Additional information on growing Arabidopsis
The following information provides more detailed instruction for growing Arabidopsis thaliana plants under more controlled conditions. (This would not be necessary for most teaching purposes, but is provided as an additional resource).
Arabidopsis can be grown in a variety of environmental settings including growth rooms, window ledges, outdoors, growth chambers and greenhouses. Peat moss-based mixes, commercial greenhouse mixes, relatively inert media watered with nutrient solutions, and defined agar media can all be employed as plant substrates. This review focuses on growth of plants on agar and soil in growth chambers and greenhouses. The plant and seed management methods are discussed in the chronological order in which they would normally be utilized.
It is necessary to use sterile conditions to grow Arabidopsis for specific experiments such as selection of transformed plants, drug resistant plants, early root and shoot phenotypes, lethal mutants, etc. Otherwise, contaminants can essentially take over plant cultures. Various shapes and sizes of containers such as Petri dishes, Magenta boxes, or culture tubes can be used, depending on the required length of the growing time (2– 3 weeks or to maturation) and characterization of phenotypes (shoot or roots). We will emphasize the use of Petri dishes. All procedures should be accomplished in a sterile hood or environment.
The most commonly used media is 0.5x or 1x Murashige and Skoog (MS) mineral salts with 0.8– 1% BactoAgarTM. Optional 0– 3% sucrose and vitamins can be added to the media. Preparation of 0.5x MS agar media is as follows:
1. Add 4.31 g of MS Salts to 1.8 L of distilled water and stir to dissolve.
2. Check and adjust pH to 5.7. Adjustments can be made with 1M KOH.
3. Dilute to final volume of 2 L and add agar (10 g / L).
4. Autoclave 15 minutes at 15 psi, 121oC.
5. Optional sucrose and vitamins should be added after agar media cools, before pouring solution into container (e.g. Petri dishes, Magenta boxes, culture tubes).
Seeds can be surfaced sterilized by soaking for 8 min in bleach (5.25– 6.15% sodium hypochlorite, or household bleach) with 0.05% Tween 20 (dish detergent can be used in place of Tween, which is a surfactant) and rinsing the seeds 3– 5 times with sterile, distilled water. Be sure that all bleach residue is removed. Maintain seeds in a small amount of water in a watch glass and plant immediately.
There are several methods for placing the seeds on medium, depending on the preferred plant density and type of container used.
a.) For planting of individual seeds in low density, a small Pasteur pipet with a latex bulb on the upper end can be used. Exhaust air from the pipet, submerge its tip and use slow release pressure on bulb to draw a single seed into the end of the pipet. The seed can be dropped at the desired location by carefully exhausting of the pipet. Do not draw seeds beyond 1– 2 cm into the pipet. Repeated pipetings are used for the remainder of the seeds.
b.) For planting at high densities with uniform distribution on agar, mix seeds in sterile distilled water (or 0.1% cooled top agar), pour onto dish, and swirl to distribute seeds evenly. A sterile Pasteur pipet tip can be used to move seeds around to adjust the distribution, and to remove excess water. Allow the water or top agar to dry slightly before replacing lid.
After planting seeds on Petri dishes with agar, replace cover and seal with Parafilm to prevent desiccation. Place dishes at 3– 4°C (refrigerator temperature) for at least 2 to 4 days to break dormancy, if needed. Dishes can be placed directly into the growth environment. A temperature of 23– 25°C, and 130– 150 mE illumination are suitable.
Different mixtures and media can be utilized for growing Arabidopsis. Growth of plants on soil includes all media that can be successfully used for non-sterile growth of plants in pots or other similar containers. Mixtures of soil that have substantial peat moss with some perlite and vermiculite for aeration can be used successfully. Peat-based commercial mixes represent a convenient and reliable base for growing plants. Mixes such as PRO-MIX BX support healthy Arabidopsis growth and have fertilizer added so that fertilization is not necessary in the very early growth phases. Seeds can be planted in various ways, however, strict control of numbers of seeds planted can be maintained, and separate rows of different lines can be planted in the same pot for critical comparisons with the techniques described here. The density of planting depends on the genetic material, the purpose of the plants and availability of seeds. For seed production, high yields are achieved utilizing densities of 10 to 20 plants per 10 cm square pot. Larger populations of plants do not necessarily reduce yield, but production per plant is reduced inversely. Larger populations are necessary for maintenance of representative proportions in a segregating population, and this can be achieved with more dense plantings in one or two 10 cm pots or in flats (approx 26 cm x 53 cm).
Preparation of pots and planting can be accomplished as follows:
- 1. Thoroughly wet soil with tap water and apply a commercially available extended time release fertilizer such as Osmocote 14– 14– 14 (14% nitrogen, 14% phosphate, 14% potassium) which feeds up to 3 months from planting (apply in amounts according to the label). Alternatively, nutrient solution can be used to wet the soil. Mix well with trowel or large spoon. Soil can be autoclaved to eliminate pests, but this is not usually necessary.
2. Place soil loosely in pots or flats, level without compressing to give a uniform and soft bed. Pots are ready for planting.
