Title:

Maximal biomass of Arabidopsis thaliana using a simple, low maintenance hydroponic method and favorable environmental conditions*

(Modified from the original article in Plant Physiology (1997) 115:317-319)

Authors:

David M. Gibeaut
John Hulett
Grant R. Cramer**
Jeffrey R. Seemann

Address:

Department of Biochemistry
University of Nevada
Reno, NV 89557

 

Footnotes:
*This research was supported by an NSF EPSCoR grant and NSF grant IBN 940709 to JRS.
**Corresponding author; e-mail
cramer@unr.edu; phone 1-775-784-4204; fax 1-775-784-1650.
 
 

The advantages of Arabidopsis thaliana (L.) Heynh. for genetic studies are well known, but its diminutive stature and associated low biomass at maturity make it a challenging species for complementary physiological and biochemical studies. Hydroponic culture can significantly increase plant growth and produce uniform, stress-free root and shoot material that can be harvested throughout the life span of the plant. However, many shy away from hydroponic culture because of perceived difficulties in set-up and maintenance (see Hydroponic Tips below). Although other methods for the hydroponic culture of arabidopsis have been reported (Rodecap et al., 1994; Delhaize and Randall, 1995; Hirai et al., 1995), they suffer from various shortcomings, including poor aeration, loss of root material, overcrowding, excess manipulation and less than favorable environmental conditions. In this paper we describe an easy, low maintenance method of hydroponic culture for arabidopsis that combines rockwool culture for uniform seedling establishment, and a closed system of solution culture for the duration of plant growth. In addition some consideration is given to temperature and light conditions which favor biomass production.

 The most difficult part of hydroponic culture for arabidopsis is the establishment of a good root system because young seedlings are prone to hypoxic stress from water logging. Rockwool (GrodanHP, Agro Dynamics Inc., East Brunswick, NJ, USA can be purchased from Hummert International, (800) 325-3055, http://www.hummert.com/) provides an excellent, well aerated rooting environment that is a far superior medium for reliable and uniform seedling establishment compared to other media we have tried including cheesecloth, blue blotter paper, brown germination paper, filter paper, fiberglass matting, agar, and soil or vermiculite filled straws. Rockwool is a mixture of igneous rock and limestone that is heated and spun into mats. Even when saturated rockwool holds about 15% air space.

Containers and tops for hydroponic culture must be opaque to produce healthy roots and discourage growth of algae. Surfaces can be painted with either epoxy or vinyl paint, or covered with aluminum foil. Where foil may be exposed to the nutrient solution, an underliner of parafilm should be used to prevent Al contamination. Black paint or aluminum foil are suitable for controlled environment chambers, but white epoxy paint is best for greenhouse conditions to reduce radiant heat load. Tops for containers should also have suitable holes for supporting the rockwool rooting medium and for removal of plants. Plastic grids, such as those used as diffusers in lighting fixtures (1.5 X 1.5 cm grid), can be cut to fit inside the lip of small, about 1-L, plastic containers. We prefer to use larger containers, such as 32-L low-density polyethylene tubs (Rubbermaid Inc. Wooster, OH, USA) covered with acrylic plastic tops (Fig. 1).

Figure 1. Three week old A. thaliana (Landsberg erecta) grown in solution culture. The upper panel shows a black-acrylic plastic top with space for 35 plants, and the plugs used to support the rockwool and plants. The lower panel shows the roots, the rockwool rooting medium, and bubbling of the solution. The tub holds 32 L of solution, and the dimensions are 53 x 38 x 22 cm. Rockwool plugs, 3 x 1.5 cm, are cored from a slab of rockwool with the grain of the rockwool in the long axis. Note how this method provides good uniformity of the shoots and roots. Bars = 5 cm.

