Applied Biological Chemistry

The Korean Society for Applied Biological Chemistry (한국응용생명화학회)
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Isolation of Protoplasts from Tomato Root by Two-step Osmotic Treatment

토마토 뿌리조직으로부터 두 단계 삼투압 처리에 의한 원형질체의 분리

  • Shin, Dae-Seop (Department of Agricultural Chemistry, Chungbuk National University) ;
  • Han, Min-Woo (Department of Agricultural Chemistry, Chungbuk National University) ;
  • Kim, Young-Kee (Department of Agricultural Chemistry, Chungbuk National University)
  • 신대섭 (충북대학교 농과대학 농화학과) ;
  • 한민우 (충북대학교 농과대학 농화학과) ;
  • 김영기 (충북대학교 농과대학 농화학과)
  • Published : 2004.06.30
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Abstract

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.

이온채널의 활성을 포함한 세포의 생리활성을 측정하기 위하여 토마토의 뿌리조직으로부터 원형질체를 분리하였다. 일반적으로 널리 사용되는 원형질체 분리법은 뿌리조직의 경우 효율이 좋지 않았다. 따라서, 다양한 조건을 변화시키며 분리효율을 조사하던 중 두 단계의 삼투압 처리로 원형질체 분리효율이 높아짐을 확인하였다. 첫 단계로 뿌리조직을 300 mM sorbitol을 포함하는 용액에서 30분간 배양한 후, 700 mM sorbitol과 세포벽 분해효소를 포함하는 용액으로 옮겼을 때, 원형질체 형성은 급격히 증가하였다. 이러한 분리방법의 최적 조건을 결정하기 위하여 pH와 삼투압, 배양시간, 세포벽 분해효소의 농도 등을 조사하였다. 원형질체 분리의 수율은 3% cellulase와 1% macerozyme, 0.1% pectolyase를 포함하는 pH 5.0의 혼합효소액에서 2시간 배양할 때 최대로 나타났다. ATPase 활성으로 평가한 원형질체의 세포활성은 뿌리조직의 시료에서 측정한 간과 유사하였다. 또한, 분리한 원형질체의 세포활성은 4시간 동안 감소하지 않아 생리활성 측정을 위한 시료로 적합하였다. 본 결과는 두 단계 삼투압 처리법이 토마토 뿌리조직으로부터 높은 수율의 원형질체 분리에 성공적임을 보인다.

Keywords

References

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