富含紫杉烷类红豆杉的离体培养

doi:10.11983/CBB22228

植物学报 ›› 2023, Vol. 58 ›› Issue (6): 917-925.DOI: 10.11983/CBB22228

富含紫杉烷类红豆杉的离体培养

冯晓晖¹^,²^,³, 闫学彤²^,³, 郑珂媛²^,³, 周强¹, 张伟中⁴, 王权勇⁴, 朱木兰²^,³^,^*()

¹吉首大学植物资源保护与利用湖南省高校重点实验室, 吉首 416000
²上海辰山植物园, 上海市资源植物功能基因组学重点实验室, 上海 201602
³中国科学院分子植物科学卓越创新中心, 上海 200032
⁴上海金和生物制药有限公司, 上海 201716

收稿日期:2022-09-29 接受日期:2023-02-28 出版日期:2023-11-01 发布日期:2023-11-27
通讯作者: * E-mail: mlzhu@cemps.ac.cn
基金资助:
上海市绿化和市容管理局科研专项(G212403)

In vitro Culture of Taxus Rich in Taxanes

Xiaohui Feng¹^,²^,³, Xuetong Yan²^,³, Keyuan Zheng²^,³, Qiang Zhou¹, Weizhong Zhang⁴, Quanyong Wang⁴, Mulan Zhu²^,³^,^*()

¹Key Laboratory of Plant Resources Conservation and Utilization, College of Hunan Province, Jishou University, Jishou 416000, China
²Shanghai Key Laboratory of Plant Functional Genomics and Resources, Shanghai Chenshan Botanical Garden, Shanghai 201602, China
³Chinese Academic Scientific Center for Excellence in Molecular Plant Science, Shanghai 200032, China
⁴Shanghai Jinhe Bio-Pharmaceutical Co., Ltd., Shanghai 201716, China

Received:2022-09-29 Accepted:2023-02-28 Online:2023-11-01 Published:2023-11-27
Contact: * E-mail: mlzhu@cemps.ac.cn

摘要/Abstract

摘要： 红豆杉(Taxus spp.)为国家一级濒危植物, 含有药用价值高的广谱抗癌成分紫杉醇, 但资源匮乏。为解决紫杉醇药源短缺问题, 该研究建立了红豆杉紫杉烷类离体富集体系。结果表明, 最佳吸收面扩大培养基配方为MS+2 mg·L^-1 6-BA+0.4 mg·L^-1 NAA+0.7 g·L^-1脯氨酸, 扩大率达90%; 高频同步诱导率最高的培养基为DCR+1 mg·L^-1 6-BA+0.1 mg·L^-1 NAA+5 mg·L^-1谷氨酰胺+1 g·L^-1活性炭, 高频同步率达82.2%; 生物量扩增I期效果最佳培养基为MS+0.7 mg·L^-16-BA+ 0.07 mg·L^-1 NAA+0.1 mg·L^-1苯丙氨酸+50 mg·L^-1间苯三酚, 芽苗生物量为每株542 mg; 生物量扩增II期效果较好的培养基为MS+0.5 mg·L^-1 6-BA+0.05 mg·L^-1 NAA+0.1 mg·L^-1苯丙氨酸+2 g·L^-1活性炭, 芽苗生物量为每株1 612 mg。富集培养后, 组培材料中紫杉烷类含量远高于天然材料, 紫杉醇含量为天然材料的6.1倍; 巴卡亭III含量为天然材料的8.2倍; 10-去乙酰基巴卡亭III含量为天然材料的68.1倍。该研究首次建立了红豆杉紫杉烷离体富集体系, 突破了紫杉醇药源短缺的瓶颈。

