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A high-throughput transient expression system for rice: Rice Protoplast Isolation and Transformation

Writer's picture: PlantHouse EnterprisePlantHouse Enterprise

This video provides a comprehensive guide to isolating and transforming rice protoplasts, a crucial technique in plant science and biotechnology.


Isolation of protoplasts

Sixty seedlings were harvested by cutting the stem at the base of the plant at soil level. Approximately 100 mm of stem and sheath tissue was retained and briefly rinsed with water to remove any adhered compost. The plant tissue was cut into ~1 mm slices using a sharp blade, directly into 40 ml 0.6 M mannitol inside a Petri dish. The tissue was incubated in the dark for 15 min at room temperature (RT) to initiate plasmolysis. After draining off the mannitol solution, the tissue was transferred to a conical flask containing 30 ml enzyme solution (20 mM 2-(N-morpholino)-ethanesulfonic acid (MSE) pH 5.7, 1.5% w/v cellulase RS, 0.75% w/v macerozyme R10, 0.6 M mannitol, 10 mM KCl, 10 mM CaCl2, 0.1% w/v bovine serum albumin (BSA)) and incubated in the dark for 4 hr at RT with gentle shaking to allow digestion of cell wall material; 30 ml W5 solution (2 mM MES pH 5.7, 154 mM NaCl, 125 mM CaCl2, 5 mM KCl) was added and the flask shaken gently by hand for 20 s to terminate digestion. Samples were filtered through 40 μm mesh by gravity to release protoplasts, and the retained tissue rinsed with an additional 60 ml W5 solution. Protoplasts were pelleted by centrifuging all flow-through from filtering and washing (120 ml total) at 250 × g for 3 min at RT. The supernatant was carefully decanted, and the pellets washed by gentle resuspension in 10 ml W5 buffer, then centrifugation at 250 × g for 3 min at RT. The supernatant was again carefully decanted, and pellets were resuspended in 2 ml MMG solution (4 mM MES pH 5.7, 0.4 M mannitol, 15 mM MgCl2).


Transformation of protoplasts

5 μg pDNA (in a volume of 10 μL, diluted with ddH2O) and 60 μL protoplast suspension were combined in a 1.5 mL microcentrifuge tube; a negative control sample (60 μL protoplasts, 10 μL ddH2O, no pDNA) was included for each round of transformations. 70 μL poly(ethylene glycol) (PEG) solution (0.2 M mannitol, 40% w/v PEG 4,000, 0.1 M CaCl2) was added, and the samples mixed gently by inversion. Tubes were incubated in the dark at RT for 25 min to allow transformation to occur. The transformation process was terminated by addition of 280 μL W5 solution followed by gentle mixing. Samples were centrifuged at 250 × g for 3 min at RT, and the supernatant carefully aspirated. Pellets were resuspended in 500 μL WI solution (4 mM MES pH 5.7, 0.5 M mannitol, 20 mM KCl) and dispensed in 125 μL aliquots into a 96-well microplate. Transformed protoplasts were incubated at RT on the lab bench for 16 hr, to allow transgenic protein(s) to accumulate.


This video is an invaluable resource for researchers engaged in gene editing, functional genomics, and plant breeding, as well as students learning essential plant cell culture and transformation techniques. Laboratory technicians responsible for executing these protocols will also find this visual guide immensely helpful.


Keywords: Protoplasts, plant transformation, rice, plant science, molecular biology, biotechnology, gene editing, cell biology, lab techniques, protocol


Citation:

Page, M. T., Parry, J., & Elizabete Carmo‐Silva. (2019). A high‐throughput transient expression system for rice. Plant Cell and Environment, 42(7), 2057–2064. https://doi.org/10.1111/pce.13542


First published: 02 April 2019


Attribution 4.0 International — CC BY 4.0 - Creative Commons

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