Materials and Reagents

250 µl pipette tips (Mettler-Toledo International, Rainin, catalog number: 17007479 )
1 ml pipette tips (Mettler-Toledo International, Rainin, catalog number: 17001121 )
50 ml SuperClear centrifuge tube (Labcon, catalog number: LAB3181 )
Filter paper (Advantec, No.1: 90 mm)
Petri dish, round, 90 x 15 mm (Alpha Plus, catalog number: 16001-1 )
50 ml syringe (Sigma-Aldrich, catalog number: Z124990 )
Syringe filter (VWR, catalog number: 89041-306 )
Micropore tape (3M, catalog number: 1530-0 )
1 ml tubes
Rice seeds: Orysa sativa spp. japonica cv. Hwayoung and OsBUL1 knockout mutant rice (osbul1)
Ethanol (Avantor Performance Materials, J.T. Baker^®, catalog number: 8006 )
Sodium hypochlorite (NaOCl, Commercial Bleach–CLOROX)
Tween 20 (Alfa Aesar, Affymetrix/USB, catalog number: J20605 )
Potassium hydroxide (KOH) (SHOWA, catalog number: 1637-0150 )
Murashige & Skoog basal medium with Vitamins (MS) (PhytoTechnology Laboratories, catalog number: M519 )
Sucrose (Alfa Aesar, Affymetrix/USB, catalog number: J21938 )
Phytogel (Sigma-Aldrich, catalog number: P8169-500G )
Brassinolide (BL) (Sigma-Aldrich, catalog number: E1641 )
Sodium hypochlorite solution (with final available chlorine of 2%) (see Recipes)
5 N potassium hydroxide (KOH) solution (see Recipes)
Murashige & Skoog (MS) media (see Recipes)
1 mM Brassinolide (BL) stock solution (see Recipes)

Equipment

Rice husker (KETT ELECTRIC LABORATORY, model: TR-130 )
Ultrasonic cleaner (Elma, model: E-30H )
Clean bench (Chu-An, model: MBH-420N )
Scissors (Basic Life, catalog number: 76000 )
Forceps (Basic Life, catalog number: BL6502 )
Growth chamber (CHANG KUANG, model: CK-68EX )
Digital camera (Sony, model: NEX-3N )
Protractor (Taiwan united stationery, catalog nunber: HA401 )
600 ml beaker (DWK Life Sciences, DURAN, catalog number: 21 106 48 )
Glass petri dish (Sun Chion, catalog number: B16A1-0090 )
Autoclave
10 ml measuring cylinders (DWK Life Sciences, DURAN, catalog number: 21 390 08 04 )
100 ml measuring cylinders (DWK Life Sciences, DURAN, catalog number: 21 390 24 02 )
500 ml measuring cylinders (DWK Life Sciences, DURAN, catalog number: 21 390 44 03 )
Vortex mixer (Vortex-Genie2, Scientific Industries, model: Model G560 )
Incubator (YIHDER TECHNOLOGY, model: LM-570RD )
Pipetmans (Gilson, models: P20 , P200 and P1000 )
RiOs^TM Essential 16 Water Purification System (EMD Millipore, model: RiOs^TM Essential 16 )
Summit Series Analytical Balance (Denver Instrument, model: SI-234 )
pH meter (UltraBasic Benchtop pH Meter, Denver Instrument, model: UB-10 )

Software

ImageJ (https://imagej.nih.gov/ij/) for lamina angle measurement

Procedure

Seedling preparation for lamina inclination
1. Surface sterilize rice seeds
  1. Remove the lemma and palea of seeds using a rice husker (Figure 1).
    
    Figure 1. Rice husker used
  2. Put 50-60 naked seeds into a 50 ml SuperClear centrifuge tube and sterilize the surface of the seeds with 30 ml of 70% ethanol for 1 min and vigorously shake by hand.
  3. Rinse the seeds with 30 ml sterile water and pour off the dirty liquid.
  4. Add 30 ml of 2% sodium hypochlorite solution (see Recipes) and place the tube in an ultrasonic cleaner for 20 min.
  5. Discard the sodium hypochlorite solution and wash the seeds with 30 ml sterile water in the clean bench. We usually repeat the washing process 10-15 times.
  6. Place the seeds on autoclaved filter paper to dry and then transfer 20 seeds into a beaker containing MS media (see Recipes) using sterile forceps in the clean bench.
2. Germinate the sterilized seeds inside a growth chamber under long days (16 h light) at 28 °C for 8 days.
3. Sample uniform seedlings (based on similar height in each genotype) by excising approximately 2 cm segments (Figure 2) containing the second-leaf lamina joint, leaf blade and leaf sheath and float excised samples on sterile water for 10 min before transferring them to BL solution.
  
