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Controlled Environment Climate Chambers |
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Within UPSC, we have a number of climate chambers available for controlled environment studies. These include 7 “walk-in” climate chambers on floor 2 and 8 “walk-in” climate chambers on floor 3, within the new high security growth facility. There are 4 smaller controlled environment cabinets located on floor 2, and 5 Percival cabinets on floor 3 (2 for in vitro culture and 3 Arabidopsis chambers for whole plant growth). The cold room on floor 2 is also available for growing and/or winter-hardening large (and small) plants.
These different controlled environment chambers and cabinets are available to all members within UPSC and can be set to a wide range of temperature and irradiance conditions to suit individual experimental requirements.
Organization
One 50% position has been set aside to provide basic services in this facility: such as record keeping, watering, fertilization and insect control. A steering committee oversees the operation of this facility and helps to ensure all UPSC groups get fair access
Steering committee
Staff
Rules and procedures
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General Rules and Access, Security Greenhouses & Arabidopsis Growth Rooms |
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Access
This facility is designed specifically for the controlled growth of transgenic plants. As a result there are special procedures in place to stop the release of transgenic pollen and seed from this facility. Before you get access to this facility you SHOULD have a guided tour and SHOULD receive information about the rules and safety regulations from one of the Arabidopsis growth managers. After this tour you can fill out the GMO safety document and give this to one of the "GMO safety officers" at the two departments, currently Ove Nilsson (SLU) and Stefan Jansson (UmU) who will grant you access.
Arabidopsis growth rooms for soil grown plants
These rooms are set up with standard growth conditions (23 ± 2ºC day temperature / 18°C night temperature either short-day (8h) or long-day (16h).
These rooms are set up on a rotating basis so that once each room is full it is closed and no new plants are put into the room until ALL plants have completed their growth cycle. The rooms are then steam-cleaned and are open again for a new cycle of plants.
In order to keep an active turnover it is absolutely necessary that you collect your dry plants in due time. Two weeks before the deadline our facility manager Britt-Marie Åkerman indicates on the door of a room when she plans to clean it. All dry plants should be harvested by then. Britt-Marie will throw away, without additional warning, all the remaining dry plants.
You have to prepare your own pots / trays with an appropriate mixture of 2/3 soil and 1/3 vermiculite. Stock of soil and vermiculite are stored on floor 3, their availability is controlled by our facility manager Britt-Marie Åkerman.
In order to limit proliferation of flies you should treat your pots / trays with a suspension of nematodes before you sow seeds or transfer seedlings. Other type of treatment for insects is the responsibility of Britt-Marie Åkerman. If you notice problems or have questions, please tell her.
Watering of plants growing in these rooms is YOUR responsibility.
There is a tap in each room but in order to avoid water leakage a timer limits the water availability. You nevertheless SHOULD close the tap in the room before leaving. In Sweden you should turn the tap clockwise (right) to close it.
Arabidopsis does not like too much humidity. Do not keep standing water in your trays. It is better to water regularly twice a week than to put a lot of water once a week. Be careful with this because not only your plants will suffer, but all plants in the growth room, from standing water in the trays!
Your tray containing plants SHOULD be properly labeled so they can easily be identified in case of problems. Use appropriate plastic label on which you indicate your name and that of your group leader in CAPITAL LETTERS so it can be deciphered.
You should indicated the number of trays on the list situated on the door
ALL MATERIAL NOT READILY IDENTIFIABLE (NO LABEL, UNREADABLE NAME, NON-VISIBLE NAME) WILL BE SYSTEMATICALLY THROWN AWAY WITHOUT CONSULTATION AND WARNING.
IT IS FORBIDEN TO TRANSFER PLANTS FROM ONE ROOM TO ANOTHER UNLESS AGREED WITH BRITT-MARIE.
At the department of Plant Physiology, non-flowering Arabidopsis plants are allowed to be brought out of the containment - but only if they have been growing in a room where no other plants are flowering! Once plants have begun to flower they MUST NOT be removed from the contained facility for any reason.
