Protoplast Isolation and Establishment of Transient Expression System of Tripterygium wilfordii Suspension Culture Cells

doi:10.11983/CBB16171

Abstract

Abstract: We aimed to find the best protoplast extraction conditions with Tripterygium wilfordii suspension culture cells and establish an efficient transient expression system. Key factors in protoplast isolation, such as concentration of enzymes, time of enzymolysis, osmotic pressure and centrifugation speed, were studied. PEG mediation was used to transfer the GFP gene into protoplasts. The optimal extraction system was with cellulose R-10 2.0%, pectinase Y-23 0.5%, macerozyme R-10 0.5%, 0.6 mol?L^-1mannitol. The suitable enzymolysis time was 10 h, and the optimal centrifugation speed was 67 ×g. Under LSCM, the protoplast emitted green fluorescence after transforming the plasmid encoding green fluorescent protein into it. We revealed the optional conditions to isolate protoplast of T. wilfordii suspension cells and established the transient transformation system; it laid a foundation for further study on the functional genes and syn- thetic biology of T. wilfordii.

Key words: protoplast, suspension culture cells, transient transformation, Tripterygium wilfordii

Hu Tianyuan, Wang Rui, Chen Shang, Ma Baowei, Gao Wei, Huang Luqi. Protoplast Isolation and Establishment of Transient Expression System of Tripterygium wilfordii Suspension Culture Cells[J]. Chinese Bulletin of Botany, 2017, 52(6): 774-782.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB16171

https://www.chinbullbotany.com/EN/Y2017/V52/I6/774

Figures/Tables 7

Table 1 Proportion of enzymic digestion to remove Triptery- gium wilfordii suspension cells cytoderm

Group	Cellulase (%)	Pectinase (%)	Macerozyme (%)
1	1.5	0.3	0.5
2	2.0	0.3	0.5
3	2.0	0.5	0.5
4	2.0	0.7	0.5
5	2.5	0.3	0.5

Figure 1 The influence of enzyme concentration on Tripterygium wilfordii suspension cells protoplast yield (A) and activity (B) Different lowercase letters indicate significant differences at P<0.05.

Figure 2 The influence of enzymatic hydrolysis time on Trip- terygium wilfordii suspension cells protoplast yield (A) and activity (B)Different lowercase letters indicate significant differences at P<0.05.

Figure 3 The influence of mannitol concentration on Tripterygium wilfordii suspension cells protoplast yield (A) and activity (B)Different lowercase letters indicate significant differences at P<0.05.

Figure 4 The influence of centrifugal speed on Tripterygium wilfordii suspension cells protoplast yield (A) and activity (B)Different lowercase letters indicate significant differences at P<0.05.

Figure 5 Morphology of Tripterygium wilfordii suspension cells protoplast under optical microscope(A) Morphology of protoplast before trypan blue staining; (B) Morphology of protoplast after trypan blue staining, and the blue cell in red box was dead. Bars=100 μm

Figure 6 Protoplast of Tripterygium wilfordii suspension cells under laser scanning confocal microscop(A) Protoplasts under the laser confocal microscope in bright field; (B) GFP fluorescence of protoplast under the laser confocal microscope in excitation 488 nm; (C) There is no red chloroplast spontaneous fluorescence of protoplast under the laser confocal microscope in excitation 488 nm; (D) Red chlorophyll fluorescence signals and GFP signals from protoplast. Bars=1 μm

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