MGH Molecular Biology DepartmentPlant Growth Facility Jenifer Bush, Greenhouse Manager 617-726-5931bush@molbio.mgh.harvard.edu

updated: August 2, 2007

I. Contacts

II. Description of the Facilitya. Simches Growth Facilityb. Thier Greenhouses

III. Rules and Regulations

IV. Standard Protocols and Proceduresa. Plant Growth Procedureb. Gnatrol Use to prevent fungus gnatsc. Agrobacterium handling/disposald. Powdery Mildew information

V. Links (coming soon)

Contact InformationPlant Growth Facility Personnel

Jenifer Bush, Greenhouse Manager (reports to Ausubel and Sheen)

617 726-5931bush@molbio.mgh.harvard.edu

Panfilo Federico, Facilities Manager617 726-5958federico@molbio.mgh.harvard.edu

Nicholas Pritchard, Assistant Facilities Manager617 643-2889pritchard@molbio.mgh.harvard.edu

Professor Fred Ausubelausubel@molbio.mgh.harvard.edu

Professor Jen Sheensheen@molbio.mgh.harvard.edu

MGH Buildings and Grounds 6-2422

Minus Eleven (repair of plant growth equipment)781-335-5557

Description of the Plant Growth Facility

IntroductionThe plant growth facility is designed primarily for growing Arabidopsis, however corn, rice, and tobacco are occasionally grown. Walk-in rooms with light, temperature and humidity control are used for experiments, production of seeds, and for general plant growth. Reach-in growth chambers are available for plant experiments that require unusual growth conditions, or that otherwise need to be separated. Two isolated rooms with reach-in growth chambers are available for plant pathogen studies. Plants can also be grown in sterile conditions such as liquid culture using lighted incubator/shakers, or they can be grown on plates in a warm room. Greenhouse space is also available in the Thier building.

Walk-in Growth Rooms There are 4 walk-in growth rooms on the 7th floor, and 2 on the 8th floor. The rooms are set to standard growth conditions. The growth conditions are posted on the front of each room. These do occasionally change if enough researchers require it. Space in the rooms is not assigned. You may place your flats on any free shelf in these rooms.

Reach-in chambers with temperature, light and humidity control are available on the 8th floor. Chambers are assigned as they become available. If you need a chamber, let Jenifer know what growth conditions you require and she will try to find you an appropriate chamber. Chambers are fully utilized, so there may be a wait before you can get a chamber. Jenifer will try to accommodate you as soon as possible.

Plant Pathogen RoomsThere is a separate room for work with the plant pathogen Erisyphe orontii (powdery mildew). There are special rules and restrictions regarding use of this room. Contact Jenifer or Julia Dewdney in the Ausubel lab if you plan to work with this organism. A second plant pathogen room is available for work with plant pathogens that do not have air borne spores, or that are otherwise of minimal risk to our other plants. This room has 3 growth chambers and a biosafety cabinet.

Warm room and tissue culture lab

Plants may be grown under sterile conditions on plates or in liquid culture. A lab with a laminar flow hood and a biosafety cabinet are available on the 8th floor. There is warm room with shelves and lights on timers for growing plants on plates. Lighted incubator/shakers are available for seedling/liquid culture.

Sun roomFor experiments that require some natural light, a “sun room” is available on the 8th floor. This room has a skylight, and a westward facing window. HID lights supplement the natural light in this room.

GreenhousesThere are 2 rooftop greenhouses in the Thier building (formerly known as Wellman). The greenhouses have some temperature control, and there are several choices for lighting and bench layouts. These greenhouses are suitable for a large-scale experiment, a mutant screens or for bulking up seed from large numbers of plants. They are also a good place to grow plants with higher light requirements such as tobacco or maize.

Rules and RegulationsThe National Institute of Health instituted a set of guidelines for working with organisms that contain recombinant DNA. The section that specifically addresses plant rDNA can be found in Appendix P.

http://www4.od.nih.gov/oba/RAC/guidelines_02/APPENDIX_P.htm

The plant growth facility here at MGH operates under a BL1-P containment level.

