• Journal of Plant Biotechnology
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• 제37권1호
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• pp.72-76
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• 2010

To develop transgenic cucumber tolerant to abiotic stress, a cotyledonary-node explants were co-cultivated with Agrobacterium tumefaciens (EHA101) carrying TPSP gene (pHC30-TPSP). After transfer to fresh medium every two week for eight weeks, putative transgenic plants were selected when shoots grown a length greater than 3 cm from the cotyledonary-node explants on selection medium supplemented with $5\;mgl^{-1}$ phospinotricin as selectable agent. The confirmation of transgenic cucumber was based on the Northern blot analysis. Thirty four shoots (5.2%) with resistance to phospinotricin were obtained from 660 explants inoculated. Of them, transformants were only confirmed from 11 plants (1.7%). Transgenic cucumber expressing TPSP gene was more synthesized at 3.8 times amounts of trehalose (0.014 mg g fresh $wt^{-1}$) than non-transformants (0.0037 mg g fresh $wt^{-1}$). However, all of transgenic plants showed abnormal morphology, including stunted growth (< height 15 cm), shrunken leaves, and sterility as compared with non-transgenic plants (> height 150 cm) under the same growth environment. These results lead us to speculate that the overproduction of trehalose was toxic for cucumber, even though that had known for rice as non-toxic.

• Journal of Plant Biotechnology
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• 제43권3호
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• pp.341-346
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• 2016

The objectives of this study were to 1) evaluate the efficiency of the protocol of Agrobacterium-mediated transformation of cucumber to introduce phytoene synthase-2a carotene desaturase (PAC genes); 2) demonstrate the integration of PAC genes into the genome of putative transgenic cucumber based on growth on selection medium, PCR and Southern analysis; 3) evaluate the expression of PAC genes in transgenic cucumber based on the analysis of RT-PCR and Northern blot hybridization. Out of 5,945 cotyledonary-node explants inoculated with Agrobacterium, 65 (1.1%) explants produced 238 shoots. Integration of PAC genes into the genome of the cucumber was demonstrated based on the analysis of gDNA-PCR, 21 out of the 238 plants regenerated; while 6 plants proved positive for Southern blot hybridization. Transgene expression was demonstrated based on analysis of RT-PCR, 6 plants proved positive out of the 6 plants analyzed; while 4 plants out of 6 proved positive during Northern blot hybridization. This study successfully demonstrated the production of transgenic cucumber, integration, and expression of the PAC gene in cucumber.

• Journal of Plant Biotechnology
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• 제40권4호
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• pp.198-202
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• 2013

환경스트레스 저항성 오이 형질전환체 생산을 위해서 오이 "Eunsung" 품종의 자엽절 절편을 Nit유전자를 포함하는 pPZP211와 pCAMBIA2300 발현벡터로 각각 형질전환된 Agrobacterium과 공동 배양하였다. 공동배양 후 형질전환체 선발, 형질전환체 유도, 신장, 유식물체 생산 등은 자엽절 절편을 이용하는 CTM방법(Jang et al. 2011)에 따라 수행하였다. 발현벡터에 따라 선발배지에 100 mg/L paromomycin을 첨가하여 선발과정을 거쳤으며, 선발배지에서 3 cm크기의 shoot를 유도한 후 PCR, Southern, RT-PCR 및 Northern분석을 통해 형질전환 여부를 확인하였다. 공동배양 한 2,547개의 자엽절 절편으로부터 105개체(4.12%)가 선발배지로부터 얻어졌으며, 그들 중 45개체(1.77%)만이 Nit유전자의 PCR product를 얻을 수 있었다. 오이 genome에 Nit유전자의 도입여부를 확인하기 위하여 45개체에 대한 Southern분석을 수행한 결과 각각 39개체(1.53%)서 확인할 수 있었으며, 이중 오직 6개체(0.24%)에서만 Nit유전자가 안정적으로 발현되고 있음을 RT-PCR과 Northern분석을 통해 확인하였다. 이러한 결과는 Nit유전자가 오이 genome에 안정적으로 도입 및 발현되고 있음을 보여 주고 있음을 알 수 있었다.

