• 한국식물생명공학회:학술대회논문집
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• 한국식물생명공학회 2005년도 추계학술대회 및 한일 식물생명공학 심포지엄
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• pp.406-406
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• 2005

• Journal of Plant Biotechnology
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• 제7권4호
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• pp.211-218
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• 2005

Genetic transformation was carried out by using biolistic gun method. The hypocotyl derived embryogenic calli (explants) of cotton (Gossypium hirsutum L.) cv. Cocker-312 were transformed with a recombinant pGreen II plasmid, in which both, bar (selection marker) and GUS (${\beta}$-glucuronidase) reporter genes were incorporated. Explants were arranged on osmoticum-containing medium (0.5M mannitol) 4 hours prior to and 16 hours after bombardment that was resulted into an increase about >80% for GUS stable expression. 3 days after bombardment, GUS assay was performed, which exhibited, $18.36{\pm}1.00$ calli showed blue spots. The transformed embryogenic calli were cultured on selection medium (@ 6 mg/L basta) for 3 months. The putative transgenic plants were developed via selective somatic embryogenesis (@1.50 mg/L basta); maximum $27.58{\pm}1.25$ somatic embryos were obtained while $17.47{\pm}1.00$ embryos developed into plantlets (@ 0.75mg/L basta). In five independent experiments, up to 7.24% transformation efficiency was recorded. The presence of the transgenes was analyzed by using PCR and southern hybridization analysis. The transgenic plants were developed with in 6-7 months, but mostly transformants were abnormal in morphology.

• Journal of Plant Biotechnology
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• 제1권1호
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• pp.31-38
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• 1999

An artificial symbiosis was established between diazotropic Azomonas insignis and strawberry (Fragaria x ananassa). The partnership was created by in vitro techniques through callus induction and organogenesis. The basis of this partnerships is the bacterial dependence on the plants metabolic activity, using maltose in the medium as a carbon and energy source which can be utilized by the plant cells only. The presence of bacteria in the intercellular spaces of the callus tissues and regenerated plants was proven by microscopic techniques. Nitrogenase activity could also be detected in the plant tissues. For successful and high frequency introduction of bacteria to the plant tissues, biolistic gun method was used. On the basis of the DNA transfer method, Azotobacter vinelandii bacteria were delivered directly into strawberry tissues by the particle bombardment. This was the first use of living bacteria as microprojectils for bombardment of plant tissues. The treatment was successful, the presence of bacteria in the developing callus tissue and regenerated plants were detected by light and electron microscopy.

• Journal of Plant Biotechnology
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• 제48권4호
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• pp.246-254
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• 2021

Environmental stresses are estimated to have reduced global crop yields of wheat by 5.5%. However, traditional approaches for the transfer of resistance to these stresses in wheat plants have yielded limited results. In this regard, genetic transformation has undoubtedly opened up new avenues to overcome crop losses due to various abiotic stresses. Particle bombardment has been successfully employed for obtaining transgenic wheat. However, most of these procedures employ immature embryos, which are not available throughout the year. Therefore, the present investigation utilized mature seeds as the starting material and used the calli raised from three Moroccan durum wheat varieties as the target tissue for genetic transformation by the biolistic approach. The pANIC-5E plasmid containing the SINA gene for drought and salinity tolerance was used for genetic transformation. To enhance the regeneration capacity and transformation efficiency of the tested genotypes, the study compared the effect of copper supplementation in the induction medium (up to 5 μM) with the standard MS medium. The results show that the genotypes displayed different sensitivities to CuSO₄, indicating that the transformation efficiency was highly genotype-dependent. The integration of transgenes in the T₀ transformants was demonstrated by polymerase chain reaction (PCR) analysis of the obtained resistant plantlets with primers specific to the SINA gene. Among the three genotypes studied, 'Isly' showed the highest efficiency of 9.75%, followed by 'Amria' with 1.25% and 'Chaoui' with 1%.

• Journal of Plant Biotechnology
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• 제5권3호
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• pp.163-168
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• 2003

A routine system based on particle bombardment of embryogenic callus for recovery of fertile transgenic rice (Oryza sativa L.) plants was developed. Embryogenic callus was established within 2-3 months from calli derived from mature seeds of Korean rice cultivar, Nagdongbyeo. The callus was bombarded with the plasmid pRQ6 containing the $\beta$-glucuronidase gene (gusA) and hygromycin phosphotransferase gene (hph, conferring resistance to hygromycin B), both driven by CaMV 35S promoter. Placement of cells on an osmoticum-containing medium (0.2 M sorbitol and 0.2 M mannitol) 4 hrs prior to and 16 hrs after bombardment resulted in a statistically significant increase with 3.2-fold in transient expression frequency gusA. In five independent experiments, the average frequency of transformation showing GUS activities was $8.86\%$. A large number of morphologically normal, fertile transgenic rice plants were obtained. Integration of foreign gene into the genome of $R_0$ transgenic plants was confirmed by Southern blot analysis. GUS and HPT were detected in $R_1$ progeny and Mendelian segregation of these genes was observed in $R_1$ progeny.