BL1-P practices 1) Access is restricted to trained plant personnel only. Users of the

facility must receive training on handling transgenic materials before they are allowed to begin working in the facilities. Visitors to the facility must sign in at the front desk and be escorted through the facility. Refresher training will be given to all users of the facility on a regular basis.

2) Records should be kept of all experiments in progress in the growth facility. Once per month, the greenhouse manager makes a list of all experiments in the growth facility. Because some experiments exist for less than one month, it is important for each researcher to also keep a record of his or her experiments in the growth facility.

3) Autoclaving is required for all plant materials, pots, and soil. Place these items in autoclave bags and leave them just inside the door of room 7302. The department kitchen staff will pick up the bags and autoclave them. Once the plants are autoclaved, they will then be placed in red biohazard bins for incineration.

4) Pest monitoring and control is carried out by the greenhouse manager who will monitor for all pest outbreaks and will implement control measures.

5) BL-1P applies to all plants in this growth facility. This includes wild type, mutant and transgenic plants.

Growth Facility Rules

Keep sink, counters, and all work surfaces clean These areas are common spaces that need to be used by everyone. Do not leave a mess in the facility. Cleaning supplies are provided.

All plants are to be grown from seed Outside plants are a potential source of unwanted pests and diseases and are not allowed in the plant growth facility. All plants should be propagated from seed.

Label all flats Name or initials, the planting date, and a one or two word description of what is planted should be on every flat. “Jenifer Bush, 3/31/04, Col. W.t.” Unlabeled flats are subject to being tossed. Use tape and a marker so that your label is easy to read.

Treat flats with Gnatrol™ In order to prevent infestations of fungus gnats, use Gnatrol™ when initially wetting down your flats. Guidelines for using this B.t. product are on the bulletin board near the door.

Record all plantings In addition to tracking plantings in your own notebooks, the walk-in rooms have a white board on the door where you can record your name, the type of planting, the date, number of flats. Wild type, mutant, and transgenic plantings need to be recorded.

Harvest seeds promptly Dead, dry plant plants should be harvested and and then autoclaved. Space is limited. Dead, dry flats can sometimes harbor thrips or spider mite pests.

Remove dead or unneeded plants For the same reasons listed above, plants that are no longer needed should be bagged up to be autoclaved.

Tie up, stake, or aracon mature plants Minimize the risk of dropping seed onto other people’s flats.

Autoclave all plants All plants includes wild type, mutant, and transgenic plantings. Place plants, soil and pots in an autoclave bag, tie, and leave inside the door of the 7th floor plant facility. The kitchen staff will pick these up for autoclaving before disposal.

Report problems to greenhouse manager Insect, disease, temperature and lighting problems should be addressed to Jenifer Bush as soon you notice them.

Walk-in Growth Rooms The growth rooms are considered common space, and members of both labs are welcome to use them. Growth conditions are labeled on the outside of each room.

Reach-in Chambers Reach chambers are available for experiments that require unusual growth conditions or that otherwise need to be isolated from other plants.

Plant Tissue Culture lab. Two hoods are available for pouring plates, planting on plates, and other procedures requiring sterile conditions. Wipe down the hood with 70% EtOH after use, and do not leave any materials behind in the hood.

Plant Warm Room The light stands in the warm room are set up on various timers. 12 hour, 16 hour, 18 hour, and 24 hour photoperiods are standard. It is possible to set up a different condition if that is needed. Label your plates, or label the shelf you are using with your name and the date. Discard plates that are no longer needed.

Lighted Incubator Shakers Six temperature controlled, incubator/shakers are available for seedling assays, cell cultures, or other plant related experiments.

Thier Greenhouse RulesKeep sink, counters, and work surfaces clean These areas are common spaces that need to be used by everyone. Do not leave a mess in the facility

Record all plantings Each researcher must keep a record of all plantings in a laboratory notebook.

Treat flats with Gnatrol™ In order to prevent infestations of fungus gnats, use Gnatrol™ when initially wetting down your flats. Guidelines for using this B.t. product are on the shelf near the sink.