You are responsible for maintaining your plants so that they do not stand dry in the rooms and that they do not contaminate other users trays with seeds. Flowering plants should be either taped to sticks or bagged to control the spread of seeds.
Arabidopsis growth rooms for in vitro culture
Two rooms with the same settings as above are available for in vitro culture. One on floor 4 (n°8) and one on floor 3.
These rooms are for in vitro culture or cell suspensions ONLY. No other type of culture is allowed.
All plates or vials SHOULD be identifiable otherwise they will be discarded without warning.
You should control regularly your plates and remove them as soon as a contamination (fungi or bacteria) is visible.
If contaminated plates accumulate they will be discarded without warning.
Arabidopsis transformation by infiltration or flower dipping
There is a bench specifically dedicated to Arabidopsis transformation with Agrobacterium. You can either perform infiltration (a vacuum pump is available) or use the dipping method.
Protect the bench with bench-coat before performing the transformation. Avoid spraying bacteria all around you. Do not leave bacteria suspension standing for ever on the bench.
Clean the bench by wiping well with 70% Ethanol. Contaminated plastic and paper can be thrown in the greenhouse waste. Sterilize all bacterial waste solutions by adding 1/100 volume of pure (5.2%) Jodopax (or correspondingly more if you use diluted Jodopax), shake thoroughly and incubate for at least 30 minutes. Then the liquid can be washed down the sink.
Selection of transgenic plants with BASTA
Do NEVER spray BASTA on your plants inside the growth chamber since you risk to spray the neighboring plants and kill them. Your colleagues will not appreciate that!
Take out your plants from the chambers or greenhouse and keep them above the sink when spraying BASTA.
Do not forget to rinse the sink properly after you finished.
Cleaning
These facilities are common and you are expected to behave socially! Think that you are not alone and there is no acceptable reason for your colleagues to clean after you.
Once you have finished preparing pots, sowing or collecting seeds, sampling etc... Please put the common tools at their place and clean the bench, the hoods, the binoculars or any other common material you have been using.
YOU SHOULD WASH THE TRAYS AFTER HARVESTING THE PLANTS!
Waste handling
There are different containers for garbage.
Glass and wooden sticks should be discarded in the cardboard containers. The other waste like Petri dishes, seeds, dry or green plants, soil, pots etc. should be thrown in the containers with either green or yellow lids. Each garbage container contains a black plastic garbage bag.
When a garbage container is full you are supposed to bring it on floor 3 and transfer the garbage bag in a thicker bag that you will seal and tape with GMO Tape. These bags will be discarded in specific containers.
You are supposed to be social and respect your colleagues' work and environment. If you notice any mis-behaviour you should inform Catherine Bellini (directly or via one of the facilities growth managers) who can issue a warning. AFTER 2 WARNINGS THE ACCESS TO THE GROWTH FACILITIES WILL BE CANCELLED.
Costs
Charges for these growth rooms is based on a flat fee per tray per growth cycle. The rooms can take 14 trays per shelf at 6 shelves per room.
Charges have been set at:
Long-day rooms: 100:- per tray
Short-day rooms: 150:- per tray
Arabidopsis greenhouse Floor 5: 100:- per tray per 3 month cycle
Sign up sheets with the names of all registered facility users will be placed on the doors when a room is open for use and you simply need to mark these sheets with the number of trays you have in the room.
Poplar greenhouses Floor 5: charges as per Wallenberg greenhouse.
20:- per table per day with watering
This also equates to 1:- per tray per day (or 100:- per tray per 3 month growth cycle for Arabidopsis)
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General Rules and Access, Controlled Environment Climate Chambers |
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The controlled environment facility is for growth of experimental material ONLY. It is not to be used to grow Arabidopsis for transformation or to be used to grow transgenic plants for seed production. The Arabidopsis growth rooms on floor 3, 4 and the greenhouses on floor 5 are available for these purposes.
