발표요지 : 더덕 ( Codonopsis lanceolata L . ) 의 실생 절편으로부터의 체세포배발생

민성란,이행순,김석원,장용만,양승균,유장렬 한국식물생명공학회 ( 구 한국식물조직배양학회 ) 1990 한국식물생명공학회 춘계학술연구발표회 Vol.1990 No.-

유자의 성숙종자 배양 및 종자유래 배발생 현탁배양으로부터 체세포배발생을 통한 유자의 식물체 재생

민성란,최명석,정원중,유장렬,Min, Sung-Ran,Choi, Myung-Suk,Jeong, Won-Joong,Liu, Jang-Ryol 한국식물생명공학회 2002 식물생명공학회지 Vol.29 No.3

유자의 성숙종자를 MS 기본배지에 치상하여 종자의 내피에서 주심조직 유래의 희고 부서지기 쉬운 배발생캘러스가 1.2%의 빈도로 형성되었다. 이 캘러스는 1 mg/L 2,4-D를 첨가한 MS배지에서 증식되었다. 증식된 캘러스를 0.1 mg/L kinetin을 첨가한 MS 배지에 옮겼을 때 많은 수의 체세포배가 형성되었다. 배발생캘러스를 1 mg/L 2,4-D를 첨가한 액체 배지에 넣어 배발생 현탁배양계를 확립하였다. 배양된 현탁배양세포를 0.5 mg/L ABA를 첨가한 고체배지에 평판하였을 때 높은 빈도로 체세포배로 발달하였으며 MS 기본배지 혹은 1 mg/L kinetin 첨가배지에서 소식물체로 발달하였다. 소식물체는 성공적으로 토양으로 옮겨서 온실에서 육성되었다. Off-white, friable embryogenic calluses were formed on the internal integument of mature seeds of yuzu (Citrus junos) cultured on Murashige and Skoog's basal medium at a frequency of 1.2%. Embryogenic calluses were proliferated when cultured on medium with 1 mg/L 2,4-D. Upon transfer to medium with 0.1 mg/L kinetin, embryogenic calluses produced numerous somatic embryos. Embryogenic suspension cultures were established by placing embryogenic calluses into liquid medium with 1 mg/L 2,4-D. When plated onto medium with 0.5 mg/L ABA, embryogenic cells developed into somatic embryos at a high frequency, and then regenerated into plantlets. Plantlets were successfully transplanted to potting soil and grown in a greenhouse.

유자의 성숙종자 배양 및 종자유래 배발생 현탁배양으로부터 체세포배발생을 통한 유자의 식물체 재생

민성란,정원중,유장렬,최명석 한국식물생명공학회 2002 JOURNAL OF PLANT BIOTECHNOLOGY Vol.29 No.3

감미단백질 모넬린 발현 딸기 형질전환 식물체 개발

민성란,고석민,유재일,박지현,이소영,이인하,김현숙,김태일,최필선,정원중,김석원,김종현,유장렬 한국식물생명공학회 2015 식물생명공학회지 Vol.42 No.3

Leaf discs from ‘Yeobong’ and ‘Maehyang’ strawberry plants were used as explants for transformation. The Agrobacterium tumefaciens strain EHA105 harboring the monellin gene under the control of the CaMV 35S promoter was used in co-cultivation experiments. The frequencies of callus formation and plant regeneration from leaf explants after co-cultivation in ‘Yeobong’ were higher than those of ‘Maehyang’. These transgenic plants showed normal growth patterns and flowering. PCR and Southern hybridization confirmed that 1 to 2 copies of the monellin gene were integrated into genome of the transgenic strawberry plants. Northern blot analysis confirm that the transcripts were expressed in transgenic strawberry plants. Although long-term subcultured transgenic strawberry plants showed a phenomenon to escape the transgene, the transformation system established in this study provides new opportunities for genetic improvement of strawberry plants.

