PROJECT 1

GENETIC SCREEN

FOR DEVELOPMENTAL AND RESPONSE MUTANTS

OBJECTIVES:

This lab will provide experience in designing and performing a mutagenesis screen in a forward genetics approach. Mutated populations will be screened to detect morphological and conditional mutants. These mutants will be used in crosses to characterize the nature of the mutation. The outcome should be an isolated mutation that the student can discuss potential characteristics of the gene or genes involved. Similar mutants are used in the molecular mapping project to illustrate how the mutants are used to isolate the gene responsible.

BACKGROUND:

Molecular genetics seeks to elucidate the molecular mechanisms that make an organism work- the biochemical functions of enzymes and macromolecular interactions that determine development and metabolism. There are two general approaches for linking a certain function with a specific gene. One is to create a mutant organism that has an altered function, and then to find which gene was altered. Another is to take a gene or gene product and then work backwards to find its function (reverse genetics). The mutational approach offers an advantage of knowing that the gene you find has a defined function. Once a mutant is found, the gene that was altered can be identified (cloned), and further tools can be used to determine how that gene product interacts with products of other genes to produce the overall function. Thus, identifying mutants in a certain process is the first important step in this approach.

Classical genetics is based upon analysis of genetic variation to dissect the components that determine a certain trait. Although natural variants can be used in genetics (e.g. different varieties of corn or different humans), there can be too many differences between them to allow easy analysis of just one gene. Induced mutations, produced through treatment of the organism with a mutagenic agent, produces genetic variants that potentially have fewer genes altered. The chemical mutagen used for many organisms is ethylmethane sulfonate (EMS). EMS adds an ethyl group to G and T residues, allowing the modified base to pair with T and G residues, respectively. The organism is usually treated at a stage where the mutation of DNA in one cell will ultimately lead to a uniformly mutated organism in a subsequent generation.

Mutation of Arabidopsis

Mutagenesis is normally performed upon seeds, providing for easy handling of large numbers of individuals and a small number of cells per mutated organism.

Two problems are encountered in mutating seed. First is the low statistical probability that any one gene will be mutated in any one plant. Thus, many plants need to be screened to detect mutations in that gene. An optimal dose of mutagen will provide as much mutagenic potential as possible, without being so great as to kill the plants. Unfortunately, the kill rate increases exponentially with dose as the mutation rate for any one gene rises linearly. The optimum dose for mutation is that which produces about 37% survival among the mutagenized seeds. The mutation rate can also be measured for a visible known phenotype, such as albinoism (lack of chlorophyll). These are often 1 albino per 1000 seed. A second problem in mutagenized seed is that there are multiple cells in the embryo of seeds that will give rise to different parts of the plant. Thus, a mosaic plant is formed. Mutations occurring in one cell in the seed may only appear on one side of the plant, if at all. Thus, multiple seeds need to be collected from each plant. Third, recessive mutations are at least 10-fold more likely to occur than dominant mutations. The recessive mutations cannot be observed in the heterozygous plant that grows form the mutagenized seed (called the M1 generation), because only one homologous chromosome bears the mutation. Therefore, this plant must be crossed with itself to produce a fraction of homozygous progeny. Arabidopsis is a good model plant since it is self fertile and this self fertilization in each flower is easy- you just let it alone. As a typical monohybrid cross, only one-quarter of the progeny (called the M2 generation) display the mutant phenotype. From all these considerations, one needs to screen about 34 M2 plants to have a 95% probability of finding a mutation that occurred in the original plant. A set of seed from one M2 plant could represent a family- related seeds that contain multiple individuals arising from the same mutagenized seed. The object is to screen as many families as possible. In practice, seeds from multiple M1 plants are collected together (pooled), and M2 plants are screened from the pool. For a pool of 50 M1 plants, one would need to screen about 1796 plants to have a 95% probability of detecting a mutation occurring in one of the seeds.

In order to save the time required for growth and self-fertilization of the M1 generation, we will use M2 seed purchased from a company that specializes in supplying mutagenized Arabidopsis seed. The envelope will show the number of plant families represented in the packet.

