Field Performance and Morphological Characterization of Transgenic Codonopsis lanceolata Expressing γ-TMT Gene.

Bimal Kumar Ghimire,Cheng Hao Li,Hyun-Young Kil,Na-Young Kim,Jung-Dae Lim,Jae-Kwang Kim,Myong-Jo Kim,Ill-Min Chung,Sun-Joo Lee,Seok-Hyun Eom,Dong-Ha Cho,Chang-Yeon Yu 한국약용작물학회 2007 한국약용작물학회지 Vol.15 No.5

Field performance and morphological characterization was conducted on seven transgenic lines of Codonopsis lanceolata expressing γ-TMT gene. The shoots were obtained from leaf explants after co-cultivation with Agrobacterium tume-faciens strain LBA 4404 harboring a binary vector pYBI 121 that carried genes encoding γ-Tocopherol methyltransferase gene (γ-TMT) and a neomycin phosphotransferase II gene (npt II) for kanamycin resistance. The transgenic plants were transferred to a green house for acclimation. Integration of T-DNA into the T0 and T1 generation of transgenic Codonopsis lanceolata genome was confirmed by the polymerase chain reaction and southern blot analysis. The progenies of transgenic plants showed phenotypic differences within the different lines and with relative to control plants. When grown in field, the transgenic plants in general exhibited increased fertility, significant improvement in the shoot weight, root weight, shoot height and rachis length with relation to the control plants. However, all seven independently derived transgenic lines produced normal flower with respect to its shape, size, color and seeds number at its maturity. Indicating that the addition of a selectable marker gene in the plant genome does not effect on seed germination and agronomic performance of transgenic Codonopsis lanceolata. T1 progenies of these plants were obtained and evaluated together with control plant in a field experiment. Overall, the agronomic performance of T1 progenies of transgenic Codonopsis lanceolata showed superior to that of the seed derived non-transgenic plant. In this study, we report on the morphological variation and agronomic performance of transgenic Codonopsis lanceolata developed by Agrobacterium transformation.

Overexpression of human erythropoietin in tobacco does not affect plant fertility or morphology

Tamba A. Musa,Chiu-Yueh Hung,Diane E. Darlington,David C. Sane,Jiahua Xie 한국식물생명공학회 2009 Plant biotechnology reports Vol.3 No.2

Human erythropoietin (EPO) is a leading product in the biopharmaceutical market, but functional EPO has only been produced in mammalian cells, which limits its application and drives up the production costs. Using plants to produce human proteins may be an alternative way to reduce the cost. However, a recent report demonstrated that overexpression of the human EPO gene (EPO) in tobacco or Arabidopsis rendered males sterile and retarded vegetative growth, which raises concern whether EPO might interfere with hormone levels in transgenic plants. In the present study, we demonstrated that overexpressing EPO with additional 50-His tag and 30 ERretention peptides in tobacco did not cause any developmental defect compared to GUS plants. With our method, all 20 transgenic plants grew on selective medium and, further confirmed by PCR, were fertile. Most of them grew similarly compared to GUS plants. Only one transgenic plant (EPO2) was shorter in plant height but had twice the life span compared to other transgenic plants. When 11 randomly selected EPO plants, along with the abnormal plant EPO2, were subjected to RT-PCR analysis, all of them had detectable EPO transcripts. However, their protein levels varied considerably; seven of them had detectable EPO proteins analyzed by western blot. Our results indicate that overexpressing human EPO protein in plants does not have detrimental effects on growth and development. Our transformation systems allow us to further explore the possibility of glycoengineering tobacco plants for producing functional EPO and its derivatives. Human erythropoietin (EPO) is a leading product in the biopharmaceutical market, but functional EPO has only been produced in mammalian cells, which limits its application and drives up the production costs. Using plants to produce human proteins may be an alternative way to reduce the cost. However, a recent report demonstrated that overexpression of the human EPO gene (EPO) in tobacco or Arabidopsis rendered males sterile and retarded vegetative growth, which raises concern whether EPO might interfere with hormone levels in transgenic plants. In the present study, we demonstrated that overexpressing EPO with additional 50-His tag and 30 ERretention peptides in tobacco did not cause any developmental defect compared to GUS plants. With our method, all 20 transgenic plants grew on selective medium and, further confirmed by PCR, were fertile. Most of them grew similarly compared to GUS plants. Only one transgenic plant (EPO2) was shorter in plant height but had twice the life span compared to other transgenic plants. When 11 randomly selected EPO plants, along with the abnormal plant EPO2, were subjected to RT-PCR analysis, all of them had detectable EPO transcripts. However, their protein levels varied considerably; seven of them had detectable EPO proteins analyzed by western blot. Our results indicate that overexpressing human EPO protein in plants does not have detrimental effects on growth and development. Our transformation systems allow us to further explore the possibility of glycoengineering tobacco plants for producing functional EPO and its derivatives.