엽록체 형질전환 유래 분자 농업의 연구 동향

민성란,정원중,김석원,이정희,정화지,유장렬,Min, Sung-Ran,Jeong, Won-Joong,Kim, Suk-Weon,Lee, Jeong-Hee,Chung, Hwa-Jee,Liu, Jang-R. 한국식물생명공학회 2010 식물생명공학회지 Vol.37 No.3

고등식물의 엽록체 형질전환은 핵 형질전환에서 기대 할 수 없는 여러 가지 이점을 가진다. 외래 단백질의 발현율을 획기적으로 높일 수 있고, 여러 유전자를 동시에 발현시킬 수 있으며, 상동재조합에 의한 부위-특이적 유전자 삽입으로 인해 유전자 침묵 및 위치효과가 없다. 더욱이, 대부분 작물은 화분을 통한 도입된 유전자의 전이가 불가능한 모계 유전을 하기 때문에 엽록체 형질전환은 환경 친화적이다. 엽록체 형질전환 시스템은 핵 형질 전환과 달리 작물에서의 성공에 제한적이었으나 지난 10년 동안 이런 한계가 극복되어 콩, 당근, 상추 및 유채 등의 작물에서도 성공하게 되었다. 그러므로 이제 작물의 엽록체 형질전환은 농업적 형질의 개선뿐 만 아니라, 고부가가치 백신과 의료용 단백질 생산을 통한 의약품 산업의 성장에 활용될 수 있을 것이다. Chloroplast transformation in higher plants offers many attractive advantages over nuclear transformation, including a high-level accumulation of foreign proteins, multi-gene expression in single transformation event via transgene stacking in operons and no position effect due to site-specific integration of transgenes by homologous recombination. Most importantly, chloroplast transgenic plants are eco-friendly because their transgenes are maternally inheritance in most crop plants. However, chloroplast transformation system has limited success in crops alike nuclear transformation. In the past two decades, great progress has been made to overcome the limitations of chloroplast transformation, thus expending chloroplast bioreactor to several important crops including soybean, carrot, lettuce, and oilseed. Therefore, it has become possible that chloroplast transformation of crops can be used not only for the improvement of agronomic traits, but also for the production of vaccines and high valuable therapeutic proteins in pharmaceutical industry.

Particle Bombardment에 의한 고구마의 형질전환

민성란,정원중,이영복,유장렬 한국식물생명공학회 1998 식물생명공학회지 Vol.25 No.5

$\beta$-Glucuronidase (GUS) gene of Escherichia coli was introduced into sweet potato (Ipomoea batatas (L.) Lam.) cells by particle bombardment and expressed in the regenerated plants. Microprojectiles coated with DNA of a binary vector pBI121 carrying CaMV35S promoter-GUS gene fusion and a neomycin phosphotransferase gene as selection marker were bombarded on embryogenic calli which originated from shoot apical meristem-derived callus and transferred to Murashige and Skoog (MS) medium supplemented with 1 mg/L 2,4-dichlorophenoxyacetic acid and 100 mg/L kanamycin. Bombarded calli were subcultured at 4 week intervals for six months. Kanamycin-resistant calli transferred to MS medium supplemented with 0.03 mg/L 2iP, 0.03 mg/L ABA, and 50 mg/L kanamycin gave rise to somatic embryos. Upon transfer to MS basal medium without kanamycin, they developed into plantlets. PCR and northern analyses of six regenerants transplanted to potting soil confirmed that the GUS gene was inserted into the genome of the six regenerated plants. A histochemical assay revealed that the GUS gene was preferentially expressed in the vascular bundle and the epidermal layer of leaf, petiole, and tuberous root. Escherichia coli의 $\beta$-glucuronidase (GUS) 유전자를 고구마의 배발생세포괴에 particle bombardment로 도입하여 재분화 식물체에 발현시켰다. CaMV35S-GUS 융합유전자와 선발표지로서 neomycin phosphotransferase유전자가 들어있는 binary 운반체 pBI121 DNA를 텅스텐 입자로 코팅하여 정단분열 조직 유래의 배발생 세포괴에 bombarding하였다. Bombarding된 세포괴를 1mg/L 2,4-D와 100mg/L kanamycin이 첨가된 MS 배지로 옮겨 한달 간격으로 6개월동안 계대배양하였다. Kanamycin 저항성 캘러스를 0.03mg/L 2iP, 0.03 mg/L ABA 및 50 mg/L kanamycin이 들어있는 MS 배지로 옮겨 체세포배를 유도하였고, kanamycin이 첨가되지 않은 MS 기본배지에서 식물체로 발달시켰다. 토양에서 생육중인 6개체의 식물을 대상으로 PCR과 northern분석을 수행한 결과 GUS 유전자가 식물체 genome에 안정적으로 도입, 발현되었음이 확인되었다. 조직화학적 분석으로 GUS 유전자가 형질전환 식물체에서 발현됨을 밝혔다.