The tops are cut from a large sheet of acrylic plastic, 0.32 cm thick, purchased from a local plastics dealer, and holes are cut in the tops to hold 35 individual plants in removable, plastic plugs. The plugs, purchased from a hardware store, are made from 3.81 cm diameter test plugs for polyvinylchloride pipe used in plumbing for drinking water. A 1.27 cm diameter hole is drilled in each plug to accept the rockwool, and the plugs are painted with vinyl or epoxy paint. Cylinders of rockwool (3 x 1.5 cm) are cored from a slab and placed in the plugs or grids to a depth that allows contact with the solution with about 2 cm remaining above the solution. Dry seeds can be sprinkled on the rockwool or wet seeds can be pipetted after treatment in cold water. After sowing, the rockwool is wetted with a wash bottle to help disperse and settle the seeds as well as aide wetting of the entire surface. Plants are thinned beginning on the second day after germination and as needed thereafter to one plant per plug.

 Solutions must be well aerated and mixed. Pumps and air stones purchased from an aquarium store are suitable for most uses and can provide a continuous air supply. Continuous aeration is not necessary and may even inhibit root growth by the constant agitation. A more desirable situation is to bubble solutions intermittently from an oil-free compressed air source. For the 32-L tubs we bubble air through a 30-cm long air stone for a 5 min duration every 30 min.

 In the often cited one-quarter strength modified Hoagland solution (Hoagland and Arnon, 1950; Johnson et al., 1957; Epstein, 1972) the macronutrients are reduced to one-quarter strength to avoid possible osmotic effects, ion toxicity and adverse interactions of certain nutrients, whereas the micronutrients are kept at full strength to prevent depletion. In comparison, the formulations used in most commercial hydroponic operations today are much closer in concentration to the full-strength Hoagland solution. With these considerations in mind we have formulated a solution with about one-third the concentration of macronutrients and the full concentration of micronutrients reported for the solution culture of lettuce (Resh, 1995). These changes were made to prevent depletion of nutrients in long running experiments while maintaining a low osmotic pressure. The hydroponic nutrient solution that we use comprises the macronutrients: 1.25 mM KNO3, 1.50 mM Ca(NO3)2, 0.75 mM MgSO4, 0.50 mM KH2PO4; and the micronutrients, 50 µM KCl, 50 µM H3BO3, 10 µM MnSO4, 2.0 µM ZnSO4, 1.5 µM CuSO4, 0.075 µM (NH4)6Mo7O24, 0.1 mM Na2O3Si and 72 µM Fe-diethylenetriamine pentaacetate (Sequestrene 330, Ciba-Geigy and can be purchased from Hummert International, (800) 325-3055, http://www.hummert.com/ as Sprint 330). The final solution pH is 6.0, and the electroconductivity is 0.66 dS m-1. Even though Si is not recognized as an essential mineral for plants, we include Si because Si is naturally present in the soil solution and in the plant cell wall. Silicon may confer benefits including mechanical strengthening, improved nutritional balance and pathogen resistance (Epstein, 1994).

 Many of the environmental effects on the growth and transition to flowering of arabidopsis have been reviewed (Martinez-Zapater et al., 1994). Arabidopsis is a facultative long-day plant and there are many interactive effects of day length, light intensity, light quality and temperature on the transition to flowering. Generally, flowering is induced by 16-h photoperiods if the plants are sufficiently mature; however, under constant light plants can be forced to flower with only 2-5 leaves. At less than inductive photoperiods, the total photon flux determines the rate of development and time of flowering. We have observed considerable shortening of the flowering time in plants grown at 400 compared to 200 µmol quanta PAR m-2 s-1 with a 10-h photoperiod. Arabidopsis is commonly grown at 100-200 µmol quanta PAR m-2 s-1; however, light saturation of CO2 fixation occurs at about 600 µmol quanta PAR m-2 s-1 (Eckardt et al., 1997). Higher temperatures tend to reduce the time to flowering and leaf number. The typical temperature used in laboratories ranges between 16 and 25C. We have observed that Landsberg erecta does well at 23C, whereas Columbia shows some signs of stress at this temperature.