Untreated plants may be disposed of in the regular trash Only wild type plants are permitted at Thier Greenhouses. These may be disposed of in the regular trash. Plants that are inoculated must be autoclaved by the researcher.

Label all flats Name or initials, the planting date, and a one or two word description of what is planted should be on every flat. “Jenifer Bush, 3/31/04, Col. W.t.” Unlabeled flats are subject to being tossed. Use tape and a marker so that your label is easy to read.

Harvest seeds/ Remove dead or unneeded plants Dead, dry plant plants should be harvested promptly and removed from the growth facility. Dead, dry flats can sometimes harbor thrips or spider mite pests.

Report problems to greenhouse manager Insect, disease, temperature and lighting problems should be addressed to Jenifer Bush as soon as possible.

All plants are to be grown from seed Outside plants are a potential source of unwanted pests and diseases and are not allowed in the plant growth facility. All plants should be propagated from seed.

Reach-in Chambers There are 4 reach-in growth chambers available. Please see Jenifer if you would like to sign up to use one.

Transgenic Plants Transgenic plants should be grown at the Simches Research Facility at 185 Cambridge St. If transgenic plants need to be grown at Thier, then special containment, labeling, and disposal rules will apply. See Jenifer if you need to grow transgenic plants at Thier. Plant Growth Procedure MGH Molecular Biology DepartmentJenifer Bush, Greenhouse Manager 617-726-5931Date: 2/16/07

Introduction:This protocol describes how most Arabidopsis plants are grown in the Plant Growth Facility in the Department of Molecular Biology at Massachusetts General Hospital.

I. Preparation of Seeds Arabidopsis seeds Tubes of any size (1.5 ml. 15 ml, 50 ml- depending on how many

seeds you are planning to plant) .1% phytoagar or other agar solution or water (using phytagar

allows the seeds to remain suspended in solution making them easier to distribute when planting).

paper folded in half and then unfolded (an index card or wax coated weighing paper both work very well.)

marker aluminum foil

1. Tap out the desired number of seeds to be planted onto the piece of paper. This should include some extra seeds if you have them because it is much easier to plant some extra and thin them later, than it is to precisely plant individual seeds.2. Add .1% phytagar to the tube. 3. Pour seeds into tube with the phytagar. Close the tube and mix.4. Label the tube with the identity of the seeds and the date.5. Wrap the tube with aluminum foil to block out light. Place the tube in a 4mC fridge for 1-4 days. (Note: some growers skip this step. Not all ecotypes or mutants require the cold treatment of seeds.)

II. Preparation of soil: Metro Mix 360, Fafard #2 Mix no hole tray tray with holes or “basket tray” insert (801 = 8 wells, 601 = 6 wells, 606 = 6 packs of 6 cells

each, 3601 = 36 cells, or strip 50 = 50 cells)

soil mix (metro mix 360 or fafard #2 mix) Gnatrol (detailed Gnatrol protocol is a separate document)

1. Assemble a “no-hole” tray, a tray with holes, and an insert. The insert can be 8 wells, 6 wells, 36 cells, or 50 cells. The tray with holes can be a lightweight “basket” tray, or a 1020 tray (a 1020 tray looks like a no hole tray but has holes in it).

2. Fill the insert with desired soil mix. Fill the insert completely but do not press the soil down. The soil should remain “fluffy.” Use your hand to level off the top so there is an even surface.

3. Wet the soil using either of these methods:a. Bottom water method

i. Place the no hole tray in the sink and add Gnatrol to it (rate is 1-8 tsp per gallon of water). Fill the tray with tap water. Mix so that the Gnatrol is in solution.

ii. Place tray with holes and soil filled insert into the no hole tray. Allow the soil to absorb the solution. This will take 10-20 minutes. Trays can be prepared in this manner and left on a shelf overnight.

b. Top water methodi. Add Gnatrol to a watering can (rate is 1-8 tsp per

gallon of water). Fill the watering can with tap water and the mix so that the Gnatrol is in solution.

