Protoplast Isolation and Establishment of Transient Expression System of Tripterygium wilfordii Suspension Culture Cells
Received date: 2016-08-21
Accepted date: 2017-01-10
Online published: 2017-01-10
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-1 mannitol. 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.
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 . DOI: 10.11983/CBB16171
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