关键词: 红豆杉, 吸收面, 高频同步, 离体富集, 紫杉烷类

Abstract: Taxus contains the anti-cancer ingredient paclitaxel, is the top-protected endangered plant in China, and has great medicinal value but scarce resources. To solve the problem of paclitaxel source shortage, in this study, an in vitro culture of a taxane-rich Taxus system was established. The results showed that MS+2 mg·L^-1 6-BA+0.4 mg·L^-1 NAA+0.7 g·L^-1 Pro was the optimal treatment for absorption surface expansion, and the expansion rate reached 90%. The adventitious bud high-frequency synchronous growth induction was in the DCR+1 mg·L^-1 6-BA+0.1 mg·L^-1 NAA+5 mg·L^-1 Glu+1 g·L^-1 AC. The best biomass amplification phase I was in MS+0.7 mg·L^-1 6-BA+0.07 mg·L^-1 NAA+0.1 mg·L^-1 Phe+50 mg·L^-1 PG, in which the biomass reached 542 mg. The optimized biomass amplification phase II was obtained in MS+0.5 mg·L^-1 6-BA+0.05 mg·L^-1 NAA+0.1 mg·L^-1 Phe+2 g·L^-1 AC, in which the biomass reached 1 612 mg. The content of taxanes in tissue culture materials is much higher than that of natural materials. The content of paclitaxel in tissue culture materials was 6.1 times that of natural materials. The contoent of baccatin III in tissue culture materials was 8.2 times that of natural materials. The content of 10-DAB in tissue culture materials was 68.1 times that of natural materials. This study established an in vitro culture system of taxane-rich Taxus, and solved the problem of paclitaxel drug shortage.

Key words: Taxus spp., absorbing surface, high-frequency synchronous growth, in vitro enrichment, taxanes

冯晓晖, 闫学彤, 郑珂媛, 周强, 张伟中, 王权勇, 朱木兰. 富含紫杉烷类红豆杉的离体培养. 植物学报, 2023, 58(6): 917-925.

Xiaohui Feng, Xuetong Yan, Keyuan Zheng, Qiang Zhou, Weizhong Zhang, Quanyong Wang, Mulan Zhu. In vitro Culture of Taxus Rich in Taxanes. Chinese Bulletin of Botany, 2023, 58(6): 917-925.

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https://www.chinbullbotany.com/CN/Y2023/V58/I6/917

图/表 11

参考文献 24

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Treatments	75% ethanol (s)	Disinfectant (min)	Pollution rate (%)	Survival rate (%)
1	10	3	68.9±5.09^a	31.1±5.09^f
2	10	5	35.6±3.85^c	57.8±3.85^bcd
3	10	7	20.0±3.33^de	62.2±3.85^bc
4	30	3	54.4±5.09^b	45.6±5.09^e
5	30	5	25.6±5.09^d	74.4±5.09^a
6	30	7	12.2±1.92^ef	64.4±8.38^b
7	50	3	41.1±5.09^c	51.1±1.92^de
8	50	5	16.7±3.33^e	64.4±5.09^b
9	50	7	5.6±1.92^f	53.3±5.77^de

Treatments	75% ethanol (s)	Disinfectant (min)	Pollution rate (%)	Survival rate (%)
1	10	3	68.9±5.09^a	31.1±5.09^f
2	10	5	35.6±3.85^c	57.8±3.85^bcd
3	10	7	20.0±3.33^de	62.2±3.85^bc
4	30	3	54.4±5.09^b	45.6±5.09^e
5	30	5	25.6±5.09^d	74.4±5.09^a
6	30	7	12.2±1.92^ef	64.4±8.38^b
7	50	3	41.1±5.09^c	51.1±1.92^de
8	50	5	16.7±3.33^e	64.4±5.09^b
9	50	7	5.6±1.92^f	53.3±5.77^de

Treatments	NAA (mg·L^-1)	6-BA (mg·L^-1)	Proline (g·L^-1)	Expansion rate (%)
1	0.1	1	0	28.9±5.09^g
2	0.1	2	0.1	53.3±8.82^ef
3	0.1	3	0.3	62.3±2.02^de
4	0.2	1	0.5	58.9±5.09^ef
5	0.2	2	0.7	76.7±6.67^abc
6	0.2	3	1	86.7±5.77^ab
7	0.3	1	0	47.8±6.93^f
8	0.3	2	0.1	51.1±15.40^ef
9	0.3	3	0.3	86.7±5.77^ab
10	0.4	1	0.5	55.6±5.09^ef
11	0.4	2	0.7	90.0±6.67^a
12	0.4	3	1	63.3±3.33^cde
13	0.5	1	0	52.2±1.92^ef
14	0.5	2	0.1	75.6±8.39^bcd
15	0.5	3	0.3	81.1±10.18^ab
16	0.6	1	0.5	64.4±11.71^cde
17	0.6	2	0.7	57.8±6.94^ef
18	0.6	3	1	46.7±5.77^f

