植物生态学报 ›› 2013, Vol. 37 ›› Issue (10): 972-977.DOI: 10.3724/SP.J.1258.2013.00100
收稿日期:
2013-02-27
接受日期:
2013-07-24
出版日期:
2013-10-01
发布日期:
2013-09-29
通讯作者:
勾晓华
基金资助:
ZHANG Jun-Zhou,GOU Xiao-Hua(),ZHAO Zhi-Qian,LIU Wen-Huo,ZHANG Fen,CAO Zong-Ying,ZHOU Fei-Fei
Received:
2013-02-27
Accepted:
2013-07-24
Online:
2013-10-01
Published:
2013-09-29
Contact:
GOU Xiao-Hua
摘要:
树轮气候学及树轮生态学研究在国内外取得了长足进展, 但对树轮与气候的响应机制缺乏深入的了解, 迫切需要进行以微树芯石蜡切片为基础的树轮生态学研究。形成层活动监测从生理生态学角度出发, 广泛用于树轮对气候变化响应的机制探讨研究中, 然而目前还没有对微树芯石蜡切片制作的方法作详细的论述和探讨。该文通过对祁连圆柏(Sabina przewalskii)等5个不同树种微树芯石蜡切片的制作实践, 对已有的植物石蜡切片技术进行改进, 增加组织软化的步骤, 并对其他步骤作了相应的调整, 详细介绍了微树芯石蜡切片制作的方法, 为树轮生态学、解剖学, 以及木材解剖学关于木质化材料石蜡切片的制作方法提供参考。
张军周, 勾晓华, 赵志千, 刘文火, 张芬, 曹宗英, 周非飞. 树轮生态学研究中微树芯石蜡切片制作的方法探讨. 植物生态学报, 2013, 37(10): 972-977. DOI: 10.3724/SP.J.1258.2013.00100
ZHANG Jun-Zhou, GOU Xiao-Hua, ZHAO Zhi-Qian, LIU Wen-Huo, ZHANG Fen, CAO Zong-Ying, ZHOU Fei-Fei. Improved method of obtaining micro-core paraffin sections in dendroecological research. Chinese Journal of Plant Ecology, 2013, 37(10): 972-977. DOI: 10.3724/SP.J.1258.2013.00100
图1 微树芯采样。A, 祁连山东部祁连圆柏采样点。B, 为避免对下次采样产生影响, 采用“Z”字形方式采样。C, 采集的微树芯。D, 微树芯采集后迅速固定。
Fig. 1 Micro-core sampling. A, Sampling points of Sabina przewalskii in eastern Qilian Mountains. B, In order not to affect the next sampling, using “Z” font sampling. C, Micro-core collection. D, Micro-core rapid fixation after collection.
步骤 Step | 试剂 Reagent | 时间 Time |
---|---|---|
1 | 30%乙醇 30% ethanol | 90-120 min |
2 | 50%乙醇 50% ethanol | 90-120 min |
3 | 70%乙醇 70% ethanol | 90-120 min |
4 | 85%乙醇 85% ethanol | 90-120 min |
5 | 95%乙醇 95% ethanol | 60-90 min |
6 | 无水乙醇I Anhydrous ethanol I | 60-90 min |
7 | 无水乙醇II Anhydrous ethanol II | 60 min |
8 | 二甲苯:乙醇= 1:1 Dimethylbenzene: ethanol = 1:1 | 60 min |
9 | 二甲苯I Dimethylbenzene I | 60 min |
10 | 二甲苯II Dimethylbenzene II | 60 min |
11 | 二甲苯:石蜡= 1:1 Dimethylbenzene: paraffin = 1:1 | 48-72 h |
12 | 石蜡I Paraffin I | 120 min |
13 | 石蜡II Paraffin II | 60 min |
14 | 石蜡III Paraffin III | 30 min |
表1 微树芯脱水、透明、浸蜡的步骤及时间
Table 1 Steps and time of micro-cores dehydration, cleaning and waxdip
步骤 Step | 试剂 Reagent | 时间 Time |
---|---|---|
1 | 30%乙醇 30% ethanol | 90-120 min |
2 | 50%乙醇 50% ethanol | 90-120 min |
3 | 70%乙醇 70% ethanol | 90-120 min |
4 | 85%乙醇 85% ethanol | 90-120 min |
5 | 95%乙醇 95% ethanol | 60-90 min |
6 | 无水乙醇I Anhydrous ethanol I | 60-90 min |
7 | 无水乙醇II Anhydrous ethanol II | 60 min |
8 | 二甲苯:乙醇= 1:1 Dimethylbenzene: ethanol = 1:1 | 60 min |
9 | 二甲苯I Dimethylbenzene I | 60 min |
