Refinement of a clearing protocol to study crassinucellate ovules of the sugar beet (Beta vulgaris L., Amaranthaceae)

Table 2 The optimized protocols for improved clearing efficacy in crassinucellate ovules of B. vulgaris (n ≥ 15)

Ovule development	Tissue processing		Notes
Ovule development	Treatment	Conditions^a	Notes
Pre-fertilization stages^b (protocol I)	Rehydration^c	On an orbital shaker^d	1, 2, 3
	1 M HCl	5 min
	1 M HCl	60 °C, 10 min
	1 M HCl	5 min
	Schiff’s reagent	30 min, in the dark
	Sulfur water^e	3 × 10 min
	dH₂O	3 × 5 min
	Dehydration^f	On an orbital shaker
	Clearing^g	On an orbital shaker, with vacuum treatment^h
Post-fertilization stages (protocol II)	Rehydration	On an orbital shaker	2, 4
	95% H₂SO₄	5 min
	dH₂O	5 min
	1 M HCl	5 min
	1 M HCl	60 °C, 10 min
	1 M HCl	5 min
	Schiff’s reagent	30 min
	Sulfur water	3 × 10 min
	dH₂O	3 × 5 min
	Dehydration	On an orbital shaker
	Clearing	On an orbital shaker, with vacuum treatment
	Manual removal of the seed coat
Post-fertilization stages (protocol III)	Rehydration	On an orbital shaker	4, 5, 6
	95% H₂SO₄	5 min
	dH₂O	5 min
	3% H₂SO₄	60 °C, 90 min
	dH₂O	3 × 5 min
	Dehydration	On an orbital shaker
	Clearing	On an orbital shaker, with vacuum treatment
	Manual removal of the seed coat

1. The tissue transparency was significantly improved
2. Schiff’s reagent stained ovules making the tissues visible—eosin treatment may be omitted
3. Longer than 24-h incubation in pure methyl salicylate improved the tissue transparency
4. The tissue transparency was improved due to the removable seed coat, caused by the use of H₂SO₄
5. In the case of ovules in pre-fertilization stages, maceration in concentrated sulfuric acid may be omitted
6. Protocols II and III may be used interchangeably, since both gave the same clearing results
^aAll the steps were performed at room temperature, unless otherwise specified
^bThe whole ovaries were cleared to prevent the material loss during its preparation process
^cRehydration = ethanol: 70%, 50%, 30%; dH₂O (5 min each)
^dParameters of shaking were set at 150 rpm
^eSulfur water was made with 5 mg mL⁻¹ K₂S₂O₅ in 0.05 M HCl
^fDehydration = ethanol: 10%, 30%, 50%, 70% (15 min each); 95%, 100% (1 h each)
^gClearing = 100% ethanol: methyl salicylate, in proportions of 3:1, 1:1, 1:3 (2 h each change); pure methyl salicylate (at least 24 h)
^hAfter 1 h of each change of the clearing solution, vacuum treatment for 5 min was applied

ISSN: 1746-4811