엽록체 형질전환 유래 분자 농업의 연구 동향

민성란,정원중,김석원,이정희,정화지,유장렬 한국식물생명공학회 2010 JOURNAL OF PLANT BIOTECHNOLOGY Vol.37 No.3

Chloroplast transformation in higher plants offers many attractive advantages over nuclear transformation, including a high-level accumulation of foreign proteins, multi-gene expression in single transformation event via transgene stacking in operons and no position effect due to site-specific integration of transgenes by homologous recombination. Most importantly, chloroplast transgenic plants are eco-friendly because their transgenes are maternally inheritance in most crop plants. However, chloroplast transformation system has limited success in crops alike nuclear transformation. In the past two decades, great progress has been made to overcome the limitations of chloroplast transformation, thus expending chloroplast bioreactor to several important crops including soybean, carrot, lettuce, and oilseed. Therefore, it has become possible that chloroplast transformation of crops can be used not only for the improvement of agronomic traits, but also for the production of vaccines and high valuable therapeutic proteins in pharmaceutical industry. 고등식물의 엽록체 형질전환은 핵 형질전환에서 기대할 수 없는 여러 가지 이점을 가진다. 외래 단백질의 발현율을 획기적으로 높일 수 있고, 여러 유전자를 동시에발현시킬 수 있으며, 상동재조합에 의한 부위-특이적 유전자 삽입으로 인해 유전자 침묵 및 위치효과가 없다. 더욱이, 대부분 작물은 화분을 통한 도입된 유전자의 전이가 불가능한 모계 유전을 하기 때문에 엽록체 형질전환은 환경 친화적이다. 엽록체 형질전환 시스템은 핵 형질전환과 달리 작물에서의 성공에 제한적이었으나 지난 10년 동안 이런 한계가 극복되어 콩, 당근, 상추 및 유채 등의 작물에서도 성공하게 되었다. 그러므로 이제 작물의엽록체 형질전환은 농업적 형질의 개선뿐 만 아니라, 고부가가치 백신과 의료용 단백질 생산을 통한 의약품 산업의 성장에 활용될 수 있을 것이다.

An episomal vector system for plastid transformation in higher plants

민성란,Seyed Javad Davarpanah,정서희,박연일,유장렬,정원중 한국식물생명공학회 2015 Plant biotechnology reports Vol.9 No.6

We developed a new plastid transformation vector system using the putative replication origin of a minicircular chromosome from the marine dinoflagellate Heterocapsa triquetra. Transplastomic tobacco plants generated with this vector properly expressed the green fluorescent protein (GFP) gene without incorporating it into the plastid genome. To construct the episomal vector, a 610-bp DNA fragment containing the putative replication origin was fused to a dicistronic expression cassette encoding the aminoglycoside 3’-adenyltransferase (aadA) and gfp genes under control of the plastid rrn promoter. The vector was delivered to plastids of tobacco leaf explants by biolistic bombardment. After 8 weeks of bombardment, episomal transformant shoots were generated from leaf explants cultured on selection media containing 500 mg/L spectinomycin. Fluorescence microscopy and northern blot analysis demonstrated GFP expression in episomal transformant plants. PCR, Southern blot analysis, recovery of episomes, and sequencing analysis showed the vector to be maintained as self-replicating extrachromosomal circular DNA molecules for at least 6 months. Using a single construct for all plants, our episomal vector system may offer an advantage over the conventional plastid vector systems, which require species-specific constructs.

