Protocols

Phenotyping of wheat plants

Germination:

Seedling vigour: (1-3 scale)

Days to heading: This is noted on the basis of days taken from date of sowing to the date when 75 per cent spikes are visible in the plot.

Days to Physiological maturity: This is recorded from the date of sowing to the day when rachis of 75% of the spikes are mature i.e have lost green colour.

Plant Height: The trait is recorded when the plants have attained physiological maturity. It is measured from base to tip of the main shoot excluding the awns. Height of five marked plants is measured and average taken.

Peduncle length: The length of peduncles of main spikes of five marked plants measured and average obtained.

Tiller number:  The trait is recorded when the plants have attained physiological maturity. Only productive tillers are counted. The tillers in the centre of the plot in the middle rows are counted i.e. in a 4 row plot the two border rows are eliminated and only two middle rows are considered. The meter scale is placed in the centre of these rows and tillers are counted separately for each of the two rows and average calculated.

Number of spikelets/spike: Spikelets are counted on main spikes of five marked plants in each plot.

We neeed to use the term Floret fertility and should be counted as percentage of the number of grains to the number of fully developed florets. Spikelet fertility: It is calculated using the grain number/spike and spikelets/spike.

Total Biomass: Biomass is recorded when plants are fully dry. Similar to grain yield, for biomass also in larger plot, central one square meter area is harvested separately and weighed. In smaller plots, central two rows are harvested separately and weighed.

Grain weight/spike: The threshed grains from main shoot spikes are weighed to obtain grain weight/ spike.

Yield: The yield is calculated after harvest. The fully dried plots are harvested. In larger plot, central one square meter area is harvested separately, threshed and after cleaning grain yield /sqm is obtained. In smaller plots, central two rows are harvested separately, threshed and grain yield obtained.

Grain number:  The main spikes of five marked plants are harvested separately, threshed with hands and cleaned. The grain so obtained are counted and averaged to obtain grain number/spike.

Grain filling duration: It is calculated as days taken from anthesis to physiological maturity of each genotype.

QUICK METHOD OF DNA EXTRACTION IN 96-WELL FORMAT

1. Use the special 2mL 96 deep well plates (Phenix-MPI-009) (always use the plate with one cut corner) for DNA extraction using quick method.
2. Cut one inch long 3 pieces of fresh leaf and place into plate well. Put one stainless steel bead of 3 mm size in each well either manually or using wide bore pipette tip or funnel (one need to be extra careful to put only one bead in each well by either way). Cover the plate with parafilm and silicon mat then put the plate into -80^oC for at least 1 hour (plates can be preserved in -80^oC for longer duration).
3. Using Qiagen clamp, place the plates into qiagen mixer mill for grinding the tissue for 1 min at 25 cycles/sec speed.
4. Using 8 or 12 channel pipette add 750 µL of CTAB buffer solution (60°C) and place the plate into water bath (preheated to 60°C) and run at 60°C for 1-1.5 hrs (can be extended maximum to 3 hrs).
5. Let the plate cool down to room temperature and add 750 µL (or equal volume) of Phenol:Chloroform:Isoamyl alcohol (25:24:1) or Chloroform:Isoamyl alcohol (24:1) into the wells. Mix thoroughly using multichannel pipette by pipetting in and out and centrifuge plate at room temperature at 3200 rpm for 15 minutes.
6. Transfer the supernatant (using 8 or 12 channel pipette with wide bore tips) to a new two cut corner plate and add 2/3 volume (500 µL) of isopropanol (room temp). Mix the samples by pipetting in and out gently using wide bore tips for precipitation.
7. Centrifuge the plate at room temperature at 3200 rpm for 10 minutes and drain the solution by retaining the DNA pellet in the plate. Wash DNA with 100 µL of 70% ethanol. Leave at room temperature for 30 minutes, plate can be left overnight in 4^oC for washing.
8. Quick spin for 4 min and drain the ethanol, air dry the DNA pellet, and add 100 µL of 1X Tris EDTA buffer and 1µL of RNAse (10mg/mL). Leave at 4°C overnight before use. This method would yield about 30-50 µg of DNA that can be used for PCR analysis for more than one year.

