Chinese Bulletin of Botany ›› 2017, Vol. 52 ›› Issue (6): 774-782.DOI: 10.11983/CBB16171

• TECHNIQUES AND METHODS • Previous Articles     Next Articles

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

Hu Tianyuan1, Wang Rui1, Chen Shang1, Ma Baowei1, Gao Wei1,2,*(), Huang Luqi3   

  1. 1School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, China
    2Key Laboratory of Collateral Diseases, Beijing 100069, China
    3Key Laboratory of Dao-di Herbs, National Resources Center for Chinese Materia Medica, China Academy of Chinese Medicinal Sciences, Beijing 100700, China
  • Received:2016-08-21 Accepted:2017-01-10 Online:2017-11-01 Published:2018-02-22
  • Contact: Gao Wei

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-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.

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