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Scalable Transfection of Maize Mesophyll Protoplasts

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Induction of efficient cell division in alfalfa protoplasts.

L A Holbrook¹, T J Reich, V N Iyer

¹Ottawa Research Station, Agriculture Canada, K1A OC6, Ottawa, Canada.

Plant Cell Reports

|November 21, 2013

Summary

Immobilizing alfalfa protoplasts in agarose significantly improves their division and plating efficiency. This method overcomes cytokinesis blocks, leading to successful callus development and a 5- to 30-fold increase in efficiency.

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A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

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Area of Science:

Plant Biotechnology
Cell Biology

Background:

Alfalfa (Medicago sativa L.) protoplasts from cell suspension cultures exhibit poor division in liquid media.
Protoplast division is often inhibited at cytokinesis, preventing complete cell division.

Purpose of the Study:

To enhance the division and plating efficiency of alfalfa protoplasts.
To overcome the cytokinesis block observed in liquid cultures.

Main Methods:

Protoplasts were cultured in liquid medium for 1-4 days.
Immobilization of protoplasts in agarose was employed to facilitate division.
Microcolonies were transferred to agar medium for callus development.

Main Results:

Immobilization in agarose overcame the inhibition of protoplast division.
Plating efficiencies of 2-10% were achieved within 30 days.
The agarose treatment resulted in a 5- to 30-fold improvement in plating efficiency.

Conclusions:

Agarose immobilization is an effective strategy to improve alfalfa protoplast division and plating.
This method facilitates successful callus development from isolated protoplasts.