2X CTAB EXTRACTION BUFFER

Stock	10 ml	50 ml	200 ml	500 ml	1000 ml
1M Tris, pH 7.5	1 ml	5 ml	20 ml	50 ml	100 ml
5M NaCl	2.8 ml	14 ml	56 ml	140 ml	280 ml
0.5M EDTA, pH 8.0	0.4 ml	2 ml	8 ml	20 ml	40 ml
CTAB	0.2 g	1 g	4 g	10 g	20 g

CTAB = Cetyl Trimethyl Ammonium Bromide

Put all the stock solutions and CTAB in less amount of ddH2O (say 500 ml for 1000 ml of CTAB buffer) and make up the volume after the dissolution of CTAB.

Add 2-mercaptoethanol to the warmed buffer just prior to use in the hood at the concentration of 0.2 ml per 100 ml of 2X CTAB buffer.

Note: 

• Rinse the tips with distilled water each time while doing mixing in the step #5 and step #6
• The plate should be tilted slowly so that the pellet is not lost while draining.

RNA EXTRACTION: HOT PHENOL

Note* =More specifically, prepare all glassware and metalware by wrapping the opening with aluminum foil and baking in dry oven at 180-220⁰C for atleast 8 hours (overnight is fine). Any items that cannot be baked, including Plasticware should be dipped in DEPC (0.1% in water) for 2 hours or overnight at 37⁰C and subsequently autoclaved to degrade the DEPC

All chemicals, bottles, work bench, pipettes used should be dedicated to RNA. 

Change the gloves frequently. 

Solutions should be prepared in 0.1% DEPC treated autoclaved water and autoclaved.

Tissue collection: 

Dip tissue in Liquid Nitrogen and then put in well.

Tissue amount should be as follows:-

Grains before 10 DPA:- 2 maximum

Grain after 10 DPA:- 1 grain

Extraction: The RNA extraction and cDNA synthesis is a two day procedure. On first day start before noon to proceed till step no.13 mentioned. On second day finish the rest of the steps and proceed with cDNA synthesis right away.

1. Set the water bath at 80⁰C. (Make sure you turn the water bath back to 37⁰C after you are done).
2. Prepare working extraction buffer solution by adding equal volumes of phenol (pH = 4.7) and stock extraction buffer (final concentration of 1:1)
3. Heat the above mixture to 80⁰C for at least 1/2 half and ensure it reaches 80⁰C with the help of thermometer.
4. Take out the your freezed samples from -80⁰C and immediately dip in pestle morter containing Liquid Nitrogen, starts crushing upto fine powder samples.
5. Quickly add 750ul of the working extraction buffer in each well with the help of 1000ul pipette. Put the plate in the water bath set at 80⁰C for 5minutes.
6. Homogenize by vigorous vortexing for 2-3 minutes.
7. Add 350ml of chloroform in each tube. and mix the samples on shaker for 30 minutes or till solution in each well turns milky. 
8. Centrifuge for 15 min @4000 rpm at 4⁰C.
9. Add 50ul (1/3^rd of the supernatant) of 8 M LiCl to each well. Transfer 150ul of the supernatant to the corresponding tubes. Vortex the tube for 1- 2mins.
10.  Leave the tubes on ice for at least 2 hrs (to precipitate RNA).
11.  Centrifuge at 4,000 rpm for 1hr at 4⁰C.
12.  Discard the supernatant (by gently tilting the tubes) and add 500ml of 2M LiCl. Then centrifuge at 4,000 RPM for 10 min (4⁰C). 
13. Discard the supernatant and add 500ml of 70% EtOH made with DEPC treated water. Keep it for overnight at -80⁰C.
14. Next day, take out the plates and keep it on ice for thawing.
15.  Centrifuge at 4,000 RPM for 10 min (4⁰C). Discard the EtOH slowly and place the  tubes in inverted position (without turning it back) on the tissue paper placed in laminar air flow. 
16. After 2-3 minutes place the tubes in a position facing the filter of laminar air flow to air dry the pellet for 5 to 10 mins. Do not over-dry otherwise the pellet will not resuspend easily.
17. Dissolve the RNA by adding 50ml of DEPC treated water and Mix it by vortexing for 2-3 mins. If the RNA is critical then it is wise to save about half at -80⁰C for future use and proceed with the rest. 
18. Treat the samples with Turbo DNAse 1ml and 5ul of DNase buffer and incubate in water bath set at 37⁰C for an hour.
19. Stop the enzyme reaction by adding 10ml of 20mM EGTA stop solution and incubate at 65⁰C for 10 min. 
20. To the total volume, add 1/10 vol (4ul) Sodium acetate (3M, pH 5.2) and 2.5 vol (100ul) of 100% ethanol, mix well. 
21. Keep it on ice for 30mins to 1 hour and centrifuge at 4,000 RPM for 1 hour at 4⁰C. 
22. Discard the supernatant and wash the pellets with 70% ethanol (DEPC water) and centrifuge at 4000rpm for 10min.
23. Drain the ethanol and air dry the pellets (Ref. point no. 16 & 17 above)
24. Dissolve the RNA by adding 20 ml of DEPC treated water and mix by vortexing and check on formaldehyde gel to see the quality and quantify the RNA.
25. Proceed for cDNA synthesis right away.

