• Journal of Plant Biotechnology
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• v.37 no.3
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• pp.275-282
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• 2010

Chloroplast transformation in higher plants offers many attractive advantages over nuclear transformation, including a high-level accumulation of foreign proteins, multi-gene expression in single transformation event via transgene stacking in operons and no position effect due to site-specific integration of transgenes by homologous recombination. Most importantly, chloroplast transgenic plants are eco-friendly because their transgenes are maternally inheritance in most crop plants. However, chloroplast transformation system has limited success in crops alike nuclear transformation. In the past two decades, great progress has been made to overcome the limitations of chloroplast transformation, thus expending chloroplast bioreactor to several important crops including soybean, carrot, lettuce, and oilseed. Therefore, it has become possible that chloroplast transformation of crops can be used not only for the improvement of agronomic traits, but also for the production of vaccines and high valuable therapeutic proteins in pharmaceutical industry.

• ALGAE
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• v.33 no.4
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• pp.351-358
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• 2018

The ability to transform the chloroplast of Cyanidioschyzon merolae was limited by lack of confirmed and reliable promoter sequences (among other reasons), capable of delivering stable or modulated DNA transcription followed by protein synthesis. Our research has confirmed the applicability of three selected chloroplast promoters in C. merolae chloroplast overexpression of the exogenous protein (i.e., chloramphenicol acetyltransferase) and genetic transformation. These results might facilitate further research on genetically modified strains of C. merolae to envisage yet unknown aspect of cellular and plastic physiology as well as C. merolae potential applications as bio-factories or sources of useful chemicals.

• Journal of Plant Biotechnology
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• v.33 no.3
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• pp.185-194
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• 2006

Chloroplasts are believed to be descended from certain cyanobacteria, which were taken up by phagocytosis into a host cell and lived there in a symbiotic relationship. In contrast to the current static concept on the chloroplast genome, its dynamism has been recently demonstrated: the chloroplast genome is active in intramolecular homolgous recombination, producing subgenomic circles when it obtains homolgous sequences via genetic transformation. Chloroplast tranformation in higher plants provides many advantages over nuclear transformation that include higher expression levels of transgenes, polycistronic expression of transgenes, and maternal transmission of transgenes. Tobacco has been used as a model for chloroplast genetic transformation. However, it is recently possible to transform the chloroplasts of other major food and economic crops including rice, soybean, and cotton. Chloroplast-transformed crops will be able to replace bioreactors using microorganisms for production of value-added proteins in future.

• Journal of Plant Biotechnology
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• v.35 no.1
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• pp.41-45
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• 2008

Laccase (EC 1.10.3.2) is a small group of enzymes that catalyze the oxidation of a broad range of phenolic compounds including hazardous and recalcitrant pollutants in the environment. This study attempted to develop an efficient system for production of a recombinant laccase by chloroplast genetic transformation of tobacco. Chloroplast transformation vector was constructed and introduced into the tobacco chloroplast genome using particle bombardment. Chloroplast-transformed plants were subsequently regenerated. PCR and southern blot analyses confirmed stable integration of the laccase gene into the chloroplast genome. Northern blot analysis revealed that mRNA of the laccase gene was highly expressed in chloroplast-transformed plants.

• Journal of Plant Biotechnology
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• v.46 no.4
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• pp.323-330
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• 2019

The high expression level of industrial and metabolically important proteins in plants can be achieved by plastid transformation. The CaIA vector, a Capsicum-specific vector harboring aadA (spectinomycin resistance), is a selectable marker controlled by the PsbA promoter, and the terminator is flanked by the trnA and trnI regions of the inverted repeat (IR) region of the plastid. The CaIA vector can introduce foreign genes into the IR region of the plastid genome. The biolistic method was used for chloroplast transformation in Scoparia dulcis with leaf explants followed by antibiotic selection on regeneration medium. Transplastomes were successfully screened, and the transformation efficiency of 3 transgenic lines from 25 bombarded leaf explants was determined. Transplastomic lines were evaluated by PCR and Southern blotting for the confirmation of aadA insertion and its integration into the chloroplast genome. Seeds collected from transplastomes were analyzed on spectinomycin medium with wild types to determine genetic stability. The increased chloroplast transformation efficiency (3 transplastomic lines from 25 bombarded explants) would be useful for expressing therapeutically and industrially important genes in Scoparia dulcis L.

