국화는 전 세계적으로 두번째로 중요한 분화식물로서 널리 재배되고 있다. 그 중 국화 ‘신마’는 몇가지 원예적 가치들로 인해 한국에서 잘 알려져는 품종 중 하나이다. 하지만 신마의 흰 화색은 소비자들에게 호감이 낮다는 문제가 있다 따라서 아그로박테리움 매개 형질전환을 이용하여 기본적으로 지닌 좋은 형질에 중요한 분화 가치 중 하나인 화색을 개량하는 것이 필요하다. 본 연구는 국화의 엽조직으로부터 신초 재분화에 미치는 영향에 대해 기술하고자 한다. 신초의 수는 6주 동안 자란 모식물체에서 분리한 엽조직을 0.5mg?L?1 6-benzyladenine (BA)과 0.5mg?L?1 α-naphthaleneaceic acid (NAA)가 포함된 MS배지에서 초기 1주일간 암처리를 한 후 재분화를 관찰하였을 때 하나의 엽조직에서 재분화된 신초개수(9.7)가 가장 많았다. 또한 아그로박테리움 매개 형질전환 연구에 이용하기 위해 신초 재분화에는 영향을 주지 않으면서 아그로박테리움을 제거할 수 있는 적절한 항생제를 찾아 형질전환체를 선발할 수 있는 최적의 농도를 밝혀내었다. 무(Raphanus sativus)에서 분리한 안토시아닌 조절 유전자 RsMYB를 포함하는 pB7WG2D를 가진 아그로박테리움 strain GV3101을 이용하여 공동배양한 엽조직으로부터 형질전환 국화를 얻었으며 플로우사이토메트리를 이용한 분석에서 모본 식물체인 비형질전환체와 형질전환체간의 배수성 차이는 없는것으로 나타났다. 본 연구에서는 더불어 몇 가지 요인을 최적화함으로서 아그로박테리움 매개 형질전환 효율을 높였음을 보여준다. 이 뿐 아니라 국화 품종 ‘신마’에서 무에서 분리한 RsMYB전사인자가 비정상적으로 발현하는 것을 보여주었다. 국화에 삽입된 RsMYB는 식물체의 길이, 잎 모양, 크기 및 화기의 수와 같은 형태적인 특성에는 아무런 영향을 주지 않았다. RT-PCR의 결과 RsMYB유전자는 형질전환 국화에서 안토시아닌 생산에 반응을 돕는 주요 생합성유전자(CmF3H, CmDFR, CmANS)의 조절을 한다는 것을 확인하였고 비 형질전환체에서는 이러한 반응이 보이지 않았다. 이것은 국화에서 MYB전사인자를 이용하여 안토시아닌 생합성 유전자의 조절 역할을 관찰한 첫번째 보고이다.
Chrysanthemum is a globally second most important ornamental plant and therefore grown worldwide. Generally, there are two types of chrysanthemum classified on the basis of plant morphology, such as standard-type and spray-type chrysanthemums. As the former type is more popular than the latter one in floricultural industry, its higher production amount is yearly higher than that of the latter one. The chrysanthemum cv. Shinma used in this thesis is a kind of standard type chrysanthemum and occupies several desirable horticultural traits as mentioned in earlier chapters, thus, it is one of well-known cultivars in Korea. However, as its white colored flower seems to be less attractive to flower lovers and consumers, it is necessary to modify flower color of this cultivar for adding the important ornamental value in addition to currently existing desirable traits. Until now, conventional breeding or mutation breeding has developed a large number of chrysanthemum cultivars; however, it is not possible to introduce desirable traits by these techniques. Only biotechnological approaches are able to perform this which is unable by these techniques. Of biotechnological approaches, Agrobacterium-mediated genetic transformation is a commonly used technique for introduction of desirable genes to the host plants. This thesis describes the development of an efficient regeneration and transformation system in order to produce transgenic chrysanthemum containing anthocyanin regulatory gene. Chapter 1 contains about chrysanthemum including its history, its role in floricultural industry, and worldwide production. In addition, role of genetic engineering in flower color modification and use of MYB transcription factors for anthocyanin production are also described. Moreover, importance of tissue culture technique in Agrobacterium-mediated genetic transformation and factors affecting the genetic transformation are also briefly described. Chapter 2 describes factors affecting shoot regeneration from leaf explants of the chrysanthemum. The maximum number of shoots per explant (9.7) was regenerated in leaf explants of 6-week-old donor plants cultured on MS medium containing a combination of 0.5 mg?L?1 6-benzyladenine (BA) and 0.5 mg?L?1 ?-naphthaleneacetic acid (NAA), with initial dark treatment for 7 days. Among the different auxins tested, indolebutyric acid (IBA) was the most effective for root induction and development, whereas the presence of ethylene inhibitors strongly suppressed shoot regeneration. Ploidy levels were analyzed by flow cytometry: there was no ploidy variation between the regenerated plants and the mother plant grown under greenhouse conditions. Chapter 3 shows that the appropriate choice of antibiotics for shoot regeneration, elimination of Agrobacterium growth, and optimization of minimal concentration of selective agents for screening putative transgenic shoots are important for Agrobacterium-mediated genetic transformation. Of four antibiotics evaluated for shoot regeneration and inhibition of Agrobacterium growth, Clavamox had the least inhibitory effect on shoot regeneration compared to timentin, carbenicillin, and cefotaxime. In addition, its concentration at 125 mg?L?1 was found to be the most suitable for shoot regeneration and production of quality shoots and suppressing the growth of Agrobacterium in explants infected with strains GV3101 or C58C1 for 3 and 4 weeks, respectively. The