유전자총 실험조건 최적화를 통한 형질전환 백합 식물체 생산

김종보,Kim, Jong Bo 한국식물생명공학회 2017 식물생명공학회지 Vol.44 No.1

유전자총을 이용한 형질전환 체계와 PPT (D-L-phosphinothricin) 선발을 통하여 나리 인편조직으로부터 형질전환 식물체가 획득되었다. 본 연구에서 나리 '레드플레임' 품종의 인편조직에 선발유전자로 제초제저항성 유전자인 bar 유전자 그리고 내염성과 내건성의 복합환경저항성을 나타내는 AtSIZ 유전자를 목적유전자로 가지고 있는 플라스미드를 금입자에 코팅해서 유전자총을 이용해서 형질전환 하였다. 이러한 형질전환 체계 확립을 위해 헬륨가스 압력은 1,100 psi, 금 입자크기는 $1.0{\mu}m$ 그리고 목적 절편체까지의 거리는 6 cm 그리고 유전자총 처리 24시간 전과 후에 0.2 M sorbitol과 0.2 M mannitol을 혼합해서 MS배지에 첨가한 프로토콜로부터 우수한 형질전환 결과를 나타내었다. 유전자총 발사 처리 후, 1주간 선발제로 사용되는 PPT가 없는 MS 배지로 이식하여 배양 후, PPT 10 mg/l이 첨가된 선발배지에서 4주 간격의 계대배양을 통해 8-12주간 선발과정을 거친다. PPT 선발 배지에서 생존한 신초가 형성된 형질전환 나리 인편 조직들을 호르몬이 없는 MS 배지로 다시 옮겨주면 발근 및 추가 생육이 이루어진다. 생존한 형질전환 나리 기내 소식물체들로부터 PCR 검정을 통해 선발유전자인 bar 유전자 그리고 목적유전자인 AtSIZ 유전자의 도입이 확인되었다. 결론적으로 100여개의 나리 인편조직을 본 연구에서 확립된 유전자총 실험프로토콜을 이용하면 대략적으로 형질전환 나리 17-18 개체를 획득할 수 있으며 본 연구에 기술된 유전자 총 매개 형질전환 체계는 추가적인 보완이 이루어지면, 향후 나리 육종 프로그램에 기여할 것이다. Transgenic lily plants have been obtained after particle bombardment, using PDS-1000/He system and scale explants of lilies, followed by PPT (D-L-phosphinothricin) selection. In this study, scales of the lily plants cv. 'red flame' were bombarded with a plasmid containing the bar gene as a selectable marker, and the AtSIZ gene as a gene of interest, showing salt tolerance and drought tolerance respectively, and both being driven by the CaMV 35S promoter. For optimization of a protocol, factors which optimized and showed a high transformation efficiency under following conditions, were considered: a bombardment pressure of 1100 psi, a target distance of 6 cm and $1.0{\mu}m$ of gold particle, and 24-h pre-culture and post-culture on MS medium containing 0.2 M sorbitol and 0.2 M mannitol as osmoticum agents. After bombardment, all the bombarded scales of lily were transferred to MS medium without selective agents, for a week. Subsequently, these bombarded scales were transferred to a selection MS medium containing 10 mg/l PPT, and incubated for a month for further selection, after which they were cultured for another 4-8 weeks with a 4-week subculture regime on the same selection medium. After transferring into hormone-free MS medium, the PPT-resistant scales with shoots were successfully rooted and regenerated into plantlets. PCR analysis revealed that the surviving putatively transformed plantlets indicated the presence of both the bar gene and the AtSIZ gene. In conclusion, when 100 scales of lily cv. Red flame are bombarded, this study produced approximately 17-18 transgenic plantlets with an optimized bombardment protocol. The protocol described here can contribute to the breeding program of lilies.

유전자총과 아그로박테리움을 이용한 여러 가지 한국 잔디류의 형질전환체계 확립

김종보,김경덕,박대섭,Kim Jong-Bo,Kim Kyong-Duck,Park Dae-Sup 한국잔디학회 2004 한국잔디학회지 Vol.18 No.3

