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.

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.

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.

한국산 고감염 Agrobacterium spp의 분리 및 연초의 형질전환에 이용

양덕춘,정재훈,이정명 한국식물생명공학회 1998 식물생명공학회지 Vol.25 No.3

한국에서 자생한 자연산 tumor 조직과 근권토양으로부터 형질전환율이 높은 hypervirulent Agrobacterium spp를 분리하기 위해서 Salix, Diospyros, Populus 및 Malus에서 형성된 tumor 조직과 근권토양을 채취하괴 Schroth 선택배지와 New and Kerr 배지를 이용하여 78 균주의 colony를 특성에 따라 분리하였다. 이중에서 48 균주가 당근 disc에서 tumor를 형성하였으며 tumor를 형성한 균주중 형성시기가 빠르며 크기가 커 hypervirulent 균주로 생각되는 A. tumefaciens SP101을 biotype 1, 그리고 A. tumefaciens SM042를 biotype 2로 동정하였다. 토양중에서 선발한 A. tumefaciens SM042와 disarmed A. tumefaciens PC2760에 kanamycin 저항성 유전자를 함유하고 있는 binary vector pGA643을 도입하여 conjugant인 A. tumefaciens SM643과 A. tumefaciens PC643을 kanamycin과 tetracycline이 함유된 최소배지에서 획득하였다. 연초의 형질전환을 위해서 conjugant Agrobacterium과 연초조직을 동시배양 후 2,4-D와 kanamycin이 함유된 선발배지에 치상하여 형질전환을 유도한 결과 A. tumefaciens SM643이 A. tumefaciens PC643보다 더 많은 캘러스가 형성되었다. 그러나 A. tumefaciens PC643을 사용한 형질전환 캘러스는 대부분 friable한 캘러스로 유도되었으며 정상적으로 식물체로 생장하였으나 A. tumefaciens SM643을 사용한 캘러스는 매우 딱딱하며 둥그런 형태의 캘러스와 friable한 캘러스가 혼재한 상태로 생장하였으며, 이중에서 friable한 캘러스는 정상적인 shoot가 형성되었으나 딱딱한 캘러스로 유도된 형질전환체는 식물체로 형성되지 않고 반구형 미색의 전형적인 tumor 캘러스로 생장하였다. 한편 형질전환시 캘러스를 유도하지 않고 직접 shoot를 형성시킨 결과 disarmed Ti-plasmid를 사용하지 않고 wild-type Ti-plasmid를 사용해도 정상적인 형질전환체를 획득할 수 있었다. Total of 78 strains were characterized based on the morphological characteristics of colonies isolated on Schroth, and New & Kerr's media for selection of hypervirulent wild-type Agrobacterium spp from galls, hairy root-like process and soil of Populus, Malus, Salix and Diopyros in Korea. Among them, 48 strains were able to induce tumors in carrot disc. Hypervirulent A. tumefaciens SP101 and SM042 were identified as biotype 1 and biotype 2, respectively, These strains formed fast growing, larger tumors as compared to those induced by other strains. The binary vector pGA643 with kanamycin resistant gene was mobilized from E. coli MC100 into A. tumefaciens strain SM042 isolated from soil, and/or disarmed vector PC2760 using a triparental mating method with E. coli HB101/pRK2013, and transconjugants, A. tumefaciens SM643 and PC643 were obtained in minimal media containing kanamycin and tetracycline. Tobacco tissues were cocultivated with conjugant Agrobacterium and then transferred to selective medium with 2,4-D and kanamycin to induce the transformants. Calli were formed more efficiently in cocultivation with A. tumefaciens SM643 than that with A. tumefaciens PC643. Most of calli transformed with A. tumefaciens PC643 were friable and regenerated into normal plantlets, while the calli transformed with A. tumefaciens SM643 were compact, hard, and mixed with friable calli. The friable calli formed normal shoots, while compact calli did not form shoots but only grew to typical compact tumor calli. When the shoots formed directly from tobacco stems without callus induction after transformation by A. tumefaciens SM643 with wild-type Ti-plasmid, normal transformed plants can be induced without using disarmed Ti-plasmid.

