이태리포푸라 Ⅰ-214 엽육조직에서 (葉肉組織) 원형질체 분리에 미치는 몇가지 요인

박용구,손성호 ( Young Goo Park,Sung Ho Son ) 한국산림과학회 1986 한국산림과학회지 Vol.74 No.1

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 0.1 ㎎/ℓ 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 × 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 엽육조직(葉肉組織)에서 원형질체(原形質體) 분리(分離)에 미치는 몇가지 요인(要因)

박용구,손성호,Park, Young Goo,Son, Sung Ho 한국산림과학회 1986 한국산림과학회지 Vol.74 No.1

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. 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.

수원포플러와 구아디 포플러 원형질체 융합에 의한 체세포잡종체 유도

박용구(Young Goo Park),김정희(Jung Hee Kim),손성호(Sung Ho Son) 한국산림과학회 1992 한국산림과학회지 Vol.81 No.3

Protoplasts isolated from leaf mesophyll tissues of Populus koreana X P. nigra var, italics were fused with those of P. euramericana cv. Guardi. Well expended healthy leaves of 5 to 7 week-old-plantlet grown in vitro were used as source materials. Leaves from P. koreana × P. nigra var, italics and P. euramericana cv. Guardi were digested in enzyme solution I (2.0% Cellulase, 1.2% Hemicellulase, 0.4% Macrozyme, 2.0% Driselase, 0.05 Pectolyase ; w/v) and enzyme solution II (1.0% Cellulase, 1.2% Hemicellulase, 0.4% Macrozyme, 2.0% Driselase, 0.05% Pectolyase : w/v), respectively, The highest frequency of fusion among the protoplasts originated from the two source materials was approximately 21% using 40% PEG or 15% dextran. In addition, fusion frequency was enhanced by incorporating 30mM of Ca^(2+) in eluting solution at pH 10.5. Dividing cells and/or mint-calli were obtained by culturing the fusion products in a liquid 8p-KM medium supplemented with 0.6M sucrose, 0.45μM 2, 4-D, and 0.5μM BA. Shoots were regenerated from the fusion product-derived calli after culture on MS medium containing 5.0μM zeatin, To verify the putative hybrid or cybrid, SDS-PAGE was carried out. From the 24 regenerants, just two plants showed intermediate protein band patterns compared with those of the original source plants.

세포배양을 이용한 현사시나무의 안토시아닌 생성

박용구(Young Goo Park),최명석(Myung Suk Choi),손성호(Sung Ho Son) 한국산림과학회 1992 한국산림과학회지 Vol.81 No.2

The influence of various levels of major medium components such as sucrose, nitrate, phosphate, plant growth regulators, and light intensity for cell growth and the production of anthocyanin content in cell cultures of Populus alba X P. glandulosa were investigated. Best results for anthocyanin yield were obtained using Mura.shige and Skoog(MS) medium containing 5% sucrose, 12.5% nitrate, 200% phosphate, 1.0㎎/ℓ indole-3-acetic acid(IAA), 1.0㎎/ℓ benaylaminopurine(BAP), and continuous illumination of 7,000 lux. On the other hand, maximum cell growth was achieved with 5% sucrose, 50% nitrate above 400% phosphate compare with that of MS basal mediumi, and 0.5mgj1 2, 4-dichlorophenoxyacetic acid(2, 4-D). Anthocyanin accumulation in a suspension cultured cells of given genotype was stimulated by subculturing onto the medium lacking 2, 4-D. Pigmented cell clusters were extracted with methanol containing 1% hydrochloric acid (HCl) and then anthocyanin was identified by thin layer chromatography (TLC) and U. V. spectrophotometer.

현사시나무 기내배양 엽육조직에서 분리된 원형질체 배양 및 식물체 재분화

박용구,손성호 ( Young Goo Park,Sung Ho Son ) 한국산림과학회 1988 한국산림과학회지 Vol.77 No.2

The leaf mesophyll protoplasts of Populass alba × glandrdosa were isolated from leaf of plantlet in vitro and cultured for plant regeneration. The MS medium (minus NH₄NO₃) with 0.5 ㎎/ℓ BAP and 2.0 ㎎/ℓ 2, 4-D showed the moderate frequency of dividing protoplasts cultured by the lipuid plating method during the first week of culture. The percentage of colony formation was revealed the highest frequency by the gauze contained semi-solid agar plating method after 5 weeks cultured. Ridding out the gauze, the micro-callus was formed on the same semi-solid medium in 8 weeks after protoplasts culture. For proliferation of callus, mini-callus was transferred on the MS solid medium with 0.5 ㎎/ℓ 2, 4-D and 0.1 ㎎/ℓ BAP 12 weeks after culture. Shoot regeneration occurred when the calli derived from protoplasts were cultured on MS medium with 1.0 ㎎/ℓ zeatin and such shoots could be readily rooted on the one half strengthen MS medium with non-phytohormone. Rooting shoots were planted in green-house 22 weeks after protoplast culture.

