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A simple and effective method to encapsulate tobacco mesophyll protoplasts to maintain cell viability.

Lei R, Qiao W, Hu F, Jiang H, Zhu S - MethodsX (2014)

Bottom Line: Protoplasts have been widely used for genetic transformation, cell fusion, and somatic mutation due to the absence of a cell wall.In this work, we propose a simple and effective silica/alginate two-step method to immobilize protoplasts with advantages in experimental manipulation and microscopic imaging, as well as in potentially studying cell biological processes such as secretion and metabolism.This simple and effective silica sol-gel/alginate two-step immobilization of protoplasts in Transwell has great potential for applications in genetic transformation, metabolite production, and migration assays.

View Article: PubMed Central - PubMed

Affiliation: Institute of Plant Quarantine, Chinese Academy of Inspection and Quarantine, Beijing, China.

ABSTRACT
Protoplasts have been widely used for genetic transformation, cell fusion, and somatic mutation due to the absence of a cell wall. However, without the protection of a cell wall, protoplasts are easy to rupture and aggregate during washing, collecting, and gene transfection. In this work, we propose a simple and effective silica/alginate two-step method to immobilize protoplasts with advantages in experimental manipulation and microscopic imaging, as well as in potentially studying cell biological processes such as secretion and metabolism. The proposed two-step immobilization method adopts Transwell with clear tissue culture-treated membrane to support protoplasts in the form of uniform thin layer, which has three unique properties. •The tissue culture-treated membrane has a good affinity for the plant cell; thus, protoplasts can spread evenly and form a very thin layer.•There are more choices for membrane pore size, depending on the application.•It is very convenient to change or collect the solution without mechanically disturbing the protoplasts. This simple and effective silica sol-gel/alginate two-step immobilization of protoplasts in Transwell has great potential for applications in genetic transformation, metabolite production, and migration assays.

No MeSH data available.


Related in: MedlinePlus

Microscopic pictures of bright field, autofluorescence, fluorescein green fluorescence and merged after FDA staining of protoplasts immobilized by different immobilization methods: (A) silica sol–gel/alginate two-step immobilization method; (B) ETAF; (C) TAL; (D) Ca-alginate droplet immobilization method.
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fig0010: Microscopic pictures of bright field, autofluorescence, fluorescein green fluorescence and merged after FDA staining of protoplasts immobilized by different immobilization methods: (A) silica sol–gel/alginate two-step immobilization method; (B) ETAF; (C) TAL; (D) Ca-alginate droplet immobilization method.

Mentions: The confocal microscopic pictures of immobilized protoplasts are shown in Fig. 2. By using silica sol–gel/alginate two-step immobilization, most of the immobilized protoplasts distribute evenly on the Transwell membrane and good quality confocal microscopic images are easily obtained, which is advantageous over other preparation methods (Fig. 2). Using the other three immobilization methods, protoplasts are observed and are distributed in the supporting matrix in a multilayer manner such that the majority of the protoplasts are not on the focal plane, making it difficult for cell observation (Fig. 2). As we know, the transparent polyethylene terephthalate (PET) membrane is tissue treated to be hydrophilic, which makes the cell better attached and spread on the surface. Therefore, a small amount of protoplast solution as few as 25–30 μl can be well spread on the Transwell membrane to form a mono-film. With the penetration of CaCl2 from the lower pores, Ca-alginate gel is evenly produced on the membrane, without exerting any force on the protoplasts. However, using ETAF to immobilize protoplasts in Ca-alginate gel, we find that pushing the coverglass would easily damage protoplasts and require very clean glasses and good operator skills to yield a success rate of approximately 4 in 10 times. For TAL method, although there is no applied force, the layer is thick and unevenly due to the quick gel formation when the alginate solution contacts with the agar; therefore, the protoplasts cannot be clearly observed in one layer (Fig. 2).


A simple and effective method to encapsulate tobacco mesophyll protoplasts to maintain cell viability.

Lei R, Qiao W, Hu F, Jiang H, Zhu S - MethodsX (2014)

Microscopic pictures of bright field, autofluorescence, fluorescein green fluorescence and merged after FDA staining of protoplasts immobilized by different immobilization methods: (A) silica sol–gel/alginate two-step immobilization method; (B) ETAF; (C) TAL; (D) Ca-alginate droplet immobilization method.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4487327&req=5

fig0010: Microscopic pictures of bright field, autofluorescence, fluorescein green fluorescence and merged after FDA staining of protoplasts immobilized by different immobilization methods: (A) silica sol–gel/alginate two-step immobilization method; (B) ETAF; (C) TAL; (D) Ca-alginate droplet immobilization method.
Mentions: The confocal microscopic pictures of immobilized protoplasts are shown in Fig. 2. By using silica sol–gel/alginate two-step immobilization, most of the immobilized protoplasts distribute evenly on the Transwell membrane and good quality confocal microscopic images are easily obtained, which is advantageous over other preparation methods (Fig. 2). Using the other three immobilization methods, protoplasts are observed and are distributed in the supporting matrix in a multilayer manner such that the majority of the protoplasts are not on the focal plane, making it difficult for cell observation (Fig. 2). As we know, the transparent polyethylene terephthalate (PET) membrane is tissue treated to be hydrophilic, which makes the cell better attached and spread on the surface. Therefore, a small amount of protoplast solution as few as 25–30 μl can be well spread on the Transwell membrane to form a mono-film. With the penetration of CaCl2 from the lower pores, Ca-alginate gel is evenly produced on the membrane, without exerting any force on the protoplasts. However, using ETAF to immobilize protoplasts in Ca-alginate gel, we find that pushing the coverglass would easily damage protoplasts and require very clean glasses and good operator skills to yield a success rate of approximately 4 in 10 times. For TAL method, although there is no applied force, the layer is thick and unevenly due to the quick gel formation when the alginate solution contacts with the agar; therefore, the protoplasts cannot be clearly observed in one layer (Fig. 2).

Bottom Line: Protoplasts have been widely used for genetic transformation, cell fusion, and somatic mutation due to the absence of a cell wall.In this work, we propose a simple and effective silica/alginate two-step method to immobilize protoplasts with advantages in experimental manipulation and microscopic imaging, as well as in potentially studying cell biological processes such as secretion and metabolism.This simple and effective silica sol-gel/alginate two-step immobilization of protoplasts in Transwell has great potential for applications in genetic transformation, metabolite production, and migration assays.

View Article: PubMed Central - PubMed

Affiliation: Institute of Plant Quarantine, Chinese Academy of Inspection and Quarantine, Beijing, China.

ABSTRACT
Protoplasts have been widely used for genetic transformation, cell fusion, and somatic mutation due to the absence of a cell wall. However, without the protection of a cell wall, protoplasts are easy to rupture and aggregate during washing, collecting, and gene transfection. In this work, we propose a simple and effective silica/alginate two-step method to immobilize protoplasts with advantages in experimental manipulation and microscopic imaging, as well as in potentially studying cell biological processes such as secretion and metabolism. The proposed two-step immobilization method adopts Transwell with clear tissue culture-treated membrane to support protoplasts in the form of uniform thin layer, which has three unique properties. •The tissue culture-treated membrane has a good affinity for the plant cell; thus, protoplasts can spread evenly and form a very thin layer.•There are more choices for membrane pore size, depending on the application.•It is very convenient to change or collect the solution without mechanically disturbing the protoplasts. This simple and effective silica sol-gel/alginate two-step immobilization of protoplasts in Transwell has great potential for applications in genetic transformation, metabolite production, and migration assays.

No MeSH data available.


Related in: MedlinePlus