Seedlings were transferred on solid MS medium with the indicated chemicals: propidium iodide (PI, 10 μM, Sigma-Aldrich or Thermofisher), hydroxyurea (HU, final concentration 5 mM, Sigma-Aldrich) for 24 hours, gibberellic acid (GA, final concentration 10 μM, Sigma-Aldrich) for 1 hour before ablation, paclobutrazol (PAC, final concentration 2 or 10 μM as indicated, Sigma-Aldrich) for 1 hour before ablation, epibrassinolide (EBL, Sigma Aldrich, final concentration 1 μM) for 1 hour before ablation, dexamethasone (DEX, Sigma Aldrich, final concentration 5 μM) for 1 hour before ablation, 1-Naphthylacetic acid f (NAA, Sigma Aldrich, final concentration 1 μM) or 1 hour before ablation, 6-Benzylaminopurine (BAP, Sigma Aldrich, final concentration 50 nM) for 1 hour before ablation.
Paclobutrazol pac
Manufactured by Merck Group
Sourced in United States
Paclobutrazol (PAC) is a plant growth regulator that is commonly used in laboratory settings. It functions by inhibiting the biosynthesis of gibberellic acid, a plant hormone responsible for stem elongation. PAC is often utilized in research to study the effects of growth regulation on various plant species.
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Lab products found in correlation
Gibberellic acid ga, by Merck Group (1 mentions)
Epibrassinolide ebl, by Merck Group (1 mentions)
6 benzylaminopurine bap, by Merck Group (1 mentions)
Hydroxyurea hu, by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Bacto agar, by Thermo Fisher Scientific (1 mentions)
Gibberellin, by Merck Group (1 mentions)
Coolpix 4500, by Nikon (1 mentions)
Bx51 camera, by Olympus (1 mentions)
Smz1500 stereomicroscope, by Nikon (1 mentions)
6 protocols using paclobutrazol pac
1
Modulation of Seedling Development
2
Characterizing Root Development in M. truncatula
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Seeds of the M. truncatula genotype Jemalong R108 were used in this study. The della1 (NF12399), della2 (NF4302) and della3 (NF10539) mutant lines are described in Fonouni-Farde et al.31 (link). Seeds were scarified and sterilized as described in Gonzalez-Rizzo et al. (2006)48 .
For in vitro pharmacological treatments, germinated R108 seeds were grown vertically on a growth paper (Mega International;http://www.megainternational.com/index.htm ) on a “i” medium49 supplemented with 1.5% Bacto-Agar (Gibco), in growth chambers at 24 °C under long-day conditions (16 h light at 150μE light intensity/8 h dark). After two days, the growth paper carrying the plants was transferred on a fresh “i” medium with or without GA3 (0,1 µM, Sigma-Aldrich) or paclobutrazol (PAC, 0,01 µM, Sigma-Aldrich), defined as the minimal concentrations leading to significant effects on root development. Root length was measured using the ImageJ software and emerged LRs were quantified two weeks post-germination.
For hormonal treatments, seedlings grown on a grid in a Magenta box containing a liquid “i” medium were pre-treated for 3 h with 10−6 M GA3 (Sigma-Aldrich) and then treated for 6 h with 10−7 M BAP (Sigma-Aldrich). Control experiments performed in parallel consisted in mock-treated samples. Roots were collected and immediately frozen in liquid nitrogen.
For in vitro pharmacological treatments, germinated R108 seeds were grown vertically on a growth paper (Mega International;
For hormonal treatments, seedlings grown on a grid in a Magenta box containing a liquid “i” medium were pre-treated for 3 h with 10−6 M GA3 (Sigma-Aldrich) and then treated for 6 h with 10−7 M BAP (Sigma-Aldrich). Control experiments performed in parallel consisted in mock-treated samples. Roots were collected and immediately frozen in liquid nitrogen.
3
GA3 and PAC Sensitivity Assay
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Gibberellic acid (GA3) and paclobutrazol (PAC) (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in ethanol. An identical volume of the blank solvent (ethanol) was used as a mock treatment. To detect GA sensitivity, sterilized seeds were first soaked in 150 μM PAC for 2 days to inhibit endogenous GA biosynthesis, and then germinated seeds were grown in DW with various concentrations of GA3. Seedling lengths were measured after 7 days of growth.
4
Germination and Hypocotyl Assays for Brassinosteroid Signaling
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For each germination assay, three independently grown seed batches of the wild type (WT), HBI1-OE, or BEE2-OE were compared. To ensure synchronous germination, seeds were imbibed at 4 °C for 3 d, then moved to a growth chamber with a 16 h/8 h light/dark cycle at 22 °C. The experiments were performed on half-strength MS medium supplemented with 1 µM 2,4-epibrassinolide (BR), 1 µM BRZ (Sigma-Aldrich, USA), 100 µM gibberellin (GA3), 1 µM paclobutrazol (PAC) (Sigma-Aldrich, USA), or 100 µM ABA (Sigma-Aldrich, USA). At least 80 seeds were imbibed for each treatment and examined for testa and endosperm rupture under a SMZ1500 stereomicroscope (Nikon, Japan), and photographed with a high-resolution digital camera (COOLPIX4500, Nikon, Japan). Germination rate was determined by calculating the percentage of testa and endosperm rupture in the control and different treatments. In the hypocotyl length assay, seeds were incubated for 36 h to attain 100% germination because BR-treated seeds germinate faster than those of the WT. After germination, testas were stripped and 50–70 embryos were photographed with a BX51 camera (Olympus, Japan). Hypocotyl length was measured using the Image J software (https://imagej.nih.gov/ij/index.html ). The SPSS software (http://www.spss.com/ ) was used for statistical analysis throughout this study.
5
Paclobutrazol Effects on Seedling Growth
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Seeds were sterilized with 10% commercial bleach including a detergent (Kitchen Haiter, Kao) for 20 min and then rinsed with sterile water three times for 5 min each. The seeds were imbibed in sterile water with gentle rotation for 3 d. The germinated seeds with emerged root of 4–6 mm were grown in 1/2 MS medium56 (link) containing 0, 1 or 10 μM paclobutrazol (PAC) (Sigma-Aldrich). The lengths of root and shoot of the grown seedlings were measured at 12 days after planting.
6
Modulation of Legume-Rhizobia Symbiosis by Gibberellin
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For Figs 1D , 5A , and B , pea seeds were nicked and treated at the time of planting with either 5 µl of ethanol (control) or 10 µg of GA3 (Merck, Australia) in 5 µl of ethanol. Seeds were planted and inoculated at 7 d as described by Foo and Davies (2011) (link). At 25 d, 5 µl of ethanol (control) or GA3 in 5 µl of ethanol was applied to the uppermost mature leaf. For Fig. 1D , roots were stained for lacZ and infection structures recorded as described above. For Fig. 5A and B , nodule number and size were recorded as described by Foo et al. (2016b (link)) at 37 d. For Fig. 5C and D , lupin seeds were nicked and treated at the time of planting with either 10 µl of ethanol (control), 10 µg of GA3, and/or 10 µg of paclobutrazol (PAC; Sigma Aldrich Pty. Ltd, Australia) in 10 µl of ethanol, planted and inoculated with appropriate bacteria as described by Foo et al., (2016a (link)), and nodule size was recorded at 28 d as described by Foo et al. (2016b (link)).
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