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연초 Crown Gall Callus유래 Teratoma Shoot의 생육과 발근에 미치는 식물호르몬과 활성탄의 영향
양덕춘,강춘기,최광태,Yang, Deok-Chun,Kang, C.K.,Choi, K.T. 한국연초학회 1992 한국연초학회지 Vol.14 No.1
These studies were conducted to investigate effects of phytohormone and activated carbon on the growth and rooting of teratoma shoots induced from Nicotiana tabacum cv. NC2326 transformed by Aerobacterium tumefaciens C58. GA was effective for shoot elongation and reduction of multiple shoots from teratoma shoot, however, leaves of teratoma shoot cultured on the medium with GA were pointed. ABA was also effective in promoting shoot elongation, but was not for reduction of multiple shoots. Teratoma shoot cultured on the medium with 1 n activated carbon promoted shoot elongation and inhibited the number of shoots differentiated, but was grown as abnormal shoot. Addition of 1% activated carbon and 0.5mg/l BA to culture media was effective for shoot elongation and reduction of multiple shoot and for formation of round leaves as normal leaves. Though these shoots were inoculated on the rooting medium, they could not from roots but formed multiple shoots. Boric acid, myo-inositol and sucrose were also ineffective on the rooting of teratoma shoots.
당근 절편을 이용한 Agrobacterium spp. 분리, 동정과 선발균주에 의한 연초의 형질전환
양덕춘,양덕조,최광태,Yang, Deok-Chun,Yang, D.C.,Choi, K.T. 한국연초학회 1991 한국연초학회지 Vol.13 No.1
These studies were carred out to obtain the transformant from tobacco cells by Agrobacterium spp. from crown gall and soil at the natural field in Korea, and identified their virulence. Kodo's and Clark's selective media were used for isolation of Agrobacterium spp. In these media, total of 99 strains were characterized based on the morphological characteristics of colonies. Among them 34 strains were able to induce on carrot discs. And hypervirulent strains C23-1 and K29-1 were identified as Agrobacterium tumefaciens biotype 1 and biotype 2, respectively. These strains formed fast growing, larger gall as compared to those induced by other strains on the carrot discs. Transformed tobacco callus was initiated on the phytohormone free MS medium with 250$\mu\textrm{g}$/ml carbenicillin after co-cultivation of tobacco stem explants and Agrobacteria. On the phytohormone free media, shoot was rarely formed from transformed callus. However, these shoot were teratoma shoots which were not grown as normal shoot, and teratoma shoot from transformant by C23-1 was smaller than that of K29-1.
인삼(Panax ginseng C. A. Meyer)접합자 배의 형질전환을 위한 재분화 및 항생제 농도 조건
양덕춘(Deok-Chun Yang),이은경(Eun-Kyung Lee),최원균(One-Kyun Choi),김무성(Moo-Sung Kim) 한국자원식물학회 2003 한국자원식물학회지 Vol.16 No.1
인삼 종자로부터 적출한 embryo로부터 multi-shoot를 얻기 위하여 CPA와 BA를 단독 및 복합처리 하여 각각의 유기 양상을 알아본 결과 CPA 0.5mg/ t 에 BA 1.0 mg/ t 복합처리가 multi-shoot를 얻는데 가장 효과적인 것으로 나타났으며, 식물호르몬 종류를 달리한 처리구에서는 특히, 2,4-D 1mg/ t 와 kinetin 0.5mg/ t 처리구에서 한 개의 embryo 당 4∼6개의 shoot를 유기시킬 수 있었다. 또한 인삼의 형질전환을 위해 인삼 자엽으로부터 pre-embryoid를 유기하기 위한 가장 최적의 조건은 2,4-D 1mg/ t 과 kinetin 0.5mg/ t 복합처리구에서 가장 효과적이었다. 기내배양에 의해 형성된 shoot로부터 잎의 포함여부에 따른 petiole, 그리고 embryo에서 kanamycin 내성정도를 조사한 결과 잎을 포함한 petiole과 embryo는 kanamycin 100㎍/ m1 농도에서도 다소간 생존하는 것으로 나타나 매우 높은 농도의 항생제를 사용해야 할 것으로 판단되었으며, 잎을 포함하지 않은 petiole의 적정 농도는 100㎍/ m1 정도인 것으로 판단되었다 또한 배배양에 의한 embryo로부터 유기된 자엽 을 2,4-D 1mg/ t 과 kinetin 0.5mg/ t 복합 처리구에서 선발할 경우와 인삼 자엽으로 부터 직접 somatic embryo를 유기하여 인삼의 형질전환을 유도하기 위해서 가장 적합한 kanamycin 농도로는 75∼100㎍/ m1이 가장 효과적인 것으로 나타났다. To obtain multi-shoot using zygotic embryos dissected from ginseng seed, the embryos were cultured on MS medium supplemented with CPA and BA. Effective multi-shoot induction was achieved on 0.5mg/ t CPA and 1.0mg/ t BA treatment. Among the various plant growth regulator treatment, MS basal medium with 1mg/ t 2,4-D and 0.5mg/ t kinetin was more competent and could be induced 4∼6 shoots per one embryo. Also, the best condition for pre-embryoid induction from ginseng cotyledon so as to ginseng transformation appeared to 1mg/ t 2,4-D and 0.5mg/ t kinetin treatment. The kanamycin level to select transformants varied greatly by different explant tyues. The petiole explants with leaf and embryo could survived up to 100㎍ / ml kanamycin concentration where as petiole explants without leaf died all at the level. Conclusionally, our results suggest that optimum kanamycin concentration for ginseng transformation using somatic embryos is about 75∼100㎍ / ml concentration.