 For plants to obtain maximal growth a balance of environmental conditions must be found that delays flowering yet produces rapid growth. We have observed that under an 8-h photoperiod plants grow large, but slowly. Whereas, under a 14-h photoperiod, plants grow more rapidly but flowering is induced early thus reducing leaf biomass. Estimates of growth under various environmental conditions are difficult to find in the literature (but see Martinez-Zapater et al. 1994, and references therein). Most studies are conducted under inductive photoperiods, 16-h to continuous light, which produce plants with about 0.1 to 0.01 the leaf biomass we report (Table 1). Although we have not thoroughly examined all possible combinations of growth conditions (i.e. photoperiod, light intensity, and temperature), we have adopted conditions which provide vigorous vegetative growth as determined by FW, leaf area and days to flowering (Table 1, Figure 2).

 

Table I. Growth of A. thaliana (Landsberg erecta) in hydroponic culture under a 10-h photoperiod (400 µmol quanta PAR m-2 s-1), 75% RH, and day/night temperatures of 21/18C.
Data were obtained from 5 separate experiments comprising 163 plants. Only selected harvest dates are shown, and values are the means for individual plants (± se).


Root


Shoot*


Days after planting


FW (g)


DW (g)


FW (g)


DW (g)


Raceme Height (cm)


Leaf area (cm2)


25 (n=10**)

0.057 ± 0.011

0.005 ± 0.001

0.204 ± 0.022

0.022 ± 0.002

0.0

8.31 ± 0.73

32 (n=9)

0.498 ± 0.057

0.032 ± 0.003

1.16 ± 0.069

0.121 ±0.006

0.0

38.9 ± 1.35

39 (n=5)

1.48 ± 0.112

0.097 ± 0.005

5.026 ± 0.213

0.508 ± 0.020

6.36 ± 0.62

130 ± 5.98

48 (n=3)

2.917 ± 0.164

0.211 ± 0.010

10.94 ± 0.499

1.195 ± 0.037

16.6 ± 1.53

258 ± 6.69


* Leaves, stem and raceme combined.
**Numbers of individuals

 

Figure 2. Hydroponic culture can produce large amounts of root and shoot biomass. The plants ages are from left to right: 16, 21, 25, and 48 d after planting. Note the massive root systems produced by hydroponic culture. These roots would undoubtedly be restricted if grown in conventional pot or tray systems. The inflorescence is approximately 16 cm in height.

Under these conditions, germination from dry seed began on 3 d, the transition to flowering began about 30-32 d, and bolting commenced about 33-34 d. It should be noted that the atmospheric concentration of CO2 was closely controlled in these experiments and that elevated CO2 (360 vs. 1000 µmol/mol) decreased the days to flowering by about 5 d.

The advantages of hydroponic culture have long been recognized in the production of larger and more uniform plants than those grown in soil. Some of the reasons for this are inherent in the ample supply of nutrients and water, and the elimination of root restriction; root restriction can significantly inhibit shoot growth (Peterson and Krizek, 1992). In addition, the excellent aeration of the root environment in hydroponic culture is considered a major factor that allows superior plant growth compared to soil grown plants. For arabidopsis a well aerated, yet uniformly wet medium is critical for good germination and seedling establishment, because the tiny seedlings are easily stressed by over- or under-watering. Rockwool provides such a medium for good seedling establishment. Beside the obvious benefits of increased growth and elimination of stress factors, hydroponic culture also allows the easy harvest of root tissues for studies of growth, physiology and biochemistry.
 
 Hydroponic Tips!

1. The rockwool can be difficult to cut with a cork borer. It cuts much easier and cleaner if you cut the rockwool while it is soaking in water or nutrient solution.

2. Some individual rockwool slabs appear to have high salt contamination. Soaking and rinsing in water prior to planting should alleviate this problem.

3. Individual seeds can be easily planted by touching the seed with the wet tip of a pencil or sharp stick.

4. To get good germination, the rockwool must be thoroughly wetted before or right after planting the seed. If you cut it out in solution then it will already be thoroughly wetted.

