• Journal of Plant Biotechnology
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• 제1권1호
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• pp.8-12
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• 1999

New winter wheat winter barley hybrids were produced (Mv9 kr1 Igri, Mv9 kr1 Osnova, Asakazekomugi Manas). The wheat-barley hybrids showed entire male sterility and were multiplied in tissue culture. Chromosome configurations were studied with GISH in meiosis in the Mv9 krl x Igri hybrid and in its progenies multiplied in vitro. Chromosome pairing between wheat and barley has been observed in some cells in the hybrids multiplied in vitro. Backcross plants with 43 and 44 chromosomes were selected with the aim of developing new wheat-barley addition lines. Wheat-barley translocations were demonstrated with GISH in backcross progenies originating from in vitro regenerated wheat (Triticum aestivum L. cv. Chinese Spring) x barley (Hordeum vulgare L. cv. Betzes) hybrids. Five different translocations were observed. Sequential N-banding and GISH analyses were performed to further identify the translocations. The N-banding pattern of the Robertsonian translocation suggests that this chromosome consists of the short arm of barley chromosome 4H translocated to the long arm of 2B of wheat. Plants with four different homozygous translocations were selected from the following BC2F3 generation.

• The Plant Pathology Journal
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• 제23권3호
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• pp.155-160
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• 2007

In order to develop chili pepper bacterial spot resistant cultivars and near-isogenic lines (NILs) to prompt the molecular mapping of the resistance gene, we have run backcross breeding program since 1994. Two resistance genes against Xanthomonas axonopodis pv. vesicatoria Bs2 from Fla. XVR 3-25 and Bs3 from our breeding line 25-11-3-2, were introduced into a land race, Chilseongcho (abbreviated to Chilseong hereafter) with good fruit guality. We report here the testing of $BC_4F_3\;to\;BC_4F_5$. We found that $BC_4F_5$ lines of the crosses were homozygous with respect to the respective genes of introduction. The lines, in which Bs2 gene was introduced, were hypersensitively resistant to both race 1 and race 3 of X. axonopodis pv. vesicatoria, whereas, those in which Bs3 was introduced were resistant to race 1.

• Plant Biotechnology Reports
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• 제2권1호
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• pp.47-58
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• 2008

To select agronomically useful transgenic plants, a large number of transgenic events are initially produced, gene transfer confirmed, and advanced to obtain homozygous lines for testing in field trials. Direct in planta assays for identifying the transgene carriers in the segregating progeny are based on the activity of selectable marker gene and are easy, simple and inexpensive. For this purpose, expression of bar gene as measured by tolerance to damage by glufosinate ammonium, the active ingredient in the herbicide BASTA, was investigated. Dose damage curves were generated by leaf paint tests with BASTA on four genotypes of sorghum. Transgenic plants were characterized in terms of sensitivity to the concentration of glufosinate ammonium. In transgenics, symptoms of BASTA swab tests at different growth stages and PCR analysis for cry1B were carried out and correlated. Germination tests could not be employed for large scale evaluation of transgenic progeny because of mortality of tolerant seedlings after transplantation to soil. Based on the above findings, a simple, inexpensive, time-saving, two-step scheme for effective evaluation of transgenics and their progeny containing bar gene as selection marker using BASTA swab tests is described.

• Journal of Plant Biotechnology
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• 제40권2호
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• pp.88-101
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• 2013

Eating quality of rice attracts more and more attention from rice-eating consumers in the recent years. Thus, improvement of eating quality of cooked rice has become one of the most important breeding goals in japonica rice. Here, the generation of transgenic japonica rice with improved eating quality and grain yield are reported. Overexpression of OsSbe1 gene encoding rice starch branching enzyme 1 was driven by 35S promoter. Eleven independent homozygous $T_3$ transgenic lines were characterized and had shown higher palatability (71.2 ~ 72.6) than wild type Gopum (70.4). Moreover, transgenic rice lines showed an increase in 1000-grain weight and number of spikelets per panicle compared with the wild type. The yield of milled rice was 562.8 ~ 596.7 kg/10a in eight $T_3$ lines, but 542.1 kg/10a in wild type. Gene expression analyses in mRNA transcription and enzyme activity levels suggest that improved eating quality is due to the up-regulation of OsSbe1 gene.

• 한국육종학회지
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• 제40권3호
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• pp.211-214
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• 2008

Haploid system by anther culture allows the development of homozygous lines when doubled. The response of anther culture to Basta (glufosinate) resistance was investigated on transgenic plants (cv. Anjungbyeo) in order to identify inheritance of bar gene associated with Basta. Most of the regenerated transgenic plants were sterile, and only a few plants produced viable seeds ($A_1$) in the greenhouse. The bar gene was analysis by PCR in basta resistant transgenic plant ($TA_0$). The transgenic seeds ($A_1$) were significantly germinated in Basta solution compared with non-transformed seeds. As a result of anther culture, in regenerated haploid plants, segregation ratio was 1:1 in five of eight cross combinations. In diploid plants, segregation ratio was 1:1 in seven of eight cross combinations. Although there was some differences in the cross combinations, most of the combinations had 1:1 segregation ratio which supports the theory. The difference may be a result of the small sample size or the difference of anther culture response caused by genotypic difference. Hence, when many cross combinations were anther-cultured the results would support the theory.

