The Gram-positive bacterium Clavibacter michiganensis subsp. michiganensis is the causal agent of canker disease in tomato. Because it is very important to control newly introduced inoculum sources from commercial materials, the specific detection of this pathogen in seeds and seedlings is essential for effective disease control. In this study, a novel and efficient assay for the detection and quantitation of C. michiganensis subsp. michiganensis in symptomless tomato and red pepper seeds was developed. A pair of polymerase chain reaction (PCR) primers (Cmm141F/R) was designed to amplify a specific 141 bp fragment on the basis of a ferredoxin reductase gene of C. michiganensis subsp. michiganensis NCPPB 382. The specificity of the primer set was evaluated using purified DNA from 16 isolates of five C. michiganensis subspecies, one other Clavibacter species, and 17 other reference bacteria. The primer set amplified a single band of expected size from the genomic DNA obtained from the C. michiganensis subsp. michiganensis strains but not from the other C. michiganensis subspecies or from other Clavibacter species. The detection limit was a single cloned copy of the ferredoxin reductase gene of C. michiganensis subsp. michiganensis. In conclusion, this quantitative direct PCR assay can be applied as a practical diagnostic method for epidemiological research and the sanitary management of seeds and seedlings with a low level or latent infection of C. michiganensis subsp. michiganensis.

Using the abundant available information about the tomato genome, we developed DNA markers that are linked to disease resistant loci and performed marker-assisted selection (MAS) to construct multi-disease resistant lines and varieties. Resistance markers of Ty-1, T2, and I2, which are linked to disease resistance to Tomato yellow leaf curl virus (TYLCV), Tomato mosaic virus (ToMV), and Fusarium wilt, respectively, were developed in a co-dominant fashion. DNA sequences near the resistance loci of TYLCV, ToMV, and Fusarium wilt were used for primer design. Reported candidate markers for powdery mildew-resistance were screened and the 32.5Cla marker was selected. All four markers (Ty-1, T2, I2, and 32.5Cla) were converted to cleavage amplification polymorphisms (CAPS) markers. Then, the CAPS markers were applied to 96 tomato lines to determine the phenetic relationships among the lines. This information yielded clusters of breeding lines illustrating the distribution of resistant and susceptible characters among lines. These data were utilized further in a MAS program for several generations, and a total of ten varieties and ten inbred lines were constructed. Among four traits, three were introduced to develop varieties and breeding lines through the MAS program; several cultivars possessed up to seven disease resistant traits. These resistant trait-related markers that were developed for the tomato MAS program could be used to select early stage seedlings, saving time and cost, and to construct multi-disease resistant lines and varieties.

The CMV (cucumber mosaic virus) is the most frequently occurring virus in chili pepper farms. A variety of peppers that are resistant to CMVP0 were developed in the middle of 1990s through a breeding program, and commercial cultivars have since been able to control the spread of CMVP0. However, a new pathotype (CMVP1) that breaks the resistance of CMVP0-resistant peppers has recently appeared and caused a heavy loss in productivity. Since no genetic source of this new pathotype was available, a traditional breeding method cannot be used to generate a CMVP1-resistant pepper variety. Therefore, we set up a transformation system of pepper using Agrobacterium that had been transfected with the coat protein gene, CMVP0-CP, with the aim of developing a new CMVP1-resistant pepper line. A large number of transgenic peppers (T(1), T(2) and T(3)) were screened for CMVP1 tolerance using CMVP1 inoculation. Transgenic peppers tolerant to CMVP1 were selected in a plastic house as well as in the field. Three independent T(3) pepper lines highly tolerant to the CMVP1 pathogen were found to also be tolerant to the CMVP0 pathogen. These selected T(3) pepper lines were phenotypically identical or close to the non-transformed lines. However, after CMVP1 infection, the height and fruit size of the non-transformed lines became shorter and smaller, respectively, while the T(3) pepper lines maintained a normal phenotype.

수박작물의 대목용으로 사용하는 수박공대에 CGiMMV-CP 유전자를 도입하여 개발된 LM 수박공대의 CGMMV 내성 정도를 격리하우스와 노지 포장내에서 조사하였다. 격리온실에서의 $T_{3}$ 형질전환 수박대목의 CGMMV 내성은 접종 후 70일까지 유지되는 반면 대조구는 접종 후 20일에 전부 이병되었다. 인위적 토양전염 포장에서 형질전환체는 접종후71일까지 약 40%의 내성률을 보였으며, 대조구는 접종 후 37일에 모두 이병되었다. 인위적 접촉전염 포장에서 형질전환체는 대조구에 비해 약 10일 정도 지연효과를 보였다. 따라서 CGMMV-CP 형질전환체는 CGMMV에 저항성을 가진 것이 아니라 감염시기를 지연시키는 부분 내성으로 나타났다. CGMMV-CP homozygous T 세대를 진전시켜서 형질전환 수박공대 계통을 $BC_{1}T_{5}$ 세대에서 선발하였다. 또한 LM 수박공대에 형질전환 되지 않은 접수 (슈퍼금천수박)를 접목하여 non-LM 수박을 생산하고 CGMMV-CP 유전자에 관련된 물질의 이동 여부를 조사하였다. PCR, northern, western 분석한 결과 수박공대 대목에서 형성되는 DNA, RNA, protein 물질이 접수로 이동되지 않음을 확인하였다. Previously developed transgenic watermelon rootstocks (gongdae) inserted by CGMMV-CP were examined to test the virus tolerance levels. In the restricted plastic house, the $T_{3}$ watermelon rootstock showed tolerance to CGMMV until 70 days after inoculation on the leaves while the non-transformed watermelon rootstock became susceptible at 20 days after inoculation. In the field, tolerance efficiency of transgenic rootstocks maintained up to 40% at 71 days after contamination with CGMMV in the soil while all of the non-transformed rootstocks became susceptible at 37 days with the same condition. In the same field, transgenic rootstocks showed more tolerance to CGMMV than the non-transformed rootstocks as those were inoculated on the leaves, but it showed only 10 days delay before being susceptible. Therefore, transgenic rootstocks have a characteristic of delay effect against CGMMV susceptibility, rather than resistance character. From $T_{3}$ rootstocks homozygous for the CGMMV-CP horticulturally favorable individuals were selected for further breeding and a transgenic line was finally obtained at the $BC_{1}T_{5}$. A material transfer experiment was conducted to find out if the DNA, RNA or expressed protein in the transgenic rootstocks could move to the grafted scion (non-transformed watermelon, Super-Kumcheon). PCR, northern, and western blot analysis were performed and no evidence of transferring of those materials from rootstock to scion was ever found.