다양한 Zoysiagrass 4가지 품종들을 식물재료로 사용하여 Agrobacterium만 이용한 방법 그리고 particle bombardment로 배발생캘러스에 상처를 낸 후, Agrobacterium으로 공동배양 시키는 2가지 다른 형질전환 방법을 비교하였다. 예비실험에서 일반적으로 형질전환에 널리 사용되는 kanamycin과 PPT(phospinitricin)의 적적선발농도에 대해서 실험하였는데, kanamycin의 경우 300mg/l 그리고 PPT의 경위 50mg/l의 농도에서 가장 효과적인 선발 효율을 나타내었다. Agrobacterium을 이용한 형질전환은 Agrobacterium을 2일간 배양시킨 다음, 박테리아 농도를 O.D 600nm=1.0-1.2로 맞추고, 배발생캘러스를 30분간 간염 시키는 방법이 효과적이었는데, particle bombardment를 이용하여 캘러스에 상처를 유발시킨 후, Agrobacterium으로 감염시키면 3배 이상 높은 형질전환 수율을 획득할 수 있었다. 이상의 결과는 한국잔디 형질전환에 있어서 particle bombardment과 Agrobacterium을 병행하여 실시한 최초의 보고이고, 이러한 시스템을 기반으로 하여 향후 한국잔디를 포함하여 다른 난지형 및 한지형 잔디의 품종개량에 널리 이용되리라 생각된다. In this report, several factors such as infection time and concentration of bacterial suspension, influencing on transient gene expression in Agrobacterium-mediated transformation were evaluated. An appropriate concentration (O.D 600nm = 1.0-1.2) of bateria and 30 min of infection time showed a higher level of GUS expression. To improve transformation efficiency (TE), friable embryogenic calli (FEC) were bombarded by tungsten particles without plasmid DNA, and then co-cultivated with A. tumefaciens LBA4404 which contains pTOK233 super binary vector, carrying neomycin phosphotransferase (NPTII), hygromycin phosphotransferase (hpt) and$\beta-glucuronidase$ (GUS) genes. Three days after co-cultivation with A. tumefaciens and particle bombardment, FEC cultures were transferred to the selection medium (SM: MS medium supplemented with BA 1mg/l, hygromycin 100mg/l, cefotaxime 250 mg/l and vancomycin 200mg/l). They were cultured for 2 weeks and then transferred to the second SM containing hygromycin 50mg/l, cefotaxime 200 mg/l and vancomycin 100mg/l. Later, stable GUS expression was detected 4 to 6 weeks after transfer to the SM. Further, TE from Agrobacterium-mediated transformation after particle bombardment increased to about 3-folds compared with Agrobacterium-mediated transformation without particle bombardment. In the present study, we established an efficient transformation protocol of zoysiagrass by using A. tumefaciens in the combination with particle bombardment for the first time.

Picloram을 이용한 효율적인 백합 캘러스 유도 체계 확립

김종보,Kim, Jong Bo 국제문화기술진흥원 2021 The Journal of the Convergence on Culture Technolo Vol.7 No.3

나리는 세계 5대 절화에 속하는 주요 절화 중의 하나이며, 아시아, 유라시아 및 북아메리카 등 다양한 지역에 서식한다. 나리 신품종개발을 위해서는 교배, 돌연변이 및 선발육종 기술 외에 조직배양 기술을 포함하는 생명공학기술 도입을 통한 신품종 육성이 필요한데 조직배양 체계확립은 나리 육종체계에 있어서 필수적인 요소 중 하나이다. 조직배양의 많은 분야 중 배발생캘러스를 이용하여 식물체 재분화 증식 체계확립이 많은 작물에서 연구 중인데 본 연구에서도 배발생캘러스 유도에 사용되는 오옥신 중 picloram을 다양한 농도로 처리하여 최적의 배발생캘러스 유도 농도를 선정하고자 실험을 수행하였다. 실험 결과, 3-4주 후부터 CEC (compact embryogenic callus), FEC (friable embryogenic callus) 및 백색캘러스 이렇게 3가지 형태 캘러스가 발생했는데 1.0 mg/l 처리구에서 CEC와 FEC 모두 높은 효율을 나타내었으나 갈변율이 높은 관계로 0.75 mg/l를 나리 배발생캘러스 유도에 적합한 최적 농도로 선정하였다. 이러한 연구결과는 향후 배발생캘러스를 이용한 나리 식물체 재분화 체계 확립 그리고 우량품종 증식체계 확립에 기여할 것이다. Lily is one of the most important 5 cut flowers in international flower market and lilies are distributed in Asia, Eurasia and North America. To develop a new lily cultivar, in addition to hybridization, mutation and selection methods, biotechnological techniques including tissue culture are also required. Establishment of tissue culture system is one of the requirement for the breeding program in Lily. Among many fields of plant tissue culture, establishment of regeneration system via embryogenic calluses are studied in many crops. In this study, research was carried out to decide the proper concentration of picloram which is used for the induction of embryogenic calluses. As a result, 3 different types of callused were observed after 3-4 weeks. They were CEC (compact embryogenic callus), FEC (friable embryogenic callus) and white callus type. 1.0 mg /l of picloram showed the best result for the production of embryogenic callus, however, due to its higher rate of browning in this concentration, 0.75 mg/l of picloram was selected as a proper concentration of picloram for the induction of CEC and FEC in Lily. These results can be contributed to the establishment of both regeneration system and mass propagation in lily in the future.