  Figure 2. Eight-day-old rice seedlings for lamina inclination assay. WT (Hwayoung cultivar) and osbul1 mutant rice were grown in beakers containing MS media (left). Leaf segments containing the second-leaf lamina joint of rice seedlings are marked by boxes outlined with dotted lines, which were used for lamina inclination assay.
Exogenous BL treatment
1. Put 20 ml of each test BL solution at designated concentrations (0 M, 10^-6 M, 10^-7 M, 10^-8 M and 10^-9 M in water) in 90 x 15 mm Petri dishes. The solution should be prepared immediately before use.
2. Float leaf lamina segments on BL solution (see Recipes) (0 M, 10^-6 M, 10^-7 M, 10^-8 M and 10^-9 M) in an incubator at 29 °C in the dark for 2 days (Figure 3).
  
  Figure 3. BR-induced lamina joint inclination in WT (Hwayoung cultivar) and OsBUL1 knock-out mutant (osbul1) rice. The photo was taken after BL (10^-6 M) treatment for 2 days.
3. After 2 days, take sample photos with a digital camera, print them and measure the angle induced between the lamina and the sheath (degree of angle of leaf blade against the axis of leaf sheath) with a protractor (Figure 4).
  
  Figure 4. Measurement of lamina angles. A. Measuring the lamina angle with a protractor; B. Representative leaf segments containing lamina joint, blade and sheath from WT (Hwayoung cultivar) and OsBUL1 KO (osbul1) plants after incubation for 48 h in the BL solution at different concentrations. C. Reduced leaf angles of OsBUL1 dsRNAi line (#2) compared to WT (TNG67 cultivar) in 0 M, 10^-8 M and 10^-6 M BL solution (*P < 0.001).

Data analysis

The same number of rice seedlings was used for each BL concentration set (n = 6-12). When ImageJ software was used for lamina angle measurement, photo files containing rice lamina fragments were opened in the program. By using the angle tool in the software (https://imagej.nih.gov/ij/docs/tools.html), 3 points, one each for the lamina blade, joint and sheath in the lamina fragment were fixed and the angle made by the three points was measured (Figure 5). Measured angle values are presented as means with standard deviations using Microsoft Office 2011 Excel (Figure 4C). The Student’s t-test is used for statistical significance. Data from at least three independent repeats were obtained.

Figure 5. Measurement of lamina angles using the ImageJ program. The three points are marked by green arrows using the angle tool. The measured angle is marked by the box with a dashed red border.

Notes

BL solution should be freshly prepared. Since the sensitivity of this test varied within the rice cultivars employed, crossed data analyses with different rice cultivars should be avoided. For lamina angle measurement, image processing computer programs such as ImageJ can also be used.

Recipes

Sodium hypochlorite solution (with final available chlorine of 2%) for 30 ml
Add 10 ml of commercial Bleach (CLOROX) into 20 ml of sterile water containing a few drops of Tween 20
5 N potassium hydroxide (KOH) solution for 30 ml
Add 8.417g KOH into 30 ml distilled water and filter the solution with a syringe filter
Murashige & Skoog (MS) media for 500 ml
2.2 g Murashige & Skoog basal medium with Vitamins
15 g sucrose
Distilled water up to 500 ml
Adjust pH to 5.7 with a few drops of 5 N KOH, and distribute 100 ml into a 600 ml beaker containing 0.3 g phytogel each, and then cover the beaker with a glass Petri dish. Seal the beaker with 3M micropore tape and autoclave
1 mM Brassinolide (BL) stock solution

Dissolve 2 mg of BL (molecular weight 480.68) in 1 ml of ethanol
Add 3.16 ml of distilled water to get 1 mM final concentration
Then split into aliquots in 1 ml tubes and store at -20 °C
Serial dilution: e.g., to make 10^-4 M BL solution, 1 ml of BL (10^-3 M) mixed with 9 ml of sterile water becomes 10 ml of 10^-4 M BL solution

Acknowledgments

This protocol was adapted from the method for rice lamina inclination assay described by Jang et al. (2017). We thank Ms. Miranda Loney for help with English editing. This work was supported in part by a core grant from BCST of ABRC, Academia Sinica.

References

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