Our screen will use soil-grown plants. These plants will be followed over several weeks of growth so that alterations in different stages of development can be observed. This should identify developmental mutants, presumably altered in a signaling pathway that coordinates development. As the plants mature, think of other conditional screens that you could set up. Conditional mutants are ones in which a phenotype is apparent only under certain circumstances. For example, the mutants may be normal-looking in typical light conditions, but may remain green in constant darkness while other plants turn yellow and elongate. The mutant may survive heat stress better than others, or conversely be more sensitive to heat stress than the other plants. Either type of mutant would provide insights into the signal pathways through which plants can tolerate heat stress.

Once a mutant plant is identified, it is marked with thread. The plant is allowed to flower. Some flowers will be used to pollinate female wild-type plants (out-crossed). Others will be allowed to self-pollinate (selfed). The seeds from these out-crossed and selfed flowers will be planted so that the phenotypes of the progeny can be determined. The segregation of progeny will be used to indicate the nature of the mutation in: genetic contribution to the mutations, dominant/recessive relationships, and number of genes contributing to the phenotype.

Mutation of C. elegans

C. elegans is a lower animal, and so separates the gamete-producing cells from somatic cells early in development. Therefore, treating embryos or adult nematodes will mostly only cause somatic mutations that are not passed on to the next generation. However, treating near-adult nematodes with EMS will mutate some of the cells that do give rise to gametes. The sperm-producing cells are most susceptible. We will treat C. elegans that are in the L4 stage while they are preparing to start producing sperm. These animals will be allowed to self-fertilize since the overwhelming majority are hermaphodites. The generation produced by this self-fertilization (M1) will be mostly heterozougous for any mutation. Therefore, like arabidopsis, the M1 generation will not display recessive mutations. We will allow the M1 generation to self-fertilize, and this will produce an M2 generation where ¼ of the progeny of any one mutated nematode could display a recessive mutation. C. elegans will reproduce so rapidly, that within a week, another generation (M3) will appear. This generation may display some additional phenotypes, uncovered through additional segregation to produce homozygous mutants. However, the numbers of nematodes on the plate will also become larger. The best chance of identifying mutants will be in the M2 generation. Since the mutagenesis was performed on sperm cells, the nematodes will not be subjected to the mosaics that arabidopsis was troubled with.

The screen for mutants in C. elegans is again for both developmental and response mutants. Developmental mutants can arise from alterations in the signaling pathways that determine cell fate- a process for which C. elegans serves as a model system. Since it is an animal, the responses of C. elegans are more behavioral and are on a fast time-scale than the arabidopsis. Behavioral phenotypes you may watch for are motility, mating, egg-laying, and response to touch. Other more conditional mutations can be seen in tolerance to different drugs or chemotaxis towards or away from certain chemicals.

Analyzing the mutants in C. elegans will be similar to that described above for arabidopsis. However, with the faster generation time of 4-5 days, the steps will be faster. Leaving a mutant alone on a plate will allow it to self-fertilize and subsequent progeny should display the mutation. The appearance of wild-type progeny will tell you something about the nature of the mutation. In order to cross it with wild-type, we will provide wild-type males that will mate with the L4 hermaphrodites and preferentially produce out-crossed progeny. Ratios of mutant vs wild-type progeny will tell you more about the mutation. Further tests can be carried out by crossing your mutants with one someone else in the lab has found, or one from the C. elegans Stock Center. This will allow you to determine if the it is the same or a different genetic locus that is creating this phenotype. The goal is to learn as much as possible about your mutants within the semester.

Lab report expectations

You are expected to follow at least one mutation you find, either in Arabidopsis or C. elegans. Characterize it as much as possible:

-Describe the phenotype, back it up with photographs or drawings

-Describe what other mutants have been found with the same phenotype in the literature. What is known about this phenotype? What gene(s) are responsible, their map locations, the enzyme(s) encoded, the type of pathway involved. This is important even if your mutation proves to not be one of these genes. Place your mutation in relation to these.