GMOs
Handling of GMOs outside of the high security containment facility
We have been granted permission by Jordbruksverket to grow GMOs in the growth rooms and cabinets on floor 2, outside of the high security containment facility, but only if we abide by strict, and strictly enforced, rules. These rules are posted on the doors of all growth rooms and they are listed below:
1. Under no circumstances are GMOs allowed to flower in the growth cabinets and growth rooms outside of the high security containment facility. Any plants found to have initiated flowering will be destroyed immediately without consultation.
2. All GMO seedlings must be marked with a RED tag on which is written your name and the identity of the transgene. This information must also be recorded in the booking sheets retained by the secretary.
3. All GMO waste generated outside of the high security containment facility must be placed in the specially marked RED containers and this material should then to be taken to Floor 3 for disposal in the container inside the containment facility.
4. All bags thrown into the GMO waste container on Floor 3 must first be marked with yellow GMO tape.
5. No GMOs can be taken out of the high security containment facility if they have been in the same room as a flowering Arabidopsis or poplar.
6. Handling of GMO seeds is not allowed outside of the high security containment facility. Therefore all planting of transgenic seed MUST be done in the high security labs on Floors 3, 4 or 5.
7. Arabidopsis and hybrid poplar that have been modified with respect of flowering time regulation must not to be handled or grown outside the high security facility from April 1 to September 30
Disposal of GMO material from all facilities
ALL garbage containing transgenic material MUST be put in special double garbage bags and before being thrown into the special container for this waste (found in the annex to the high security facility on floor 3), the garbage bag must be marked with "GMO tape". The "GMO safety officers" at the two departments, currently Ove Nilsson and Stefan Jansson, control all GMO growth. If you have questions relating to handling of GMO materials, please ask the GMO officers.
Access
In order to use the controlled environment facility you must fill in a Request form, where you state what type of chamber you require, the specific conditions and the duration of the planned experiment. The group leader must be informed before you submit your application. You have to submit this form at least two weeks in advance to Jan Karlsson. However, with the current pressure on this facility it is advisable to plan well in advance of 3 months.
Once the request form has been completed and space allocated, all users must SIGN IN when they begin their experiments and SIGN OUT when the experiment is completed. Users will be charged for space until they have signed out and the chambers, pots, trays etc must be cleaned before they can sign out.
Identification
All material in the growth facility must be CLEARLY (to others, not just for the sake of the user) identified both with the name of the person running the experiment and the research group.
Material not readily identifiable may be thrown out without warning and without consultation.
Costs
Growth chambers (Klimatkammare):
80:- per day
N.B. the full cost is charged regardless of whether you have 1 plant or 1000. If there is more than one user the costs are shared.
Growth cabinets (Klimatskåp):
40:- per day
Cold room:
15:- per m2 per week |
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Procedures for Arabidopsis transformation using Agrobacterium |
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The following guidelines are intended to limit the possibility of cross contamination of plants with agrobacterium and to maintain a clean laboratory environment. They are designed for the benefit of all users of the facility.
The most commonly used procedure for Arabidopsis transformation is floral dip (see attached information on the protocol)
- All Arabidopsis transformation should be carried out ONLY at the designated area on level 4 of the UPSC laboratory.
- The transformation should be carried out on a tray to contain any spills.
- The agrobacterium to be used in the transformation should be placed in a suitable beaker or other container and should not be more than half full. When transporting the agrobacterium from one laboratory to another it should be in a sealed plastic container.
- Following dipping the plants should be placed inside one of the plastic ‘cut flower’ bags to eliminate the possibility of contact with other plants and the spread of agrobacterium. It is useful to fold the top of the bag over and hold it in place with a paper clip or small piece of tape for 2 days after transformation to allow the plants to recover.