10 | 二甲苯II Dimethylbenzene II | 60 min |
11 | 二甲苯:石蜡= 1:1 Dimethylbenzene: paraffin = 1:1 | 48-72 h |
12 | 石蜡I Paraffin I | 120 min |
13 | 石蜡II Paraffin II | 60 min |
14 | 石蜡III Paraffin III | 30 min |
步骤 Step | 试剂 Reagent | 染色时间 Staining time |
---|---|---|
1 | 二甲苯 Dimethylbenzene | 12 min |
2 | 无水乙醇:二甲苯= 1:1 Anhydrous ethanol:dimethylbenzene = 1:1 | 6 min |
3 | 无水乙醇 Anhydrous ethanol | 6 min |
4 | 95%乙醇 95% ethanol | 6 min |
5 | 85%乙醇 85% ethanol | 6 min |
6 | 70%乙醇 70% ethanol | 6 min |
7 | 50%乙醇 50% ethanol | 6 min |
8 | 30%乙醇 30% ethanol | 6 min |
9 | 水 Water | 6 min |
10 | 1%番红水溶液 1% safranine water solution | 3-5 h |
11 | 水 Water | 60 s |
12 | 30%乙醇 30% ethanol | 60 s |
13 | 50%乙醇 50% ethanol | 60 s |
14 | 70%乙醇 70% ethanol | 60 s |
15 | 85%乙醇 85% ethanol | 60 s |
16 | 0.5%固绿乙醇溶液 0.5% fast green ethanol solution | 30 s |
17 | 95%乙醇 95% ethanol | 60 s |
18 | 无水乙醇 Anhydrous ethanol | 60 s |
19 | 无水乙醇:二甲苯=1:1 Anhydrous ethanol:dimethylbenzene = 1:1 | 60 s |
20 | 二甲苯 Dimethylbenzene | 60 s |
表2 微树芯石蜡切片番红-固绿染色步骤及时间
Table 2 Steps and time of micro-cores staining with safranine and fast green
步骤 Step | 试剂 Reagent | 染色时间 Staining time |
---|---|---|
1 | 二甲苯 Dimethylbenzene | 12 min |
2 | 无水乙醇:二甲苯= 1:1 Anhydrous ethanol:dimethylbenzene = 1:1 | 6 min |
3 | 无水乙醇 Anhydrous ethanol | 6 min |
4 | 95%乙醇 95% ethanol | 6 min |
5 | 85%乙醇 85% ethanol | 6 min |
6 | 70%乙醇 70% ethanol | 6 min |
7 | 50%乙醇 50% ethanol | 6 min |
8 | 30%乙醇 30% ethanol | 6 min |
9 | 水 Water | 6 min |
10 | 1%番红水溶液 1% safranine water solution | 3-5 h |
11 | 水 Water | 60 s |
12 | 30%乙醇 30% ethanol | 60 s |
13 | 50%乙醇 50% ethanol | 60 s |
14 | 70%乙醇 70% ethanol | 60 s |
15 | 85%乙醇 85% ethanol | 60 s |
16 | 0.5%固绿乙醇溶液 0.5% fast green ethanol solution | 30 s |
17 | 95%乙醇 95% ethanol | 60 s |
18 | 无水乙醇 Anhydrous ethanol | 60 s |
19 | 无水乙醇:二甲苯=1:1 Anhydrous ethanol:dimethylbenzene = 1:1 | 60 s |
20 | 二甲苯 Dimethylbenzene | 60 s |
图3 番红-固绿染色后的祁连圆柏微树芯显微图片。C, 形成层区域; L, 木质化的细胞; nX, 当年新形成的木质部; P, 韧皮部; X, 上一年的木质部。
Fig. 3 Micro-cores micrograph of Sabina przewalskii stained with safranin-fast green. C, cambial zone; L, lignified cells; nX, current year newly formed xylem; P, phloem; X, last year xylem.
图4 不同树种微树芯石蜡切片显微图片。A, 红桦。B, 油松。C, 青海云杉。D, 青杄。标尺=100 μm。
Fig. 4 Micro-cores paraffin section micrograph of different tree species. A, Betula albo-sinensis. B, Pinus tabuliformis. C, Picea crassifolia. D, Picea wilsonii. Bar = 100 μm.
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