수원포플러와 구아디 포플러 원형질체(原形質體) 융합(融合)에 의한 체세포잡종체(體細胞雜種體) 유도(誘導)

박용구,김정희,손성호,Park, Young Goo,Kim, Jung Hee,Son, Sung Ho 한국산림과학회 1992 한국산림과학회지 Vol.81 No.3

유망(有望) 속성수(速成樹)로 개발중(開發中)인 수원포플러(P. koreana ${\times}$ nigra var. italica)와 포플러 낙엽병(落葉病)에 내성(耐性)을 가진 구아디포플러(P. euramericana cv. Guardi)의 엽육조직(葉肉組織)에서 원형질체(原形質體)를 분리융합(分離融合)하여 잡종식물체(雜種植物體)를 생산(生?)하였다. 실험재료(實驗材料)인 수원포플러는 BA $0.5{\mu}M$, 구아디포플러는 BA $2.0{\mu}M$ 처리(處理)된 MS 배지(培地)에서 대량(大量) 증식(增殖)시킨 후 1/2 MS배지에서 계대배양(繼代培養)하여 전개(展開)된 엽조직(葉組織)을 사용(使用)하였다. 수원포플러는 효소용액(酵素溶液) I (Cellulase 2.0%, Macerozyme 0.4%, Hemicelluase 1.2%, Driselase 2.0%, Pectolyase 0.05%)에서 구아디포플러는 효소용액(酵素溶液) II (Cellulase 1.0%, Macerozyme 0.4%, Hemicellulase 1.2%, Driselase 2.0%, Pectolyase 0.05%)에서 원형질체(原形質體)를 분리(分離)하여 각각 $4.04{\times}10^7$, $2.45{\times}10^7$개의 높은 수율(收率)을 얻었다. 양수종간(兩樹種間)의 원형질체(原形質體) 융합율(融合率)은 PEG 40%가 포함(包含)된 융합용액(融合溶液)에 20분 처리(處理)하고 $Ca^{2+}$ 30mM 첨가(添加)된 희석액(稀釋液)(pH 10.5)을 사용(使用)하였을때 양수종간(兩樹種間) 1:1 융합율(融合率)이 30%정도로 높게 나타났다. 융합(融合)된 원형질체(原形質體)는 0.6M sucrose, $4.5{\mu}M$ 2, 4-D, $0.5{\mu}M$ BA가 첨가(添加)된 8p-KM에서 정치(定置) 혹은 진탕배양(震湯培養)하여 2개월후 캘루스를 얻을 수 있었으며, $5.0{\mu}M$ zeatin 처리구(處理區)에서 평균 4개의 식물체(植物體)가 재분화(再分化)되었다. 융합산물(融合?物)에서 유래(由來)한 식물체(植物體)의 잡종성(雜種性) 여부(與否)를 확인(確認)하기 위해 SDS-PAGE를 실시(實施)하였다. 모수(母樹)인 수원포플러와 구아디포플러간에는 단백질형에 차이(差異)가 있었으며 재분화(再分化) 개체중(個體中) 양친(兩親)의 중간형(中間型)을 나타내는 개체(個體)는 세포융합(細胞融合)에 의한 재분화개체(再分化個體)로 추정(推定)할 수 있었다. Protoplasts isolated from leaf mesophyll tissues of Populus koreana ${\times}$ P. nigra var. italica were fused with those of P. euramericana cv. Guardi. Well expended healthy leaves of 5 to 7 week-old-plantlet grown in vitro were used as source materials. Leaves from P. koreana ${\times}$ P. nigra var. italica and P. euramericana cv. Guardi were digested in enzyme solution I (2.0% Cellulase, 1.2% Hemicellulase, 0.4% Macrozyme, 2.0% Driselase, 0.05% Pectolyase ; w/v) and enzyme solution II (1.0% Cellulase, 1.2% Hemicellulase, 0.4% Macrozyme, 2.0% Driselase, 0.05% Pectolyase ; w/v), respectively, The highest frequency of fusion among the protoplasts originated from the two source materials was approximately 21% using 40% PEG or 15% dextran. In addition, fusion frequency was enhanced by incorporating 30mM of $Ca^{2+}$ in eluting solution at pH 10.5. Dividing cells and/or mint-calli were obtained by culturing the fusion products in a liquid 8p-KM medium supplemented with 0.6M sucrose, $0.45{\mu}M$ 2, 4-D, and $0.5{\mu}M$ BA. Shoots were regenerated from the fusion product-derived calli after culture on MS medium containing $5.0{\mu}M$ zeatin. To verify the putative hybrid or cybrid, SDS-PAGE was carried out. From the 24 regenerants, just two plants showed intermediate protein band patterns compared with those of the original source plants.

