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Controlled Environment Climate Chambers

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 5 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.
growth_fac

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

Britt-Marie Åkerman

Rules and procedures

Request form

General Rules and Access, Security Greenhouses & Arabidopsis Growth Rooms

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 into the environment. Before you get access to this facility you must first go over these procedures with one of the "GMO safety officers" at the two departments, currently Ove Nilsson (SLU) and Stefan Jansson (UmU), and sign a form stating that you have read and understood the rules.

N.B.: Failure to comply with these rules may result in your access to this facility being revoked.

Arabidopsis growth rooms
These rooms are set up with standard growth conditions: approx. 23ºC day and 18ºC night temperature and 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 before being re-opened for a new cycle of plants.

Plants can be removed from these rooms for sampling and other experimental reasons throughout their growth cycle – but once plants have begun to flower they MUST NOT be removed from the high security facility for any reason. Furthermore, while it is OK to take plants out of these rooms for experimental reasons - under no circumstances are plants to be shifted between growth rooms. If plants need to be moved for seed collection they can be taken to the greenhouse set aside for Arabidopsis on floor 5.

Watering
Watering of plants growing in these rooms is generally the responsibility of the individual. There is a timer that controls the water flow, in the lab area close to the exit. Press this once and you will have water available. It will turn off automatically. To get water in the rooms, turn on the water tap, turning anti-clockwise. After usage, turn it off clockwise, and release the pressure in the hose by letting excess water out.

Insecticide treatment routines
Treatment for insects is the responsibility of the facility manager (Britt-Marie Åkerman). To control ongoing problems with thrip and fungus gnat (blomflugor) infestations in the controlled growth facility, please observe the following routines:

1. In the growth rooms located on floors 3, 4 and 5, you must flag ALL new pots with a RED tag on the day you put the pots into the growth room. You do not need to write anything on this tag; it is only to alert Britt-Marie that the pot needs to be treated.

2. Once per week Britt-Marie will drench all flagged pots with “Confidor WG70”, which is a systemic insecticide. She will then remove the RED tag.

3. Britt-Marie will give subsequent treatments as required – as either a spray or drench as appropriate.

Obviously, this means that no one should use red tags for any other purpose when growing plants within the controlled growth facility.
Transgenic plants being grown on floor 2 should still be marked with a red tag as usual.

Arabidopsis infertility
A number of groups have experienced problems with low seed production. It is possible that this is caused by inadequate nutrition. A concentrated nutrient solution is available in the lab on floor 5 – along with instructions for diluting it prior to use. For optimum growth, you should fertilize your plants once per week. For seed production, you should certainly fertilize your plants at least once just as they begin to flower.

Soil
At present, we are purchasing different soil for poplar (Torv) and Arabidopsis (Blomjord). The small bags we use for Arabidopsis growth is expensive “ecologically friendly” soil – and hence prone to contain more bugs. Britt-Marie will not be purchasing this anymore – and neither should anyone else. Vermiculite and perlite are available to mix with the soil to lighten it and make it more suitable for Arabidopsis.

Plant maintenance
Users are responsible for maintaining their plants. Flowering plants should be either taped to sticks or bagged to control the spread of seed and to minimize the risk of contaminating other peoples trays.

Activities such as seed collecting, potting and plant transformation should only be done at the designated areas in the laboratory on floor 4 to prevent the possibility of contamination. Once you have finished working at an area it should be cleaned thoroughly either by sweeping up, using the hand held vacuums or with a cloth. Under no circumstances should there be any stray seed or soil left on bench tops.

Waste handling
All garbage containing transgenic materials MUST be put in special double garbage bags. Before being thrown into the container (found in the annex to the growth facility on floor 3), the garbage bag must be marked with "GMO tape".

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
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)

General Rules and Access, Controlled Environment Climate Chambers

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 2 weeks.

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

Procedures for Arabidopsis transformation using Agrobacterium

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.

September 2010

Thursday, September 02, 2010
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