Establishment of transient gene expression systems in protoplasts from Liriodendron hybrid mesophyll cells.
Liriodendron is a genus of the magnolia family comprised of two flowering tree species that produce hardwoods of great ecological and economic value. However, only a limited amount of genetic research has been performed on the Liriodendron genus partly because transient or stable transgenic trees have been difficult to produce. In general, transient expression systems are indispensable for rapid, high-throughput screening and systematic characterization of gene functions at a low cost; therefore, development of such a system for Liriodendron would provide a necessary step forward for research on Magnoliaceae and other woody trees. Herein, we describe an efficient and rapid protocol for preparing protoplasts from the leaf mesophyll tissue of a Liriodendron hybrid and an optimized system for polyethylene glycol-mediated transient transfection of the protoplasts. Because the leaves of the Liriodendron hybrid are waxy, we formulated an enzyme mix containing 1.5% (w/v) Cellulase R-10, 0.5% (w/v) Macerozyme R-10, and 0.1% (w/v) Pectolyase Y-23 to efficiently isolate protoplasts from the Liriodendron hybrid leaf mesophyll tissue in 3 h. We optimized Liriodendron protoplast transfection efficiency by including 20 μg plasmid DNA per 104 protoplasts, a transformation time of 20 min, and inclusion of 20% (w/v) polyethylene glycol 4000. After integrating the Liriodendron WOX1 gene into pJIT166-GFP to produce a WOX1-GFP fusion product and transfecting it into isolated protoplasts, LhWOX1-GFP was found to localize to the nucleus according to its green fluorescence.
Co-Innovation Center for the Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, China.|School of Life Science, Huaiyin Normal University, Huaian, China.
Ailing
A
Huo
Co-Innovation Center for the Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, China.|Key Laboratory of Forest Genetics and Biotechnology, Ministry of Education, Nanjing Forestry University, Nanjing, China.
Zhenyu
Z
Chen
Co-Innovation Center for the Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, China.|Key Laboratory of Forest Genetics and Biotechnology, Ministry of Education, Nanjing Forestry University, Nanjing, China.
Pengkai
P
Wang
School of Life Science, Huaiyin Normal University, Huaian, China.
Liming
L
Yang
Co-Innovation Center for the Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, China.|Key Laboratory of Forest Genetics and Biotechnology, Ministry of Education, Nanjing Forestry University, Nanjing, China.
Guangping
G
Wang
Co-Innovation Center for the Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, China.|Key Laboratory of Forest Genetics and Biotechnology, Ministry of Education, Nanjing Forestry University, Nanjing, China.
Dandan
D
Wang
Co-Innovation Center for the Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, China.
Suchan
S
Liao
Co-Innovation Center for the Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, China.|College of Biology and the Environment, Nanjing Forestry University, Nanjing, China.
Tielong
T
Cheng
Co-Innovation Center for the Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, China.|Key Laboratory of Forest Genetics and Biotechnology, Ministry of Education, Nanjing Forestry University, Nanjing, China.
Jinhui
J
Chen
Co-Innovation Center for the Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, China.|Key Laboratory of Forest Genetics and Biotechnology, Ministry of Education, Nanjing Forestry University, Nanjing, China.
Jisen
J
Shi
D018929:Cell Culture Techniques; D015870:Gene Expression; D005822:Genetic Vectors; D031567:Liriodendron; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D058503:Mesophyll Cells / Q000378:metabolism; D018515:Plant Leaves; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D011523:Protoplasts / Q000378:metabolism; D014162:Transfection
D008164:Luminescent Proteins; D010940:Plant Proteins