-The genetic properties of your mutation: Is it genetically determined or is it due to environmental variation? Is it dominant or recessive? Does complementation testing tell if it is the same gene or different gene than another mutation?

OVERVIEW

Mutagenesis 1: Plant M2 seeds on agar and soil

Each student/group will perform a screen for morphological mutants, planting seeds in pots in soilless mix.

Read Instructions: Planting mutagenized seed in soilless mix

Web resources:

Dated pictures of Arabidopsis growth- get an idea of the timeframe (click on different dates):

http://www.arabidopsis.com/main/vgr/job0065.html

Movies of Arabidopsis and other plants- germination, growth, responses

http://sunflower.bio.indiana.edu/~rhangart/plantmotion/PlantsInMotion.html

(Choose Plant movies in frame on left- needs Quicktime)

Arabidopsis handling tips:

http://www.biosci.ohio-state.edu/~plantbio/Facilities/abrc/handling.htm

Questions to consider:

The density at which plants are sowed might affect the growth of some of the plants. What might you expect if the seeds are sown too densely or in clumps of seeds?

What phenotypes will you be looking for in arabidopsis? Consider that a number of phenotypes are apparent only under certain conditions (e.g. etiolation of seedling in darkness) or certain stresses (e.g. dryness, salt spray or in soil, disease organism). Is there any treatment that you could think would be testable? See the resources in labs below.

Mutagenesis 2: Mutagenesis of C. elegans

The nematodes will be treated with the mutagenic EMS before class- it takes about 4 hours of soaking. Afterwards, we will wash off the EMS and you will plate 4-10 M0 (L4 larvae) onto a plate with bacterial lawns. This will give you a feeling for the transfer of C. elegans. These will lay eggs that represent the F1 generation. We will pick off the M0 adults the next day to limit the number of F1 individuals. Those will lay eggs for the F2 generation.

Read instructions: Transferring C. elegans

Web Resources:

Handbook of worm anatomy: http://www.wormatlas.org/

C. elegans as an experimental system: Encyclopedia of life: http://mrw.interscience.wiley.com/emrw/9780470015902/els/article/a0000564/current/html

Anatomy and Larval Stages: http://thalamus.wustl.edu/nonetlab/ResearchF/elegans.html

Overviews of C. elegans:

A Biochemist’s Guide to C. elegans 2007: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1855192

http://www.dsls.usra.edu/biologycourse/workbook/Unit4.2.pdf (from worms in space initiative- go to pg. 3)

Flyover: http://130.15.90.245/BIRDSEYEMOVIE.qt

C. elegans movies: http://www.bio.unc.edu/faculty/goldstein/lab/movies.html http://www.wormclassroom.org/ge.html#movie http://130.15.90.245/c__elegans_movies.htm

Mutagenesis 3: Elimination of M0 parents from plates. After 2 days, the L4 larvae that you put on your plate will have matured and started to lay eggs that represent an M1 generation. You will pick off the M0 adults to limit the number of M1 individuals. Those will lay eggs for the M2 generation that we want to screen next week. We will also look at different known mutants of C. elegans.

Mutagenesis 4: Screening for mutations in C. elegans

The plates you made last lab have been pooled and transferred to many large plates that now have a population of F2 progeny. Look through the plates for behavioral or developmental mutants.

IMPORTANT RESOURCES!!!! WORM BOOK online: http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=wormbook
Chapters on basic systems- muscles, nervous system, reproduction, development. Very useful.

Worm BOOK II online: http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=ce2
advanced topics in specific aspects of C. elegans

Worm Breeding for Dummies- tips for crosses and techniques
http://courses.biology.utah.edu/peters/5265/References/WBFD copy.pdf

Some different types of mutants are: (movies at: http://130.15.90.245/c__elegans_movies.htm)

Shape- e.g. Dumpy (dpy), long/stringy (lon), blistered (bli)

               Classic Paper: Park E-C;Horvitz HR 1986. Mutations with dominant effects on the behavior and morphology of the nematode C. elegans. Genetics 113: 821-                                                                             52.http://www.genetics.org/cgi/reprint/113/4/821

