OK? At what temp
should the PRMT be
stored, and for how long
will it last?
Agar (Sigma-Aldrich A9799)
CaCl2 <!>
Diammonium tartrate
D-Mannitol
BCD medium, liquid <R>
Quantity (for X X m l )
Final concentration
4g
1.1 g
920 mg
80 g
to 1 liter
0.4% (w/v)
10 mM
5 mM
8% (w/v)
Most high-grade agars can be used, but some may affect the pH of the medium. If an agar other than SigmaAldrich A9799 is used, the quantity (4 g) may need to be altered. Sterilize the medium by autoclaving for 40
minutes at 121°C.
PROTOPLAST REGENERATION MEDIUM, LIQUID (PRML)
Reagent
AU: Deleted the agar
from the PRML recipe
– OK? I added CaCl2
to table – OK? At what
temp should the
PRML be stored, and
for how long will it
last?
CaCl2 <!>
Diammonium tartrate
D-Mannitol
BCD medium, liquid <R>
Quantity (for 1 liter)
Final concentration
1.1 g
920 mg
85 g
to 1 liter
10 mM
5 mM
8.5% (w/v)
Most high-grade agars can be used, but some may affect the pH of the medium. If an agar other than
Sigma-Aldrich A9799 is used, the quantity (1.1 g) may need to be altered. Sterilize the medium by autoclaving for 40 minutes at 121°C.
PROTOPLAST WASH (PW) SOLUTION
AU: At what temp
should the PW
solution be stored,
and for how long
will it last?
Reagent
CaCl2 • 6H2O <!>
D-Mannitol
H2O
Quantity (for 1 liter)
Final concentration
2.19 g
85 g
to 1 liter
10 mM
8.5% (w/v)
Sterilize by autoclaving for 40 minutes at 121°C. This recipe contains CaCl2 at 10 mM, but different laboratories use concentrations between 0 mM and 50 mM with no apparent differences in protoplast viability.
PROTOPLAST WASH SOLUTION FOR AGROBACTERIUM TRANSFORMATION (APW)
AU: Do you need to
prepare this solution
fresh? If so, please
indicate here. If not,
please indicate at what
temp you should store
this solution and how
long it will last.
Reagent
CaCl2 • 6H2O <!>
1000x Vancomycin stock
solution (50 mg/ml) <!>
1000x Cefotaxime stock solution
(200 mg/ml) <!>
D-Mannitol solution (8.5% w/v)
Quantity (for 100 ml)
Final concentration
219 mg
100 µl
10 mM
1x
100 µl
1x
100 ml
102
/
Chapter 3
5x SHORTY BUFFER STOCK SOLUTION
Reagent
Quantity (for 500 ml)
Final concentration
25 ml
100 ml
50 ml
100 ml
225 ml
25 mM
400 mM
1% (w/v)
200 mM
EDTA (500 mM)
LiCl (2 M) <!>
Sodium dodecyl sulfate (SDS) (10% w/v) <!>
Tris-Cl (1 M, pH 9.0)
H2O
Store at 4°C AU: for up to how long?. Dilute fivefold with H2O as required.
SOLUTION B
Reagent
Quantity (for 1 liter)
Final concentration
25 g
to 1 liter
0.1 M
Reagent
Quantity (for 1 liter)
Final concentration
KH2PO4
H2O
25 g
to 1 liter
184 mM
MgSO4 • 7H2O <!>
H2O
SOLUTION C
AU: FOR SOLUTIONS
B, C, AND D: If you
should sterilize this
solution, please specify
how. Do you need to
prepare this solution
fresh? If so, please indicate here. If not, please
indicate at what temp
you should store this
Dissolve the KH2PO4 in 500 ml of H2O and adjust the pH to 6.5 with a minimal volume of 4 M KOH <!>.
Bring the final volume to 1 liter with additional H2O.
SOLUTION D
solution and how long
it will last.
Reagent
KNO3 <!>
H2O
Quantity (for 1 liter)
Final concentration
101 g
to 1 liter
1M
Quantity (for 1 liter)
Final concentration
6g
6g
50 mM
50 mM
TRIS-MALEATE BUFFER
Reagent
Maleic acid <!>
Tris-(hydroxymethyl)aminomethane <!>
H2O
to 1 liter
Adjust the pH to 6.0 with 10 M NaOH <!> or KOH <!>. Store at 4°C AU: for up to how long?.
The Moss Physcomitrella patens
/
103
REFERENCES
Ashton, N.W. and Cove, D.J. 1977. The isolation and preliminary
characterization of auxotrophic and analogue resistant mutants
in the moss Physcomitrella patens. Mol. Gen. Genet. 154: 87–95.
Axtell, M.J., Snyder, J.A., and Bartel, D.P. 2007. Common functions
for diverse small RNAs of land plants. Plant Cell 19: 1750–1769.
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