5. Water level is crucial for good germination. We find that the germination is best 1.5 to 2 cm below the seed level. If the water level is too high, the seeds become too wet and won't germinate properly. If the water level is too low, the water level will drop below the plugs and will dry out causing poor germination or seedling death. Once roots extend into solution, it is desirable to let the solution drop below the plugs. This will help reduce potential algae growth on the plugs without adverse affects on plant growth.

6. To prevent algae build up on top of the rockwool plugs, sprinkle a little bit of supersoil (dark soil mix) on top of the rockwool. This will cut out the light and completely inhibit algae growth.

7. We regularly us continuous aeration with aeration stones.

8. The rockwool plugs can slip down into the solution. Some people use cut off 15 mL Falcon tubes with enough of the cone edge at the bottom to add support for the plug so that it doesn't fall through.

9. You can also grow lots of plants on a thin slab for screening purposes.

10. How to make Gibeaut's solution. Note: the macronutrient stock solutions are kept in separate containers. The Fe stock solution is kept in aluminum-foil-covered or dark-brown bottle to prevent light degradation. The micronutrient stock solution is a mixture of all micronutrients combined together in one container. Be sure to make with distilled-deionized water.After making the complete Gibeaut's solution, the pH will be quite high and must be adjusted to approximately 6.0 with 10 M HNO3. A general rule of thumb is to change the solution when it goes above pH 7.0 or add HNO3 to reduce pH back to pH 6.0.

Gib Macronutrients
FW
[STOCK]

STOCK

(g/L)

mL (solution)/

L (water)

Ca(NO3)2 x 4H2O
236.15
1M
236.15
1.50
KNO3
101.11
1M
101.11
1.25
Mg(SO4) x 7H2O
246.48
1M
246.48
0.75
KH2PO4
136.09
1M
136.09
0.50
Na2O3Si x 9H2O
284.20
0.1M
28.420
1.00
Fe (Sprint 330)
10% (w/w)
0.072 M
40.0
1.00
Gib Micronutrients
KCl
74.56
50mM
3.728
1.00
MnSO4 x H2O
169.01
10mM
1.690
CuSO4 x 5H2O
249.68
1.5mM
0.375
ZnSO4 x 7H2O
287.54
2mM
0.575
H3BO3
61.83
50mM
3.092
(NH4)6Mo7O24
1235.9
0.075mM
0.093

11. Download new improved microfuge tube method here: Microfuge Hydroponics Method

If you have other suggestions, please email me at cramer@unr.edu.

LITERATURE CITED

Delhaize E, Randall PJ (1995) Plant Physiol 107: 207-213
Eckardt NA, Snyder GW, Portis Jr AR, Ogren WL (1997) Plant Physiol 113: 575-586
Epstein E (1972) Mineral Nutrition of Plants: Principles and Perspectives. John Wiley and Sons, New York
Epstein E (1994) Proc Natl Acad Sci USA 91: 11-17
Hirai MY, Fujiwara T, Chino M, Naito S (1995) Plant Cell Physiol 36: 1331-1339
Hoagland DR, Arnon DI (1950) Circ. 347. Berkeley, CA: Agric Exp Stn, Univ of Calif
Johnson CM, Stout PR, Broyer TC, Carlton AB (1957) Plant and Soil 8: 337-353
Martinez-Zapater JM, Coupland G, Dean C, Koorneef M (1994) Arabidopsis. Cold Spring Harbor Laboratory Press. pp 403-433
Peterson TA, Krizek DT (1992) J Plant Nutrition 15: 893-911.
Resh HM (1995) Hydroponic Food Production: a Definitive Guidebook for the Advanced Home Gardener and the Commercial Hydroponic Grower. 5th ed Woodbridge Press Publishing Co, Santa Barbara CA
Rodecap KD, Tingey DT, Lee EH (1994) J Environ Quality 23: 239-246
 

 

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