PCR-aided RFLP기술을 이용한 인삼의 DNA분석
양덕춘(Deok-Chun Yang),김무성(Moo-Sung Kim) 고려인삼학회 2003 Journal of Ginseng Research Vol.27 No.3
This study was carried out to obtain basic information on breeding using PCR-aided RFLP technology which can identify the variation inter- and intra-species of ginseng in the level of DNA. It was intended to investigate banding pattern on psbA and rbcL genes of chloroplast DNA in ginseng after treating with restriction enzymes. To isolate psbA and rbcL genes of chloroplast, both psbA-N, psbA-C primer and rbcL-N, PX-1 primer were used. As a result, 1,008 bp<br/> band of psbA gene and 1, 336bp band of rbcL gene were appeared, which was optimal and expected molecular weight. In addition, primers to isolate atpB, rpoB, trnL, and trnF genes were used, resulting in the expected 1366, 900, 1500 and 1008 bp bands. Genes of psbA and rbcL isolated by PCR were cut by restriction enzymes, Sau3A, TaqI, AluI, HaeIII, and RFLP pattern was investigated. KG line and other species of ginseng were cut by TaqI treatment, and bands were located in 800 bp. The treatment treated by AluI also showed the same 800 bp band in KG line and other species. In HaeIII treatment, 500 bp of faint bands were shown in case of KG line, whereas any bands were not observed in other species. All<br/> chloroplast genes formed bands by PCR amplification. However, it was not evident to distinguish intra-or inter-species of ginseng after restriction enzyme treatment. Therefore, more restriction enzyme treatment or sequence comparison method should be considered for further experiment.
내병성 관련유전자의 운반체 재조합 및 인삼 (Panax ginseng C.A. Meyer)의 형질전환
양덕춘(Deok Chun Yang),이은경(Eun Kyung Lee),김무성(Moo Sung Kim) 고려인삼학회 2003 Journal of Ginseng Research Vol.27 No.1
For study about introduce of gene connected with disease and transformation system of gingseng, chitinase gene cloned from soybene and disease resistant gene were carried out for expression and transformation of plant using Agrobacterium. The disease resistance gene(DR-49), 35S-35S-AMV, has been constructed. The disease resistance gene and chitinase gene were introduced into the binary vector pRD 400, which were mobilized into Agrobacterium tumefaciens strain MP 90 and LBA 4404 harboring disarmed Ti-plasmid. As a result of induce transformants using ginseng embryo and petiole, multi shoots were formed on MS medium supplemented 1 mg/l 2,4-D and 0.5 mg/l kinetin. Also transformation by cotyledonwas effective on MS medium supplemented 1 mg/l 2,4-D and 0.5 mg/l kinetin, transformation percent of disease resistant gene and chitinase gene were showed 18%, 14% respectively. As transformed tissue is under pre-embryoid condition, normal shoot is required through the process of matured embryo.
Chitinase 유전자 도입 형질전환 감자식물체의 역병저항성
최경화,양덕춘,김현순,최경자,조광연,정혁,Choi, Kyung-Hwa,Yang, Duk-Chun,Kim, Hyun-Soon,Choi, Kyung-Ja,Cho, Kwang-Yeon,Jung, Hyuk 한국식물생명공학회 1999 식물생명공학회지 Vol.26 No.3
Chitinase 유전자가 삽입된 감자 Belchip 품종의 형질전환체와 대조구에 곰팡이 병원균을 접종하였다. 7개 계통의 형질전환식물체를 12cm정도 키운 후 병원균인 Phytophthora infestans의 zoospore를 접종하여 인공적으로 역병을 유발시켰다. 그 결과 발병율에 따라서 세 그룹으로 분리되었는데 대조구에 비하여 감염 정도가 심한 것 2개 계통. 비슷한 3개 계통, 발병 정도가 약한 2개 계통으로 구분되었다. 대조구에 비하여 저항성이 높았던 2개 계통과 발병이 심했던 1개 계통만을 대상으로 하여 2차실험을 실시한 결과, 1차실험과 비슷한 경향을 나타냈다. 포장에서 생육된 2개의 저항성 계통은 자연적으로 발생한 역병에 대해서도 역시 대조구에 비하여 역병저항성이 더 높았다. A fungal infection assay between normal and transgenic potato harboring chitinase gene in cultivar Belchip was investigated. In the first stage of experiment, seven transgenic lines having 12cm tall were tested for their resistance against potato late blight pathogen Phytophthora infestans by infection with the zoospores, artificially, Susceptibility to potato late blight infection could be classified into three types based on the rate. In terms of resistance to the disease, two lines were higher, two lines were more suppressive, and three lines were similar as compared with the control. In the following experiment, only 2 risistant lines and 1 suppressed line were used to confirm the resistance again. The results of both experiments were similar. Furthermore, two highly resistant transgenic lines grown in field exhibited a higher resistance than control under the conditions of natural ocurrence of the fungal disease.