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KMID : 0903520040470020192
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Journal of the Korean Society of Agricultural Chemistry and Biotechnology
2004 Volume.47 No. 2 p.192 ~ p.196
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Isolation of Protoplasts from Tomato Root by Two-step Osmotic Treatment
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Shin Dae-Seop
Han Min-Woo
Kim Young-Kee
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Abstract
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In order to measure cellular physiological activity including ion channel activity, protoplasts were isolated from the root tissue of tomato plant. The general methods recommended were not efficient enough to make protoplasts from the root tissue. Among various conditions tested, we found that a two-step treatment of osmosis is very efficient for the isolation of protoplasts. In this procedure, root tissues were preincubated in a solution containing 300 mM sorbitol for 30 min. Then, they moved to the reaction solution containing 700 mM sorbitol as well as cell wall-digesting enzymes. The formation of protoplast was greatly increased by this method. In order to find the optimal condition of the two-step method, various conditions of pH, osmotic pressure, incubation time, and the concentrations of cell wall-digesting enzymes were tested. The yield of protoplast isolation was maximal at pH 5.0 after 2 hr incubation. Mixed enzymes of 3% cellulase, 1% macerozyme, and 0.1% pectolyase showed maximal protoplast isolation. The physiological activity of isolated protoplast evaluated by measuring the cellular ATPase activity was as high as that measured from the preparation of root tissue. The protoplasts isolated by this method were remained healthy up to 4 hrs which is enough time to measure the cellular physiological activity. These results show that the two-step treatment of osmotic pressure was successful to obtain high yield of healthy protoplast from tomato root tissue.
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KEYWORD
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protoplast, tomato roots, ATPase, osmotic treatment
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