ii. Add a nozzle to the spout of the watering can. iii. Place the flat of soil in the sink. iv. Using the watering can, wet down the flat from

above. The water/ gnatrol solution will “rain” onto the soil. You will likely need to pause watering a few times to allow the solution to soak into the soil. Make sure that the soil is wet all the way through. Flat is now ready for planting

Jiffy 7’s no hole tray tray with holes or “basket tray” insert (801 = 8 wells, 601 = 6 wells, 606 = 6 packs of 6 cells

each, 3601 = 36 cells, or strip 50 = 50 cells) Jiffy 7 pellets (small, medium or large) Gnatrol (detailed Gnatrol protocol is a separate document)

1. Prepare Gnatrol Solution in a no hole tray. (Gnatrol rate is 1-8 tsp/gal).

2. Assemble an insert with a tray with holes or basket tray. Place Jiffy 7 pellets in each cell or well of the insert. The hole of the Jiffy 7 should face up.

3. Submerge the assembly in the no hole tray. The Jiffy 7’s may float at first, but will sink as they absorb the solution. It takes approximately 30 minutes for the Jiffy 7’s to absorb the solution.

They often need to be repositioned after swelling. Use a pair of forceps to upright any pellets that may be on their sides.

4. The Jiffy 7’s are designed for cuttings in the horticulture industry and once swelled, will have a hole in the middle. With your finger, or a spatula, gently close the hole. (This is an optional step.)

III. Sowing Seeds

Glass Pasteur pipette and bulb Prepared seeds Prepared flat Tape and marker Clear plastic propagation dome

1. Using the pipette, take up some liquid containing seeds. For each planting position in the flat, place a drop containing 2-4 seeds on top of the soil. Arabidopsis prefers light to germinate, so placing the seed on the soil surface is ideal. Plant the entire flat.2. Label the flat with your initials, the planting date, and the identity of the seeds. For example “1/16/07, JB, Col. w.t.”3. Cover the flat with a clear plastic propagation dome.4. Place the flat in the growth room or growth chamber.

IV. Growth ConditionsThe walk-in growth rooms are available to all the plant researchers and you can find a space in any of them. The growth conditions for each room are posted on the outside of each door.

As of July, 2007 the conditions in the department’s 6 walk-in growth rooms were:

Room 1 (Protoplasts, VIGS, etc)

temp. 22/18RH 60%light 75 uE13 hour day/ 11 hour nightlights on 7 am

Room 2

temp. 22/18RH 60%light 75 uE12 hour day/ 12 hour nightlights on 9 am

Room 3 (Protoplasts, etc)

temp. 23/20RH 65 %light 70 uE12 hour day/ 12 hour nightlights on 7 am

Room 4 (Seed Production)

temp. 22RH 60%light 100 uE16 hour day/ 8 hour nightlights on 8 am

Room 5 (Protoplasts, etc) temp. 22/18

Room 6

temp. 23/20

RH 60%light 100 uE 12 hour day/ 12 hour night lights on 9 am

RH 60%light 100 uE16 hour day/ 8 hour nightlights on 8 am

Other growth conditions are possible using reach-in growth chambers. See Jenifer if you would like to sign up to use one of these.

V. Thinning SeedlingsAfter 7-10 days, remove the dome. Using forceps, remove the extra seedlings so that only one plant remains in each position.

VI. Watering Plants and Fertilizing PlantsPlants should be checked frequently during the growth cycle. The best way to determine how wet or dry a flat is to lift it. Allow the soil to dry slightly before watering again. Bottom water the flats by filling the no hole tray with water approximately half way. Allow the flat to soak for about 30 minutes and then pour off the excess water. In most cases it is sufficient to water the flats twice per week.

The soil comes with a small amount of nutrient that will easily sustain Arabidopsis for 4-5 weeks. If seeds are desired, then it is a good idea to fertilize the plants once they have begun to flower. ¼x Hoagland fertilizer is available in the large tanks on both the 7th and 8th floors. Water flowering plants with 1/4x Hoagland once every week or two, until plants stop flowering.