• 한국식물병리학회지
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• 제11권1호
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• pp.80-86
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• 1995

The cDNA of CMV-As satellite RNA was introduced into tobacco plants (Nicotiana tabacum cv. Samsun NN) using a binary Ti plasmid vector system of Agrobacterium tumefaciens. The cDNA of satellite RNA introduced into tobacco plants was detected by polymerase chain reaction (PCR) and molecular hybridization analyses. Symptom development was distinctly suppressed in the transgenic tobacco plants when inoculated with CMV-Co. CMV concentration in the transgenic tobacco plants was decreased to 1/40 of non-transgenic tobacco plants. The kanamycin resistance gene of the transgenic tobacco plants was also detected in the progeny.

• Journal of Plant Biotechnology
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• 제32권3호
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• pp.161-165
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• 2005

Agrobacterium과 자엽절편의 공동배양으로 박과작물인 오이의 형질전환체를 생산하였다. 오이 배양재료는 "은침"의 자엽절편을 사용하였으며, reporter유전자로서 gus유전자와 선발표지로서 bar 또는 nptII유전자로 각각 제작된 pPTN289와 pPTN290벡터를 GV3101, LBA4404, EHA101에 형질전환하여 공동배양하였다. 형질전환빈도는 Agrobacterium의 종류에 따라 현저한 차이가 있었으며, 특히 사용한 균주중 EHA101에서 0.35%로 가장 높았다. 선발배지에서 형성된 오이 식물체중 제초제 저항성 (12개체)과 paromomycin 저항성 (3개체)을 얻었고, 이들 모두 gus양성반응 나타냈다. Southern분석에 의하여 오이 형질전환체의 genome에 gus유전자가 도입되어 있음을 확인 하였다.

• Journal of Plant Biotechnology
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• 제38권3호
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• pp.198-202
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• 2011

Agrobacterium과 자엽절 절편 공동배양으로 오이 형질전환체를 생산하였다. 오이 자엽절 절편 (c.v. Eunsung)은 선발 마커로서 nptII유전자와 유용유전자로서 DQ유전자가 포함된 pPZP211를 EHA101에 형질전환하여 공동 배양하였다. 3회 반복실험으로부터 평균 형질전환 빈도는 4.01%를, 최대 빈도는 5.97%를 보여 주었다. Paromomycin항생제 저항성을 갖는 9개의 식물체를 선발과정을 통해 얻었으며, Southern blot 분석에 의해 6개 식물체의 genome에 nptII유전자자 안정적으로 도입되어 있음을 확인할 수 있었다.

• 식물조직배양학회지
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• 제25권3호
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• pp.201-206
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• 1998

Superoxide dismutase (SOD)를 많이 생산하는 신기능성 오이(Cucumis sativus L.) 과실을 개발하기 위하여, 발아 7일째의 자엽절편에 완두콩(Pisum sativum) 유래 MnSOD 유전자를 Agrobacterium tumefeciens LBA4404의 매개로 형질 전환시켰다. 1mg/L zeatin, 0.1mg/L IAA, 300mg/L claforan, 100mg/L kanamycin이 포함된 선발배지에서 부정아를 유도한 후, 1주일 간격으로 계대배양하면서 kanamycin 저항성 형질전환체를 선발하였다. 선발배지 배양 6주 후 kanamycin 저항성을 가진 부정아만을 절단하여 0.2mg/L NAA가 함유된 발근배지에 옮겨 뿌리를 유도한 후, 화분에서 순화시켰다. 소식물체의 잎에서 DNA를 분리하여 PCR 로 분석한 결과, 3개의 식물체에서 선발마커유전자인 NPTII 유전자가 존재함을 확인하여 SOD 유전자가 오이에 도입됨을 확인할 수 있었다. 이중 한 개체의 과실에서 SOD 함량이 무처리개체에 비해 약 3.2배 높았으며 native gel 전기영동에서 도입한 SOD isoenzyme 밴드가 강하게 검출되었다.

