Isolation of Haustorium Protoplasts Optimized by Orthogonal Design for Transient Gene Expression in <i>Phelipanche aegyptiaca</i>
Xiaojian Zeng,
Xiaolei Cao,
Qiuyue Zhao,
Siyuan Hou,
Xin Hu,
Zheyu Yang,
Tingli Hao,
Sifeng Zhao,
Zhaoqun Yao
Affiliations
Xiaojian Zeng
Key Laboratory at the Universities of Xinjiang Uygur Autonomous Region for Oasis Agricultural Pest Management and Plant Protection Resource Utilization, Agriculture College, Shihezi University, Shihezi 832003, China
Xiaolei Cao
Key Laboratory at the Universities of Xinjiang Uygur Autonomous Region for Oasis Agricultural Pest Management and Plant Protection Resource Utilization, Agriculture College, Shihezi University, Shihezi 832003, China
Qiuyue Zhao
Key Laboratory at the Universities of Xinjiang Uygur Autonomous Region for Oasis Agricultural Pest Management and Plant Protection Resource Utilization, Agriculture College, Shihezi University, Shihezi 832003, China
Siyuan Hou
Key Laboratory at the Universities of Xinjiang Uygur Autonomous Region for Oasis Agricultural Pest Management and Plant Protection Resource Utilization, Agriculture College, Shihezi University, Shihezi 832003, China
Xin Hu
Key Laboratory at the Universities of Xinjiang Uygur Autonomous Region for Oasis Agricultural Pest Management and Plant Protection Resource Utilization, Agriculture College, Shihezi University, Shihezi 832003, China
Zheyu Yang
Key Laboratory at the Universities of Xinjiang Uygur Autonomous Region for Oasis Agricultural Pest Management and Plant Protection Resource Utilization, Agriculture College, Shihezi University, Shihezi 832003, China
Tingli Hao
Key Laboratory at the Universities of Xinjiang Uygur Autonomous Region for Oasis Agricultural Pest Management and Plant Protection Resource Utilization, Agriculture College, Shihezi University, Shihezi 832003, China
Sifeng Zhao
Key Laboratory at the Universities of Xinjiang Uygur Autonomous Region for Oasis Agricultural Pest Management and Plant Protection Resource Utilization, Agriculture College, Shihezi University, Shihezi 832003, China
Zhaoqun Yao
Key Laboratory at the Universities of Xinjiang Uygur Autonomous Region for Oasis Agricultural Pest Management and Plant Protection Resource Utilization, Agriculture College, Shihezi University, Shihezi 832003, China
The efficient protoplast transient transformation system in plants is an important tool to study gene expression, metabolic pathways, and various mutagenic parameters, but it has not been established in Phelipanche aegyptiaca. As a root parasitic weed that endangers the growth of 29 species of plants in 12 families around the world, there is still no good control method for P. aegyptiaca. Even the parasitic mechanisms of P. aegyptiaca and the related genes regulating parasitism are not yet understood. In this study, by comparing the factors related to protoplast isolation and transfection, we developed the optimal protocol for protoplast isolation and transfection in Phelipanche aegyptiaca haustorium. The optimal protoplast yield and activity were 6.2 × 106 protoplasts/g fresh weight [FW] and 87.85%, respectively, by using 0.5 mol/L mannitol, enzyme concentrations of 2.5% cellulase R-10 and 0.8% Macerozyme R-10 at 24 °C for 4 h. At the same time, transfection efficiency of protoplasts was up to 78.49% when using 30 μg plasmid, 40% polyethylene glycol (PEG) concentration, 24 °C incubation temperature, and 20 min transfection time. This is the first efficient protoplasts’ isolation and transient transformation system of Phelipanche aegyptiaca haustorium, laying a foundation for future studies on the gene function and mechanisms of haustorium formation in parasitic plants.