이태리포푸라 I-214 엽내조직에서 원형질체 분리에 미치는 몇가지 요인

손성호,박용구 경북대학교 유전공학연구소 1995 遺傳工學硏究所報 Vol.10 No.1

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 0.1 ㎎/ℓ 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 0.6M Mannitol in addition to DTT and MES buffer adjusted to pH 5.6. Over 2.4×10^6 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.

이태리 포푸라 엽육조직에서의 원형질체분리에 미치는 몇가지 요인

손성호,박용구,우종호,신동일 경북대학교 유전공학연구소 1986 遺傳工學硏究所報 Vol.1 No.1

A method of isolating Populus euramericana cv. I-214 mesophyll protoplast was developed to facilitate the eventual use of genetic engineering techniques in this species. The suitable medium for shoot multiplication an vitro was MS basal medium with 0.1㎎/1 BAP. The effects of several factors influencing protoplast isolation could be evaluated quickly by using leaves in vitro and know volumes of maceration and washing media. The best yields of mesophyll protoplasts were obtained using leaves do vitro in 2.0% Cellulase R-10, 0.8% Macerozyme R-10, 1.2% Hemicellulase, 2.0% Driselase, 0.05% Pectolyase Y-23, and 0.6M mannitol in addition to DTT and MES buffer adjusted pH 5.6. Over 2.4×10^6 protoplasts per gram of leaves were produced using these condition. For protoplast purification, the most favorable sucrose concentration of floating solution was 0.6M after washing them by CPW solution.

은백양 (Populus alba) 엽육조직에서 원형질체 분리

손성호,박용구,우종호,설일환,신동일 경북대학교 유전공학연구소 1995 遺傳工學硏究所報 Vol.10 No.1

Factors affecting the isolation of protoplasts from mesophyll were investigated in P. alba. The yield of mesophyll protoplasts was 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 0.6M mannitol in addition to DTT and MES buffer adjusted pH 5.6. Over 11.0×10^6 protoplasts per gram of leaf were produced using the condition. For protoplasts purification, the most favorable sucrose concentration of floating solution was 0.6M after washing them by CPW solution. This method of screening factors affecting protoplast isolation could be applicable to other species.

세포배양을 이용한 현사시나무의 안토시아닌 생성

손성호,최명석,박용구 경북대학교 유전공학연구소 1995 遺傳工學硏究所報 Vol.10 No.1

The influence of various levels of major medium components such as sucrose, nitrate, phosphate. plant growth regulators, and light intensity for cell growth and the production of anthocyanin content in cell cultures of Populus alba XP. glandulosa were investigated. Best results for anthocyanin yield were obtained using Murashige and Skoog MS medium containing 5% sucrose. 12.5% nitrate. 200% Phosphate. 1.0㎎ l indole-3-acetic acid IAA. 1.0㎎ l benzylaminopurine BAP, and continuous illumination of 7.000 lux. On the other hand, maximum cell growth was achieved with 5% sucrose. 50% nitrate above 400% phosphate compare with that of MS basal medium, and 0.5㎎ l 2, 4-dichlorophenoxyacetic acid 2, 4-D. Anthocyanin accumulation in a suspension cultured cells of given genotype was stimulated by subculturing onto the medium lacking 2, 4-D. Pigmented cell clusters were extracted with methanol containing 1% hydrochloric acid, HCl and then anthocyanin was identified by thin layer chromatography TLC and U.V. spectrophotometer.