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Arabidopsis Growing Protocol – A General Guide

Plant Science > Plant physiology > Plant growth
Author: Xiyan Li
Vol 1, Iss 17, 9/5/2011, 6540 views, 1 Q&A

[Abstract] Arabidopsis as the model organism for higher plants is widely studied among plant biology labs around the world. However, taking care of this tiny plant may not be trivial. Here is a general guide used for the Heven Sze lab at the University of Maryland, College Park. A lot of efforts have been taken by the Sze lab and fellow lab members to formulate a general guide for Arabidopsis plant growth in the lab.

Procedure

 

1.        Preparing Soil for Planting Soil

We use Miracle-Gro? Potting Mix with Fertilizer (2 cubic feet bag)

Item #: 156581 Model: 74278300 from Lowe’s) and Miracle-Gro? 8 Qt. Miracle Gro? Perlite

(Item #: 68468  Model: 70752300 from Lowe’s)

 

 

 

To make Arabidopsis-friendly soil,  mix well 1 bag of potting mix (2 cb ft) with half bag of perlite (4 qt) in the big bin used to store soil.  The big bin can hold barely 2 bags of potting mix in total.

Note: Sze lab always sterilize soil before putting it into planting pots !!! (see below)

1)        Scoop dry soil into an autoclave bin.  Fill until the soil is approximately one inch from the top after packing down with the scooper.

2)        Cover the bins with aluminum foil and put a piece of autoclave tape on each one.  Make sure the outsides of the bins are dirt free before putting in the autoclave.

3)        Autoclave on normal/flash/quick mode for 30-50min depending on how many bins are being autoclaved.

4)        Once cool, the soil can be placed into pots, packing down lightly.  Remember to cover the autoclaved soil well again to prevent any seed/fungi contamination.  Once opened, autoclaved soil can be good for use for up to 2 weeks.  Please don’t autoclave more than needed.

5)        Fill the flat with pots (NOT on the top of soil!) with deionized water, cover with humidomes and let soak for 3-6 hours or overnight.  There should be standing water (~ 1 inch) after soaking, add more water if not.

6)        The soil in pots is now ready for growing plants.

2.        Seeds sterilization

1)         Method A

a)       Put certain amount of seeds (wild type, mutant, or complements lines) into an eppendorf tube. Add 1 ml sterilization solution.

Sterilization solution:

20% Chlorox bleach

0.05% Tween-20

in sterile water, must be freshly made.

b)      Vortex at maximal speed for 20-30 seconds.

c)       Stand for 7-10 min with occasional vortex.

d)      Spin in a microcentrifuge at 8,000 rpm for 5 seconds.

e)       Pour off the water carefully.

f)        Add 1 ml sterile water and vortex to suspend the fluffy seeds.

g)      Spin in a microcentrifuge at 8,000 rpm for 5 seconds and pour off the supernatant.

h)       Repeat step 6-7 for another 4 times.

i)         Move the sterile seeds to small Fisher dishes (100  15 mm size) with a wet Whatman filter paper (sterile or autoclaved) on its bottom.

j)         Transfer the seeds to agar medium plate or to soil with a tweezer that has been soaked in 70% ethanol for >30min.

2)         Method B

a)       Put certain amount of seeds (no more than 0.) into 1.5ml eppendorf  tubes. Add 1 ml 70% ethanol.

b)      Pour off water and add 5ml 70%Ethanol, incubate for 5min.

c)       Rinse with sterile D.I. water four times.

d)      Add 0.1% Triton X-100 (or 30% Bleach + 0.1% Triton X-100) 10min.

e)       Rinse thoroughly with sterile D.I.water (at least 5 times).

f)        Move the sterile seeds to small Fisher dishes (100  15 mm size) with a wet Whatman filter paper on its bottom.

 

3.        Planting

 

General concern

Please keep in mind your seeds can be contaminated with fungi and bacteria when they were produced on parental plants, seed sterilization is recommended for all plants (up to bolting time) and necessary for all flower-producing/seed-collecting plants and seeds from other labs. The nasty chamber contamination is usually from dirty seeds in our lab (since we always use autoclaved soil)   Flower parts are very attractive to insects and pest, please keep an eye on the flowers.

Grow healthy Arabidopsis plants

Before start, the trays must be cleaned/brushed free of old seeds or fungus.

1)        Fill pots (148.56cm) to the top with autoclaved soil.

2)        Add dI water to the tray which contains the pots, about 1/3-1/2 the height of a pot. It usually takes at least 3h to let the soil soak thoroughly. Then spray the surface of the soil with a spray bottle (dI water), to make sure that the seeds will fall on a wet surface.

3)        Transfer seeds to soil (8-10 seeds/pot, to space seeds apart).

4)        After planting, cover the tray with a humidome, taping it to the tray, to keep the necessary humidity.

5)        Put the tray in 4°C cold room for 3-5 days (5 day is better) at dark.

6)        Move to growth chamber with the humidome still covering.

7)        Plants should germinate after 3-4 days in chamber with long day light cycle. About 5-7 days leave the humidome half-open for anther 2-3 days when 2 pieces of cotyledons have developed completely.

8)        Remove the humidome and water with dI water twice a week (Tuesday and Friday, for example).

Note:

1)        Arabidopsis is sensitive to all stress conditions, so please water only when there is no standing water in the tray.

2)        If the plants are desired to proceed to flowering, water with ? strength of Hoagland solution (recipe attached) once every other week until they start bolting (twice in total is enough).  Hoagland is nitrogen-rich media that promotes the transition to reproductive growth.

