발표요지 : Defence relative gene 의 도입에 의한 식물세포의 형질전환

양덕춘,최광태,한성수 한국식물생명공학회 ( 구 한국식물조직배양학회 ) 1990 한국식물생명공학회 춘계학술연구발표회 Vol.1990 No.-

Ti-plasmid에 依하여 形質轉換된 煙草組織의 特性과 再分化 樣相

梁德春,李政明 경희대학교 부설 식량자원개발연구소 1987 硏究論文集 Vol.8 No.-

Studies were conducted to examine the characteristics of teratoma shoots obtained from diploid and haploid tobacco crown gall tumor tissues induced by the inoculation of Agrobacterium tumefaciens and the effects of plant hormones and active carbon in the cultural media on the growth and regeneration patterns of the teratoma shoots. Results can be summarized as follows. 1. Teratoma shoots could be obtained when the crown gall tumor, induced by the inoculation of A. tumefuciens strain C-58 to the stem, was successively grown on hormone-free medium. 2. The growth of tumor callus induced by C-58 strain was inhibited when several plant hormones were added to the cultural media. Plant regeneration could not be obtained from the tumor callus or the teratomas. 3. Addition of active carbon and benzyl adenine to cultural media was effective for shoot elongation and reduction in multiple shoot formation. 4. The growth of tumor callus obtained from the haploid plant was promoted by the addition of plant hormones to the media. Whole plants were also regenerated from the teratoma shoots formed from tumor callus. 5. Normal or transformed plants were regenerated from the tumor callus formed by the inoculation of a strain of A. tumefaciens collected from field soil. The transformed plants are characterized by having twisted dark-green leaves, shorter internodes, and abundant axillary shoots.

Randomly Amplified Polymorphic DNA를 이용한 인삼의 DNA 분석

양덕춘,김무성 한국국제농업개발학회 2002 韓國國際農業開發學會誌 Vol.14 No.4

본 연구는 인삼의 종 및 변종간의 유전적 다양성을 식물의 분류학적 연구에 이용하여 품종육성을 위한 기초자료로 활용하고자 인삼 조직의 DNA를 사용하여 RAPD primer를 선정하고 RAPD양상을 조사하고자 수행되었다. 인삼의 RAPD를 수행함에 있어서 DNA분리에 이용되는 조직의 차이가 PCR이후 DNA band 양상에 어떠한 영향을 미치는 지를 조사하고자 포장에서 정상적으로 생육하고 있는 자경종에서 조직별로 시료를 채취한 후 primer를 사용하여 PCR을 수행하여 나타난 band의 양상은 모든 조직에서 동일한 band를 형성하였다. 50개의 서로 다른 10-mer random primer를 이용하여 band가 확실히 나타나는 18개의 primer를 선발하여 자경종, 풍기황숙, 중국삼, 미국삼의 DNA를 사용하여 PCR 증폭을 하였다. 그러한 PCR product를 1.2%의 Agarose gel에 전기영동하여 banding patter에 따라 분석하였다. 이러한 RAPD 결과를 Cluster analysis 방법을 사용하여 similarity index(SI)로서 원연관계를 조사한 결과 자경종과 풍기황숙이 근연관계를 가졌고 중국삼은 자경종과 약간의 차이를 가졌으며 또한 자경종, 풍기황숙, 중국삼은 미국삼과 변이의 차이를 나타냈다. This study was carried out to analyse genetic variability among different ginseng cultivars, and to use it for the basic information on rearing ginseng cultivars. In performing RAPD of ginseng, DNA was extracted from the different tissues of 'Jakyung' growing normally in the field. PCR was performed to check if there is difference among different tissues, resulting in the same banding pattern, Eighteen of 50 different 10-mer UBC primers showing clear bands were selected, and PCR amplification was performed using DNA extracted from ginseng cultivars, Jakyung, Punggi Hwangsuk, China, and America. The PCR product amplified was analysed according to banding patter after electrophoresis in 1.2% agarose gel. Genetic difference based on RAPD result was investigated by similarity index (SI) of Cluster analysis. As a result, Jakyung and Punggi Hwangsuk had close relationship, whereas Chinese ginseng differed a little from Jakyung. Jakyung, Punggi Hwangsuk, and Chinese ginseng showed genetic difference from America ginseng.

