Factors Affecting the Isolation of Mesophyll Protoplasts from Populus euramericana cv. I-214

이태리포푸라 I-214 엽육조직(葉肉組織)에서 원형질체(原形質體) 분리(分離)에 미치는 몇가지 요인(要因)

  • Park, Young Goo (College of Agriculture, Kyungpook National University) ;
  • Son, Sung Ho (College of Agriculture, Kyungpook National University)
  • Received : 1986.03.04
  • Published : 1986.09.30

Abstract

A method isolating Populus euramericana cv. I-214 mesophyll protoplasts was developed to facilitate application of genetic engineering techniques to this species. The suitable medium for shoot multiplication in vitro was MS basal medium with $0.1mg/{\ell}$ BAP. The effects of several factors influencing protoplast isolation could be evaluated quickly by using leaf in vitro and known volumes of maceration and washing media. The best yields of mesophyll protoplasts were obtained using leaves in vitro in 2.0% Cellulase R-10, 0.8% Macerozyme R-10, 1.2% Hemicellulase, 2.0% Driselase, 0.05% Pectolyase Y-23, and O.6M Mannitol in addition to DTT and MES buffer adjusted to pH 5.6. Over $2.4{\times}10^6$ protoplasts per gram of leaf were produced using these conditions. For protoplast purification, the most favorable sucrose concentration of floating solution was 0.6M after washing them with CPW solution. This method of screening factors affecting protoplast isolation could be applicable to other species.

이태리포푸라 I-214 (Populus euramericana cv. I-214)의 기내배양(器內培養)한 엽육조직(葉肉組織)에서 원형질체(原形質體) 분리(分離)에 미치는 몇가지 요인(要因)에 대(對)해 조사(調査), 검토(檢討)하였다. 기내(器內)에서 배양(培養)된 아(芽)를 다량(多量)으로 증식(增植)하기 위한 배지(培地)는 MS 기본배지(基本培地)에 $0.1mg/{\ell}$의 BAP를 첨가(添加)한 것이 가상 좋은 성적을 나타냈다. 엽(葉) 1g 당(當) $2.4{\times}10^6$개의 가장 높은 원형질체(原形質體) 분리(分離) 빈도를 나타낸 것은 Cellulase R-10 2 %, Macerozyme R-10 0.8 %, Hemicellulase 1.2 %, Driselase 2.0 %, Pectolyase Y-23 0.05 %에 DTT와 MES 완충액을 첨가(添加)한 후 삼투압 안정제로 0.6 M의 Mannitol을 넣고 pH를 5.6으로 조정한 효소용액(酵素溶液)이었다. CPW 용액(溶液)으로 세정(洗淨)한 후 0.6 M의 Sucrose 용액(溶液)에 처리(處理)한 것이 회수율(回收率) 51.8 %로 가장 높게 나타났다.

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