optimal minimal concentration of phosphinothricin (PPT) was found to be 1.0 mg?L?1 for screening of putative transgenic shoots. Moreover, transgenic plants of the chrysanthemum were obtained by culturing explants co-cultivated with A. tumefaciens strain GV3101 harboring plasmid pB7WG2D with an anthocyanin regulatory gene RsMYB isolated from radish (Raphanus sativus), which was placed under the control of cauliflower mosaic virus promoter (CaMV) 35S and petal specific promoter InMYB1 isolated from the morning glory (Ipomoea nil), on shoot regeneration medium supplemented with an antibiotic (125 mg?L?1) and a selective agent (1.0 mg?L?1). Flow cytometry analysis revealed that there was no variation in ploidy level between transgenic plants and mother plants (non-transformants). The transformation efficiency obtained from this study (Chapter 3) was relatively low (1%), thus, it is necessary to improve transformation efficiency by assessing various factors. Chapter 4 describes necessity to optimize factors involving in Agrobacterium-mediated transformation for enhanced genetic transformation efficiency of this chrysanthemum cultivar. It was found that optimal conditions of individual factors tested in this study have more or less effect on enhancement of transformation efficiency, whereas co-cultivation temperature and Agrobacterium strains were main factors affecting higher transformation efficiency. However, the protocol developed by combination of all optimized factors gave 8 folds more transformation efficiency than the simple (un-optimized) protocol or approximately 4 folds higher than those of individually optimized factors. Transformation efficiency was calculated as number of explants divided by number of PCR positive shoots. Herbicide resistant assay revealed that PCR positive transgenic shoots have stronger resistance to BastaTM spraying than that of non-transformant (control). Chapter 5 describes role of RsMYB anthocyanin regulatory gene inserted in the transgenic chrysanthemum. This study focused ectopic expression of an RsMYB transcription factor isolated from radish (Raphanus sativa) in chrysanthemum cv. Shinma. It was found that presence of the RsMYB in the chrysanthemum did not affect any morphological characteristics such as plant height, leaf shape or size, and number of flowers. Results of RT-PCR confirmed that RsMYB could regulate the three key biosynthetic genes (CmF3H, CmDFR, and CmANS) which are responsible for anthocyanin production in transgenic chrysanthemum, while which were not detected in non-transgenic line. In comparison of two promoters (a constitutive promoter, CaMV 35S of cauliflower mosaic virus and petal specific promoter of InMYB1 isolated from morning glory Ipomoea nil), transgenic lines harboring the petal specific promoter showed higher expression levels in key biosynthetic genes than those with CaMV 35S. However, no anthocyanin accumulation was visually observed in both leaves and floral tissue from any transgenic lines obtained, which was further confirmed by anthocyanin contents. This is the first report of use of MYB transcription factor in chrysanthemum to investigate its role in regulation of anthocyanin biosyenthetic genes. Chapter 6 discusses general aspects of factors affecting shoot regeneration, appropriate choice of antibiotic for genetic transformation, and factor affecting enhanced transformation efficiency. Moreover, regulatory function of RsMYB gene on expression of anthocyanin biosynthetic genes is described. Furthermore, possible explanations on why anthocyanin is not expressed in the transgenic lines are also given. In conclusion, I believe that the efficient regeneration and genetic transformation system of chrysanthemum cv. Shinma described in this thesis will be helpful to introduce other foreign interest genes to this cultivar for the further improvement of desirable horticultural traits.
CHAPTER I 1General Introduction 1CHAPTER Ⅱ 11Optimization of factors affecting efficient shoot regeneration in Chrysanthemum (Chrysanthemum morifolium cv. Shinma) 11CHAPTER Ⅲ 34Evaluation of antibiotics and selective agents for Agrobacterium-mediated genetic transformation of Chrysanthemum 34CHAPTER Ⅳ 57Assessments of factors affecting efficient Agrobacterium-mediated genetic transformation of the Chrysanthemum 57CHAPTER Ⅴ 76Characterization of morphology of transgenic plant containing RsMYB gene and regulatory function of the transgene 76CHAPTER Ⅵ 91General Discussion 91Summary 103Reference …….………….107Abstract in Korean 125