Motility- uncoordinated (unc) classic papers: Park and Horvitz above
Brenner 1974 Genetics 77:71-94 http://dev.wormbase.org/papers/31_Brenner74.pdf

See the C. elegans movies (Quicktime) http://www.wormclassroom.org/ge.html#movie

Encyclopedia of life: C. elegans nervous system http://mrw.interscience.wiley.com/emrw/9780470015902/els/article/a0000110/current/html

Rolling (rol)-
(http://www.bioone.org/bioone/?request=get-document&issn=0022-3395&volume=086&issue=02&page=0269)

Classic paper: Cox GN;Laufer JS;Kusch M;Edgar RS 1980.Genetic and phenotypic characterization of roller mutants of C. elegans. Genetics 95: 317-339 http://www.genetics.org/cgi/reprint/113/4/821

Response- mechanical touch (mec) (http://sq.ucsd.edu/sq1_marika_orlov.pdf)

Encyclopedia of life: C. elegans nervous system http://mrw.interscience.wiley.com/emrw/9780470015902/els/article/a0000110/current/html

Development-eggless (egl), bag of worms , vulvaless, multivulva

Classic paper: Trent C;Tsung N;Horvitz HR 1983. Egg-laying defective mutants of the nematode C. elegans. Genetics 104: 619-647.
http://www.genetics.org/cgi/reprint/91/1/6

Vulval mutants-often are eggless: Ferguson EL, Horvitz HR. 1985 Identification and characterization of 22 genes that affect the vulval cell lineages of the nematode Caenorhabditis elegans. http://www.genetics.org/cgi/reprint/110/1/17

Encylopedia of Life: C. elegans vulval development: http://mrw.interscience.wiley.com/emrw/9780470015902/els/article/a0001146/current/html

How to make a vulva:
http://pharyngula.org/index/weblog/comments/how_to_make_a_vulva/&e=10401

everted vulva: Seydoux G, Savage C, Greenwald I. Isolation and characterization of mutations causing abnormal eversion of the vulva in Caenorhabditis elegans.
Dev Biol. 1993 Jun;157(2):423-36.

Dauer formation-dau mutants

Classic paper: Malone EA;Thomas JH 1994. A screen for nonconditional dauer-constitutive mutations in Caenorhabditis elegans. Genetics 136: 879-886.http://www.genetics.org/cgi/reprint/136/3/879

High frequency of males- (him)

Classic papers: Hodgkin J;Horvitz HR;Brenner S. 1979. Nondisjunction mutants of the nematode C. elegans. Genetics 91: 67-94. http://www.genetics.org/cgi/reprint/91/1/67

                            Hodgkin JA;Brenner S. 1977. Mutations causing transformation of sexual phenotype in the nematode C. elegans.  Genetics 86: 275-287 http://www.genetics.org/cgi/reprint/86/2/275

Examples of other screens:

Ethanol tolerance: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=420456

Aging: http://wormpage.tripod.com/aging.html

http://www.botany.utoronto.ca/courses/bio260/Munoz&Riddle_2003_Genetics_163_171.pdf

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=ce2.section.946

Dauer formation: http://www.cals.ncsu.edu/course/zo402/celegans.html

Levamisole Resistance (Lev is an acetylcholine esterase inhibitor) http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=7203008

Mutagenesis 5: Screening for morphological seedling mutations of Arabidopsis

Screens on soilless mix-grown plants will observe plants with altered phenotypes. See Arabidopsis : An Atlas of Morphology and Development in the lab for reference to what wild-type looks like.

Questions to consider:

How many plants of any one mutation type do you expect to find?

How can you tell if the phenotype of a “mutant” you are seeing is from environmental conditions (e.g. crowding, lack of water, shading) rather than from a genetic defect?

Look again at the web resources below (on the web site)- what types of mutants might you be able to find? Do some require that the plant is older than it is now?