체세포배발생에 의한 한국 고구마 품종의 고빈도 식물체 재분화

민성란,유장렬,노태홍,김칠현,주정일 한국식물생명공학회 1994 식물생명공학회지 Vol.21 No.3

국내 두 품종의 고구마를 대상으로 경단분열조직 배양에 의한 체세포배발생과 이들로부터 식물체 재분화 체계를 확립하였다. '부여재래' 나 '율미'의 경단분열조직 절편을 1mg/L 2,4-D가 첨가된 MS배지에서 배양하여 6주후에 80% 이상의 절편으로부터 배발생 캘러스를 얻었다. 이 캘러스를 0.1 mg/L 24-D와 0.1 mg/L kinetin이 첨가된 배지에 옮겼을때 '부여재래' 와 '율미'의 체세포배 발생률이 각각 71% 및 63%로 다른 처리에 비해 현저히 높았다. 형성된 체세포배를 상하로 이등분하여 1 mg/L 2,4-D가 첨가된 MS배지에 치상하였을 경우 품종에 관계없이 길이가 짧은 체세포배의 상반부에서 이차배가 더 많이 발생하였다. 이들 체세포배는 MS 기본배지에서 90% 이상이 완전한 식물체로 발달하였다. 이러한 결과는 우리가 이전에 확립하였던 고구마의 경단분열조직으로부터의 체세포배발생 방법(SABRAO J 21:93-101)이 광범위한 고구마 계통에 적용될 수 있음을 시사한다. Culture conditions for high Sequency somatic embryogenesis and plant regeneration in tissue cultures of sweet potato of two Korean cultivars 'Puyojaerae' and 'Yulmi' are described. Shoot apical meristem explants (height 150 $\mu\textrm{m}$; base: 350 $\mu\textrm{m}$) were cultured on MS medium supplemented with 1 mg/L 2,4-D. After 6 weeks of culture, greater than 80% of the survived explants produced embryogenic calli. When transferred onto MS medium with 0.1 mg/L each of 2,4-D and kinetin, the calli gave rise to somatic embryos at frequencies of 71% ('Puyojaerae') and 63% ('Yulmi'), respectively: When somatic embryos at various developmental stages measured in length were transversely cut into two halves and cultured on MS medium with 1 mg/L 2,L-D, the upper halves produced secondary embryos more frequently than the lower ones, and halves of somatic embryos less than 1 mm in length had a higher competence for secondary embryo formation than longer ones of either cultivar. However 'Puyojaerae' somatic embryo halves showed a higher frequency of secondary embryo formation than 'Yulmi' ones on the whole. Upon transfer onto MS basal medium, most of the primary and secondary somatic embryos underwent development into plantlets. The plantlets were transplanted to potting soil and grown to maturity in a phytotron. The overall results suggest that the shoot apical meristem culture system for somatic embryo formation in sweet potato previously established by us (SABRAOJ 21: 93-101) may be applicable regardless of it genotypes.

안정적 감자 엽록체 형질전환 식물체 생산

민성란,정원중,박지현,류재일,이정희,오광훈,정화지,유장렬 한국식물생명공학회 2011 식물생명공학회지 Vol.38 No.1

Chloroplast genetic engineering of higher plants offers several unique advantages compared with nuclear genome transformation, such as high levels of transgene expression, a lack of position effect due to site-specific transgene integration by homologous recombination, multigene engineering in a single transformation event and reducing risks of gene flow via pollen due to maternal inheritance. We established a reproducible chloroplast ransformation system of potato using a tobacco specific plastid transformation vector, pCtVG (trnI-Prrn-aadA-mgfp-TpsbA-trnA). Stable transgene integration into chloroplast genomes and the homoplasmic state of the transgenome were confirmed by PCR and Southern blot analyses. Northern, immunoblot analysis, and GFP fluorescence imaging revealed high expression and accumulation of GFP in the plastids of potato leaves. This system would provide new opportunities for genetic improvement and mass production of value added foreign proteins in this crop.