拟南芥种植手册_tair(2)

原版拟南芥规范种植手册

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) with 0.05% Tween 20 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, 130-150 (E m-2 sec-1 illumination are suitable. 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 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 "Sunshine LC1 mix" support healthy

Arabidopsis growth and have fertilizer added so that fertilization is not necessary in the very early growth phases.