Web Resources: (links on web site)

-Nottingham Arabidopsis Stock Center Picture Book of Arabidopsis Mutants:

http://seeds.nottingham.ac.uk/Nasc/action.lasso?-response=picbook/picture_book.lasso&-token.user=11639328

-Descriptions of characterized mutants available from Arabidopsis stock center –you can search in the description, eg. Round leaves: http://arabidopsis.org/servlets/Search?action=new_search&type=germplasm

-Search the locus names or keyword on the Arabidopsis.org site:

http://www.arabidopsis.org/servlets/Search?type=general&action=new_search

-List of Arabidopsis Lab websites- organized by topic:

http://www.arabidopsis.org/info/lab.jsp

Lab web pages describing certain mutants:

Various morphological mutants-nice pictures!:

http://vannocke.user.msu.edu/pages/811%20lab/mutants.html

Glucose insensitive: http://www.ag.ohio-state.edu/~jcjang/Mutants/gim.html

Embryo-defective: http://mutant.lse.okstate.edu/embryopage/embryopage.html

http://www.plantsci.cam.ac.uk/Haseloff/teaching/MCBPart1B/Lecture1.html

(Great background for embryo development mutants)

Flower mutants:

http://www-biology.ucsd.edu/others/yanofsky/flower/intro_to_flower_dev.htm

http://www.its.caltech.edu/~plantlab/html/.index.html

Root morphology:

http://www.biologists.org/serve.cgi?Development/119/01/dev6015.pdf

background:

http://www.plantsci.cam.ac.uk/Haseloff/teaching/MCBPart1B/Lecture2.html

Meristem mutations:

http://www.plantsci.cam.ac.uk/Haseloff/teaching/MCBPart1B/Lecture3.html

What will you name your mutants? Guidelines

http://www.arabidopsis.org/info/guidelines.html

Mutagenesis 6: Crossing C. elegans mutants

Making crosses in C. elegans takes a bit of preparation. One needs to find or create males. Males will occur in about 1/500 worms. To increase your chances of finding it in a culture of your mutant, take 6-10 L4 onto a new plate and heat shock it at 30^oC for 6 hr.. Then look at the next generation; the frequency should be 2-5%. Another method is to cross in the him-8 mutation. This mutation increases the frequency of males to 30-40%. We will have a strain of him-8 mutants in the lab that will produce a high-frequency of males. Pick a male, preferably L4, from this plate and cross it with several of your mutant hermaphrodites (pick L4 ones). The progeny from this cross will be heterozyogous for your mutation. Pick a male again, and cross it with a hermaphrodite mutant again. The progeny from this cross should all be mutant.

You will want to set up several crosses.

Mutant x Mutant: This cross is going on as you grow your mutant alone because the mutant is self-fertilizing. Check the phenotypes of the progeny that arise from the plate. Are all mutant or is a percentage appearing wild type. If the latter, is there a correlation of age with the phenotype? What is the ratio. Separate out progeny on separate plates- do the ratios reappear?

Mutant x WT: This cross takes a bit of matchmaking on your part. Pick a him-8 male (or several) and cross it with your mutant. Try to take an L4 mutant and males so that self-fertilization is minimized. Check this possibility by looking at the males in the subsequent generation- are there a high proportion as expected? There should be approximately 50% males if the cross worked. Does your mutation show up in this generation? The males should be heterozygous in most cases. If your mutated gene is on chromosome X, then the male has only the mutant allele since it is hemizygous for the X chromosome. Be cautious about the hermaphrodites since self fertilization of the hempahrotite parent may have occured. Test several. If they are heterozygous for your mutation, then setting them by themselves should produce 1/4 homozygous mutant progeny again.

Mutant 1 x Mutant 2: This is a complementation test. Do this for testing the allelism of different mutants with a similar phenotype. For this, you will need to make males from your mutant strains as described above. You can use the males isolated from the mutant x wt cross above. Use as many other strains as possible- others of yours, others in the lab, or ones we have from the stock center.

Mutant x GFP carrying strains: We have strains that are carrying the GFP reporter gene expressed from promoters that express GFP in nerves or muscles. Crossing one of your male mutants with these strains will allow you to work towards finding a homozygous mutant with the GFP construct in it. Examining such worm under the fluorescence microscope and comparing it to the regular wt GFP worms will allow you to visualize if muscle or nerve development has been altered in your mutant.