          Extraction Buffer: 

            Make 1 liter Stock Extraction buffer as follows (in a baked bottle) and kept it at room temperature.

1M Tris-Hcl (pH = 8.0) = 100 ml

8M Licl                          = 12.5 ml               

0.5M EDTA (pH = 8.0) = 20 ml

20% SDS (pH=7.2)        = 50 ml

DEPC treated water       = 817.4 ml

Total                               = 1000 ml

Note:-

• Materials like tips, beakers coming in contact with DEPC should be autoclaved before using again for other purposes or disposing off.
• Chemicals used for solution preparation should be directly weighed in baked bottles.
• Spatula should be flame sterilized and the bulb of pH meter should be wiped with chloroform prior to measuring the pH of the solutions.

Chemicals calculations

• Step-by-step for 100 mL of 20 mM EGTA:

• Add ~80 mL of DI water to a beaker with a stir bar.
• Add 0.76 g EGTA to the water and begin stirring.
• Adjust the pH to ~7.0–8.0 using NaOH or KOH:
• Transfer the solution to a 100 mL volumetric flask and bring the final volume up to 100 mL with DI water.

2.         8M Licl converted to 2M Licl (Take 25 mL of 8 M LiCl, Add distilled water up to a final volume of 100 mL)

3. To Make 100 mL of 3 M Sodium Acetate: Using Anhydrous Sodium Acetate, Weigh 24.6 g anhydrous sodium acetate

• Dissolve in ~70 mL distilled water
• Adjust pH if needed (typically pH ~4.0–5.6)
• Bring the volume up to 100 mL with distilled water

4. To make 1M Tris-Hcl (pH = 8.0) = 100 ml = Weigh out 12.11 g Tris and add to a 100 mL

5.   To make 0.5M EDTA= Weigh out 3.72 g of Na₂EDTA, Add it to ~15 mL of distilled water in a beaker, Stir, EDTA will start dissolving as pH approaches 8, bring the volume up to 20 mL with distilled water.

6.  To Make 20%SDS= Weigh 10 g of SDS powder.  Add it to ~40 mL of warm distilled water in a beaker, SDS dissolves slowly at room temperature; using a warm water bath (~37–50°C) helps dissolve it faster, Stir gently until fully dissolved, Adjust the pH to 7.2 using 1 N HCl, dropwise. Transfer the solution to a 50 mL volumetric flask or measuring cylinder and bring the volume up to 50 mL with distilled water.