TITLE: Maize hydroponics & Root multiple ion uptake kinetics [sterilization, germination, sowing, growth & uptake]
AUTHORS: Marcus Griffiths, Sonali Roy, Haichao Guo, Anand  Seethepalli, David Huhman, Yaxin Ge, Robert E Sharp, Felix B Fritschi, Larry M York

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Nutrient uptake is critical for crop growth and is determined by root foraging in soil. Growth and branching of roots lead to effective root placement to acquire nutrients, but relatively little is known about absorption of nutrients at the root surface from the soil solution. This knowledge gap could be alleviated by understanding sources of genetic variation for short-term nutrient uptake on a root length basis. A modular platform called RhizoFlux was developed for high- throughput phenotyping of multiple ion-uptake rates in maize (Zea mays L.). Using this system, uptake rates were characterized for the crop macronutrients nitrate, ammonium, potassium, phosphate, and sulfate among the Nested Association Mapping (NAM) population founder lines. The data revealed substantial genetic variation for multiple ion- uptake rates in maize. Interestingly, specific nutrient uptake rates (nutrient uptake rate per length of root) were found to be both heritable and distinct from total uptake and plant size. The specific uptake rates of each nutrient were positively correlated with one another and with specific root respiration (root respiration rate per length of root), indicating that up- take is governed by shared mechanisms. We selected maize lines with high and low specific uptake rates and performed an RNA-seq analysis, which identified key regulatory components involved in nutrient uptake. The high-throughput multiple ion-uptake kinetics pipeline will help further our understanding of nutrient uptake, parameterize holistic plant models, and identify breeding targets for crops with more efficient nutrient acquisition.</div></div>

[STEPS]
?. [Seed sieving to consistent size]
(*Optional)For each experiment or series of experiments sieve seeds to mean seed size of parents or population used
?. [Seed sterilization (Bleach only method)]
12 minutes in
[Bleach (Sodium hypochlorite)]
?. [Seed sterilization (Bleach only method)]
Rinse x3 with autoclaved dH2O and then leave with a dH2O cap
?. [Seed pre-germinatation]
(*Optional.) Note: stratification step was not used in the manuscript associated with this work on maize as germination was already consistentStratify seeds at 4 °C dark for 5 days
[dark for 5 days]
?. [Seed pre-germinatation]
Soak germination paper with 0.2 mM CaSO4. Lay seeds on soaked germination paper soaked 2” from top. Stapel genoype ID label to paper. Roll germination paper side to side & tie with cable ties. Part-submerge paper rolls in 0.2 mM CaSO4, ensuring seeds are not submerged, & cover in foil to prevent drying out.
[0.2 mM CaSO4]
Using a 4L jug approximately 2.5 L of CaSO4 should allow a 2" gap from the line of seeds
?. [Seed pre-germinatation]
Transfer seeds to incubator at 28 °C for 3-5 days for pre-germination
[dark for 3-5 days]
?. [Seedling growth in hydroponic tanks]
Set up growth chamber 28/20°C, 16/8h, 50% humidity,  210 μmol m-2 s-1 light intensity full light both fluorescent and incandescent with 1 hour dusk and dawn
[day temperature 16 h]
[night temperature 8 h]
?. [Seedling growth in hydroponic tanks]
Make plant hydroponics conePlastic mesh plant baskets (1.5” × 2”, Shenzhen Skywalker Electronic Limited, Shenzhen, China)Plant cone-tainers (SC10 Super RL98 cell, Stuewe & Sons Inc., OR, USA). A slot about 3 mm wide was cut from the bottom of the cone to about 5 cm from the top in order to accommodate the tubing from the sampling platform described below.Pot Lid 2" where shoot will emerge (Gro Pro Net)PVC clear tubing cut into 2" lengths used to guide shoot through lid hole
?. [Seedling growth in hydroponic tanks]
Make hydroponics tankTank - Quantum DG92080 Dividable Grid Container. 10.88" x 16.5" x 8"Inlay - 250mm x 395mm acrylic inlay with rounded corners. Hole design - 24 holes 1.75" diameter (44.45mm), plus 2 x holes for aeration XX diamter.