3)        Miracle-Gro potting soil has Micracle-Gro plant food ( slowly-released fertilizer), which usually support plant growth for up to 3 months.  So restrain the impulse to over-fertilize them.

4)        Keep an eye when plants start flowering.  Use any possible pest control as needed.

5)        Aphids is a common insect problem. Spray 1:100 “Orthene” on the infected plants under hood, repeat spray for 3 days. Be careful not to over-spray them, otherwise it will kill the plants.

6)        When plants stop flowering, move them out to a ventilated area for seeds to dry. Collect seeds promptly as soon as the siliques look yellow or brown, break with little or no applied pressure.

7)        Note: First, you should clean up some area free seeds of other species, to make sure all the seeds here are no contamination with other seeds you do not want. Rub your fingers around the silique, allow the seeds to fall on a big piece of paper below. Filter seeds through nylon mesh into another piece of paper. Store seeds in eppendorf tubes, poke a hole on the cap of the tube. The collected seeds should be left at Room Temperature for one more week to dry. Then place them in a sealed container with Drierite.

Recipes

 

1.        1/4 Strength Hoagland’s solution (used to water Arabidopsis plant)

 

   

FW

Stock

g/250ml stock

final

ml stock/4L final

KNO3

101.11

2M

50.6

1.25mM

2.5

FeNa-EDTA

367.1

20mM

1.8355

12.5uM

2.5

KH2PO4

136.1

1M

34.0

0.5mM

2.0

MgSO4

120.4

 2M

60.2

0.5mM

1.0

Ca(NO3)2.4H2O*

236.2

2M

118.1

0.5mM

1.0

Minors

 

 

 

 

1.0

 

*:add last when volume is almost full.








 

 

Minors:

Chemical

FW

Stock (mM)

Final (uM)

g/100ml stock

H3BO3

61.83

70

17.5

0.43g

MnCl2

197.9

14

3.5

0.28g

CuSO4

159.6

0.5

0.125

0.08g

ZnSO4

287.5

1

0.25

0.03g

Na2MoO4.2H2O

241.9

0.2

0.05

0.005g

 

Note:  start from >3.9L water, then add every thing and stir thoroughly

           Ca2+ may precipitate with SO42-.

 

 

2.        Homemade MS (Murashige and Skoog) medium for Arabidopsis plants under different Ca2+/Mn2+ conditions

(ref: Murashige T. & Skoog F. , Physiol Plant 15, 473-97 1962)

 

Stock:

1. Macronutrients (10x)

mM (1x MS)

Salt

10x stock (g/l)

41.2

NH4NO3

16.5

18.8

KNO3

19

3.0

CaCl2 anhydrous*

3.3

1.5

MgSO4.7H2O

3.7

1.25

KH2PO4

1.7

* CaCl2 is dropped out for Ca related medium.

 

2. Fe-EDTA (100x)

Na 0.2

Na2.EDTA

3.73

Fe 0.1

FeSO4. 7H2O

2.78

 

3. Micronutrients (100x)

uM (1x MS)

Salt

100x stock (g/L)

100.0

H3BO3

0.62

100.0

MnSO4.H2O

1.69

30.0

ZnSO4.7H2O

0.86

5.0

KI

0.083

1.0

Na2MoO4.2H2O

0.025

0.1

CuSO4. 5H2O

0.0025

0.1

CoCl2. 6H2O

0.0025

 

4. CaCl2  0.5 M

 

Make the Media:

For ? MS 1 L, add:

 

50 ml

macronutrient 10x stock

 

 

5ml

Fe-EDTA 100x stock

 

 

5ml

micronutrient 100x stock

 

 

 

proper CaCl2 0.5M (add KCl to –Ca medium as control for Cl-)

 

 

0.5g

MES (always use 0.5g for 1 liter medium regardless of MS strength)

 

 

adjust pH to 5.7

 

 

add agar to 1% or 0.8%

 

 

autoclave for 20-30 min

 

 

Composition of Plant Media   (mg/L, 1x)

Component

Gamborg’s B5

Murashige and Skoog

 

 

 

Total Weight

3,300

4,620

 

 

 

Inorganic Salts

 

 

CaCl2 anhydrous

113.23

332.16

CoCl2·6H2O

0.025

0.025

CuSO4·5H2O

0.025

0.025

FeSO4·7H2O

27.8

27.8

H3BO3

3

6.2

KH2PO4

 

170

KI

0.75

0.83

KNO3

2,500

1,900

MgSO4·7H2O

246

370

MnSO4·H2O

10

16.9

NaH2PO4·H2O

150

 

Na2-EDTA

 

37.3

Na2MoO4·2H2O

0.25

0.25

NH4NO3

 

1,650

(NH4)2SO4

134

 

ZnSO4·7H2O

2

8.6

 

 

 

Vitamins

 

 

i-Inositol

10

100

Nicotinic acid

1

 

Pantothenic acid.Ca-salt

0.874

 

Pyridoxine·HCl

1

 

Riboflavine

0.015

 

Thiamine·HCl

10

0.4 *

 

* The original formulation contains 0.1 mg/l thiamine.HCl.

 

References

 

1.          Hoagland D.R., Arnon D.I. (1950). The water culture method for growing plants without soil. California Agric Exp Stn Circ 347: 1-32.

 

 



How to cite this protocol: Li, X. (2011). Arabidopsis Growing Protocol – A General Guide. Bio-protocol 1(17): e126. http://www.bio-protocol.org/e126



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