VII. Preparing plants for the collection of seeds Twist-ties and stakes

or Aracon tubes and bases

If bulk seeds are desired, multiple plants of the same line can be tied together. If seeds from individual plants need to be collected, then each can be staked and tied, or aracons can be used.

To tie plants for bulk seed harvest, allow all the plants in the pot to bolt and then bundle them together with a twist-tie. As the plants continue to grow, they may need to be tied again. This can be done several times as long as the siliques (seed pods) are still green. Once the siliques turn yellow and begin to senesce, there is the risk that they will drop their seeds. At this point, it is better to no longer manipulate the plants until you are ready to harvest seeds.

To tie individual plants, allow each plant to bolt and place a stake beside each plant. Use a twist-tie to loosely tie the plant to the stake. As the plant continues to grow, it will likely need to be re-tied a few times. Once the siliques begin to turn yellow and senesce, do not handle them any longer.

Another way to collect individual plants is to use aracons. These come in two pieces: a plastic base catches the seeds, and a clear plastic cylindrical top which supports the bolts and keeps them separated from each other. When the plant first begins to bolt, place the base over it, threading the bolt through the hole in the center. When all (or most) of the plants in the flat have bolted, and have bases in place, fit the top over the base. Tops can be placed on the bases earlier, but it is a little bit easier to wait until most of the bases are in place. As the plants grow, they sometimes will grow out of the holes in the sides or out the top. Trim these off, or stuff the plants down into the top. You want to keep the plants separated so that their seeds do not contaminate their neighbors.

VIII. When to stop watering

At approximately 8-10 weeks (depending on growth condition), the plants will discontinue producing new flowers. When the siliques start to turn yellow, and the plant has begun to senesce, reduce watering. Once the majority of siliques on the plant have filled out, and many are turning yellow, it is okay to stop watering. Allow the plant to dry down.

IX. Harvesting Seeds

Sieve (or sieve with funnel- see Jenifer about this item)TubesPermanent markerClean sheets of paperScissorsAutoclave bag

Fold a few sheets of paper in half, and then unfold them. Carefully cut the plant at the base of the stem and place it on the folded paper. Fold the paper again, squeezing the plant, and releasing the seeds. Place the sieve on another sheet of folded paper, and pour the plant debris into the sieve.

If aracons were used, then cut the plant away from the flat and remove the whole aracon assembly. Over the folded paper, pull the plant through the hole in the base. Dump the contents of the base into the sieve.

Sift the plant debris. Reddish brown seeds will drop through onto the paper. Pour the seeds into the tube. Use a permanent marker to label the identity of the seeds.

Gnatrol™ Use to Prevent/Control Fungus GnatsGnatrol™is a biological control product used to control fungus gnats, which are one of the most serious Arabidopsis pests. Eggs of the gnats come in on our soil and once they hatch and grow into adults, the gnats can quickly spread throughout the growth facility. The active ingredient in Gnatrol™ is a toxin produced by the bacteria Bacillus thuringiensis israelensis (B.t.). This B.t. toxin, when ingested by the fungus gnat larva, paralyzes the gut and the larva stops feeding. Gnatrol™ works by targeting the larval stage of the fungus gnat. Gnatrol™ does not appear to affect the growth of Arabidopsis.

SafetyThe only potential hazard listed for Gnatrol™ is that direct contact with the skin may cause mild irritation. Gnatrol™ contains 0.6% B.t. ITU (international toxic units); the remainder is inert ingredients. A specimen label and MSDS are posted on the bulletin board inside the door of the 7th floor growth facility.

Application ProcedurePrevention: It is most important to apply Gnatrol™ to flats when you are initially wetting them down prior to planting.

Existing Fungus Gnat Populations: The amount of Gnatrol™ to apply depends upon the level of infestation. There is a wide suggested range. Gnatrol™ is only effective for 48 hours, so applications may need to be repeated.

Mix Gnatrol™ with clean water using the following rates as guidelines.

Preventative/Light Infestation Heavy InfestationGnatrol™ 1-2 tsp. per gallon H2O 4-8 tsp. per gallon H2O

Metric conversion 1 tsp per gallon = 1.3 ml per liter.