Growth and Yield Response of Transgenic Rice Plants Expressing Protoporphyrinogen Oxidase Gene from Bacillus subtilis

Jung Sung Chung,Yong In Kuk,Sunyo Jung,Kyoungwhan Back,Han Yong Kim,Ja Ock Guh 韓國作物學會 2003 Korean journal of crop science Vol.48 No.4

Transgenic rice plants expressing a Bacillus subtilis protoporphyrinogen oxidase (Protox), the last shared enzyme of the porphyrin pathway in the expressed cytoplasm or the plastids, were compared with non-trangenic rice plants in their growth characteristics such as tiller number, plant height, biomass, and yield. Transgenic rice plants of ~textrmT3 generation had 8 to 15 % and 25 to 43% increases in tiller number compared to non-transgenic rice plants at 4 and 8 weeks after transplanting(WAT); similar values were observed for ~textrmT4 generation at 4 and 8 WAT. However, the plant height in both ~textrmT3 and ~textrmT4 generations was similar between transgenic rice plants and non-transgenic rice plants at 4 and 8 WAT. Transgenic rice plants had 13 to 32% increase in above-ground biomass and 9 to 28% increase in grain yield compared to non-transgenic rice plants, demonstrating that biomass and yield correlate with each other. The increased grain yield of the transgenic rice plants was closely associated with the increased panicle number per plant. The percent of filled grain, thousand grains and spikelet number per panicle were similar between transgenic and non-transgenic rice plants. Generally, the growth and yield of transgenic generations (~textrmT2 , ~textrmT3 , and ~textrmT4 ) and gene expressing sites (cytoplasm-expressed and plastid-targeted transgenic rice plants) were similar, although they slightly varied with generations as well as with gene expressing sites. The transgenic rice plants had promotive effects, indicating that regulation of the porphyrin pathway by expression of B. subtilis Protox in rice influences plant growth and yield.

Field Performance and Morphological Characterization of Transgenic Codonopsis lanceolata Expressing $\gamma-TMT$ Gene.

Ghimire, Bimal Kumar,Li, Cheng Hao,Kil, Hyun-Young,Kim, Na-Young,Lim, Jung-Dae,Kim, Jae-Kwang,Kim, Myong-Jo,Chung, Ill-Min,Lee, Sun-Joo,Eom, Seok-Hyun,Cho, Dong-Ha,Yu, Chang-Yeon The Korean Society of Medicinal Crop Science 2007 韓國藥用作物學會誌 Vol.15 No.5

Field performance and morphological characterization was conducted on seven transgenic lines of Codonopsis lanceolata expressing ${\gamma}-TMT$ gene. The shoots were obtained from leaf explants after co-cultivation with Agrobacterium tume-faciens strain LBA 4404 harboring a binary vector pYBI 121 that carried genes encoding ${\gamma}-Tocopherol$ methyltransferase gene (${\gamma}-TMT$) and a neomycin phosphotransferase II gene (npt II) for kanamycin resistance. The transgenic plants were transferred to a green house for acclimation. Integration of T-DNA into the $T_0\;and\;T_1$ generation of transgenic Codonopsis lanceolata genome was confirmed by the polymerase chain reaction and southern blot analysis. The progenies of transgenic plants showed phenotypic differences within the different lines and with relative to control plants. When grown in field, the transgenic plants in general exhibited increased fertility, significant improvement in the shoot weight, root weight, shoot height and rachis length with relation to the control plants. However, all seven independently derived transgenic lines produced normal flower with respect to its shape, size, color and seeds number at its maturity. Indicating that the addition of a selectable marker gene in the plant genome does not effect on seed germination and agronomic performance of transgenic Codonopsis lanceolata. $T_1$ progenies of these plants were obtained and evaluated together with control plant in a field experiment. Overall, the agronomic performance of $T_1$ progenies of transgenic Codonopsis lanceolata showed superior to that of the seed derived non-transgenic plant. In this study, we report on the morphological variation and agronomic performance of transgenic Codonopsis lanceolata developed by Agrobacterium transformation.

Simultaneous overexpression of both CuZn superoxide dismutase and ascorbate peroxidase in transgenic tall fescue plants confers increased tolerance to a wide range of abiotic stresses

Lee, Sang-Hoon,Ahsan, Nagib,Lee, Ki-Won,Kim, Do-Hyun,Lee, Dong-Gi,Kwak, Sang-Soo,Kwon, Suk-Yoon,Kim, Tae-Hwan,Lee, Byung-Hyun G. Fischer 2007 Journal of plant physiology Vol. No.

