Materials and Reagents

1. Regular weight (brown) germination paper 48.5” x 36.5”, custom-sized to 12” x 24” (Anchor Paper Company, catalog number: SD3836S )
   
2. Small kitchen wire mesh strainer/sieve with handle (20 cm diameter)
   
3. Small plastic cups, measuring boats (Fisherbrand^TM Hexagonal Polystyrene Weighing dishes) (Thermo Fisher Scientific, Fisher Scientific, catalog number: 02-202-101 ), or double-faced filter paper for drying seed- should be the same number as the number of entries
   
4. Waterproof pencil art grip aquarelle black (Faber-Castell, catalog number: 114299 ) and permanent marker (Sharpie^®, Fine Point Permanent Marker, black)
   Note: Black marker works better as other colors fade faster.
   
5. Plastic tags (5” x 5/8”) (International Greenhouse, catalog number: CN-1000 ) for labeling (optional if rolls are labeled)
   
6. Rubber bands (OfficeMax Extra Long Rubber bands, or any other brand)
   
7. Glassine bags (Seedburo S411 shoot bags, treated, 4” x 2-1/2” x 11”)
   
8. Personal protective items: latex gloves, lab coat, closed shoes, mask goggles
   
9. Maize seeds: Genotypes used in this protocol are pure lines obtained from the North Central Regional Plant Introduction Station in Ames, Iowa (Abdel-Ghani et al., 2013).
   Notes:
   
   1. Seeds should be multiplied under the same conditions to avoid differences due to the environment on the seed size.
      
   2. Seeds should display high germination percentages to keep a similar number of biological replications within experimental units.
      
10. Chlorox^® solution (6% sodium hypochlorite), household bleach (USA)
    
11. Deionized sterile distilled water (ddH₂O)
    
12. Potassium nitrate (KNO₃) (Thermo Fisher Scientific, Fisher Scientific, catalog number: P-263-500 )
    
13. Calcium nitrate [Ca(NO3)₂] (Thermo Fisher Scientific, Fisher Scientific, catalog number: C109-3 )
    
14. Monopotassium phosphate or potassium phosphate monobasic (KH₂PO₄) (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP-362-500 )
    
15. Magnesium sulfate (Thermo Fisher Scientific, Fisher Scientific, catalog number: M65-500 )
    
16. Iron from iron chelate [Fe-EDTA, (Sigma-Aldrich, catalog number: E6760-100G ), Fe-DTPA, or Fe-EDDHA]
    
17. Monocalcium phosphate or calcium phosphate monobasic [Ca(H₂PO₄)₂] (MP Biomedicals, catalog number: 193803 )
    
18. Calcium sulfate dihydrate (CaSO₄·2H₂O) (MP Biomedicals, catalog number: 191414 )
    
19. Potassium sulfate (K₂SO₄) (Sigma-Aldrich, catalog number: P0772-1kg )
    
20. Boric acid (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP168-1 )
    
21. Manganese chloride-4 hydrate (MnCl₂·4H₂O) (Sigma-Aldrich, catalog number: 221279-100g )
    
22. Zinc sulfate-7 hydrate (ZnSO₄·7H₂O) (Sigma-Aldrich, catalog number: Z0251-100G )
    
23. Copper sulfate-5 hydrate (Thermo Fisher Scientific, Fisher Science, catalog number: S25287A )
    
24. Molybdic acid (H₂MoO₄) (Sigma-Aldrich, catalog number: 232084-100G )
    
25. Hoagland’s nutrient solution
    
    1. High N (15 mM NO₃^-) Hoagland’s solution (see Recipes)
       
    2. Low N (1.5 mM NO₃^-) Hoagland’s solution (see Recipes)
       
    3. Micronutrient stock solution (1 L) (see Recipes)
       
26. 30% ethanol (C₂H₆O) (commercial grade from any brand) (see Recipes)
    
27. 2.5 g/L Fungicide solution Captan^® (Bonide Products Inc.) (see Recipes)
    

Equipment

1. 2 L capacity beakers (each beaker holds 8-10 paper rolls) (Coring, Pyrex^® Griffin Beakers, catalog number: 10000-2L )
   
2. 50 ml capacity beakers (Pyrex^®, Griffin Beakers), for sterilizing and washing seeds (Sigma-Aldrich, catalog number: CLS100050 )
   
3. Plant growth chamber (Conviron, model: PGC FLEX )
   
4. Cold room (Rheem Puffer Hubbard environmental chamber) or refrigerator
   
5. Autoclave (PRIMUS Sterilizer , model: PSS5 )
   
6. Sensitive balance (Ohaus, model: Adventurer^TM AR0640 )
   Note: This product has been discontinued.
   
7. Flatbed scanner (Epson, model: Expression 10000 XL , or any other brand)
   
8. Computer with flash drive and Windows operating system
   
9. Fisher Scientific^TM Isotemp^TM general purpose heating and drying oven (Thermo Fisher Scientific, Fisher Scientific, model: 15-103-0503) or any constant temperature oven/dryer
   
10. Ruler or yardstick (Acme Westcott 15728 36” Aluminum Yard Stick)
    

Software

1. WinRhizo (Regent Instruments, model: WinRhizo Pro 2009) or ARIA (Automatic Root Image Analysis) (Pace et al., 2014)

Procedure