Most of our green watering cans hold 11 liters.

To initially wet down a flat takes approximately 2-3 liters of water/Gnatrol™ solution.

Before planting, thoroughly saturate flats with Gnatrol™ solution Young seedlings and small plants- bottom water with Gnatrol™

solution. Older plants- top water older plants if possible.

The fungus gnat larvae live near the soil surface. Bottom watering is also acceptable.

For existing infestations- repeat Gnatrol™ application weekly.

Biosafety Guidelines for Handling Agrobacterium for Plant Transformation MGH Molecular Biology DepartmentJenifer Bush, Greenhouse Manager 617-726-5931

The Agrobacterium strain that we use in the laboratory has been altered from the form found in nature to substantially reduce its virulence and any environmental harm. For example, it is not able to form tumors on host plants because the majority of crown gall tumor-forming related genes have been deleted from the Ti plasmid.

The ability of A. tumefaciens to insert genes into a plant is what makes it such a useful tool in the laboratory. However, this trait may still be a risk factor for the environment.

To prevent any unintentional release of A. tumefaciens strains into the environment, adhere to these guidelines.

1. Only use disarmed non-tumorigenic Ti plasmid vectors and non-pathogenic viral vectors in A. tumefaciens for stable or transient plant transformation.

2. The leftover A. tumefaciens cultures must be rendered biologically inactive by either autoclaving them or mixing them with an equal volume of bleach for 1 hour.

3. Any spilled A. tumefaciens culture should be wiped up, and the

surface cleaned with 70% ethanol or 50% concentration bleach. Towels or pads used to clean the spill should then be autoclaved.

4. Plants that are treated or infiltrated with A. tumefaciens should be autoclaved after seed harvest or when the experiment is complete.

5. Consider using a minimal amount of A. tumefaciens by pipetting A. tumefaciens cultures onto flowers instead of dipping whole plants.

Powdery Mildew

MGH Molecular Biology DepartmentJenifer Bush, Greenhouse Manager 617-726-5931

Powdery mildew is a common plant pathogen found in agricultural settings, greenhouses, and even controlled environment growth facilities such as ours at MGH. Powdery mildew has air borne spores that are easily spread. To minimize outbreaks of infection within our own shared plant growth facility, it is important for all users of the facility to be able to recognize powdery mildew and know exactly what to do (and what not to do) if they encounter it.

Symptoms:

White powdery patches appear on the upper surface and leaf tips of rosette leaves

Plants typically are 4-6 weeks old (symptoms usually not visible on younger plants

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When you encounter a powdery mildew infected plant, the spores are already in the air, and you should consider yourself, your clothing, your shoes, your hair are now entirely covered in spores. Spores of powdery mildew can remain viable for a few days. Fortunately, the spores immediately die when they come into contact with water.Immediate steps to take if you see an infected plant:

Cover the sporulating leaf with a damp paper towel. This will reduce the number of spores that will become airborne. Cover the flat with a dome.

If the plant(s) can be tossed, then place the pots and or tray in an autoclave bag. Spray the outside of the autoclave bag with water, and then put that in a second autoclave bag. Deliver the bag to the autoclave. (Do not leave the bag in the growth facility).

Leave the growth facility, turn in your lab coat to the laundry, and do not enter any part of the growth facility again that day. You may need to ask some one to help care for your other plants that day. Take a shower and wash your hair. Wash your clothing. Water will kill any viable spores that are clinging to you.

Contact Jenifer Bush (bush@molbio.mgh.harvard.edu) or (617-726-5931). If she is away, or you cannot reach her, put a sign on the chamber or room where you found the powdery mildew, warning others not to enter the room. Contact one of the other lab members with expertise in handling powdery mildew (Julia

Dewdney, Julia Plotnikov, or Carine Denoux).

Preventative steps to minimize the risk of a powdery mildew outbreak:

Wear a clean lab coat to reduce the amount of spores brought into the facility from the outside.

Work in “clean” plant areas first. If you must enter a plant area where there has been an outbreak of powdery mildew, plan to do that work at the very end of the day and go home directly afterwards.

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