연초의 형질전환을 위한 새로운 표지유전자로서 Mouse Adenosine Deaminase 유전자의 이용가능성

양덕춘,한성수,윤의수 한국식물생명공학회 1995 식물생명공학회지 Vol.22 No.4

The development of selectable markers for transformation has been a major factor in the successful genetic manipulation of plant. We established a new selectable marker system for tobacco transformation using chimeric adenosine deaminase (ADA) gene, which confers resistance to cytotoxic adenosine analogues, 9-$\beta$-D-arabinofuranosyl adenine(Ara-A) and cordycepin. The transformants with the chimeric ADA gene in tobacco grew in the presence of normally lethal level of cytotoxic adenosine analogues, 100 $\mu$M Ara-A and 50 $\mu$M cordycepin. We successfully distinguished transformed shoot from non-transformed shoot on the same selectable media with cytotoxic adenosine analogues. In this selectable media, we were able to select seeds with/ without ADA gene from transgenic tobacco seeds. Theses results show that the mammalian ADA gene may serve as a new selectable marker for tobacco transformation. 식물세포의 형질전환을 위한 새로운 표지유전자의 개발은 식물유전공학의 중요한 관건이 되고 있다. 본 실험은 독성 adenosine 유도체인 9-$\beta$-D-arabinofuranosyl adenine (Ara-A)과 cordycepin등에 저항을 나타내는 adenosine deaminase (ADA) 유전자를 새로운 식물세포의 형질전환용 표지유전자로 사용코자 수행하였다. 정상식물체에서는 치사하는 농도인 Ara-A 100 $\mu$M과 cordycepin 50 $\mu$M이 함유된 선발배지에서 ADA 유전자에 의하여 형질전환된 연초식물체는 생존이 가능하였으며 성공적으로 형질전환체를 선발할 수 있었다. 또한 형질전환된 연초식물체에서 획득한 종자도 동일한 선발배지에서 ADA 유전자가 유전된 종자와 유전되지 않은 종자를 쉽게 구별할 수 있었다. 이런 결과는 동물유전자인ADA 유전자를 연초조직의 새로운 형질전환용 표지유전자로 사용할 수 있음을 시사한다.

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.

형질전환 연초(Nicotiana tabacum L.)의 Mouse Adenosine Deaminase 유전자 발현

양덕춘,박지창,최광태,이정명 한국식물생명공학회 1995 식물생명공학회지 Vol.22 No.4

The mammalian adenosine deaminase(ADA) gene was stably expressed in transgenic tobacco plane. The chimeric ADA gene 35S/35S/AMV/ADA/Tnos, has been constructed. This chimeric gene was introduced into the binary vector pRD400, which was thereafter mobilized into Agrobacterium tumefaiens strain MP90 harboring disarmed Ti-plasmid. The resulting strains were used to transform Nicofiana tabacum L. using the leaf disc. Incorporation of the chimeric gene into plant were confirmed by PCR and Northern blot analyses. Immunoblot analysis showed that ADA protein was successfully synthesized in the transgenic tobacco plants. 동물유전자인 mouse adenosine deaminase (ADA)유전자가 안정적으로 연초 형질전환체에서 발현되었다. ADA cDNA 을 연초에서 강력히 발현시키기 위해서 35S/35S/AMV promoter을 부착시켰으며 식물세포에 도입을 위해서 binary vector인 pRD400을 이용하였다. 연초의 형질전환은 Tri- parental mating에 의해서 도입된 ADA 유전자 함유 binary vector와 disarmed Ti-plasmid을 함유하고 있는 Agrobacterium tmefacience MP9O을 사용하였다. 동시배양은 연초의 잎 disc을 이용해서 kanamycin이 첨가된 배지로부터 직접 shoots을 선발하여 형질전환체로 유도하였다. 형질전환 체에 ADA 유전자의 삽입여부는 PCR을 이용하였으며, 실험결과 ADA 유전자를 확인할 수 있었다. 또한 도입된 mouse ADA 유전자로부터 mRNA 및 단백질 합성여부를 각각 northern blot 및 immunoblot 분석한 결과 형질전환체 에서는 공히 확인되었다.

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.

인삼, 약용작물의 기능성 개발 현황 및 산업화 방안

양덕춘 한국약용작물학회 2018 한국약용작물학술대회 발표집 Vol.2018 No.05