?. [Seedling growth in hydroponic tanks]
Prepare 1/2 Hoagland's growth solution at pH 6 using stock solutions.  ABCDEF1MacronutrientsChemical nameMW (g/mol)Conc (μM)Stock x100 (g sal / 1L)mL stock to add / 1L&nbsp;2KH2PO4Potassium phosphate monobasic136.0865006.80103KNO3Potassium nitrate101.1032570058104CaCl2Calcium chloride110.984200022.20105MgSO4Magnesium sulfate120.3676100012.04106(NH4)(NO3)Ammonium nitrate&nbsp;80.04343002.40107MicronutrientsChemical nameMW (g/mol)Conc (μM)Stock x1000 (g sal / 1L)ml stock to add / 1L 8H3BO3Boric acid61.833462.8419ZnSO4·7H2OZinc sulfate heptahydrate287.549672.01110MnCl2&nbsp;·4H2OManganese(II) chloride tetrahydrate197.905291.78111CuSO4·5H2OCopper(II) sulfate pentahydrate249.6850.320.08112(NH4)6Mo7O24·4H2OAmmonium molybdate1235.99750.1140.14113Fe(III)-EDTA (C10H12N2NaFeO8)Ethylenediaminetetraacetic acid iron(III) sodium saltmol)367.045615028.261.94MacronutrientsMicronutrientsFor 18 L Hoagland's solution add 1.2 mL 1M KOH
ABCDEF1MacronutrientsChemical nameMW (g/mol)Conc (μM)Stock x100 (g sal / 1L)mL stock to add / 1L&nbsp;2KH2PO4Potassium phosphate monobasic136.0865006.80103KNO3Potassium nitrate101.1032570058104CaCl2Calcium chloride110.984200022.20105MgSO4Magnesium sulfate120.3676100012.04106(NH4)(NO3)Ammonium nitrate&nbsp;80.04343002.40107MicronutrientsChemical nameMW (g/mol)Conc (μM)Stock x1000 (g sal / 1L)ml stock to add / 1L 8H3BO3Boric acid61.833462.8419ZnSO4·7H2OZinc sulfate heptahydrate287.549672.01110MnCl2&nbsp;·4H2OManganese(II) chloride tetrahydrate197.905291.78111CuSO4·5H2OCopper(II) sulfate pentahydrate249.6850.320.08112(NH4)6Mo7O24·4H2OAmmonium molybdate1235.99750.1140.14113Fe(III)-EDTA (C10H12N2NaFeO8)Ethylenediaminetetraacetic acid iron(III) sodium saltmol)367.045615028.261.94
[per tank]
[KH2PO4 Potassium phosphate monobasic (mw 136.086)]
[KNO3 Potassium nitrate (mw 101.1032)]
[CaCl2 Calcium chloride (mw 110.984)]
[MgSO4 Magnesium sulfate (mw 120.3676)]
[(NH4)(NO3) Ammonium nitrate (mw 80.0434)]
[H3BO3 Boric acid (mw 61.833)]
[ZnSO4·7H2O	 Zinc sulfate heptahydrate (mw 287.5496)]
[MnCl2 ·4H2O Manganese(II) chloride tetrahydrate (mw 197.9052)]
[CuSO4·5H2O Copper(II) sulfate pentahydrate (mw 249.685)]
[(NH4)6Mo7O24·4H2O Ammonium molybdate (mw 1235.9975)]
[Fe(III)-EDTA (C10H12N2NaFeO8) Ethylenediaminetetraacetic acid iron(III) sodium saltmol (mw 367.0456)]
[1M KOH]
?. [Seedling growth in hydroponic tanks]
Fill growth tank with prepared Hoaglan'ds solution pH 6. Sow plants ensuring roots are in contact with solution. Label lid with genotype information.Provide aeration to the hydroponic tank using an airpumpEcoPlus 1030 GPH (3900 LPH, 35W) Commercial Air PumpEcoPlus Air Manifold T-Style 3/8" Inlet, 8-Valve Outlet3/8" tubing3/16" tubing
?. [Seedling growth in hydroponic tanks]
Maintain plants in hydroponicsCheck pH & add iron chelate as required (eg. every few days / weekly)Replace Hoagland's solution with fresh as required (eg. every week / 2 weeks)
?. [Nutrient deprivation of plants before uptake assay]