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.148.2-149
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• 2003

Cucumber fruit mottle mosaic tobamovirus (CFMMV) causes severe mosaic symptoms with yellow mottling on leaves and fruits, and occasionally severe wilting of cucumber plants. No genetic source of resistance against this virus has been identified. The genes coding for the coat protein or the putative 54-kDa replicase were cloned into binary vectors under control of the SVBV promoter. Agrobacterium-mediated transformation was peformed on cotyledon explants of a parthenocarpic cucumber cultivar with superior competence for transformation. R1 seedlings were evaluated for resistance to CFMMV infection by lack of symptom expression, back inoculation on an alternative host and ELISA. From a total of 14 replicase-containing R1 lines, 8 exhibited immunity, while only 3 resistant lines were found among a total of 9 CP-containing lines. Line 144 homozygous for the 54-kDa replicase was selected for further resistance analysis. Line 144 was immune to CFMMV infection by mechanical and graft inoculation, or by root infection following planting in CFMMV-contaminated soil. Additionally, line 144 showed delay of symptom appearance following infection by other cucurbit-infecting tobamoviruses. Infection of line 144 plants with various potyviruses and cucumber mosaic cucumovirus did not break the resistance to CFMMV. The mechanism of resistance of line 144 appears to be RNA-mediated, however the means is apparently different from the gene silencing phenomenon. Homozygote line 144 cucumber as rootstock demonstrated for the first time protection of a non-transformed scion from soil inoculation with a soil borne pathogen, CFMMV.

• 식물조직배양학회지
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• 제28권1호
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• pp.53-58
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• 2001

제초제 저항성 오이 (Cucumis sativus L. cv Green angel)를 생산하기 위하여 배발생 현탁배양세포와 binary vector pGA-bar을 지닌 Agrobacterium tumefacians (LBA4404)를 공동배양하였다. 형질전환 벡터의 T-DNA부분에는 kanamycin에 저항성을 나타내는 neomycin phosphotrans ferase (npt II) 유전자와 phosphinothricin (PPT)에 저항성을 나타내는 phosphinothricin acetyltransferase (bar) 유전자를 지니고 있다. 48시간의 공동배양 후 배발생 캘러스는 20mg/L PPT가 함유된 성숙배지에서 배양하였다. 약 200개체의 형질전환 유식물체를 40mg/L PPT가 첨가된 호르몬이 없는 배지에서 생산하였다. 5개의 오이 형질전환 식물체의 염색체에 bar유전자가 도입되어 발현되는 것을 northern blot 분석을 통하여 확인하였다. 형질전환 오이 식물체가 토양에서 성숙되었다. 성숙한 오이 식물체는 PPT가 함유된 상업적 제초제 (Basta)를 일반적인 사용 농도 (3ml/L)처리시에도 저항성을 나타내며 생장하였다.

• Journal of Ecology and Environment
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• 제29권4호
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• pp.323-330
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• 2006

We investigated the impact of watermelon grafted onto Cucumber Green Mottle Mosaic Virus (CGMMV)-resistant transgenic watermelon rootstock on insects as non-target organisms in a greenhouse in 2005. We quantitatively collected insect assemblages living on leaves and flowers, and we used sticky traps to collect alate insects. We compared the patterns of insect assemblages and community composition, cotton aphid (Aphis gossypii Glover) on watermelon leaves and western flower thrip (Frankliniella occidentalis Trybom) on watermelon male flowers, between CGMMV-resistant transgenic watermelon (TR) and non-transgenic watermelon (nTR). Non-parametric multidimensional scaling (NMS) ordination verified that insect assemblages on leaves and sticky traps were different between TR and nTR (P<0.05). The insect assemblages on male flowers were not statistically significant. Multi-response permutation procedures proofed our results from NMS results (P>0.05). Conclusively, TR watermelons appear to have some adverse effects on the population of cotton aphids on leaves and sticky traps, but watermelon male flowers do not show an adverse effect. Further research is required to assess the effect of TR on the aphid and western flower thrip. Life table experiments might support the specific reason for the adverse effects from leaf assemblages. Assessment of non-target impacts is an essential part of the risk assessment of non-target insects for the impact of transgenic organisms.