• Applied Biological Chemistry
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• 제39권4호
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• pp.260-264
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• 1996

Cymbidium속 난의 형질전환계를 확립하기 위해서, microprojectile bombardment 방법을 이용하여 춘란(Cymbidium virescence)의 rhizome 조직세포에 외래 유전자를 도입하는 조건을 검토하였다. 각 parameter별 적정조건으로서 텅스텐 입자의 크기는 $1.11\;{\mu}m$, He 가스 압력은 $77.33kg/cm^2$, gap distance는 6.35mm, target distance는 7.0cm 이었다. DNA피복입자를 투사한 $400{\mu}m$ 두께의 rhizome 절편을 2개월간 배양한 뒤 GUS 활성을 조사한 결과 이 유전자가 발현되는 세포들이 관찰되었다. Kanamycin (100 mg/L)을 첨가한 배지에서 6개월 동안 선택배양을 통해 얻은 rhizome의 DNA를 PCR로 분석한 결과 nptII 유전자의 삽입을 확인할 수 있었다.

• Journal of Plant Biotechnology
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• 제6권1호
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• pp.9-13
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• 2004

Transient uidA expression was used to optimize parameters required for biolistic transformation of suspension cells of Easter lily, Lilium longiflourm. Maximum uidA expression occurred following bombardment with gold particles as compared to tungsten. A 3hr pre-treatment of suspension cells with 0.125M osmoticum resulted in a 1.5X increase in uidA expression. A helium pressure of 1550 psi combined with a particle travelling distance of 6cm resulted in maximum uidA expression as compared to either 1100, 1200, or 1800 psi. Transient transformation resulted in up to 493 uidA expressing cells/Petri plate. For stable transformation suspension cells of Lilium longiflorum, were co-bombarded with plasmid DNA containing cucumber mosaic virus (CMV) replicase under the rice actin (Act1) promoter and either the bar or PAT genes under the cauliflower mosaic virus (CaMV 355) promoter. Ten regenerated plants contained the transgene as analyzed by PCR, and two of the ten plants were confirmed to contain the transgene by Southern hybridization. The two transgenic plants were independent transformants, one containing the bar gene and the other both the CMV replicase and bar genes. Plants were sprayed at the rosette stage and found to be resistant to 1000 mg/L of phosphinothricin (Trade name-Ignite) indicating expression of the bar gene throughout the leaves when bar was under control of the CaMV 35S promoter.

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.81.2-82
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• 2003

Plants have evolved along with pathogens, and they have developed sophisticated defense systems against specific microorganisms to survive. G-protons are considered one of the upstream signaling components working as a key for the defense signal transduction pathway. For activation and inactivation of G-protein, GTP-biding proteins are involved. GTP -binding proteins are found in all organisms. Small GTP-binding proteins, having masses of 21 to 30kD, belong to a superfamily, often named the Ras supefamily because the founding members are encoded by human Ras genes initially discovered as cellular homologs of the viral ras oncogene. Members of this supefamily share several common structural features, including several guanine nucleotide binding domains and an effector binding domain. However, exhibiting a remarkable diversity in both structure and function. They are important molecular switches that cycle between the GDP-bound inactive form into the GTP-bound active form through GDP/GTP replacement. In addition, most GTP-binding proteins cycle between membrane-bound and cytosolic forms. such as the RAC family are cytosolic signal transduction proteins that often are involved in processing of extracellular stimuli. Plant RAC proteins are implicated in regulation of plant cell architecture secondary wall formation, meristem signaling, and defense against pathogens. But their molecular mechanisms and functions are not well known. We isolated a RacB homolog from rice to study its role of defense against pathogens. We introduced the constitutively active and the dominant negative forms of the GTP-hinging protein OsRacB into the wild type rice. The dominant negative foms are using two forms (full-sequence and specific RNA interference with RacB). Employing southern, and protein analysis, we examine to different things between the wild type and the transformed plant. And analyzing biolistic bombardment of onion epidermal cell with GFP-RacB fusion protein revealed association with the nucle.

• Molecules and Cells
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• 제40권11호
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• pp.828-836
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• 2017

Eukaryotic cells consist of a complex network of thousands of proteins present in different organelles where organelle-specific cellular processes occur. Identification of the subcellular localization of a protein is important for understanding its potential biochemical functions. In the post-genomic era, localization of unknown proteins is achieved using multiple tools including a fluorescent-tagged protein approach. Several fluorescent-tagged protein organelle markers have been introduced into dicot plants, but its use is still limited in monocot plants. Here, we generated a set of multicolored organelle markers (fluorescent-tagged proteins) based on well-established targeting sequences. We used a series of pGWBs binary vectors to ameliorate localization and co-localization experiments using monocot plants. We constructed different fluorescent-tagged markers to visualize rice cell organelles, i.e., nucleus, plastids, mitochondria, peroxisomes, golgi body, endoplasmic reticulum, plasma membrane, and tonoplast, with four different fluorescent proteins (FPs) (G3GFP, mRFP, YFP, and CFP). Visualization of FP-tagged markers in their respective compartments has been reported for dicot and monocot plants. The comparative localization of the nucleus marker with a nucleus localizing sequence, and the similar, characteristic morphology of mCherry-tagged Arabidopsis organelle markers and our generated organelle markers in onion cells, provide further evidence for the correct subcellular localization of the Oryza sativa (rice) organelle marker. The set of eight different rice organelle markers with four different FPs provides a valuable resource for determining the subcellular localization of newly identified proteins, conducting co-localization assays, and generating stable transgenic localization in monocot plants.