Simple Sequence Repeat Primer에 의한 딸기의 Genomic DNA 분석

양덕춘,최성민,김무성,민병훈 경희대학교 생명자원과학연구원 2002 硏究論文集 Vol.22 No.-

본 연구는 진핵세포에 널리 산재되어있는 repeat sequence를 primer로 사용하여 딸기의repeat sequence 좌위를 증폭시켜서 분석하고 이를 같은 종일지라도 지역간의 차이로 인한 환경의 변화 또는 재배방법의 차이에 의해서 일어나는 변이를 분석하는 방법에 활용할 수 있도록 하기 위하여 수행하였다. GGC repeat sequence의 딸기 genomic DNA에 존재여부를 판명하기 위하여 (GGC)_(6) probe를 DIG을 사용하여 labeling하여 딸기 genomic DNA에 hybridization한 결과 확실한 spot이 나타났다. 또한 repeat sequence로 이루어진 UBC SSR primer 100개중에서 4가지 서로 다른 primer에서 선명한 band를 확인할 수 있어서 딸기의 genomic DNA내에 여러 종류의 repeat sequence가 있는 것으로 판단되었다. 따라서 각 품종별 표지로서 이용성을 알아보고자 PCR을 수행하였는 바 (ACC)_(6)와 (CCG)_(6)의 primer에서는 다른 band에 비해 매우 확실한 밴드를 형성하였다 이러한 PCR product의 염기서열분석을 위해 우선 대량증식을 위한 PCR의 적정 조건은 primer 10p㏖, DNA 50ng으로서 수행하는 것이 DNA의 증폭에 좋았다. 증폭된 1 Kb DNA 절편을 이용하여 염기서열을 조사하였던 바, 808bp의 서열을 알아냈고 그 반복염기서열은 효모와 사람에 존재하는 유전자이었으며 Cdontella sinensis와의 유의성 검정결과 ACC 반복염기에 62% 동일성을 나타내었다. This study was carried out to investigate genetic variation caused by environmental change or cultural method difference, which was performed by amplifying repeat sequence loci of strawberry widely dispersed in eukaryotic cell. DIG labeled(GGC)_(6), probe was used to test if GGC repeat sequence exists in strawberry genomic DNA, resulting in clear spot on the filter. Among the 100 UBC SSR primers which contain repeat sequence, four different primers amplified clear DNA fragments, suggesting that the repeat sequence exist in the genome of strawberry. PCR was also performed to test whether these primers could be used for a marker among strawberry cultivars. (ACC)_(6), (No. 861) and (CCG)_(6) (No. 865) primers showed very clear bands compared to the other primers. The amplified 808 bp of 1 kb DNA was sequenced, and database search using the sequence information showed that the repeat sequence was the gene existed both in yeast and human, and a significant sequence homology to Odontella sinensis was found.

형질전환된 고추( Capsicum annum L.) 식물체의 Mouse Adenosine Deaminas 유전자 발현

양덕춘,이계연,유영숙,최경화,임학태 한국식물생명공학회 1997 식물생명공학회지 Vol.24 No.1

고추의 형질전환율을 높이기 위하여 우선적으로 효율적인 재분화조건을 구명하였다. 고추의 하배축과 자엽 모두 2mg/L zeatin과 0.1 mg/L NAA ( I )에서 51%, 1.0 mg/L BAP와 10.0 mg/L IBA ( II)는 45.1%의 재분화율을 보였으며, 두 배지에 5 $\mu$M AgNO$_3$을 첨가하였을 때 I의 배지에서 보다 건강한 식물체를, II의 배지에서는 재분화율이 약 8%로 증가함을 보였다. 따라서 II의 배지 조건은 가격이 비싼 zeatin의 효과를 대치할 수 있었다. 이렇게 얻어진 효율적인 재분화배지에 고추의 하배축과 자엽을 ADA와 NPT II 유전자를 함유한 Agrobacterium tumefaciens pDY183을 이용하여 형질전환을 유도하여, kanamycin 100 mg/L에서 선발하여 성공적으로 형질전환체를 얻었다. 식물체내로의 ADA와 NPT II 유전자의 도입은 PCR을 이용하여 확인하였으며, Northern blot에 의하여 ADA 유전자의 전사여부를 확인하였다. ADA 효소의 활성도는 spectrophotometer를 이용하여 측정하여 본 결과 고추세포내에서 정상적으로 발현하였으므로 동물유전자인 ADA가 식물체 형질전환시 표시 유전자로서의 사용가능성이 확인되었다. The in vitro regeneration and genetic transformation systems in hot pepper(Capsicum annuum L.) have not been routinely available, which has been a major limiting factor in the application of new genetic manipulations. An efficient procedure to regenerate whole pepper plants and to generate transgenic plants expressing a foreign gene was established. A relatively high frequency of plant regeneration was observed when hypocotyl and cotyledon explants were cultured on MS medium supplemented with NAA 0.1 mg/L plus zeatin 2.0 mg/L or IBA 10.0 mg/L plus BAP 1.0 mg/L. Addition of AgNO$_3$5 $\mu$M to these media improved the regeneration frequency up to 8%. For plant transformation, hypocotyl and cotyledon explants of hot pepper were precultured on shoot induction media without kanamycin added for 2 days, and then cocultured with Agrobacterium tumefaciens pDY183 for 2 days. Putative transformants were obtained from selection media containing 100 mg/L kanamycin sulfate and 500 mg/L carbenicillin. Putatively selected transformants were confirmed by amplification of selectable marker genes (ADA and NPT II) by polymerase chain reacion. Successful transcripts of ADA gene were detected by Northern blot analysis. Enzyme activity of ADA was also examined by spectrophotometric analysis, and expression of ADA gene in hot pepper suggests the potential application of ADA gene as a selectable marker in plants.