Read Instructions: Crossing C. elegans

Web resources:

Are there other known mutants with a similar phenotype? What is known about these other mutants?

Search Wormbase: http://www.wormbase.org/

Look in Chapters of Wormbook http://www.wormbook.org/ (e.g. behavior in http://www.wormbook.org/chapters/preprints/WormMethods/Behavior.pdf)

Look in chapters of the Cold Spring Harbor book C. elegans II online http://www.ncbi.nlm.nih.gov/books/bv.fcgi?call=bv.View..ShowTOC&rid=ce2.TOC

Search in ACEView (NCBI): http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/index.html?worm

For example see searching with unc (uncoordinated)

http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/av.cgi?exdb=AceView&db=worm&term=unc

PubMed: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi

Worm literature database: http://elegans.swmed.edu/wli/

Chi-squared calculator: http://www.graphpad.com/quickcalcs/chisquared1.cfm

Mutagenesis 7: Cross pollination of arabidopsis mutants with wild-type

Mutants isolated in the previous screen will be used to perform three types of crosses: i) a self-pollination to test genetic stability of the trait, ii) a cross with wild-type plants (imp1) in order to characterize the dominance of the mutation, and iii) a cross with other plants with a similar phenotype to determine if the different mutants represent different genes. For self-pollination, Arabidopsis takes care of the pollination itself- just leave the flowers to produce seed. For cross pollination, pollen will be taken from the mutant plant and be used to pollinate an emasculated flower from the other parent. The timing of catching flowering is important for setting up a cross. To make manipulations easier, the wild-type parent will be a male-sterile mutant that does not need to be emasculated.

Read instructions: Performing a plant cross.

Questions to consider:

What would be the result if you had chosen a flower to pollinate that had already self-pollinated?

Would the result be different whether you pollinated a female flower on the mutant or on wild-type?

Mutagenesis 8: Plant seeds from cross

Seeds developing on the flowers cross-pollinated will be harvested and planted- on soil or on agar, the same as originally used for the screen.

Read Instructions: Planting mutagenized seed in soilless mix

Questions to consider:

How many plants do you need to test in order to determine the segregation ratios?

If some of the seeds you plant do not grow, perhaps due to a phenotype linked to your mutation, how would this affect your ratios?

Lab 17 and 19 Mutagenesis 6: Screen seedlings for phenotype. Test F1 for phenotypes and determine segregation. The number of seedlings with the wild-type or mutant phenotypes will be counted. These will be used to determine segregation frequencies. The plants may need to grow until lab 19 until phenotypes appear.

Read instructions: Planting mutagenized seed in soilless mix

Web resources:

Segregation ratios:

http://www.cc.ndsu.nodak.edu/instruct/mcclean/plsc431/mendel/mendel1.htm

Chi-squared test descriptions:

http://www.cc.ndsu.nodak.edu/instruct/mcclean/plsc431/mendel/mendel4.htm

http://www.ruf.rice.edu/~bioslabs/tools/stats/chisquare.html

Chi-squared table with more degrees of freedom:

http://www.uvm.edu/~golivett/introbio/lab_reports/chi.html

Analysis:

What is the ratio of mutant to wild-type and what does it imply about:

Recessive/dominance of the mutant allele in relation to the wild-type allele

Number of genes that may be involved in the phenotype

Genetic inheritance of trait

How sure are you about this ratio?

What would be the next step in characterizing the mutation?

Could it be the same as an already defined mutation?

Search genes and databases for similar phenotypes

-What cloned genes are associated with the phenotype?

Do keyword search on Genebank:

http://www.ncbi.nlm.nih.gov/genbank/query_form.html

-Do site search on the Arabidopsis database

http://www.arabidopsis.org/search/

-Searchable list of characterized mutants of Arabidopsis

http://mutant.lse.okstate.edu/genepage/genepage.html

-List of mutants available at stock center (do “find word in page” in browser) http://aims.cps.msu.edu/aims/catalog97/II-A1.html#IIA1

http://aims.cps.msu.edu/aims/catalog97/II-C1a.html

http://aims.cps.msu.edu/aims/catalog97/II-D1.html

-Perform search on Agricola, CAB or Medline for publications

PubMed: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi

Agricola-through http://www.lib.umd.edu/ Research Port

-How could you test if it is the same or different than any of these?

