본 연구는 청색 카네이션 계통을 육성하기 위하여, 페튜니아에서 클로닝된 화색관련 유전자인 dfr과 f3'5'h 그리고 카네이션에서 클로닝된 f3'h antisense를 카네이션에 형질전환하기 위하여 수행되었다. Dfr 돌연변이인 카네이션 4품종(cv. Virginia, White charotte, West crystal, Crystal)의 경정과 엽편에 Agrobacterium LBA4404 strain, pMBP-1 ...
본 연구는 청색 카네이션 계통을 육성하기 위하여, 페튜니아에서 클로닝된 화색관련 유전자인 dfr과 f3'5'h 그리고 카네이션에서 클로닝된 f3'h antisense를 카네이션에 형질전환하기 위하여 수행되었다. Dfr 돌연변이인 카네이션 4품종(cv. Virginia, White charotte, West crystal, Crystal)의 경정과 엽편에 Agrobacterium LBA4404 strain, pMBP-1 binary vector, pSB1-pCAMBIA F3'5'H_antisenseF3'H binary vector를 사용하여 페튜니아 dfr, f3'5'h_antisensef3'h를 형질전환하고 300mg/L kanamycin(경정 형질전환체 선발), 100mg/L kanamycin(엽편 형질전환체 선발), 5mg/L hygromycin(엽편 형질전환체 선발)에서 5번 선발ㆍ계대배양하였는데, kanamycin과 hygromycin에 대하여 저항성이 없는 것은 신초발생이 억제되거나 어린잎부터 백화되어 고사하는 경향을 보이는 반면, 저항성을 보이는 개체들은 신초발생과 신장ㆍ발근에서 정상적인 모습을 보이며 생장하였다. Virginia 품종의 경정을 이용한 형질전환의 경우 총 1800개를 접종하여 5번의 선발을 거쳐 ‘Virginia'에서 30개체가 발근되었으며, 기외순화 후 최종 27계통을 획득하여 약 2.45%의 형질전환 효율을 보였고, genomic DNA를 추출하여 PCR 분석을 통해 페튜니아 dfr과 nptII의 도입을 확인하였다. 또한 3800개의 엽편을 co-transformation 방법을 통하여 Agrobacterium으로 형질전환한 후 5번의 선발을 거쳐 ‘Virginia'와 'White charotte' 2품종에서 40개체가 발근되었다. 기외순화 후 최종 35계통을 획득하여 1.45%의 형질전환 효율을 보였으며, genomic DNA를 추출하여 PCR 분석을 통해 페튜니아 dfr과 f3'5'h의 도입을 확인하였다. 이로써 델피니딘 색소발현과 관련된 유전자인 dfr, f3'5'h 그리고 f3'h가 도입된 62계통의 형질전환체가 육성되어 화색 표현형 검정에 활용할 수 있게 되었다.
본 연구는 청색 카네이션 계통을 육성하기 위하여, 페튜니아에서 클로닝된 화색관련 유전자인 dfr과 f3'5'h 그리고 카네이션에서 클로닝된 f3'h antisense를 카네이션에 형질전환하기 위하여 수행되었다. Dfr 돌연변이인 카네이션 4품종(cv. Virginia, White charotte, West crystal, Crystal)의 경정과 엽편에 Agrobacterium LBA4404 strain, pMBP-1 binary vector, pSB1-pCAMBIA F3'5'H_antisenseF3'H binary vector를 사용하여 페튜니아 dfr, f3'5'h_antisensef3'h를 형질전환하고 300mg/L kanamycin(경정 형질전환체 선발), 100mg/L kanamycin(엽편 형질전환체 선발), 5mg/L hygromycin(엽편 형질전환체 선발)에서 5번 선발ㆍ계대배양하였는데, kanamycin과 hygromycin에 대하여 저항성이 없는 것은 신초발생이 억제되거나 어린잎부터 백화되어 고사하는 경향을 보이는 반면, 저항성을 보이는 개체들은 신초발생과 신장ㆍ발근에서 정상적인 모습을 보이며 생장하였다. Virginia 품종의 경정을 이용한 형질전환의 경우 총 1800개를 접종하여 5번의 선발을 거쳐 ‘Virginia'에서 30개체가 발근되었으며, 기외순화 후 최종 27계통을 획득하여 약 2.45%의 형질전환 효율을 보였고, genomic DNA를 추출하여 PCR 분석을 통해 페튜니아 dfr과 nptII의 도입을 확인하였다. 또한 3800개의 엽편을 co-transformation 방법을 통하여 Agrobacterium으로 형질전환한 후 5번의 선발을 거쳐 ‘Virginia'와 'White charotte' 2품종에서 40개체가 발근되었다. 기외순화 후 최종 35계통을 획득하여 1.45%의 형질전환 효율을 보였으며, genomic DNA를 추출하여 PCR 분석을 통해 페튜니아 dfr과 f3'5'h의 도입을 확인하였다. 이로써 델피니딘 색소발현과 관련된 유전자인 dfr, f3'5'h 그리고 f3'h가 도입된 62계통의 형질전환체가 육성되어 화색 표현형 검정에 활용할 수 있게 되었다.