- Transformed plants should be placed in the greenhouse and NOT in the growth rooms. Placing recently dipped plants in the growth room causes ‘burning’ of the flowers and hence is likely to reduce transformation efficiency. This also reduces the possibility of cross contamination of other plants.
- You should tidy the transformation area carefully after use. Make sure any spills are cleaned and the area disinfected. Ensure the tray is cleaned. Autoclave or chemically sterilize the agrobacterium and dispose.
Simplified Arabidopsis Transformation Protocol
(from the Bent website - Steve Clough and Andrew Bent)
Our present protocol (Clough and Bent, 1998; modified from Bechtold et al. 1993) is extremely simple. We have found that the MS salts, hormone, etc. make no difference, that OD of bacteria doesn't make much of a difference, that vacuum doesn't even make much of a difference as long as you have a decent amount of surfactant present. Plant health is still a major factor - healthy fecund plants make a big difference! With this method you should be able to achieve transformation rates above 1% (one transformant for every 100 seed harvested from Agrobacterium-treated plants).
- Grow healthy Arabidopsis plants until they are flowering. Grow under long days in pots in soil covered with bridal veil, window screen or cheesecloth.
- (optional) Clip first bolts to encourage proliferation of many secondary bolts. Plants will be ready roughly 4-6 days after clipping. Clipping can be repeated to delay plants. Optimal plants have many immature flower clusters and not many fertilized siliques, although a range of plant stages can be successfully transformed.
- Prepare Agrobacterium tumefaciens strain carrying gene of interest on a binary vector. Grow a large liquid culture @ 28°C in LB with antibiotics to select for the binary plasmid, or grow in other media. You can use mid-log cells or a recently stationary culture.
- Spin down Agrobacterium, resuspend to OD600 = 0.8 (can be higher or lower) in 5% Sucrose solution (if made fresh, no need to autoclave). You will need 100-200 ml for each two or three small pots to be dipped, or 400-500 ml for each two or three 3.5" (9cm) pots.
- Before dipping, add Silwet L-77 to a concentration of 0.05% (500 µl/L) and mix well. If there are problems with L-77 toxicity, use 0.02% or as low as 0.005%.
- Dip above-ground parts of plant in Agrobacterium solution for 2 to 3 seconds, with gentle agitation. You should then see a film of liquid coating plant. Some investigators dip inflorescence only, while others also dip rosette to hit the shorter axillary inflorescences.
- Place dipped plants under a dome or cover for 16 to 24 hours to maintain high humidity (plants can be laid on their side if necessary). Do not expose to excessive sunlight (air under dome can get hot).
- Water and grow plants normally, tying up loose bolts with wax paper, tape, stakes, twist-ties, or other means. Stop watering as seeds become mature.
- Harvest dry seed. Transformants are usually all independent, but are guaranteed to be independent if they come off of separate plants.
- Select for transformants using antibiotic or herbicide selectable marker. For example, vapor-phase sterilize and plate 40 mg = 2000 seed (resuspended in 4 ml 0.1% agarose) on 0.5X MS/0.8% tissue culture Agar plates with 50 µg/ml Kanamycin, cold treat for 2 days, and grow under continuous light (50-100 microEinsteins) for 7-10 days.
- Transplant putative transformants to soil. Grow, test, and use!
For higher rates of transformation, plants may be dipped two or three times at seven day intervals. We suggest one dip two days after clipping, and a second dip one week later. Do not dip less than 6 days apart.
NOTE: Obtain proper approval for transformation work from institutional authorities. Autoclave and properly dispose of all materials.
References:
Bechtold, N., Ellis, J., and Pelletier, G. (1993)
In planta Agrobacterium-mediated gene transfer by infiltration of adult Arabidopsis thaliana plants.
C. R. Acad. Sci. Paris, Life Sciences 316:1194-1199.
Clough SJ and Bent AF, 1998.
Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.
Plant J 16:735-43.
Additional commentary can be found by searching the Arabidopsis newsgroup archives. |
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March 2012 |
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