본 연구는 온시디움에 있어서 particle bombardment 및 아그로박테리움 매개 형질전환 최적 조건을 구명하 고자 실시하였다. 실험재료는 ‘Sharry Baby’의 PLBs를 사용하였으며, 유전자는 pCAMBIA3301 vector에 삽입 되어 있는 bar와 intron-gus를 사용하였다. Particle bombardment 법에 있어서는 직경이 0.6 μm인 금 입자 에 유전자를 혼합하여 9 cm의 거리에서 1,100 psi 헬륨 가스 압으로 발사하는 것이 유전자 도입에 효과적이었 다. 아그로박테리움 매개법에 있어서는 미성숙 PLBs를 O.D 0.6 정도인 균주에 접종 후 5일간 광조건 없이 공동 배양하는 것이 유전자 도입에 효과적이었다.

An efficient method for Agrobacterium-mediated transformation of forage crop alfalfa (Medicago sativa L.) was established using secondary somatic embryogenic calli. Agrobacterium tumefaciens strain EHAlOl and a binary vector pIG121-Hm which has selection

벼에 있어서 엽록체 small heat shock protein(small HSP)의 기능을 밝히기 위하여 Agrobacterium을 이용한 형질전환법을 이용하여 벼로부터 분리한 small HSP cDNA를 도입하였다. Agrobacterium의 감염에는 벼의 미성숙 배로부터 유도한 callus를 이용하였다. 형질전환 후의 재분화율은 약 30%였다. 형질전환을 통하여 얻어진 식물체의 genomic DNA로부터 PCR 분석과 Southern blot 분석으로 엽록체 small HSP 유전자의 도입을 확인하였다. 도입 유전자의 형질전환 벼에 있어서 유전자의 발현 양상을 northern blot 분석으로 조사하였다. 그 결과 도입된 유전자는 상온에서의 발현량이 서로 다르게 나타났으며 항상적으로 발현하고 있음을 확인하였다.

Agrobacterium을 이용한 Saccharomyces cerevisiae Acid Phosphatse 유전자 (PHO5) 의 식물체로의 도입

With the purpose of improving ginsenoside production in Korean wild ginseng (Panax ginseng Meyer) mutant adventitious root lines, a synthetic gene encoding squalene synthase (PgSS2) was placed under the control of 35S promoter and transferred to Panax ginseng. Embryogenic callus obtained from ginseng adventitious root lines were transformed by infection with A. tumefaciens strain EHA105 containing the PgSS2 gene. Ten phosphinothricin-resistant plants were generated on selection medium, and the transgene integration and expression in these plants were confirmed by PAT test strip, RT-PCR and Southern hybridization. Ginsenoside analysis by HPLC revealed that the total contents of the 8 ginsenoside types (Rg1, Re, Rf, Rh1, Rb1, Rc, Rb2, Rd) in transgenic adventitious root lines were about 1.6-fold higher than that of the mutant control line (MCL1). This transformation method may facilitate the improvement of Panax ginseng in terms of the accumulation levels of ginsenoside.

Plant transformation systems have been developed using several different approaches, and the development of reliable and efficient transformation systems is still one of the most important breakthroughs in molecular biology and biotechnology of rice. However, there are many cultivars in rice and transformation efficiency is dependent on the cultivar. The conventional Agrobacterium-mediated rice transformation system using secondary calli requires 14-16 weeks for establishing transgenic plantlets, and it is thought that somaclonal variation through the Tos17 retrotransposon occurs during the tissue culture stage. Here, we modified an Agrobacterium-mediated transformation system in rice cultivar Dongjin, adding an endosperm removal step that is critical to transformation frequency and changing the Agrobacterium strain and media composition. This method increased transformation efficiency and stability. These results will provide valuable information for scientists to use a reliable and efficient rice transformation system.

Agrobacterium vector를 이용한 세포사멸 억제유전자인 AtBI-1(Arabidopsis thaliana Bax Inhibitor-1)을 벼에 도입하기 위하여, 벼품종별 식물체 재분화 능력과 AtBI-1 유전자가 도입된 형질전환체 벼에 관한 연구를 수행하여 얻어진 결과를 요약하면 다음과 같다. 벼의 품종별 식물체 재분화 능력을 조사한바 자포니카형 벼가 인디카형 및 통일형 벼에 비해 식물체 재분화능력이 높게 나타났다. 공시 품종 중에서 일품벼의 식물체 재분화율(7%)이 가장 높았다. 배양용기별 식물체 재분화 능력을 조사한바, petri-dish에서 보다 시험관에서 식물체 재분화율이 높았다. 4주동안 자란 ‘일품’ 유래의 callus를 Agrobacterium과 co-cultivation한 후 kanamycin과 carbenicillin이 첨가된 재분화 배지에 이식하였을 때 12.0%의 가장 높은 형질전환율을 나타내었다. 형질전환된 식물체의 genomic DNA를 이용한 PCR 결과는 Bin-AtBI-1-GFP 유전자는 1 kb 부근에 삽입된 것이 확인되었으며, Southern blot 분석에서도 확인이 되었다. 형질전환체의 CLSM(confocal laser scanning microscope) 분석에서 잎, 줄기, 뿌리에서 GFP가 발현되어 형광색을 띄고 있음을 확인 하였다.

Italian ryegrass (Lolium multiflorum Lam.) is one of important forage crop grass widely cultivated in Korea. The genetic manipulation of Italian ryegrass (Lolium multiflorum Lam.) necessitates a reliable and efficient, genotype-independent method of transformation. We are interested in developing molecular breeding methods to improve its nutritional quality and abiotic stress resistance. Development of a rapid and efficient transformation system is the basis for genetic manipulation of Italian ryegrass. In order to establish an efficient Agrobacterium-mediated transformation system was applied to transfer genes into seven genotypes of Italian ryegress, namely cv. 'Kogreen', 'Kopeed', 'Kowinearly', 'Kowinmaster', 'Hwasan 101', 'Hwasan104' and 'Kowinner.' The transformation system developed in this study would be useful for Agrobacterium-mediated genetic transformation of Italian ryegrass plants with genes of agronomic importance.

Herbicide resistance is the most common trait being tested and thus herbicide‐resistant genetically modified plants are now the most widely cultivated worldwide. Here we developed herbicide‐resistant transgenic Agrostis mongolica Roshev. by employing an efficient Agrobacterium‐mediated transformation procedure with 25.2% of transformation efficiency. The identification and employment of regenerable and reproducible type of callus was one of the most critical factors to ensure success in this study. PCR analysis confirmed that the bar transgene was integrated into the genome of transgenic plants. The expression of 35S‐bar gene was confirmed by Northern blot analysis. The transgenic plants showed complete resistance to herbicide, indicating that the bar gene is functional in transgenic plants.