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.

The fate of extrachromosomal DNAs in the progeny of plastid-transformed tobacco plants

민성란,정서희,유장렬,정원중 한국식물생명공학회 2015 Plant biotechnology reports Vol.9 No.6

Plastid transformation is conducted by homologous recombination. Plastid transformation using a vector- containing promoter and/or terminator sequences homologous to the plastid DNA for transgene expression often results in the generation of unintended extrachromosomal DNA molecules derived from the initial plastid DNA molecule via intramolecular recombination. All the extrachromosomal DNA molecules, including those lacking the ori sequence, found in the T0 plastid-transformed tobacco plants were still detected in the T3 plants. These results suggest that the extrachromosomal DNA molecules are newly generated from the initially transformed plastid DNA that is transmitted to the progeny during each generation. In addition, inward, outward, and overlap extension PCR of the extrachromosomal DNA molecules confirmed that these molecules were circular.

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.

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.

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

Fourier 변환 적외선 분광분석법에 의한 딸기 과육의 성숙도 측정 가능성

민성란,곽철원,김석원,정원중,정화지,최필선,고석민,박상규,정회일,유장렬,Min, Sung-Ran,Kwak, Chul-Won,Kim, Suk-Weon,Jeong, Won-Joong,Chung, Hwa-Jee,Choi, Pil-Son,Ko, Suk-Min,Park, Sang-Kyu,Chung, Hoe-Il,Liu, Jang, R. 한국식물생명공학회 2006 식물생명공학회지 Vol.33 No.4

Fourier transform - infrared spectroscopy (FT-IR) provides biochemical profiles containing overlapping signals from a majority of the compounds that are present when whole cell extracts are analyzed. We attempted to determine the ripeness of strawberry fruit flesh by FT-IR. Fruit ripeness was divided into four developmental stages based on fruit skin color: 'yellow-green', 'pink-green', 'pink', and 'red' stages. Principal component analysis of FT-IR data of inside fruit flesh extracts clustered samples of four different developmental stages into three discrete groups: (1) 'yellow-green' group, (2) 'pink-green' group, and (3) 'pink' and 'red' group. The most remarkable difference between four different developmental stages was found in the carbohydrate fingerprint region $(1,000-1,100cm^{-1})$ of the FT-IR spectrum, indicating that differences in carbohydrate compounds represented the ripeness of strawberry fruit. Overall results indicate that FT-IR in combination with PCA enables discrimination of the ripeness of strawberry fruit flesh.

인체 락토페린 생산 형질전환 고구마 개발

민성란,김재화,정원중,이영복,유장렬 한국식물생명공학회 2009 식물생명공학회지 Vol.36 No.3

면역강화와 같은 생리활성 기능을 가지는 인체 락토페린 cDNA가 들어있는 pLSM1 벡터를 이용하여 고구마배발생 캘러스에 particle bombardment 방법으로 형질전환을 수행하여 인체 락토페린 생산 고구마를 개발하였다. 선발배지에서 형성된 배발생 캘러스로부터 체세포배발생 과정을 거쳐 식물체로 재분화되었고 토양에서 정상적으로 생장하여 괴근을 형성하였다. 재분화된 식물체를대상으로 PCR과 Southern 분석을 실시한 결과 고구마의염색체 게놈에 인체 락토페린 유전자가 도입되었음을 확인하였다. 또한, northern blot 분석을 통해 락토페린 유전자의 발현을 확인하였으며 immunoblot 분석을 통해 고구마 괴근에서도 80 kDa 크기의 락토페린 단백질이 생산됨을 확인할 수 있었다. 본 연구결과 고구마는 치료용 인체단백질을 생산하는 숙주로서 매우 우수하다는 것을 입증하였다. Human lactoferrin is an iron-binding glycoproteinwith many biological activities, including the protectionagainst microbial and virus infection and stimulation of theimmune system. We introduced a human lactoferrin (hLf)cDNA under the control of 35S promoter into sweet potatoby particle bombardment. Transgenic plants were regeneratedvia somatic embryogenesis. Transgenic plants wereproduced typical tuberous roots in soil. PCR, Southern andnorthern analyses confirmed that the hLf cDNA was incorporatedinto the plant genome and was properly expressed inplants. Western blot analysis showed that the 80 kDa fulllength hLf protein was produced in transgenic tuberous roots. Overall results indicated that sweet potato would be anexcellent host to produce human therapeutic proteins.

種球의 低溫處理 및 培地의 組成이 마늘의 캘러스增殖 및 幼植物體 分化에 미치는 영향

閔成蘭,李殷模,羅相煜,盧泰弘,李永馥 충남대학교부설 생명공학연구소 1992 생물공학연구지 Vol.2 No.-

Effects of low temperature treatment and medium composition on callus proliferation and shoot differentiation of garlic seed bulbs were investigated. The calli derived from shoot-tip explants of bulbs which had been stored at 4℃ for 2 months were proliferated 1.9 times more vigorously by fresh weight on callus induction medium and regenerated 11 times more efficiently in number on shoot differentiation medium supplemented with 2 ㎎/L kinetin and 0.1 ㎎/L NAA than the calli from those at room temperature. Modified MS media containing KNO_3 and (NH_4)_2SO_4 of 40 mM each or containing KNO_3, (NH_4)_2SO_4, and KCI of 10 mM each were more effective than the original MS in callus proliferation and shoot differentiation. We believe that this culture system can be used for the mass production of virus-free stocks garlic.