<P><B>Summary</B></P><P>To mitigate the oxidative damage inflicted by biotic or abiotic stresses, plants have evolved complex anti-oxidative defense mechanisms that involve induction of antioxidant and anti-oxidative enzymes, such as superoxide dismutase (SOD) and ascorbate peroxidase (APX). To determine whether overexpression of the genes encoding copper–zinc SOD (CuZnSOD) and APX in plants is capable of decreasing reactive oxygen species (ROS) produced in response to abiotic stresses, we generated transgenic tall fescue plants expressing the CuZnSOD and APX genes in chloroplasts under the control of the oxidative stress-inducible promoter, sweet potato peroxidase anionic 2 (SWPA2). Transgenic plants were generated by <I>Agrobacterium</I>-mediated genetic transformation, and genotypes were confirmed by DNA blot analysis. Transgenic plants were exposed to several ROS-generating abiotic stresses, such as methyl viologen (MV), H<SUB>2</SUB>O<SUB>2</SUB>, and the heavy metals copper, cadmium, and arsenic, and their tolerance was evaluated. High levels of CuZnSOD and APX gene transcripts in the transgenic plants under these treatments suggested that the transgenes were functionally expressed. Compared to transgenic plants, higher amounts of ROS were generated in the leaves of control plants exposed to abiotic stresses, resulting in increased thiobarbituric acid reactive substances (TBARS), ion leakage, and chlorophyll degradation. These parameters were significantly lower in transgenic plants. Enzyme activity assays and native polyacrylamide gel electrophoresis (PAGE) showed that total SOD and APX were highly active in transgenic plants under the abiotic stresses examined. We conclude that one of the mechanisms of increased anti-oxidative defense in transgenic tall fescue plants is overexpression of the CuZnSOD and APX genes, which are utilized in scavenging ROS and thus provide improved tolerance to abiotic stresses.</P>

Generation and molecular characterization of marker-free Bt transgenic rice plants by selectable marker-less transformation

우희종,이승범,Yang Qin,임명호,이진형,신공식,조현석,박순기 한국식물생명공학회 2015 Plant biotechnology reports Vol.9 No.6

Public concern about the safety of genetically modified (GM) crops and potential pleiotropic effects of selectable marker gene insertion and expression can be avoided by developing a selectable marker-free transformation system. Various techniques have been developed to produce marker-free transgenic plants; however, an approach that does not require the use of selectable marker genes would not require post-transformation processes. We report such a method for generating of GM rice plants. To develop transgenic rice resistant to lepidopteran insects, a marker-free binary vector harboring the Bacillus thuringiensis mcyr1Ac gene was constructed and used for Agrobacterium tumefaciens-mediated transformation of rice scutellar calli. Initial screening of putative T0 transgenic rice plants was performed using polymerase chain reaction (PCR) on pools of DNA from 15 to 30 regenerated shoots, followed by genomic DNA PCR and Cry1Ac ImmunoStrip assays of individual plant extracts. Although the overall rice transformation efficiency of the non-selection approach is lower than that of traditional rice transformation by using marker genes, we derived six independent homozygous T3 events from Cry+ T0 shoots. Transgenes were detected and inheritance confirmed using DNA PCR, RT-PCR, and Southern blot analysis, and insertion sites were characterized by the sequencing of DNA flanking the transgenes. The transgene expression of cry1Ac was also stable and effective against rice leaf folder at levels comparable to those of a transgenic plant that had been derived by selection. This method could be applied to other plant species with similar transformation efficiencies to create selectable-marker-free transgenic plants for research and commercial uses.

Transgenic Tobacco Plants Expressing a Mutant VU-4 Calmodulin Have Altered Nicotinamide Co-Enzyme Levels and Hydrogen Peroxide Levels

Oh, Suk-Heung,Park, Yoon-Sick,Yang, Moon-Sik Korean Society for Biochemistry and Molecular Biol 1999 Journal of biochemistry and molecular biology Vol.32 No.1

In order to understand the biological role of calmodulin in plants, transgenic tobacco plants expressing a calmodulin mutant (VU-4 calmodulin, lys to ile-115) gene have been analyzed. SDS-PAGE and Western-blot analyses showed that the foreign calmodulin mutant is stably and highly expressed in the transgenic tobacco plants. The levels of $H_2O_2$were elevated approximately 2-fold in the transgenic plants. Furthermore, the transgenic tobacco plants have more than 6-fold higher levels of NADPH compared to control tobacco plants. The present findings, combined with previous data showing differences in the susceptibility of the transgenic tobacco seeds and normal tobacco seeds to fungal contamination (Oh and Yang, 1996), suggest that the expression of the calmodulin derivative gene in tobacco plants could increase resistance to infection by fungal pathogens.

