New Protocols for Paraffin Sections of Heterogeneous Tissues of Woody Plants

doi:10.11983/CBB18051

Abstract

Abstract: In view of the characteristics of highly lignified, large hardness and heterogeneous of various parts of woody plants, the stem segments and healing areas of bud grafting of Juglans regia were used as materials with the conventional method of paraffin sectioning. The aspects of fixation, softening, dehydration and other critical steps were modified to obtain cross-section slices with complete organizational structure and clear staining. The morphological and developmental characteristics of the healing period could be observed clearly and shortened the experimental period. The paraffin sections of stem segments of apple, peach, apricot, plum and poplar were obtained by the improved experimental process. The study provides a technical basis for studying the growth mechanism and morphological development of stem segments of woody plants and provides a reference for making paraffin sections of heterogeneous plant materials.

Key words: heterogeneous plant tissue, improved protocol, Juglans regia, paraffin section

Zhou Naifu, Zhang Junpei, Liu Hao, Zha Weiwei, Pei Dong. New Protocols for Paraffin Sections of Heterogeneous Tissues of Woody Plants[J]. Chinese Bulletin of Botany, 2018, 53(5): 653-660.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB18051

https://www.chinbullbotany.com/EN/Y2018/V53/I5/653

Figures/Tables 6

Figure 1 The intact cross-section slice of walnut stem segment tissue(A) Traditional FAA fixative, according to softening method 1 treatment; (B) Improve fixative, according to softening method 1 treatment; (C) Traditional FAA fixative, according to softening method 2 treatment; (D) Improve fixative, according to softening method 2 treatment. Bars=1 mm

Table 1 Effect of different types of fixation and softening on paraffin section of Juglans regia

	Combination 1	Combination 2	Combination 3	Combination 4
	FAA fixative solution Softening method 1	Improved fixative solution Softening method 1	FAA fixative solution Softening method 2	Improved fixative solution Softening method 2
Paraffin section	Partial cavity, divided and ripples	Continuous and ripples	Continuous and tidy	Continuous and tidy
Cortex	Brokenness and incomplete	Rupture and incomplete	Partial rupture and complete	Complete
Pith	Partial cavity and removed	Partly dehiscent, not detached	Complete	Complete
Transverse section	Fragmentary and incomplete with unclear outline	Shrinkage deformation, unclear outline	Partly complete with clear outline	Complete and smooth with clear outline

Table 2 Comparison of key steps of two different slicing methods

Key steps of slicing	Traditional slicing method (method 1)		Improved slicing method (method 2)
Key steps of slicing	Reagent	Time (h)	Reagent	Time (h)
Fixation	FAA fixative	48	Improved fixative	48
Softening	70% ethanol-glycerol	288	Hydrogen peroxide-glacial acetic acid, 70% ethanol-glycerol	48
Dehydration	Different concentrations of ethanol solution	8.5	Different concentrations of tert butanol solution	6.5
Transparency	Ethanol, xylene	3	-	-

Figure 2 The stem segment tissue slice of walnut with different treatment methods(A), (B) Traditional FAA fixative, according to softening method 1 treatment; (C), (D) Improve fixative, according to softening method 2 treatment. (B) and (D) are local amplification of (A) and (C). (A), (C) Bars=200 μm; (B), (D) Bars=100 μm. CZ: Cambium zone; Ph: Phloem; Xy: Xylem; Pi: Pith; PC: Parenchyma cell

Figure 3 The sliced section of walnut bud healing area using the improved paraffin section method(A)-(C) 6 days after grafted; (D)-(F) 12 days after grafted. (B), (C) and (E), (F) are local amplification of (A) and (D). (A), (D) Bars=1 mm; (B), (C), (E), (F) Bars=500 μm. CF: Callus formation; SC: Scion; ST: Stock; NL: Necrotic layer; DVB: Developing vascular bundles

Figure 4 The sliced sections of walnut, apple, peach, apricot, plum and poplar were obtained by improved paraffin section method(A)-(F) Paraffin sections of stems of walnut, apple, peach, apricot, plum and poplar; (G) and (H), (I) and (J), (K) and (L), (M) and (N), (O) and (P), (Q) and (R) are local amplification of (A)-(F). (A), (D), (E) Bars=1 mm; (G), (M), (O) Bars=500 μm; (H)-(L), (N), (P)-(R) Bars= 200 μm

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