Isolation and recovery of protoplasts from young leaf tissue

Working from the literature, we have obtained high yields of viable protoplasts using the following scheme with single cross hybrid maize, grown under standard greenhouse conditions.

At 21 days after seeding, plants are placed in a dark humid chamber for 16 hours.

Unrolled leaves surrounding the growing point are carefully dissected out, and leaf sections are floated on a solution of: 0.5% Onozuka R-10, 9% mannitol, and salts (Zapata et al., Plant Sci. Lett. 8:119, 1977) at pH 5.5.

The solution is shaken at 125 rpm for 2.5 to 3 hours at 23 C, and leaf sections are separated from the enzyme solution containing the protoplasts by passage through a double layer of cheesecloth.

Additional debris is removed by passage of the solution containing the protoplasts through an 85µ nylon filter, and protoplasts are collected by centrifugation at low speed (300xg for 5 minutes).

Protoplasts are washed twice in Zapata salts, 9% mannitol, and the final pellet is resuspended in the same solution.

This scheme yields 1 x 107 protoplasts/gm fr. wt. of tissue. This yield compares favorably with earlier reports of protoplast yields from maize: 2.5 x 104 protoplasts/gm (K. L. Giles, Plant Cell Phys. 15:281), 2 x 106 protoplasts/gm (H. C. Aldace et al., Tissue and Cell 9:167, 1977).

Gail Meadows and D. B. Walden


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