Agrobacterium spp. 에 의하여 형질전환된 감자조직의 생장특성

양덕춘 한국자원식물학회 1998 한국자원식물학회지 Vol.11 No.2

This study was carried out to obtain the information for growth characteristics of crown gall tumor and hairy root transformed by Agrobacterium spp,. on the media with phytohormones, casein hydrolysate and activated charoal. Crown gall tumors and hairly roots were formed respectively on potato tuber discs infected by tumerfaciens A ch 5 and A.rhizogenes ATCC15834. These tumors and roots could be grown on the phytohormone free media. PCR analysis of Rol C and Vir C gene fragments confirmed that crown gall root was prompted on the medium containing 2,4-D 2mg/l with casein hydrolysate lg/l. The survival ration of crown gall tumor callus derived from potato increased on medium containing the activated charcoal 0.5∼0.2mg/l because of the prevention, on the other hand, hairly roots were necrosis on the same medium. Callus derived from hairly root were excellently grown for a short time by suspension culture on liquid medium containing 2.4-d 2mg/L and casein hydrolysate lg/l.

딸기의 RAPD를 위한 PCR의 최적조건

양덕춘,최성민,강태진,이미애,송남현,민병훈 한국자원식물학회 2001 한국자원식물학회지 Vol.14 No.1

본 연구는 random primer를 이용하여 PCR을 수행하기 위한 딸기 DNA증폭의 최적조건을 구명하여 조직배양된 딸기 배양묘와 모본과의 유전적인 동일성의 여부 및 품종을 판별할 수 있는 marker를 개발하기 위하여 수행하였다. 추출한 딸기 커(\ulcorner)잣 DNA를 proteinase-K나 RNase-H를 처리하였을때 깨끗하고 순수한 DNA band를 확인할 수 있었으며, 50ng의 template DNA, 10pmol의 primer, 37oC annealing 온도로 45 cycle로서 PCR을 행하는 것이 가장 효율적이었다. 상기 실험결과로서 PCR적정조건을 확립한 후, UBC primer를 대상으로 딸기 여봉 DNA에서 PCR를 수행하여 RAPD의 양상을 조사한 결과 총 90개의 primer 중에서 딸기 genomic DNA에서 PCR product를 형성한 것은 46개였으며, 총 형성된 band의 수는 158개로 나타났다. Band를 형성한 primer와 band를 형성하지 않은 primer간의 GC content를 비교하면 band를 형성한 primer의 경우 GC content는 평균 67.4%이었다. 그러나 band를 형성하지 못한 primer의 경우에는GC함량이 평균58%이었다. This study was performed to select marker which can identify genetic variation between mother plant and in vitro cultured plantlets of strawberry by PCR using random primer. When 'Yeobong' DNA extracted was treated with proteinase-K and RNase-H, clear DNA bands were shown. The optimal condition for RAPD in strawberry was to use 50ng of template DNA, 10pmol of primer,37oC of annealing temperature, and 45 cycles of PCR. After establishing above PCR optimal condition, RAPD pattern was investigated by using UBC primers. PCR was performed, and 46 of 90 primers produced PCR product showing 158 total bands. GC content was compared between the primers forming bands and no bands. The GC content showing bands was average 67.4%, whereas primers showing no bands 58%.

