穿心莲不同材料来源原生质体制备及瞬时转化体系建立

doi:10.7525/j.issn.1673-5102.2026.03.007

摘要/Abstract

摘要：

优化穿心莲（Andrographis paniculata）子叶与根愈伤组织原生质体的制备方法，并探究影响其瞬时转化的关键因素，为后续开展穿心莲遗传转化及基因功能研究奠定技术基础。本研究采用酶解法研究不同甘露醇浓度、酶浓度及酶解时间对原生质体产量与活力的影响；通过PEG介导法考察PEG浓度、转化时间等因素对瞬时转化效率的影响，并建立穿心莲根愈伤组织原生质体的瞬时转化体系。穿心莲子叶原生质体制备的最佳条件：取0.2 g子叶，在含4.0%纤维素酶R-10、1.5%离析酶R-10和0.6 mol⋅L^-1甘露醇的酶解液中，26 ℃、55 r⋅min⁻¹黑暗酶解4 h，原生质体产量可达3.32×10⁵ 个⋅mL^-1，活力达90.73%。根愈伤组织原生质体制备的最佳条件：取0.4 g培养15 d的根愈伤组织，在含4.0%纤维素酶R-10、1.0%离析酶R-10和0.8 mol⋅L^-1甘露醇的酶解液中，相同条件下酶解9 h，产量可达2.18×10⁶ 个⋅mL^-1，活力达94.19%。PEG介导的瞬时转化试验表明，40% PEG 4000处理30 min、质粒用量10 μg、甘露醇浓度0.6 mol⋅L^-1为最优转化条件，转化效率可达58.03%。不同来源的穿心莲材料在原生质体制备条件上存在显著差异，需要针对材料特性进行条件优化。本研究建立了高效的穿心莲子叶和根愈伤组织原生质体制备体系及根愈伤组织原生质体的瞬时转化体系，为后续开展穿心莲基因功能解析与细胞生物学研究提供了可靠的技术支持。

关键词: 穿心莲, 原生质体, 根愈伤组织, 子叶, 瞬时转化

Abstract:

This study aimed to optimize the preparation methods of protoplasts from cotyledons and root callus of Andrographis paniculata， and to investigate the key factors affecting their transient transformation， thereby laying a technical foundation for subsequent genetic transformation and gene function research in A. paniculata. The effects of different mannitol concentrations， enzyme concentrations， and enzymatic hydrolysis time on protoplast yield and viability were studied using the enzymatic hydrolysis method. The PEG-mediated transformation method was employed to examine the influence of factors such as PEG concentration and transformation time on transient transformation efficiency， and a transient transformation system for A. paniculata root callus protoplasts was established. The optimal conditions for protoplast preparation from A. paniculata cotyledons were as follows： 0.2 g cotyledons were enzymatically hydrolyzed in a solution containing 4.0% cellulase R-10， 1.5% macerozyme R-10， and 0.6 mol⋅L^-1 mannitol at 26 ℃ and 55 r⋅min^-1 in darkness for 4 h. Under these conditions， the protoplast yield reached 3.32×10⁵ cells⋅mL^-1， with a viability of 90.73%. For root callus protoplasts， the optimal conditions were： 0.4 g 15‑day‑old callus was enzymatically hydrolyzed in a solution containing 4.0% cellulase R-10， 1.0% macerozyme R-10， and 0.8 mol⋅L^-1 mannitol under the same conditions for 9 h， yielding 2.18×10⁶ cells⋅mL^-1 with a viability of 94.19%. In PEG-mediated transient transformation， the optimal conditions were determined to be 40% PEG 4000 treatment for 30 min， 10 μg of plasmid DNA， and 0.6 mol⋅L^-1 mannitol. Under these conditions， the transformation efficiency reached 58.03%. Significant differences existed in the optimal preparation conditions for protoplasts derived from different tissues of A. paniculata， emphasizing the need for tissue-specific optimization. This study established an efficient protoplast preparation and transient transformation system forboth cotyledons and root callus of A. paniculata， providing a reliable technical platform for future gene function analysis and cell biology research in this medicinal plant.

Key words: Andrographis paniculata, protoplast, root callus, cotyledon, transient transformation

中图分类号:

S567.9

丁妹, 杨帆, 刘安然, 杜勤. 穿心莲不同材料来源原生质体制备及瞬时转化体系建立[J]. 植物研究, 2026, 46(3): 458-470.

Mei DING, Fan YANG, Anran LIU, Qin DU. Establishment of a Protoplast Isolation and Transient Transformation System from Different Explant Sources of Andrographis paniculata[J]. Bulletin of Botanical Research, 2026, 46(3): 458-470.

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编号 Number	A	B	C	产量 Protoplast yield/ （×10⁵ 个·mL^-1）
1	1	2	3	1.90
2	1	3	2	3.32
3	1	1	1	1.35
4	2	3	3	1.73
5	2	2	1	1.63
6	2	1	2	0.75
7	3	3	1	1.57
8	3	1	3	1.60
9	3	2	2	2.97
K₁	6.57	3.70	4.55
K₂	4.11	6.50	7.04
K₃	6.14	6.62	5.23
R	0.82	0.97	0.83

编号 Number	A	B	C	产量 Protoplast yield/ （×10⁵个·mL^-1）
1	1	2	3	1.51
2	1	3	2	0.44
3	1	1	1	2.74
4	2	3	3	4.46
5	2	2	1	1.53
6	2	1	2	5.67
7	3	3	1	2.00
8	3	1	3	6.23
9	3	2	2	4.92
K₁	4.69	14.64	6.26
K₂	11.66	7.96	11.03
K₃	13.15	6.90	12.20
R	8.46	7.74	5.94