Transgenic Tobacco Plants Expressing a Mutant VU-4 Calmodulin Have Altered Nicotinamide Co-Enzyme Levels and Hydrogen Peroxide Levels

Oh, Suk-Heung,Park, Yoon-Sick,Yang, Moon-Sik The Korea Science and Technology Center 1999 BMB Reports Vol.32 No.1

In order to understand the biological role of calmodulin in plants, transgenic tobacco plants expressing a calmodulin mutant (VU-4 calmodulin, lys to ile-115) gene have been analyzed. SDS-PAGE and Western-blot analyses showed that the foreign calmodulin mutant is stably and highly expressed in the transgenic tobacco plants. The levels of H₂O₂ were elevated approximately 2-fold in the transgenic plants. Furthermore, the transgenic tobacco plants have more than 6-fold higher levels of NADPH compared to control tobacco plants. The present findings, combined with previous data showing differences in the susceptibility of the transgenic tobacco seeds and normal tobacco seeds to fungal contamination (Oh and yang,1996), suggest that the expression of the calmodulin derivative gene in tobacco plants could increase resistance to infection by fungal pathogens.

토양 중 고농도 카드뮴에 노출된 MuS1 형질전환 담배 (Nicotiana tabacum cv. Xanthi)의 생리적 반응 및 카드뮴 축적

정윤화(Yoon-hwa Jeoung),김영남(Young-Nam Kim),김권래(Kwon-Rae Kim),김계훈(Kye-Hoon Kim) 한국토양비료학회 2013 한국토양비료학회지 Vol.46 No.1

The objective of this study was to understand the physiological response and cadmium accumulation of MuS1 transgenic tobacco exposed to high concentration of Cd in soil. For this, a pot experiment was carried out in a greenhouse for a month, with two lines of MuS1 transgenic tobaccos (S4 and S6) and non-transgenic tobacco cultivated in the soils spiked at three different Cd concentrations (0, 60 and 180 mg kg^-1). Both transgenic and non-transgenic tobacco showed visible toxic symptoms such as chlorosis and leaf roll as treated concentration increased. The net photosynthetic rates of MuS1 plants (S4 and S6) exposed at 180 mg kg^-1 Cd were 6.3 and 7.7 μmol m^-2s^-1, being higher than those of the non-transgenic plant (4.8 μmol m^-2s^-1). Values of stomatal conductance of MuS1 transgenic plants (0.05 and 0.008 mmol H₂O m^-2s^-1) were also higher than those of non-transgenic plant (0.03 mmol H₂O m^-2s^-1). In addition, fresh and dry weights of MuS1 transgenic plants were heavier than those of non-transgenic plant. Likewise, MuS1 transgenic plants appeared to be better physiological performance than non-transgenic tobacco when exposed at high concentration of Cd in soil. With regard to metal accumulation, MuS1 transgenic tobaccos accumulated more Cd in their roots than non-transgenic tobacco implying that MuS1 transgenic tobacco is suggested to be used for phytostabilization of heavy metals.

The Burholderia pyrrocinia Purple Acid Phosphatase Pap9 Mediates Phosphate Acquisition in Plants

Xiaoli Zhu,이승엽,양원태,이선우,백동원,Ming Shun Li,김도훈 한국식물학회 2019 Journal of Plant Biology Vol.62 No.5

Increasing the expression level of purple acidphosphatases (PAPs), which hydrolyze organic phosphate toinorganic phosphate (Pi), is an important response to Pi starvationin plants. PAPs are widely distributed in eukaryotes likemammals and plants but distributed in limited microorganisms. A PAP-like protein named Pap9 from Burkholderia pyrrociniaCH-67 was isolated. The enzyme had optimal activity atatypical pH of 8.5 and temperature of 85°C. When pap9 wasoverexpressed in Arabidopsis thaliana, the APase activity inthe transgenic plants was about 15% to 40% higher underacidic conditions and had an approximately four-fold increaseunder alkaline conditions compared with wild type. Overexpression of pap9 in plants enhanced APase activityunder Pi-deficient conditions more than under Pi-sufficientconditions. In-gel assays revealed that Pap9 exists as amonomer in transgenic plants. Root surface-associatedAPase activity in transgenic plants increased dramaticallyduring acidic and Pi-deficient conditions. These resultsindicated that bacterial Pap9 in transgenic plants wassecreted onto the root surface and released into the rhizosphere. The transgenic plants have significantly enhanced biomassthat have the potential for use in biotechnologicalapplications.