Materials and Reagents

Arabidopsis seeds
Soil
Perlite
Tap water
Aluminium foil

Equipment

50 ml Falcon tubes
Tweezers
Burner
Milligram balance with a resolution of minimum 3 decimal place (0.001 g) readability
Transparent, tight container with a cover
Cold room/fridge
Growing chamber/phytotron
Incubator (with a possibility to set temperature 105 °C or above)

Procedure

In a container, prepare a mixture of soil and perlite in a 1:1 proportion.
Fill in 50 ml Falcon tubes with soil-perlite mixture to the 70-80% of Falcon volume.
To each Falcon pour out 35 ml of water and wait till the water sinks into soil-perlite mixture. 35 ml is normally sufficient to grow plants to the stage of 4-week-old.
In a separate container, moisten soil with water in a 1:1 ratio.
Full the Falcon tubes to the brim with well-moistened soil.
Using a burner, heat the tweezers and use them to make a hole (1.5 to 2 mm wide) in a central part of the Falcon tube cap. Another hole can be made closer to the cap edge (see Figure 2) in order to supplement the system with water (in case of growing plants longer than 4 weeks).
Note: While making a whole, move the tweezers’ end from the upper side of a cap towards the bottom side to avoid protrusion of plastic scraps that could wound the seedling.
Turn off each brim-filled Falcon tube with the cap.
Wrap Falcon tube with aluminium foil or other non-transparent material to avoid algae growth.
In the central hole place 5 to 10 Arabidopsis seeds.
Store falcon tubes in 4 °C in a humid environment (for example in a closed transparent container with a water-covered bottom) for two days.
After two days, move the whole system (Falcon tubes placed in a transparent water-containing container) to the growing chamber/phytotron.
After plants germinate (after about 5-7 days), remove excessive seedlings (using tweezers), leaving only one seedling in a hole (Figure 1).

Figure 1. Falcon tubes with single seedling each – ready to determine Falcon weight at the beginning of experiment (W0)
Weigh each falcon tube and write down the falcon weight (g) at the beginning of experiment (W0).
Grow plants for 4 weeks in a growing chamber/phytotron.
After 4 weeks (Figure 2), decapitate the rosettes and dry them for 3 h at 105 °C. Weigh dry mass for each rosette (mg) (DW).

Figure 2. Rosettes after 4 weeks of experiment – ready for decapitation
After removal of rosette, weigh each falcon tube and write down the falcon weight (g) at the end of the experiment (W).
Calculate the water loss: W0 (g) – W (g) = Water used (g). For water 1 g = 1 ml.
Calculate dry weight per unit of water used (mg DW x /ml of water used).

Acknowledgments

Please also cite the following publication when citing this protocol: Wituszyńska et al. (2013). This work was supported by the Welcome/2008/1 Program operated within the framework of the Foundation for Polish Science and co-financed by the European Regional Development Fund.

References

Wituszynska, W., Slesak, I., Vanderauwera, S., Szechynska-Hebda, M., Kornas, A., Van Der Kelen, K., Muhlenbock, P., Karpinska, B., Mackowski, S., Van Breusegem, F. and Karpinski, S. (2013). Lesion simulating disease1, enhanced disease susceptibility1, and phytoalexin deficient4 conditionally regulate cellular signaling homeostasis, photosynthesis, water use efficiency, and seed yield in Arabidopsis. Plant Physiol 161(4): 1795-1805.