HANDLING ARABIDOPSIS PLANTS AND SEEDScrawl
Methods ud by the Arabidopsis Biological Resource Center GROWTH OF PLANTS
Growth of plants in sterile conditions
Growth of plants on soil
•Planting on soil
•Growth conditions
•Control of pests
20xx•Plant isolation and harvesting
豆干怎么做好吃SEED HANDLING AND PRESERVATION
Threshing
Seed drying
Seed moisture content determination
Seed packaging for storage
Seed storage and prervation
Seed viability
The methods ud by the ABRC for handling plants and eds are outlined below. The procedures are designed to generate healthy plants that give maximum t of pure eds and to prerve the in the safest and most convenient manner. Many other approaches may be equally as good, especially in specific experimental situations.
GROWTH OF PLANTS
股票手续费怎么算Arabidopsis can be grown in a variety of environmental ttings including growth rooms, window ledges, outdoors, growth chambers and greenhous.
Peat moss-bad mixes, commercial greenhou mixes, relatively inert media watered with nutrient solutions, and defined agar media can all be employed as plant substrates.
Our focus will be on growth of plants on agar and soil in growth chambers and greenhous. The plant and ed management methods are discusd in the chronological order in which they would normally be utilized.
Growth of plants in sterile conditions
It is necessary to u sterile conditions to grow Arabidopsis for specific experiments such as lection of transformed plants, drug resistant plants, early root and shoot phenotypes, lethal mutants, etc. Otherwi, contaminants can esntially take over plant cultures. Various shapes and sizes of containers such as petri dishes, 'Magenta' boxes, or culture tubes can be ud, 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
u of petri dishes. All procedures should be accomplished in a sterile hood or environment.
The most commonly ud media is 0.5x or 1x Murashige and Skoog (MS) mineral salts with 0.8-1% BactoAgar TM. Optional 0-3% sucro 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, 121o C.
5. Optional sucro 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) with 0.05% Tween 20 and rinsing the eds 3-5 times with sterile, distilled water. Be sure that all bleach residue is removed. Maintain eds in a small amount of water in a watch glass and plant immediately.
There are veral methods for placing the eds on medium, depending on the preferred plant density and type of container ud:文化休闲
a.) For planting of individual eds in low density, a small pasteur pipet with a latex bulb on the upper end can be ud. Exhaust air from the pipet, submerge its tip and u slow relea pressure on bulb to draw a single ed into the end of the pipet. The ed can be dropped at the desired locat
ion by carefully exhausting of the pipet. Do not draw eds beyond 1-2 cm into the pipet. Repeated pipetings are ud for the remainder of the eds.
b.) For planting at high densities with uniform distribution on agar, mix eds in sterile distilled water (or 0.1% cooled top agar), pour onto dish, and swirl to distribute eds evenly. A sterile Pasteur pipet tip can be ud to move eds around to adjust the distribution, and to remove excess water. Allow the water or top agar to dry slightly before replacing lid.
After planting eds on Petri dishes with agar, replace cover and al 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, 130-150 (E m-2 c-1 illumination are suitable. Growth of plants on soil
•Planting on soil
Different mixtures and media can be utilized for growing Arabidopsis. Growth of plants on soil includes all media that can be successfully ud 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 ud
successfully. Peat-bad commercial mixes reprent a convenient and reliable
ba for growing plants. Mixes such as "Sunshine LC1 mix" support healthy
Arabidopsis growth and have fertilizer added so that fertilization is not necessary in the very early growth phas.
Seeds can be planted in various ways, however, strict control of numbers of eds planted can be maintained, and parate 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 purpo of the plants and availability of eds. For ed 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 inverly. Larger populations are necessary for maintenance of reprentative proportions in a gregating population, and this can be achieved with more den 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 relea 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 ud 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 looly in pots or flats, level without compressing to give a uniform and soft bed. Pots are ready for planting.
3. When planting many eds in a pot, scatter them carefully from a folded piece of filter paper (weighing paper or other paper) distributing eds evenly onto the surface of the soil.
4. When planting individual eds in low density, u a Pasteur pipet with a latex bulb on the upper end. Exhaust air from the pipet, submerge its tip and u slow relea pressure on bulb to draw a single ed into the end of the pipet. The ed can be dropped at the desired location in the pot by carefully exhausting of the pipet. Repeated pipetings are ud for the remainder of the eds.
5. Planted eds should not be covered with additional soil, becau Arabidopsis eds need light for germination.
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6. If veral pots are planted, they can be placed in a tray or other similar container and covered with clear plastic wrap. In all cas the plastic wrap should not be allowed to contact the soil surface. Cut veral small slits in the plastic with a knife in order to provide some aeration, but still maintain enough humidity for germination and also avoid ed desiccation. Clear plastic domes are available for covering flats, but should not be tightly aled.
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 u of
a cold treatment to break dormancy of eds, also called stratification, is very important for plantings utilizing freshly harvested eds, which have more pronounced dormancy. Most widely ud lines have moderate dormancy, and cold treatment may not be required when planting older eds of the lines. For certain lines, as many as 7 days of cold treatment is necessary. Cold treatment of dry eds is normally not effective in breaking dormancy.
8. After cold treatment, place pots in growth area (growth chamber, growth room, greenhou, etc) and maintain approx 2 cm of water around ba of pots during the germination pha. Leave plastic wrap on for plants grown in growth chamber.
TASKLIST
•Growth conditions
In general, the growth and development of Arabidopsis plants, including time to flowering and time to harvest depend on veral 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 eds. Under continuous light, 25°C, good water supply and good nutrition, eds of the commonly ud 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 eds. 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 greenhou. The first method is dangerous in the greenhou tting, due to the potential for overheating underneath the plastic covering on sunny days, killing the germinating edlings. After germination, plants are watered as needed to avoid wat
er 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 greenhou 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 increas dramatically during silique filling. Daily watering at this stage is necessary for good ed production.
Water requirement is strongly influenced by relative humidity. Arabidopsis plants, including edlings, tolerate low humidity (e.g., 20-30%) although incread humidity (e.g., 50-60%) greatly reduces the risk of accidental drying of the soil surface and subquent desiccation of the fragile, germinating edlings. 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 ed t. If an extended time relea fertilizer was not utilized before, a mild mineral nutrient solution can be applied to the pots at
孔子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).
Light
Optimum light is approx 130-150 uE m-2 c-1. Very high output or cool white (VHO or SHO) fluorescent lamps, supplemented by incandescent lighting are ud for growth chambers. Older plants tolerate higher light intensity, up to full sun, although the u of 60% shade cloth in summer greenhous helps with light intensity control and temperature regulation. Supplemental evening and morning light is provided in the greenhou during winter since the plants generally require a long photoperiod (at least 12 hours) for flowering. Photoperiods of 16 hours work well for greenhou 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 ud to accelerate the reproductive cycle.
Temperature
The optimum growth temperature range for Arabidopsis is 23-25°C. In general, high temperatures favor a reduced number of leaves and flowers, and fertility is reduced. At lower temperatures, growth is slow and flowering is delayed. Lower temperatures are permissible, but higher temperatures are not recommended, especially for germination through early rotte development. Older plants tolerate higher temperatures, at least up to 30°C. It is advisable to t the greenhou temperature at 21-23°C to avoid fluctuations to higher temperatures. It is recommended that night temperatures be maintained 2-4°C lower than the day temperature. Some late flowering natural accessions (ecotypes) require an additional 4°C incubation (vernalization) of young rottes for 3-4 weeks to induce flowering.
•Control of pests
