종의 포플라수종에서 리그닌생합성에 관계된 OMT유전자의 발현

박용구,박희성,최장원,설일환,정일경,신동일,Park, Young-Goo,Park, Hee Sung,Choi, Jang Won,Sul, Ill Whan,Chung, Il Kyung,Shin, Dong Ill 한국생명과학회 1998 생명과학회지 Vol.8 No.4

We analyzed lignin content and wxpression of OMT gene during growth season in two hybrid poplar species. OMT gene expression was observed mainy in the developing secondary xylem where major quantity of lignin occurs. Lignin content in the xylem tissue increased as plant resumed growth in the spring and reached the highest in the late August. Change in lignin content was concurrent with that of OMT gene expression, indicating OMT is a key enzyme in lignin biosynthesis. 리그닌함량이 감소한 펄프소재의 육성을 위해 OMT유전자와 리그닌생합성 간의 관계를 2종의 포플라 수종을 이용하여 실험하였다. OMT유전자의 발현은 리그닌생합성이 가장 많이 일어나는 developing secondary xylem에서 높앗다. 이 사부조직에서의 리그닌함량의 증가는 봄에 성장을 개시할 때부터 증가하기 시작하여 8월 말 경에 가장 높았다. 이러한 리그닌의 증가는 OMT유전자의 발현 증가와 일치하였으며, 이는 OMT유전자가 리그닌생합성에서 매우 중요한 역할을 함을 나타내는 것이다.

Isolation and Culture of Mesophyll Protoplasts from in vitro Cultured Populus alba × P . glandulosa

박용구,한경환 ( Young Goo Park,Kyung Hwan Han ) 한국산림과학회 1986 한국산림과학회지 Vol.73 No.1

N/A This study was carried out to investigate the optimum conditions for isolation and culture of mesophyll protoplasts from Populus alba × P. glandulosa. The results obtained from the experiments are as follows; 1) The suitable concentration of BAP for shoot multiplication was 0.4 ㎎/ℓ. 2) High yield and viability of isolated protoplasts were obtained by our high enzyme-short time incubation method. 3) Optimum enzyme concentrations for mesophyll protoplast isolation were Cellulase 2%, Macerozyme 0.8%, Hemicellulase 1.2%, Driselase 2%, and Pectolyase Y-23 0.05%. 4) 0.6M mannitol in enzyme solution was the most effective for protoplast isolation and viability. 5) The most adequate pH level of enzyme solution was pH 5.6. 6) The effect of DTT and MES buffer was significant. 7) For protoplast purification, 0.6M sucrose was the most proper concentration. 8) The adding effect of Dextran T40 in floating solution was important. 9) The mesophyll protoplasts isolated through our high enzyme-short time incubation method revealed successful response to culture condition over 3 weeks of culture.

잣나무의 기내배양에 관한 연구 (Ⅰ) - Callus 의 유발과 생장 -

박용구,김우룡 ( Young Goo Park,Oue Ryong Kim ) 한국산림과학회 1983 한국산림과학회지 Vol.59 No.1

Embryo tissue from Pinus koraiensis was established in culture on G.D. and W.S. basal medium supplemented with the auxin (NAA, 2,4-D, IBA) and kinetin. The various combinations of growth regulators were studied in order to determine the specific requirements of the callus tissue in vitro. The inorganic nutrient combination of G.D. medium was found to be better than that of W,S. medium. G,D. basal medium supplemented with 0.1 ppm N.AA and 0.1 ppm kinetin was the most successful nutrient combination for the growth of callus induced from the embryo of P. koraiensis.

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.

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.

임목 원형질체 배양기법과 응용

박용구,손승호 ( Young Goo Park,Sung Ho Son,Richard B . Hall ) 한국유전학회 1990 Genes & Genomics Vol.12 No.2

Methods of isolation, culture and fusion of protoplasts are reviewed with emphasis on the critical factors affecting successful performances of protoplast systems. This paper in association with work on somaclonal variation, somatic hybridization, and recombinant DNA technology through protoplasts is potentially applicable to a wide variety of improvement programs for woody plants.

몇가지 삼림수종 (森林樹種) 종자발아에 (種子發芽) 대한 낙엽송엽 (落葉松葉) 추출물의 Allelopathic 효과

박용구,강군수,신동일 ( Young Goo Park,Goon Su Kang,Dong Ill Shin ) 한국산림과학회 1988 한국산림과학회지 Vol.77 No.1

Inhibitory effect of L. leptolepis fallen leaf extracts on the germination of Pinus densiflora, × Pinus rigitaeda, Pinus rigida, Pinus thunbergii and Larix leptolepis was investigated. Germination of those seeds in culture room and in pot at field showed the highly significant inhibitory effects by the aqueous extracts from L. leptolepis fallen leaves. Among them L. leptolepis was the most severely inhibited by the aqueous extracts used. To identify allelopathic substances, thin-layer chromatography was employed. Gallic, ferulic, t-cinnamic and vanillic acids were identified from fallen leaves of L. leptolepis. From the results, it is assumed that autotoxicity of L . leptolepis may act as inhibitory factors on germination of the species in natural stands.

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.

Agrobacterium tumefaciens 에 의한 양황철나무의 형질전환 요인

박용구(Young Goo Park),신동원(Dong Won Shin),김정희(Joung Hee Kim) 한국산림과학회 1990 한국산림과학회지 Vol.79 No.3

We have demonstrated expression of bacterial genes transferred into cells of Populrrs nigra × P. maximowiczii by A. tumefaciens strain 6044 (pGA 472). We determined the optimum concentration of kanamycin sulfate for effective selection of punctured leaf transformed using Agrobacterium binary vector pGA 472 containing a neomycine phosphotransferase gene (NPT-II) which confers kanamycin resistance. The combination of cefotaxime (200㎎/ℓ) and carbenicillin (300㎎/ℓ) showed good performance of discarding Agrobacterirrm from inoculated punctured leaf. A relatively low concentration (10㎎/ℓ) of kanamycin sulfate inhibited callus and shoots induction from punctured leaf. umber of shoots regenerated from co-cultured punctured leaf was 3.0 on MS basal medium supplemented with 10 ㎎/ℓ kanamycin sulfate, while that of not co-cultured punctured leaf was none, The regeneration rate was 10% from the punctured Leaf co-cultured on VIS medium with 10 ㎎/ℓ kanamycin. Regenerated shoots are developing from micropropagation for Southern blot analysis and inheritance of the kanamycin resistance trait (NPT-II).