-What can you interpret/speculate about the component that was altered in your mutant? Think of the phenotype, type of mutation (dominant vs recessive), and what is in the literature.

DETAILED PROCEEDURES:

Planting mutagenized seed in soilless mix

1. Scoop the sterilized potting mix (“soil”- but there is no soil, just a mix of peat moss and vermiculite) into 9 white plant pots. The soilless mix has been autoclaved to avoid insect contamination. Try to get the soilless mix even with the top and smooth the surface of the planting mix. Place the filled pots in the green or black trays.

2. Wet the planting mix. First add 2 liters of tap water to the tray. Second, to help the planting mix pick up water and to wet the top layer, use the mister to spray the surface of the planting mix to soak it. Do this before sprinkling on the seed.

3. Plant the seeds. Arabidopis seeds are tiny. Shake out a small pile onto a folded card or piece of paper. Try to sprinkle them from the folded card. Each pot should get at most 25 - 50 seeds for a screen- otherwise they get too crowded. Do not try to cover the seeds with soil, they will germinate best on the surface and touching the soil will probably stick the seeds to your hands.

For subsequent manipulations where such large numbers are not important, use fewer seeds per pot since the fewer plants in a pot, the better they will grow. Planting 6 or fewer seeds in a pot leads to healthy plants but is somewhat inconvenient. 18 (3 rows of 6) gives reasonably good growth. More than that can be troublesome.

4. Cover the trays with Saran wrap. The seeds and soil need to be fully hydrated and covering the entire pot will help the water wick up to the top and prevent drying by evaporation. Rip off a piece of Saran wrap the size of the tray and place it over the tray. Tape down the edges.

5. If the seeds were not pre-chilled, provide a cold-treatment. Breaking the dormancy of the seeds requires a 2-3 day cold treatment. Place the planted trays in the cold room (4^oC, 40^oF). After 2-3 days, carry out and place under the fluorescent lights. Leave the Saran wrap on the trays. Take off the Saran wrap when the first true leaves appear on the seedlings. This will take about 1 week after taking the tray out of the cold room.

6. Watering. Initial watering should be with very dilute fertilizer, perhaps 5-10 ml per gallon jug. Be careful not to overfertilize. The first sign of underfertilization is when the rosette leaves or sepals of young flowers turn purple. The signs of overfertilization are leaves and inflorescences that turn yellow and inflorescences that become weak, flop over, and die. It is better to underfertilize than overfertilize, since overfertilized plants are unrecoverable. Generally, use very dilute fertilizer until the plants are just about to bolt, then add fertilizer later only if needed.

6. Observe plants for mutants. Characters to look for: seedling color (e.g. white seedling (albino)), stature, flower patterns, sterility in flowers.

Crossing Arabidopsis

1. Isolate mutant by weeding away other plants out of pot. Define the pollen donor (male) and the recipient (female). A parent that will show a different phenotype when outcrossed makes the identification of potential selfed-seed easier. You can immobilize the inflorescence with tape (gently).

2. Emasculate the recipient plant (under dissecting microscope, using sharp forceps):

a) select healthy inflorescence on the recipient plant

b) remove the old flowers on the inflorescence that has already opened or are even showing white petals.

d) select the next 3-4 flower buds that are at a stage (flower bud is about to open) which has good stigmatic papillae. There should not be white petals showing on the outside. Emasculate target flowers by using forceps to slightly squeeze open the bud at a level about a third from the bottom, then remove all 6 stamens (greenish in color) from all these flowers. Make sure that no pollen is present. If in doubt, do not use that floral bud. If the stigma is really fuzzy at the top, the flower may have matured too much to allow it to have already self-fertilized. Pictures are at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1945212&rendertype=figure&id=F1

Pluck out the younger buds that you are not fertilizing to avoid confusion.