• Journal of Plant Biotechnology
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• v.33 no.4
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• pp.233-236
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• 2006

Human serum albumin (HSA) is the most abundant protein in plasma and is the most often used intravenous protein in many human therapies. However, HSA is currently extracted only from plasma because commercially feasible recombinant expression systems are not available. This study attempted to develop an efficient system for recombinant HSA production by chloroplast transformation of tobacco. A HSA cDNA was isolated from a cDNA library constructed with human liver tissue. Chloroplast transformation vectors were constructed by introducing various regulatory elements to HSA regulatory sequences. Vectors were delivered by particle bombardment into leaf explants and chloroplast-transformed plants were subsequently regenerated into whole plants. Southern blot analysis confirmed that the HSA cDNA was incorporated between rps12 and orf70B of the chloroplast genome as designed. Western blot analysis revealed that hyper-expression and increasing the stability of HSA were achieved by modification of the regulatory sequences using the psbA5'UTRs in combination with elements of the 14 N-terminal amino acids of the GFP and the FLAG tag. However, only plants transformed with the vector containing all of these elements were able to accumulate HSA.

• Journal of Plant Biotechnology
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• v.34 no.3
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• pp.271-275
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• 2007

In the chloroplast transformation process, a chloroplast containing transformed chloroplast genome copies should be selected over wild-type chloroplasts on selection medium. It is more effective for a cell to become homoplasmic if the cell contains smaller number of chloroplasts. Therefore, to reduce the number of chloroplasts in mesophyll cells in tobacco, we overexpressed FtsZ to generate transgenic plants, of which mesophyll cell contained a few enlarged chloroplasts contrast to a wild-type mesophyll cell containing approximately 100 chloroplasts. It was demonstrated that transgenic leaf tissues comprising cells with a few enlarged chloroplasts gave rise to approximately 40% higher frequency of chloroplast-transformed adventitious shoots.

• Journal of Plant Biotechnology
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• v.38 no.1
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• pp.42-48
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• 2011

Chloroplast genetic engineering of higher plants offers several unique advantages compared with nuclear genome transformation, such as high levels of transgene expression, a lack of position effect due to site-specific transgene integration by homologous recombination, multigene engineering in a single transformation event and reducing risks of gene flow via pollen due to maternal inheritance. We established a reproducible chloroplast transformation system of potato using a tobacco specific plastid transformation vector, pCtVG (trnI-Prrn-aadA-mgfp-TpsbA-trnA). Stable transgene integration into chloroplast genomes and the homoplasmic state of the transgenome were confirmed by PCR and Southern blot analyses. Northern, immunoblot analysis, and GFP fluorescence imaging revealed high expression and accumulation of GFP in the plastids of potato leaves. This system would provide new opportunities for genetic improvement and mass production of value added foreign proteins in this crop.

• Journal of Plant Biotechnology
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• v.3 no.3
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• pp.113-121
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• 2001

The use of a marker gene in a transformation process aims to give a selective advantage to the transformed cells, allowing them to grow faster and better, and to kill the non-transformed cells. In general, the selective gene is introduced into plant genome along with the genes of interest. In some cases, the marker gene can be the gene of interest that will confer an agronomic characteristic, such as herbicide resistance. In this review we list and discuss the use of the most common selective marker genes on plant transformation and the effects of their respective selective agents. These genes could be divided in categories according their mode of action: genes that confer resistance to antibiotics and herbicides; and genes for positive selection. The contention of the marker gene flow through chloroplast transformation is further discussed. Moreover, strategies to recover marker-free transgenic plants, involving multi-auto-transformation (MAT), co-transformation, site specific recombination and intragenomic relocation of transgenes through transposable elements, are also reviewed.

• Proceedings of the Botanical Society of Korea Conference
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• 1999.07a
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• pp.79-84
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• 1999

Oxidative stress is one of the major environmental stresses to plants. Reactive oxygen species (ROS) generated during metabolic processes damage cellular functions and consequently lead to cell death. Fortunately plants have in vivo defense system by which the ROS is scavenged by enzymes such as superoxide dismutase (SOD) and ascorbate peroxidase (APX). In attempts to understand the protection mechanism of plant against oxidative stress, we developed transgenic tobacco (Nicotiana tabacum cv. Xanthi) plansts thet expressed both SOD and APX in chloroplast using Agrobacterum-mediated transformation and evaluated their protection capabilities against methyl viologen (MV, paraquat) -mediated oxidative damage. Three double transformants (CAI, CA2, and CA3) expressed the chimeric CuZnSOD and chimeric APX in chloroplast, and one transformant (AM) expressed the chimeric APX and chimeric MnSOD in chloroplast. In addition, we obtained three lines of transformants (C/Al, C/A2, and A/C) that expressed the APX and SOD than control plants, and more resistant to oxidative stress caused by MV. TRansformants (C/A and A/C) overexpressing MnSOD, CuZnSOD and APX at the same time showed the highest resistance to MV-mediated oxidative stress among the transformants.