Establishment of a Protoplast Isolation and Transient Transformation System from Different Explant Sources of Andrographis paniculata

doi:10.7525/j.issn.1673-5102.2026.03.007

Abstract

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

CLC Number:

S567.9

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