다중회귀분석을 이용한 악취 관리지역에서의 복합취기강도와 개별악취물질들의 관계에 대한 연구

김종보,정상진,Kim, Jong-Bo,Jeong, Sang-Jin 한국환경보건학회 2009 한국환경보건학회지 Vol.35 No.3

We investigated a trait of odor and the relationship between odor unit and odorous compounds using multiple regression analysis based on data compiled from Sihwa (SIC), Banwol (BIC), Banwol plating (BPIC) and Poseung industrial complex (PIC). These areas are odor control areas in Gyeonggi province. It was revealed that $NH_3$ and styrene concentrations in SIC and BPIC were relatively higher and $H_2S$ concentration especially in mc was more than five times higher than other areas. As a result of regression analysis using SAS, intensity of odor unit was highly related to concentrations of $H_2S$, TMA, styrene and n-valeraldehyde in SIC, $H_2S$, acetaldehyde, and butyraldehyde in BPIC and $NH_3$ in BIC.

제지폐수의 슬러지의 탈수 및 소각특성

김종보,은종극,박승조 ( Jong Bo Kim,Jong Keug Eun,Seung Cho Park ) 한국폐기물자원순환학회(구 한국폐기물학회) 1993 한국폐기물자원순환학회지 Vol.10 No.2

행정법학의 개념과그 외연(外延)

김종보(Kim Jong-Bo) 행정법이론실무학회 2008 행정법연구 Vol.- No.21

In the study of administrative law, often the terms administrative law and public law are used interchangeably but there seems to be fundamental differences in the concept and scope between the two. While the concept of the latter derived from the distinction between private and public law, that of the former emerged in the course of the generalization of its study. Such usage of the terms appears to be rooted in the fact that a certain fine-tuning process for the two terms has not yet been properly carried out. Administrative law is a typical branch of public law. It is a field of law whose main purpose is to understand various public law systems of a nation and a community. Therefore, such a purpose needs not be limited to control only the litigable of the systems through administrative litigation and/or judicial review. Rather, the primary task of the study of administrative law should be to understand and to provide bases to design each system, relatively independent from Iitigability and reviewability. For this reason, the methodology of public law should take, as its subject matters, both administrative process which can be captured by a certain degree of litigation and/or judicial review and objective systems which are hard to be captured in such ways. If this approach is followed, the litigation-oriented legal methodology and the system-oriented legal methodology will mutually organically put together the areas of public law and this will enable a comprehensive understanding of its system. Therefore, the study of administrative law should be a system of legal theory which deals not only with laws relating to 'administration' but also with 'national systems, administrative process and laws relating to public systems.' The national systems which include the legislative and judical systems extend from the legislations relating to the prestige of public law such as national contracts, national property and national liability to various kinds of public law status of local governments. The administrative process, having been the primary subject matter of the general study of administrative law, is the most important subject matter of this field and remains as a logic system focused on administrative dispositions, Lastly, the public systems, despite the limited role of the administrative agencies, do have a very important meaning in public law and include legal personality under public law and the union system.