인공합성 Phosphinothricin Acetyltransferase 유전자에 의한 Basta 내성 연초식물체의 개발

양덕춘 한국자원식물학회 1998 한국자원식물학회지 Vol.11 No.2

This experiment was conducted to introduce phosphinothricin acetyl -transferase(PAT) gene, resistant to basta and non-selective herbidide, into tobacco(Nicotiana tabacum cv.BY4). For shoot formation,tobacco leaf disks were placed on the MS medium supplemented with 2.0mg/L BA and 0.1mg/L NAA. In this medium condition, tobacco leaf disces were cocultivated with A. tumefaciens MP90 containing NPT IIand PAT resistant to kanamycin and Basta, respectively. Shoots were obtained in the medium containing antibiotics, and those were transferred to rooting medium supplemented with 0.1mg/L NAA and antibiotics. The plants obtaining roots were transplanted into soil. Phenotype of transgenic tobacco plant was mostly as normal plant. However, about 5% was abnormal plant, which did not set seeds. PCR analysis and southern blot were performed to determine transformation. As the results, it was confirmed that PAT gene was stably integrated into tobacco genome.When herbicide, basta, was sprayed to the plants confirmed by PCR, the transgenic plants showed normal growth, whereas normal plants died. Therefore, the result of this experiment show that tobacco transformation for the resistance to basta, non-selective herbicide, was successful because PAT gene was stably integrated into tobacco.

고생장 인삼 모상근의 선발

양덕춘,김용해,양덕조,민병훈,신성련,최광태 한국식물생명공학회 1998 식물생명공학회지 Vol.25 No.6

인삼의 뿌리절편, 줄기, 엽병에 Agrobacterium을 접종하여 고생장 모상근을 선발하고자 수행하였다. 인삼 뿌리 절편에서 A. rhizogenes R1000과 A. rhizogenes $A_4$에 의하여 모상근이 유도되었으며, 인삼의 줄기 및 엽병에서도 모상근이 유도되었다. 유도된 모상근은 rolC및 virC DNA절편의 확인으로 형질전환체임을 확인하였으며, 균이 제거된 모상근을 선발하였다. 선발된 모상근 300 세포주중 성장과 형태적 특징이 뚜렷한 11 모상근을 선발하였다. 이들 모상근은 주근이 비후된 것, 주근이 가는 것, 측근의 돌기가 많은 것, 측근의 생장이 높은 것 등의 특징을 나타내었다. 이들 11 모상근중 주근이 가늘고 측근의 생장이 왕성한 형태가 모상근의 생장이 좋았으며, 주근이 비후되고 측근의 돌기가 많은 형태는 생장이 낮았다. 본 연구에서 인삼 뿌리절편으로부터 고생장 모상근인 KGHR-1, KGHR-5, KGHR-8을 선발하였다. These studies were carried out to select the active grow hairy root lines induced from various ginseng(Panax ginseng C. A. Meyer) parts. Hairy roots were induced in root explants, stem and petiole in vitro by A. rhizogenes R1000 or A. rhizogenes $A_4$. These hairy roots could be grown on the phytohormone free medium, and PCR analysis of rol C and vir C gene fragments confirmed that hairy roots were transgenic tissues. We have selected 11 hairy root lines with active growing characters among 300 hairy root lines selected based on growth and morphological characteristics on 1/2MS solid media with 250 mg/L carbenicillin. Morphological characteristics of selected 11 hairy root lines were thickness and thiness of main roots, and many projection for lateral roots, active grow of lateral roots. Among selected 11 hair root lines prominent characteristics of hairy roots with active growing characters were thiness of main roots and active grow of lateral roots. But characteristics of low growing hairy roots were thickness of main roots and low grow of lateral roots. Finally we have selected actively growing hairy roots, KGHR-1, KGHR-5, KGHR-8 among 11 hairy root lines.

Agrobacterium tumefaciens C58에 依하여 形質轉換된 人蔘組織에서의 saponin 生産

梁德春,李政明 경희대학교 부설 식량자원개발연구소 1988 硏究論文集 Vol.9 No.-

The present study was conducted to obtain some basic information on the transformation of ginseng tissue, identification of opine compound and protein, and saponin production from ginseng tissue transformed with pTiC58. Ginseng tumor callus induced by pTiC58 could be continuously cultured on the phytohormone-free basic medium. The transformation was reconfirmed by the detection and identification of opine compounds from the tumor callus. The transformed ginseng calli contained higher amount of protein than normal calli and the protein pattern of transformed calli was quite different from that of normal callus. Higher saponin content was found in the transformed callus than the normal callus, and ginsenoside composition of the transformed callus was similar to that of cultivated ginseng root, but quite different from that of normal callus.