TITLE: Salt stress experiment in supported hydroponics examining the salt stress tolerance of durum wheat
AUTHORS: Magdalena Julkowska

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a standard protocol for durum wheat treatment, which was initially developed by Genc et al., 2008 and Genc et al., 2010. As durum wheat is suffering from the Ca2+ deprivation during the salt stress exposure, this needs to be compensated for, by calculating the Ca2+ activity using Visual Minteq. The wheat seedlings in this protocol are exposed to salt stress at 3rd leaf stage (xxx days after germination) with 75 mM NaCl - applied in steps of 25 mM NaCl every 12 h. </div></div>

[STEPS]
?. Sterilize wheat seedsPut the wheat seeds in 50% household bleech for 10 minutes.Wash with sterile MiliQ water in laminar hood for 3-5 timesPut the seeds in 10 ml of sterile MQ and leave at 4C for over night to break the dormancy
?. Germinate the wheat seedsPut the sterilized seeds on autoclaved germination paper (e.g. whatmann) in sterile petri dish (7 cm diameter) and pour approximately 3-5 ml of sterile MiliQ water for germinationSeal the petridish with leukopor tape and put in the growth chamber for germination (22/20C 12/12h light/dark with 60% humidity)
?. Transfer the seedlings to larger plates (14 cm diameter) with fresh germination paper, pour 10 ml MiliQ water, seal the plates and return to the growth chamber
?. Seven days after the germination transfer the seedlings to supported hydroponics During the "flush" (pumps on) interval, gently transfer the wheat seedlings to the supported hydroponics"Brush" the plastic beads off with a spoon / spatula, put the seedling in the beads and make sure that the roots are covered with the solution
?. One day before transfer - Prepare the supported hydroponics setup
Fill in the PCV tubes, sealed at one end with mesh, with black plastic beads to approximately 2-3 cm from the top of the tube and place in the hydroponic racks  Connect the pumps to the switch board and to the current adapter, which is connected to the time plug - make sure to install the timing on/off for 30 min. each (00:30:00 - on digital time switches)Add 100 L of tap water to the tanks below the hydroponics and let the entire system run and flush with tap water for 24hAfter flushing the system - unplug the switchboard and drain the tanks from tap waterAs it will take long time - at this point you also might want to collect all the MiliQ water that you will be using in the experiment - I collect 200 L for one experiment in 30 L tanks that are stored in the greenhouse compartment
?. One hour before the seedlings transfer - Prepare the modified Hoogland solution media in the tank by mixing the following into 100L of MQ water  ABCDE1media (mM)stock (M)Mole per 100 Lml to add to tank2NH4NO30.210.02203KNO352.50.52004Ca(NO3)220.40.25005MgSO420.40.25006KH2PO40.10.10.011007NaSiO30.510.05508NaFe(III)EDTA0.050.050.0051009H3BO30.010.050.0012010MnCl20.00510.00050.511ZnSO40.0050.010.00055012CuSO40.0050.250.0005213Na2MoO30.00010.010.00001114total volume of
  nutrient solutions1543.515NaCl7557.5150016CaCl21.210.12120
ABCDE1media (mM)stock (M)Mole per 100 Lml to add to tank2NH4NO30.210.02203KNO352.50.52004Ca(NO3)220.40.25005MgSO420.40.25006KH2PO40.10.10.011007NaSiO30.510.05508NaFe(III)EDTA0.050.050.0051009H3BO30.010.050.0012010MnCl20.00510.00050.511ZnSO40.0050.010.00055012CuSO40.0050.250.0005213Na2MoO30.00010.010.00001114total volume of
  nutrient solutions1543.515NaCl7557.5150016CaCl21.210.12120
?. After emergence of 3rd leaf (approximately 1.5 weeks after transfer) replace the meda and add the first dose of salt stress at the swapBe carefull as now you have to replace the media during half an hour of the drain cycle - so make sure to have everything ready at the end of "flood" cycle and drain the tank and add 100 L od media.For the tank that gets the salt stress treatment, add the salt to reach final concentration of 25 mM NaCl and 0.4 mM of CaCl2. Keep adding 25 mM NaCl and 0.4 mM CaCl2 every 12h until final concentration of 75 mM NaCl and 1.2 mM CaCl2 has been reached.
?. Make sure to check on your plants every day, to control whether the tanks are not leaking and whether the cycle of flood and drain is ok
?. Harvest the plants 2 weeks after the application of the salt stress. In order to prevent plants for being out of the flood-and-drain cycle for too long, the plants that are to be measured are collected into a 30 L bucket and transported to the measuring station.The typical traits we score during the harvesting campaign are:- pictures using PhotoSimile from 4 angles of each tube containing one plant- Fresh Weight of the entire shoot- Fresh Weight of the entire rootIon accumulation (Na+ and K+) of:- the entire root- the last fully expanded leaf (determined by - if the leaf above is shorter - your leaf-of-interest is the last fully expanded leaf)For ion accumulation you need to know fresh weight and dry weight of your tissue of interest. We usually do it by weighing prepared and labeled tubes individually, then weigh the tubes with fresh sample and weigh it again after at least 2 days at 65 C oven.The ion accumulation is measured using the flame photometer by prior extraction of ions using 1% nitric acid (10 ml used for root and shoot) and incubating the tissue with nitric acid at 65C oven for at least 2 days