강학상 인가와 정비조합 설립인가

김종보(Kim Jong-Bo) 행정법이론실무학회 2003 행정법연구 Vol.- No.9

환경권의 헌법적 의미와 실현방법

김종보(Kim Jong-Bo),김배원(Kim Bae-Won) 부산대학교 법학연구소 2012 법학연구 Vol.53 No.1

우리 헌법에서는 환경규정을 기본권의 형식으로 규정하고 있다. 이는 여타 외국들이 환경규정을 국가목표조항으로 규정하고 있는 것보다 더 강하게 환경을 보호하려는 헌법제정권자의 의지를 표현하고 있다고 이해할 수 있지만, 실제로 환경권이 어떠한 효력을 발휘하는지는 단순히 규정의 형식만을 가지고 논할 문제가 아니라 헌법에서 말하는 환경이란 무엇이고 헌법이 환경권을 규정하는 목적이 무엇이며, 헌법에 규정됨으로써 어떠한 헌법적 효력을 발휘하게 되는가를 밝힘으로써 가능하다고 본다. 헌법상 환경은 다른 헌법 규정과의 관계와 환경권 규정의 실효성을 고려할 때 이른바 사회적 환경은 제외되는 것이 옳다. 그리고 환경권의 특성과 법적성격을 고려하여 자연환경과 생활환경을 모두 포함하는 것으로 이해한다. 환경권의 법적 성격은 방어권적 요소를 가지고 있지 않는 순수한 사회적 기본권으로 본다. 그러나 환경권이 다른 사회적 기본권과 구별되는 점은 현재의 규정으로는 환경권의 주체를 직접 헌법에서 도출하는 것이 어렵기 때문에 환경소송에서 당사자적격을 인정하기 위해서는 헌법에 구체적으로 당사자에 관한 규정을 삽입하는 것이 필요하다. 그렇다고 해도 가치관이 첨예하게 대립되는 환경문제를 사법부가 결정하는 것은 권력분립원칙상 한계가 있으므로 환경권의 내용과 행사에 대해서는 입법부가 결정하는 것이 의회민주주의 원칙에 부합한다. 결국 궁극적인 환경권의 실현방법은 의회민주주의가 가지는 의사결정의 방식을 존중하면서 지속적인 사회적 논의를 통해서 환경권의 내용을 확고하게 다져나가는 것이다. Korea provides protection of environment as a form of fundamental right. But it is not occur subjective rights no matter how provide fundamental rights. Because the legal nature is decided by the character of environment and harmonious interpretation of other constitution provisions. The legal nature of environmental right is objectivity just as social rights. Therefore the realization of environmental right is the national obligation includes that of courts. But the lawsuit of envirnment is objective lawsuit. So to have standing to lawsuit, it should be specified in the law. According to the interpretation korea constitution, it is difficult to acknowledge the subjective rights directly by American public trust doctrine. For this reason, the National Assembly should enact such provisions. The court has some limits to decide details of environmental rights. Because the court as not has democratic legitimacy as National Assembly. This task is obligation of National Assembly. So the National Assembly has a lot of legislative discretion. Above all, the realization of environmental rights depends upon environmental attitudes of people ultimately.

가설건축물의 개념과 법적 성격

김종보(Kim Jong-Bo) 행정법이론실무학회 2004 행정법연구 Vol.- No.12

It is prescribed in the building code that building is a thing which have a roof and column or wall among a structure fixed on the land. Therefore the fixation on the land is the important concept of the building element. The temporary building is analogous to a building but its structure is not thoroughgoing enough to be comprised in a building category. Nevertheless it does not classified into a simple structure because the temporary building has a point of similarity to form as well as function of building. Building code Article IS provide the temporary building concisely distinguishing from a building by building code Article8. The temporary building is not fixed on the land stronger than a ordinary building, therefore it has lower requirement of the public legal regulation than a building which is fixed on the land and guaranteed with continuance. Building code Article 15 which provides the temporary building does not define a concept of it specially. But defining the concept of temporary building is a criterion to divide sorts of the criminal penalties about an offence against building code and the first step of the construction of public legal effect provision connected with the temporary building. So that it is most important to define notion of the temporary building. It is necessary to propose to reform the provision of the temporary building premising distinction between a period of maintenance and fixation on land. It is desirable that a regular building which have a period of maintenance in the city planning site is banned from calling the temporary building. In addition, to prepare provision regarding the word "the term building", the provision of regular building with respect to a building perm it shall apply in this case. In case of the temporary building ordained building code Article 15②, and also, a structure fixed on land is classified into the term building, in the same breath the temporary building is reorganized a structure-centered which is not fixed on the land.

도시재정비촉진을 위한 특별법의 제정경위와 법적 한계

김종보(Kim Jong Bo) 한국토지공법학회 2007 土地公法硏究 Vol.35 No.-

Arguments for improving the lag behind urban including the area north of the Han river begin with designation of New Town and establishment of ‘Seoul City’s balanced development support ordinance’. After that time for improving the area north of the Han river ‘Special Act on Urban Improvement Promotion’ was established and took effect on July, 2006. But now, it is indifferent to people. This shows that Special Act on Urban Improvement Promotion functions contrary to expectations. Before Establishment of Special Act on Urban Improvement Promotion, there are many problems in areas designated as New Town: rising of land price, division of land and buildings, absence of restitution of development gains, conflicts of residents, absence of development measure. To solve this problems and develop the area north of the Han river, Special Act on Urban Improvement Promotion was established, but it doesn’t meet the anticipations. Furthermore, many limitations are disclosed. The grave issues in legislating process are necessity of great-sphere development, application of the act, constitution of the act, choice of main measure and development subject. The ministry of construction and transportation, the Seoul City, the national assembly had a different view about that. As a result, Special Act is legislated according to proposal of the ministry of construction and transportation, however those issues is still under discussions. The exclusion of urban development, reconstruction and the private subject, the absence of regulation regarding maintained areas are also seen as barrier to Special Act application.