Final published version
Licence: CC BY: Creative Commons Attribution 4.0 International License
Research output: Contribution to Journal/Magazine › Journal article › peer-review
Research output: Contribution to Journal/Magazine › Journal article › peer-review
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TY - JOUR
T1 - A high-throughput transient expression system for rice
AU - Page, Michael
AU - Parry, Martin Afan John
AU - Carmo-Silva, Ana Elizabete
PY - 2019/7/1
Y1 - 2019/7/1
N2 - Rice is an important global crop and represents a vital source of calories for many food insecure regions. Efforts to improve this crop by improving yield, nutritional content, stress tolerance, or resilience to climate change are certain to include biotechnological approaches, which rely on the expression of transgenes in planta. The throughput and cost of currently available transgenic expression systems is frequently incompatible with modern, high-throughput molecular cloning methods. Here, we present a protocol for isolating high yields of green rice protoplasts and for PEG-mediated transformation of isolated protoplasts. Factors affecting transformation efficiency were investigated, and the resulting protocol is fast, cheap, robust, high-throughput, and does not require specialist equipment. When coupled to a high-throughput modular cloning system such as Golden Gate, this transient expression system provides a valuable resource to help break the "design-build-test" bottleneck by permitting the rapid screening of large numbers of transgenic expression cassettes prior to stable plant transformation. We used this system to rapidly assess the expression level, subcellular localisation, and protein aggregation pattern of nine single-gene expression cassettes, which represent the essential component parts of the beta-cyanobacterial carboxysome.
AB - Rice is an important global crop and represents a vital source of calories for many food insecure regions. Efforts to improve this crop by improving yield, nutritional content, stress tolerance, or resilience to climate change are certain to include biotechnological approaches, which rely on the expression of transgenes in planta. The throughput and cost of currently available transgenic expression systems is frequently incompatible with modern, high-throughput molecular cloning methods. Here, we present a protocol for isolating high yields of green rice protoplasts and for PEG-mediated transformation of isolated protoplasts. Factors affecting transformation efficiency were investigated, and the resulting protocol is fast, cheap, robust, high-throughput, and does not require specialist equipment. When coupled to a high-throughput modular cloning system such as Golden Gate, this transient expression system provides a valuable resource to help break the "design-build-test" bottleneck by permitting the rapid screening of large numbers of transgenic expression cassettes prior to stable plant transformation. We used this system to rapidly assess the expression level, subcellular localisation, and protein aggregation pattern of nine single-gene expression cassettes, which represent the essential component parts of the beta-cyanobacterial carboxysome.
KW - Rice
KW - Oryza sativa
KW - protoplasts
KW - transient expression
KW - confocal microscopy
KW - cellular localisation
KW - high-throughput
KW - synthetic biology
KW - Golden Gate
KW - carboxysome
U2 - 10.1111/pce.13542
DO - 10.1111/pce.13542
M3 - Journal article
VL - 42
SP - 2057
EP - 2064
JO - Plant, Cell and Environment
JF - Plant, Cell and Environment
SN - 0140-7791
IS - 7
ER -