Change nutrient solution 48hours prior to uptake assay with a largely macronutrient-free solution.  ABCDEF1MacronutrientsChemical nameMW (g/mol)Conc (μM)Stock x100 (g sal / 1L)mL stock to add / 1L 2CaCl2Calcium chloride110.9845005.549103MicronutrientsChemical nameMW (g/mol)Conc (μM)Stock x1000 (g sal / 1L)ml stock to add / 1L 4H3BO3Boric acid61.833462.8415ZnSO4·7H2OZinc sulfate heptahydrate287.549672.0116MnCl2 ·4H2OManganese(II) chloride tetrahydrate197.905291.7817CuSO4·5H2OCopper(II) sulfate pentahydrate249.6850.320.0818(NH4)6Mo7O24·4H2OAmmonium molybdate1235.99750.1140.1419Fe(III)-EDTA (C10H12N2NaFeO8)Ethylenediaminetetraacetic acid iron(III) sodium saltmol)367.045615028.261.94MacronutrientsMicronutrients solution should already be approximately pH6
ABCDEF1MacronutrientsChemical nameMW (g/mol)Conc (μM)Stock x100 (g sal / 1L)mL stock to add / 1L 2CaCl2Calcium chloride110.9845005.549103MicronutrientsChemical nameMW (g/mol)Conc (μM)Stock x1000 (g sal / 1L)ml stock to add / 1L 4H3BO3Boric acid61.833462.8415ZnSO4·7H2OZinc sulfate heptahydrate287.549672.0116MnCl2 ·4H2OManganese(II) chloride tetrahydrate197.905291.7817CuSO4·5H2OCopper(II) sulfate pentahydrate249.6850.320.0818(NH4)6Mo7O24·4H2OAmmonium molybdate1235.99750.1140.1419Fe(III)-EDTA (C10H12N2NaFeO8)Ethylenediaminetetraacetic acid iron(III) sodium saltmol)367.045615028.261.94
[per tank]
[CaCl2 Calcium chloride (mw 110.984)]
[H3BO3 Boric acid (mw 61.833)]
[ZnSO4·7H2O	 Zinc sulfate heptahydrate (mw 287.5496)]
[MnCl2 ·4H2O Manganese(II) chloride tetrahydrate (mw 197.9052)]
[CuSO4·5H2O Copper(II) sulfate pentahydrate (mw 249.685)]
[(NH4)6Mo7O24·4H2O Ammonium molybdate (mw 1235.9975)]
[Fe(III)-EDTA (C10H12N2NaFeO8) Ethylenediaminetetraacetic acid iron(III) sodium saltmol (mw 367.0456)]
?. [Ion uptake kinetics assay]
Make uptake chambers with a volume capacity of 250 mL.1.5” ID PVC Schedule 40 pipe Foundry Company, NC, USA)1.5” hub cap fitting1.5" to 2" adapter3/23" hole in side for nutrient sampling pipe, tubing samples from middle of chamber, sealed with clear waterproof silicone3/16" hole at bottom for nutrient filling/emptying,tubing flush with bottom of chamber for emptying, sealed with clear waterproof silicone  Assemble tubing per chamberLarge tubing (pump1)3.17 mm ID tubing (Ismatec SC0222-LT 2-Stop 0; Masterflex SC0223-LT Tygon)Masterflex Hose Barb Union 1/8" (Cole-Parmer Instrument Company LLC., IL, USA)CPC ID 1/8" hose barb PMCD1702 & insert (PMC2202, Colder Products Company, MN, USA)Small tubing (pump2)0.51 mm ID tubing (Ismatec SC0005-LT 2-Stop 0; Masterflex SC0029-LT Tygon, Cole-Parmer Instrument Company LLC., IL, USA)Diba MicroBarb® Adapter, 1/4" to 0.02" ID (Diba Industries Inc., CT, USA)Assemble tubing per chamberLarge tubing (pump1)3.17 mm ID tubing (Ismatec SC0222-LT 2-Stop 0; Masterflex SC0223-LT Tygon)Masterflex Hose Barb Union 1/8" (Cole-Parmer Instrument Company LLC., IL, USA)CPC ID 1/8" hose barb PMCD1702 & insert (PMC2202, Colder Products Company, MN, USA)Small tubing (pump2)0.51 mm ID tubing (Ismatec SC0005-LT 2-Stop 0; Masterflex SC0029-LT Tygon, Cole-Parmer Instrument Company LLC., IL, USA)Diba MicroBarb® Adapter, 1/4" to 0.02" ID (Diba Industries Inc., CT, USA)Small tubing (pump2)0.51 mm ID tubing (Ismatec SC0005-LT 2-Stop 0; Masterflex SC0029-LT Tygon, Cole-Parmer Instrument Company LLC., IL, USA)Diba MicroBarb® Adapter, 1/4" to 0.02" ID (Diba Industries Inc., CT, USA)
