Protocol

 

Authors

Carol Blake, Rana Munns

Overview

This protocol describes how to grow wheat or other species in salt solution, to measure differences in the rate of salt accumulation in leaves, or differences in growth rates over time. It is suitable for screening a large number of genotypes for genetic variation in Na+ exclusion from leaves, or a smaller number of genotypes for differences in “salinity tolerance”, that is, biomass production in saline versus non-saline solution.

Background

New sources of salinity tolerance are needed for crops grown on salt-affected land. Screening large numbers of genotypes for salinity tolerance in the field is difficult, due to spatial heterogeneity of soil chemical and physical properties, and to seasonal fluctations in rainfall.

Materials/Equipment

Equipment for salt tanks:

Small Scale experiment

Pots – 6.5 by 6.5 cm square by 15.8 cm high

Tanks (45 lt) holding 40 pots, with hole in bottom and overflow pipe. A raised layer at bottom is helpful to drain pots.

Reservoir – 65lt hydroponics tank Pumps; for small tank, aquarium pumps (such as the Aqua one Moray model ($40) 710 lt/hr capacity

Tubing – to connect pumps and overflow pipe Timers – need to be activated every 30 min, for 0.5 – 5 min

Substrate – gravel (~800g per pot, so ~32kg per small tank).

Compressed air for stirring solution

Large scale experiment.

Pots – 6.5 by 6.5 cm square by 15.8 cm high

Tanks – large plastic molded trays (90 L) tanks holding 153 pots Resevevoir – 44gallon drums or 3200lt water tanks with piping to fit

Pumps 2 Davey XF92 models works well (~$500 each)

Tubing – irrigation pipe of suitable diameter for fitting to tank and pump Timers as above

Substrate – gravel (0.5mm-1cm diameter, ~800g per pot so ~125kg per tankful)

For making up solutions:

2 L plastic beaker

2 L volumetric flask

Magnetic stirrer

Electronic balance which measures to 4 decimal places.

Funnel

Deionised or distilled water

Other :Thiram or bleach for sterilizing seed

Insulation material for covering pots before coleoptile emerges

Units, terms, definitions

All concentrations are given in mM (the same as mmol L-1 or mol m-3)

Procedure

Make up Hoaglands solution according to protocol.

Method for preparing seed for germination.

Equipment required.

Petri dishes

Electronic balance which weighs to at least 2 decimal places

Tweezers

Small beakers and marker pen

Filter papers

Thiaram fungicide 1 L Schott bottle

Method.

1) Select required number of genotypes for your experiment. Check the seeds are in good condition (ie. no shriveled, cracked or small seed). Weigh seed of each genotype, to within a 5mg weight range.

2) Work out the number of rep’s you will need for your experiment and weigh more seed than you will use, to ensure you have sufficient if germination is poor.

3) Make up 1L of Thiaram. Weigh 1.4g of the powdered Thiram into a Schott bottle and dissolve in water, make up to the 1L line. (This keeps for a long time, so don’t worry if you only need a few mls.)

4) Place seeds by genotype in individually labeled beakers, cover with tap water and leave to imbibe for 1.5 – 2 hours.

5) Drain seeds.

6) Swirl seeds for a few seconds in a beaker of Thiram solution, then remove and plate up on sterile petri dishes lined with two filter papers, dampened with Thiram solution. You can reuse the thiaram for more than one set of seeds.

7) Orientate seeds with the embryo facing up, the “seamed” side down, with a space between each seed for coleoptile and root emergence.

8) Cover with a single filter paper and using a squeeze bottle filled with Thiram solution, dampen the top filter paper to ensure adequate moisture.

9) Seal petri dishes with Parafilm and refrigerate at 4^∘c for 2-3 days if seed is fresh. For older seed that doesn’t require vernilisation, you can put the petri dishes straight into a dark cupboard at room temperature for 24 hours.

10) Remove seeds from fridge and leave in a warm, dark place to germinate. Germination can take 24-48 hours, and if there are differences in the germination progress of your varieties, you can slow down the more advanced ones by placing them back in the fridge until the other lines are at the same stage.

Method for planting up an experiment.

Tank preparation for hydroponics system.

1. Work out capacity of tank and fill to appropriate level (50lt in 65l reservoir) with water.

2. Attach pump to base of reservoir firmly and set overflow tube so that it’s top is at least 5cm shorter than the top of the reservoir.

3. Place an extension tube into the overflow tube which will extend the height of the water initially as the plants establish.