3. Take mature stamens from the pollen donor flowers (use several). Gently touch the stigmatic papillae of the emasculated flowers with the anther of these stamens. The pollen will stick to the papillae and the papillae will turn yellow if the cross is good.

4. mark the crossed flowers by a thread tie on the flower petiole.

5. Repeat with another inflorescence if you can- damage to the flower may prevent its survival.

Remarks:

When emasculating the flowers or doing the crosses take care not to keep the flowers too long under the dissecting scope. They tend to dry out relatively quickly when kept under the strong light. In addition, don't squeeze the plants between your fingers during handling! The same holds true when labeling the plants.

If the cross is to the wt plants that are male-sterile (imp1), you do not have to dissect unopened flowers to avoid self fertilization. Just grab a newly opened mutant flower and rub it against an imp1 flower’s stigma. Mark the flower on the imp1 plant with thread. Treat these plants carefully since students are using other flowers.

If your mutant is not self-fertilizing, it may lack male or female function. Try cross pollination (no flower dissection) of a flower from another of your plants (wt) onto your mutant plant, and pollination of a mutant flower onto the imp1.

Seed collection:

As plants mature, they will need less water, but watering should continue while seeds are setting. It is always best to collect seeds promptly, as soon as the silliques break with little or no applied pressure.

Seed collection can be accomplished by rubbing your fingers around the sillique, and allowing the seeds to fall on a piece of paper placed below. Filter the seeds through a section of cheese cloth onto another piece of paper, to get rid of the silliques. If silliques are stored along with the seeds, fungus growth can result. If you are harvesting a single valuable silique (e.g. after a cross cross), then slip the silique into an eppendorf tube and then snip it from the stalk. Open the silique in the tube with forceps and remove the brown pod. Seeds can be stored in eppendorf tubes.

Collected seeds should be left in the tubes for approximately one week, then a hole should be poked in the top of the tube, and the tube should then be placed in a sealed container with Drierite. After another week or so the seeds may be planted. Note that seeds can be planted earlier than this, but the germination rate goes down the earlier you plant them after collecting them.

Transferring C. elegans

There are several ways of transferring C. elegans. In all, you must be careful of three things:

1) Do not poke the worm-any damage to its cuticle is going to kill it. A dead mutant is useless.

2) Do not allow the worm to dry out- they are robust on the plates, but sitting on a wire for more than a minute will allow it to dry out.

3) Avoid poking the surface of the agar. C. elegans loves to burrow into the agar and it is difficult to get that little bugger out of the agar. Bubbles or cuts in the agar promote burrowing.

In transferring one worm, the best way is to use a platinum wire. We have placed platinum wires into pasterur pipettes and have flattened the end. Sterilize the wire by placing it into the alcohol lamp flame until it glows. Let it cool outside the flame. To pick up the worm, do not use the wire as a shepard’s crook. Instead, pick up a glob of bacteria from the lawn onto the end, and use this glob to stick the worm to the wire. Place the worm onto the new plate without inserting the wire into the agar. You might want to check on the worm after transfer to make sure it is still alive.

In transferring cultures, you can cut a small chunk of agar out of one plate and place it on a new plate. This will transfer several worms, eggs, and juveniles. This is easiest for keeping your culture fresh.

Crossing C. elegans

-Use of a petri plate in which the bacteria have been streaked in a grid works best, but a lawn is fine too.

-Place an excess of males on the plate. You can never have too many males- they tend to lose interest and wander off. For example 4 hermaphrodites and 6-10 males would work well.

-make sure you are not carrying over any larvae or eggs on the worms- you might look at it for a bit to see that any unwanted guests don’t appear.

-Let the plate incubate for 3-4 days to see progeny. Since crosses with males produce about half males, you should see about half males if the cross worked. Otherwise, mostly hermaphrodites indicates that the progeny are the products of a hermaphrodite self-fertilization.

-Pick progeny off the plate and onto a new plate. You want to get a hermaphrodite that is at most an L4-with so many males around, any older stage worm may already have mated and your progeny would show an extra cross.