Other tubing sizes and dimensions can be used. A larger tubing size for rapid filling and a small diameter for precise sampling was what we decided was useful.
?. [Ion uptake kinetics assay]
Make uptake assay frameTechnical drawing of 24 hole lidParts list for frame 80/20 extruded aluminium
?. [Ion uptake kinetics assay]
Set up peristaltic pumpsAttach tubing to Pump casettesInsert casettes into Peristatlic pumpsSet tortuosity of the pump using the handles so that a steady and accurate flow rate is achieved (~12-15 notches depends on tubing size used and pump model)A 96-well microplate cover (VWR 580 International, LLC, PA, USA) was cut to size and holes drilled into the wells to make a sample tubing holder for sample collection into a deep-well collection plate. Tape was used to fasten the holder in place, so all tubings could be moved between samples easily
?. [Ion uptake kinetics assay]
Experiment setupPump 1 is used to fill the chambers with nutrient solution from a header tank at max RPM via tubing connected to the bottom of each chamber. The length of time can be predetermined since the pump speed and tubing length afect this.After chamber filling with nutrient solution. Pump 1 was used to provide continuous aeration by pumping only air into the chambers (24 rotations per minute) by lifting out the intake tubing from the header tank. (Factor for this timing during chamber filling)Pump 2 was used for periodic sampling of the nutrient solution from the middle of the chamber into a 2 mL 96-well collection plate. 1/2 mL is required for Ion chromatography. Optionally sample for double that ammount so spare sample can be stored in the freezer. The correct sample volume was achieved by running the pump for a predetermined time
?. [Ion uptake kinetics assay]
Transfer plants to uptake setupTake care that the cone-tainer slot aligns with the sampling tubing inside the chamber (as described in step8)Wait two minutes for plants to equibrilate.Start Pump2 and timer, collect first solution sample into collection plate.Once collected, reverse the flow of Pump 2 to ensure all solution has been expelled back into the chamber.Pause Pump2Move sample tubings to second row of collection plate ready for next sample.Repeat for as many samples as needed. Typical timings will be every hour, but this depends on the sample volume, solution concentration used, and plant size.
?. [Ion uptake kinetics assay]
After all samples have been collected clean out tubing ready for next experiment.Pump out all remaining solution from system.Run through tubing 3x rinse with dH2ORun through tubing 3x rinse with dH2O
[Bleach (Sodium hypochlorite)]
[HCl]