Treatments	NAA (mg·L^-1)	6-BA (mg·L^-1)	Proline (g·L^-1)	Expansion rate (%)
1	0.1	1	0	28.9±5.09^g
2	0.1	2	0.1	53.3±8.82^ef
3	0.1	3	0.3	62.3±2.02^de
4	0.2	1	0.5	58.9±5.09^ef
5	0.2	2	0.7	76.7±6.67^abc
6	0.2	3	1	86.7±5.77^ab
7	0.3	1	0	47.8±6.93^f
8	0.3	2	0.1	51.1±15.40^ef
9	0.3	3	0.3	86.7±5.77^ab
10	0.4	1	0.5	55.6±5.09^ef
11	0.4	2	0.7	90.0±6.67^a
12	0.4	3	1	63.3±3.33^cde
13	0.5	1	0	52.2±1.92^ef
14	0.5	2	0.1	75.6±8.39^bcd
15	0.5	3	0.3	81.1±10.18^ab
16	0.6	1	0.5	64.4±11.71^cde
17	0.6	2	0.7	57.8±6.94^ef
18	0.6	3	1	46.7±5.77^f

Treatments	6-BA (mg·L^-1)	NAA (mg·L^-1)	Axillary buds induction rate (%)
1	0.5	0.05	63.3±3.33^ef
2	0.5	0.1	48.9±5.09^g
3	1	0.05	81.1±1.92^ab
4	1	0.1	88.9±5.09^a
5	1	0.2	68.9±5.09^cde
6	1	0.3	64.4±5.09^ef
7	2	0.05	73.3±3.34^bcd
8	2	0.1	75.6±3.85^bc
9	2	0.2	65.6±5.09^def
10	2	0.3	58.9±5.09^f
11	3	0.1	60.0±3.33^f
12	3	0.2	43.3±3.34^g

富含紫杉烷类红豆杉的离体培养

In vitro Culture of Taxus Rich in Taxanes

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Treatments	6-BA (mg·L^-1)	NAA (mg·L^-1)	Adventitious buds induction rate (%)
1	0.2	0.02	32.2±5.09^e
2	0.4	0.04	47.8±3.85^d
3	0.6	0.06	62.2±1.92^bc
4	0.8	0.08	68.9±3.84^b
5	1.0	0.10	77.8±1.92^a
6	1.2	0.12	67.8±5.09^bc
7	1.4	0.14	61.1±3.84^c

Treatments	NAA (mg·L^-1)	6-BA (mg·L^-1)	High-frequency synchronous growth rate (%)	Average number of adventitious buds
1	0.05	0.25	38.9±5.09^e	12.2±2.65^c
2	0.05	0.5	55.6±5.09^cd	14.2±2.78^bc
3	0.1	0.5	50.0±6.67^d	20.1±3.66^b
4	0.1	1	82.2±1.92^a	31.5±2.93^a
5	0.2	1	72.2±3.85^b	33.4±3.67^a
6	0.2	2	63.3±3.34^bc	29.7±2.94^a

Treatments	6-BA (mg·L^-1)	NAA (mg·L^-1)	Adventitious bud elongation rate (%)	Average seedling height (mm)
1	0.1	0.01	35.5±6.94^c	17.3±0.85^d
2	0.3	0.03	40.0±3.33^c	22.7±1.34^c
3	0.5	0.05	74.4±5.09^b	28.4±0.94^b
4	0.7	0.07	92.2±1.92^a	33.4±1.56^a
5	1	0.1	80.0±5.77^b	29.9±0.69^ab

Treatments	PG (mg·L^-1)	Average stem thickness (mm)	Average seedling biomass (mg)
1	0	0.6±0.05^c	283±15^d
2	20	0.8±0.10^b	483±12^b
3	50	1.1±0.15^a	542±13^a
4	80	0.9±0.06^ab	490±15^b
5	100	0.8±0.15^bc	353±20^c

Treatments	AC (g·L^-1)	Average seedling height (mm)	Average seedling biomass (mg)
1	0	7.6±0.15^d	1318±12^d
2	1	7.9±0.21^d	1434±18^c
3	1.5	9.8±0.06^b	1496±24^b
4	2	10.4±0.21^a	1612±23^a
5	2.5	9.9±0.15^b	1531±15^b
6	3	8.7±0.12^c	1423±18^c

	Paclitaxel (μg·g^-1)	Baccatin III (μg·g^-1)	10-DAB (μg·g^-1)
Natural materials	15.7	16.6	29.9
Tissue culture materials	95.4	135.9	2036.0

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