3. When planting many seeds in a pot, scatter them carefully from a folded piece of filter paper (weighing paper or other paper) distributing seeds evenly onto the surface of the soil.
4. When planting individual seeds in low density, use a Pasteur pipet with a latex bulb on the upper end. Exhaust air from the pipet, submerge its tip and use slow release pressure on bulb to draw a single seed into the end of the pipet. The seed can be dropped at the desired location in the pot by carefully exhausting of the pipet. Repeated pipetings are used for the remainder of the seeds.
5. Planted seeds should not be covered with additional soil, because Arabidopsis seeds need light for germination.
6. If several pots are planted, they can be placed in a tray or other similar container and covered with clear plastic wrap. In all cases the plastic wrap should not be allowed to contact the soil surface. Cut several small slits in the plastic with a knife in order to provide some aeration, but still maintain enough humidity for germination and also avoid seed desiccation. Clear plastic domes are available for covering flats, but should not be tightly sealed.
7. Pots can be placed at 3– 4°C (refrigerator temperature) for at least 2– 4 days to eliminate any dormancy, improve germination rate and its synchrony. The use of a cold treatment to break dormancy of seeds, also called stratification, is very important for plantings utilizing freshly harvested seeds, which have more pronounced dormancy. Most widely used lines have moderate dormancy, and cold treatment may not be required when planting older seeds of these lines. For certain lines, as many as 7 days of cold treatment is necessary. Cold treatment of dry seeds is normally not effective in breaking dormancy.
8. After cold treatment, place pots in growth area (growth chamber, growth room, greenhouse, etc.) and maintain approx 2 cm of water around base of pots during the germination phase. Leave plastic wrap on for plants grown in growth chamber.
In general, the growth and development of Arabidopsis plants, including time to flowering and time to harvest, depend on several growth conditions in addition to the genetic background. Management of water, nutrition, light and temperature will ensure that healthy plants develop and produce high quality and quantity of seeds. Under continuous light, 25°C, good water supply and good nutrition, seeds of the commonly used lines germinate within 3– 5 days, bolt and flower around 3– 4 weeks, and can be harvested within 8– 12 weeks.
Water and nutrition
Maintenance of soil moisture is imperative for successful germination of seeds. This can be ensured in one of two ways: a) leaving the plastic with small perforations over the pots or tubs, or b) placing the pots in flats without the plastic cover and maintaining a depth of 1– 3 cm of water, which is maintained continually until all plants germinate and have expanded cotyledons. We prefer the former for growth chamber and the latter for the greenhouse. The first method is dangerous in the greenhouse setting, due to the potential for overheating underneath the plastic covering on sunny days, killing the germinating seedlings.
After germination, plants are watered as needed to avoid water stress. Water is best applied by sub-irrigation when the soil begins to dry. Sub-irrigation can be achieved by placing pots into flats or trays, allowing proper drainage of the soil. Over-watering should be avoided due to the potential for algal or fungal growth on the soil surface. Over-watering of greenhouse plants also provides favorable soil conditions for fungus gnat larvae. More frequent watering may be necessary during the first few days, as it is necessary to avoid any drying before the first two true leaves begin expanding. After plants have developed true leaves, watering frequency may be reduced to as low as once or twice per week until the plants flower. The water requirement of plants increases dramatically during silique filling. Daily watering at this stage is necessary for good seed production.
Water requirement is strongly influenced by relative humidity. Arabidopsis plants, including seedlings, tolerate low humidity (e.g., 20– 30%) although increased humidity (e.g., 50– 60%) greatly reduces the risk of accidental drying of the soil surface and subsequent desiccation of the fragile, germinating seedlings. Very high humidity (more than 90%) can induce the formation of mold. Low humidity (less than 50%) is desirable when siliques begin to mature.
Poor nutrition can lead to rapid flowering, short growth period and low seed set. If an extended time release fertilizer was not utilized before, a mild mineral nutrient solution can be applied to the pots at 2-week intervals (5 mM KNO3, 2.5 mM KH2PO4 (adjusted to pH 6.5), 2.0 mM MgSO4, 2.0 mM Ca (NO3)2, 50 microM Fe-EDTA, 70 microM H3BO3, 14 microM MnCl2, 0.5 microM CuSO4, 1 microM ZnSO4, 0.2 microM Na2MoO4, 10 microM NaCl, 0.01 microM CoCl2, pH 6.5).
Optimum light is approx 130– 150 uE m-2 sec-1. Very high output or cool white (VHO or SHO) fluorescent lamps, supplemented by incandescent lighting are used for growth chambers. Older plants tolerate higher light intensity, up to full sun, although the use of 60% shade cloth in summer greenhouses helps with light intensity control and temperature regulation. Supplemental evening and morning light is provided in the greenhouse during winter since the plants generally require a long photoperiod (at least 12 hours) for flowering. Photoperiods of 16 hours work well for greenhouse growth. Plants flower rapidly under continuous light or long days, while under short days flowering is prevented or delayed, favoring growth of vegetative tissue. Continuous light is well-tolerated and can be used to accelerate the reproductive cycle.