Experiments were carried out to introduce flavonoid biosynthesis related genes in carnation (Dianthus caryophyllus) for flower color alteration. Four cultivars(cv. 'Virginia', 'White charotte', 'West crystal', 'Crystal') of dfr single mutant carnation plants were transformed with a petunia dfr encod...
Experiments were carried out to introduce flavonoid biosynthesis related genes in carnation (Dianthus caryophyllus) for flower color alteration. Four cultivars(cv. 'Virginia', 'White charotte', 'West crystal', 'Crystal') of dfr single mutant carnation plants were transformed with a petunia dfr encoding petunia dihydroflavonol 4-reductase and f3'5'h_antisensef3'h encoding flavonoid 3',5'-hydroxylase and antisense of flavonoid 3'-hydroxylase. Total 1800 'Virginia' shoot-tips were transformed with Agrobacterium tumefaciens containing pMBP-1 binary vector of petunia dfr, 35s promoter and nptII selection marker gene. Co-cultures of carnation shoot-tip explants were maintained in the light at 24hrs/day in 1/2 MS medium supplemented with 1.0mg/L BA for 10days. After 10days of coculture, shoot-tip explants were transferred to a selection medium containing the 300mg/L kanamycin, 250mg/L cefotaxime/carbenicillin. Also 3800 leaf explants were co-transformed Agrobacterium tumefaciens. Strains which harbored pMBP-1 binary vector of petunia dfr with nptII selection marker gene and pSB1-pCAMBIA F3'5'H_antisenseF3'H binary vector with hpt selection marker gene respectively. Co-cultures of carnation leaf explants were maintained in the light at 24hr/day in MS medium supplemented with 0.3mg/L TDZ and 0.03mg/L NAA for 3days. After 3days of co-culture, leaf explants were transferred to a selection medium containing the 100mg/L kanamycin, 5mg/L hygromycin, 250mg/L cefotaxime/carbenicillin. They were subcultured every two weeks for over 5 times and regenerated to plantlets. Thirty transgenic plants, obtained through a shoot-tip culture method, were selected in vitro, and twenty seven were acclimated to soil in greenhouse. Integrations of the transgene in the putative transformants were confirmed through genomic DNA PCR analysis using primer for the inserted genes of nptII and petunia dfr. Forty plants, transformed baced on a leaf segment culture, were selected in vitro, and thirty five were acclimated to soil in greenhouse. Integrations of the transgene in the putative transformants were confirmed through genomic DNA PCR analysis using primer for the inserted genes of petunia dfr and petunia f3'5'h. Consequently, sixty two lines of transgenic 'Virginia and White charotte' carnation plants, incorporating flavonoid biosynthesis related genes, were obtained and are available to investigate flower color modifications at blooming stages in a greenhouse.
Experiments were carried out to introduce flavonoid biosynthesis related genes in carnation (Dianthus caryophyllus) for flower color alteration. Four cultivars(cv. 'Virginia', 'White charotte', 'West crystal', 'Crystal') of dfr single mutant carnation plants were transformed with a petunia dfr encoding petunia dihydroflavonol 4-reductase and f3'5'h_antisensef3'h encoding flavonoid 3',5'-hydroxylase and antisense of flavonoid 3'-hydroxylase. Total 1800 'Virginia' shoot-tips were transformed with Agrobacterium tumefaciens containing pMBP-1 binary vector of petunia dfr, 35s promoter and nptII selection marker gene. Co-cultures of carnation shoot-tip explants were maintained in the light at 24hrs/day in 1/2 MS medium supplemented with 1.0mg/L BA for 10days. After 10days of coculture, shoot-tip explants were transferred to a selection medium containing the 300mg/L kanamycin, 250mg/L cefotaxime/carbenicillin. Also 3800 leaf explants were co-transformed Agrobacterium tumefaciens. Strains which harbored pMBP-1 binary vector of petunia dfr with nptII selection marker gene and pSB1-pCAMBIA F3'5'H_antisenseF3'H binary vector with hpt selection marker gene respectively. Co-cultures of carnation leaf explants were maintained in the light at 24hr/day in MS medium supplemented with 0.3mg/L TDZ and 0.03mg/L NAA for 3days. After 3days of co-culture, leaf explants were transferred to a selection medium containing the 100mg/L kanamycin, 5mg/L hygromycin, 250mg/L cefotaxime/carbenicillin. They were subcultured every two weeks for over 5 times and regenerated to plantlets. Thirty transgenic plants, obtained through a shoot-tip culture method, were selected in vitro, and twenty seven were acclimated to soil in greenhouse. Integrations of the transgene in the putative transformants were confirmed through genomic DNA PCR analysis using primer for the inserted genes of nptII and petunia dfr. Forty plants, transformed baced on a leaf segment culture, were selected in vitro, and thirty five were acclimated to soil in greenhouse. Integrations of the transgene in the putative transformants were confirmed through genomic DNA PCR analysis using primer for the inserted genes of petunia dfr and petunia f3'5'h. Consequently, sixty two lines of transgenic 'Virginia and White charotte' carnation plants, incorporating flavonoid biosynthesis related genes, were obtained and are available to investigate flower color modifications at blooming stages in a greenhouse.
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