• Plant Breeding and Biotechnology
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• 제6권4호
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• pp.434-443
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• 2018

Genotyping-by-sequencing (GBS) has been used as a viable single nucleotide polymorphism (SNP) validation method that provides reduced representation sequencing by using restriction endonucleases. Although GBS makes it possible to perform marker discovery and genotyping simultaneously with reasonable costs and a simple molecular biology workflow, the standard TASSEL-GBS pipeline was designed for homozygous groups, and genotyping of heterozygous groups is more complicated. To addresses this problem, we developed a GBS pipeline for heterozygous groups that called KNU-GBS pipeline, specifically for apple (Malus domestica). Using KNU-GBS pipeline, we constructed a genetic linkage map consisting of 1,053 SNP markers distributed over 17 linkage groups encompassing a total of 1350.1 cM. The novel GBS pipeline for heterozygous groups will be useful for marker-assisted breeding programs, and diverse heterozygous genome analyses.

• 원예과학기술지
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• 제29권2호
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• pp.123-129
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• 2011

To identify unintended vertical gene-transfer rates from the developed transgenic plants, rapid and unequivocal techniques are needed to identify event-specific markers based on flanking sequences around the transgene and to distinguish zygosity such as homo- and hetero-zygosity. To facilitate evaluation of zygosity, a polymerase chain reaction technique was used to analyze a transgenic pepper line B20 (homozygote), P915 wild type (null zygote), and their F1 hybrids, which were used as transgene contaminated plants. First, we sequenced the 3'-flanking region of the T-DNA (1,277 bp) in the transgenic pepper event B20. Based on sequence information for the 3'- and 5'-flanking region of T-DNA provided in a previous study, a primer pair was designed to amplify full length T-DNA in B20. We successfully amplified the full length T-DNA containing 986 bp from the flanking regions of B20. In addition, a 1,040 bp PCR product, which was where the T-DNA was inserted, was amplified from P915. Finally, both full length T-DNA and the 1,040 bp fragment were simultaneously amplified in the F1 hybrids; P915 ${\times}$ B20, Pungchon ${\times}$ B20, Gumtap ${\times}$ B20. In the present study, we were able to identify zygosity among homozygous transgenic event B20, its wild type P915, and hemizygous F1 hybrids. Therefore, this novel zygosity identification technique, which is based on PCR, can be effectively used to examine gene flow for transgenic pepper event B20.

• Journal of Plant Biotechnology
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• 제37권4호
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• pp.567-573
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• 2010

To ascertain the effect of over-expressed maize calreticulin in tomato plant on tobamovirus movement in addition to validating potentiality of the gene (ZmCRT) as a means for the virus-resistance resource, four ZmCRT-expressing homozygous lines were generated from the T0 plants as using an Agrobacterium-mediated transformation, nucleic acid analyses, and a conventional breeding method. Of them, a line was subjected to the bioassay for tolerances to tobacco mosaic virus-U1 (TMV-U1) and tomato mosaic virus (ToMV) followed by RT-PCR and a chlorophyll fluorescence quenching analyses. Both transgenic plants transcribing ZmCRT and wild-type plants showed no symptom by 20 days after viruses inoculation, however the photosystem II quantum yield parameter measured from the upper leaves of ToMV-inoculated plants revealed that ZmCRT transgenic plants have higher photosynthetic ability than wild-type ones at that time, which indirectly implies that over-expressed ZmCRT product acts as a barrier to the cell-to-cell and/or systemic movement of ToMV. Moreover, ZmCRT transgenic plants showed remarkably longer shoot length than wild-type ones in 40 days after TMV-U1 or ToMV inoculation each, which might be resulted from higher photosynthetic ability during the phase not yet showing any external symptoms. Collectively, over-expressed ZmCRT protein in tomato plants is able to interrupt the systemic movement of infected TMV-U1 and ToMV even though not perfect.

• Journal of Plant Biotechnology
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• 제34권1호
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• pp.11-17
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• 2007

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.

• Journal of Plant Biotechnology
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• 제42권3호
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• pp.186-195
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• 2015

Anthranilate synthase (AS)는 트립토판(Trp)과 indole-3-acetic acid, indole alkaloids의 생합성 경로에서 중요한 효소로 작용한다. 트립토판 생합성 상에서 feedback inhibition에 민감하게 반응하는 AS alpha-subunit 관련 OASA2 유전자 영역의 single (F124V) 및 double (S126F/L530D) 점돌연변이로 변형된 유전자의 재조합운반체를 작성하고 이들 유전자들을 Agrobacterium 방법으로 동진벼에 도입하여 형질전환체를 육성하였다. Single 및 double 돌연변이 OsASA2 유전자가 도입된 형질전환 벼 계통들은 nos gene probe를 이용한 TaqMan PCR 방법으로 single copy를 선발하였고, intergenic 계통을 선발하기 위해서 Bfa I 제한효소를 이용하여 RB와 LB 인접서열로부터 IPCR을 통한 FST 분석을 수행하여 4 개의 intergenic 계통을 선발하였다. 도입된 유전자의 발현으로 형질전환 벼는 Trp, IAN 및 IAA가 잎에 가장 많이 축적되었고, 종자의 트립토판 함량도 증가되었다. 후대에서 tryptophan 함량이 높은 S-TG와 D-TG의 두 호모 이벤트 계통을 육성하여 트립토판 함량을 분석한 결과 대조구에 비하여 13~30배 이상 높게 나타났으며, 유리아미노산의 함량도 증가하였다. 이벤트 계통을 이용하여 microarray 분석을 수행한 결과 세포 내 이온 수송, 영양분 공급 등에 영향을 주는 유전자군들이 up-regulation 되었고, 세포 내 기능유전자의 역할을 담당하는 조효소 등이 down-regulation 된 것을 확인 할 수 있었다. 이러한 결과는 선발된 두개의 상동성 이벤트 계통들이 고함량의 유리 트립토판 생산 벼의 육종에 효과적으로 이용될 수 있음을 보여준 결과로 생각된다.