Plant up as follows

• Take a pot and tip it to a 45 degree angle and remove a little gravel.
• Take a germinated seed carefully with the tweezers and place it coleoptile up, about 2-3cm deep in the centre of the pot and cover gently with a layer of gravel.
• Insert label into or onto the pot.
• Repeat with all seeds.
• Gently water the pots to dampen everything and cover with a wet cloth or towel, and then a sheet of insulation or other dense material to prevent light penetration.
• Leave with covers on for 24 hours then remove and the coleoptile should be emerging from the gravel. If you have planted a bit deeper, emergence may take a few hours longer.
• Set pump to come on every 30 minutes and watch the water level. Initially you want to water to flood the pots every time the pump is turned on, but it only needs to run for a few seconds with the water high, before the pump should cut off again. As the plants establish, push the extension pipe back into the overflow pipe to lower the level and once leaf 1 is out you can remove the extension pipe altogether. Add ¼ hoaglands at this stage.
• Once the plants have leaf 2 emerging, ensure the water level is not coming up above the height at which the seed is planted in the pot. It is really important to ensure that the seed and crown stay dry once the roots have reached depth in the pot, as wet seeds get diseased and compromise the plants growth, interfering with results.
• Move your reps into the appropriate tank (Salt or control) and randomize. Ensure you have equal numbers of reps in each treatment.
• Once leaf 2 is emerging increase to 1/2 strength Hoagland’s in both tanks and add the CaCl2 solution to the salt tank only. Ensure that the overflow pipe has been pushed down or removed as once the solutions go on you don’t want to water to come up too high. Aim to keep the seed dry but the roots wet.
• Mix the solution thoroughly every time you add Hoagland’s or salt. To and to do this, either insert a pipe attached to an airline for a few minute to bubble the mixture, or place a second aquarium pump in the tank (without pipes attached) to circulate the mixture during the addition of nutrients or salts.
• Monitor leaf 3 emergence and score the label on each pot with a mark signifying which day L3 appeared. (examples days 0-2 below). This is the time to start adding salt to the salt tank. Do this gradually over 2-3 days. Each addition should be spaced several hours apart and equate to 25mM (which for 50lt of water in the tank is 250ml of the 5M concentrate you have made up).
• Try to add salt around 9am and again around 4pm each day until your final salt concentration is reached. If you notice any wilting, slow down the rate of salt addition to give the plants time to adjust.
• Monitor pH and conductivity as you go so that you do not make the salt solution more concentrated than you desired level and optimize pH at around 6.5. If your experiment is going to exceed more than 3 weeks you will need to change the solutions completely, around the 15-20 day mark. Since the plants are already adjusted to their salinity level you can add the whole compliment in one go, and mix well.

Salt solutions.

For most experiments, solutions of up to 250mM NaCl are used to stress plants and it is useful to have a stock of 5M NaCl made up, from which you can make the desired concentration for you experiment. To help plants adjust to the sodium salt, you also need to make up a Calcium salt solution to 2M strength.

NaCl molecular weight 58.44 so 5M made up in 2 lt is 58.4 x 5 x 2 = 584.4g)

CaCl2 molecular weight 147.02 (2M CaCl2 in 2lt is 147.02 x 2 x 2 = 588.08g)

Method to make up 5M NaCl solution.

Weigh 584.4g of NaCl into a 2lt container and top up with water to around 1.9lt and stir to dissolve. This take quite a few minutes as it is very concentrated. Pour through funnel into the volumetric flask and top up to the line accurately. Label and store in a container with a lid, which should always be securely shut.

Method to make up CaCl2 solution.

Weigh 588.08g of the CaCl2 into a 2lt beaker and top up to 1900ml with dH2O and stir to dissolve. Pour through funnel into volumetric flask and make up to the line accurately.

The table below indicates how to make up to desired concentration from the stock NaCl solution and the Calcium solution.

Stock solutions

Final Concentration of NaCl	Corresponding added CaCl2	Volume of 5M NaCl per litre (ml)	Volume of 2M CaCl2 per litre (ml)
NaCl (mM)	Added CaCl2 (mM)	5M NaCl	2M CaCl2
1		0.2	–
10		2	–
25	–	5	–
50	+2	10	0.5
100	+4	20	2
150	+8	30	4
200	+11	40	5.5
250	+15	50	7.5

• Note:- 1/2 FSH already contains 2mM Ca2+

For 20:1 ad 2.75 ml for 150 mM or 5.25 for 250 mM NaCl.

Image of four 65lt tanks set up for an experiment, with genotypes labelled and randomised through the tank and marked for Leaf 3 emergence and rep numbers.

You will need to work up a schedule of plant development and when to add solutions for your experiment. An example is attached

				Treatment: 150mM NaCl + CaCl2 at 10:1 (65lt tanks)
Date	Day	DAE	Emerg	Notes
			Leaf 3
3rd Nov	Friday			Seeds sterilized, imbibed and stored at 4 C
4th Nov	Saturday
5th Nov	Sunday
6th Nov	Monday			Seeds out of fridge into dark cupboard.
7th Nov	Tuesday			Plant up and cover.
8th Nov	Wednesday	0		Emergence 1/4 hoaglands in tanks and pumps on
9th Nov	Thursday	1
10th Nov	Friday	2		Re-arrange plants in tanks
11th Nov	Saturday	3
12th Nov	Sunday	4		L2 starting to emerge Adjust timer and pipe height
13th Nov	Monday	5		extra 1/4 hoaglands + 260 ml CaCl2
14th Nov	Tuesday	6	0 #	check L3 emergence – start salt 25mM x 2
15th Nov	Wednesday	7	1	salt and L3 scoring 25mM x 2
16th Nov	Thursday	8	2	salt and L3 scoring 25mM x 2
17th Nov	Friday	9	3	L3 scoring
18th Nov	Saturday	10	4
19th Nov	Sunday	11	5
20th Nov	Monday	12	6
21st Nov	Tuesday	13	7
22nd Nov	Wednesday	14	8	Check pH, cond’ and do envelopes
23rd Nov	Thursday	15	9
24th Nov	Friday	16	10	L3 harvest
25th Nov	Saturday	17		L3 harvest
26th Nov	Sunday	18		L3 harvest
27th Nov	Monday	19		L3 and L2 harvest
28th Nov	Tuesday	20		Start DW and cleaning tanks
29th Nov	Wednesday	21		DW
30th Nov	Thursday	22		DW
1st Dec	Friday	23
2nd Dec	Saturday

Notes and troubleshooting tips

Tap water may be used.

Check pH which may need to be lowered to pH 6.5 with HCl. If using deonised or distilled, check pH which may need to be raised with KOH. .

Note: Germination tests on the lines you are using is a good idea as it minimize waste. Good germination means you will only need to germinate a couple of extra seeds, rather than double the number you intend to use but be as generous as you can be with lines which have poor germination.

Literature references

Munns and James 2003 (picture large tanks); Boyer et al. FPB (picture small tanks)