Antibiotic-free chloroplast genetic engineering – an environmentally friendly approach

Daniell, Henry; Wiebe, Peter O.; Millán, Alicia Fernández‐San

doi:10.1016/s1360-1385(01)01949-5

Cited by 35 publications

(16 citation statements)

References 15 publications

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“…Moreover, several methods have been reported to generate selection marker-free transgenic plants, including site-specific recombination and intrachromosomal recombination to remove the selection marker and cotransformation and transposable elements to segregate the selection marker (15,16). In the chloroplast transformation system, a method that did not use an antibiotic resistance gene was established (17). However, such a method is generally time-consuming and inefficient.…”

Section: Resultsmentioning

confidence: 99%

Isolation of a rice regeneration quantitative trait loci gene and its application to transformation systems

Nishimura

Ashikari

Lin

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

146

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Regeneration of plant organs is often the essential step in genetic transformation; however, the regeneration ability of a plant varies depending on the genetic background. By conventional crosses of low-regeneration rice strain Koshihikari with high-regeneration rice strain Kasalath, we identified some quantitative trait loci, which control the regeneration ability in rice. Using a map-based cloning strategy, we isolated a main quantitative trait loci gene encoding ferredoxin-nitrite reductase (NiR) that determines regeneration ability in rice. Molecular analyses revealed that the poor regeneration ability of Koshihikari is caused by lower expression than in Kasalath and the specific activity of NiR. Using the NiR gene as a selection marker, we succeeded in selectively transforming a foreign gene into rice without exogenous marker genes. Our results demonstrate that nitrate assimilation is an important process in rice regeneration and also provide an additional selectable marker for rice transformation.regeneration ability ͉ ferredoxin-nitrate reductase ͉ selectable marker R egeneration of plants from cell culture is a critical step in the production of novel varieties of plants. Generally, it is not easy to culture and regenerate monocot plants, including agronomically important crops such as rice, wheat, and maize. In rice, an efficient culture system using mature seeds has been established based on research with model varieties such as Nipponbare (Japonica) and Kasalath (Indica). However, many leading varieties used for food production, such as Koshihikari in Japan and IR64 in tropical countries, have low regeneration ability in the mature seed culture system, resulting in a serious obstacle to efficient production of transgenic plants. It has been indicated that regeneration ability depends mainly on a few key genes (1-7), but no gene has yet been identified in any plant species. To understand the regeneration process and resolve the low regeneration ability of a leading Japanese variety, Koshihikari, we have attempted to isolate major quantitative trait loci (QTL), which would increase the regeneration ability of Koshihikari. Materials and MethodsCulture Conditions and Regeneration Test. Mature seeds were dehusked and surface-sterilized in 70% ethanol for 30 s, vigorously shaken in 1.5% sodium hypochlorite for 30 min, and rinsed five times in sterilized water. For the induction of calli, sterilized seeds were placed on the surface of an agar medium containing CHU (N 6 ) basal salt mixture (Sigma), 2 mg͞liter glycine, 0.5 mg͞liter nicotinic acid, 0.5 mg͞liter pyridoxine-HCl, 1 mg͞liter thiamine-HCl, 0.1 g͞liter myo-inositol, 0.3 g͞liter casamino acid, 2.878 g͞liter proline, 2 mg͞liter 2,4-dichlorophenoxyacetic acid, 30 g͞liter sucrose, and 3 g͞liter gelrite. The medium was adjusted to pH 5.8. Seeds were incubated in the medium at 29.5°C. Four weeks after inoculation calli formed from seeds were transferred onto regeneration medium containing MS plant salt mixture (Wako), 5 mg͞liter nicotinic acid, 10 mg͞lit...

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Section: Resultsmentioning

confidence: 99%

Isolation of a rice regeneration quantitative trait loci gene and its application to transformation systems

Nishimura

Ashikari

Lin

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

146

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“…Several recent advances should make chloroplasts even more attractive as biopharmaceutical reactors. These include engineering multiple foreign genes as operons [3]; transformation without use of antibiotic markers [4,5]; elimination of resistance genes subsequent to transformation [18,22,23]; and the transformation of edible crops such as potato and tomato [24,25]. This review highlights these and other recent achievements.…”

Section: Brief History Of Chloroplast Genetic Engineeringmentioning

confidence: 99%

“…Gene silencing, frequently observed in nuclear transgenic plants, has not been observed in genetically engineered chloroplasts. The ability to express foreign proteins at high levels in chloroplasts and chromoplasts, and to engineer foreign genes without the use of antibiotic resistant genes [4,5],make this compartment ideal for the development of edible vaccines [6]. Moreover, the ability of chloroplasts to form disulfide bonds and to fold human proteins has opened the door to high-level production of biopharmaceuticals in plants [7].…”

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confidence: 99%

Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology

Daniell

Khan

Allison

2002

Trends in Plant Science

322

153

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Chloroplast genomes defied the laws of Mendelian inheritance at the dawn of plant genetics, and continue to defy the mainstream approach to biotechnology, leading the field in an environmentally friendly direction. Recent success in engineering the chloroplast genome for resistance to herbicides, insects, disease and drought, and for production of biopharmaceuticals, has opened the door to a new era in biotechnology. The successful engineering of tomato chromoplasts for high-level transgene expression in fruits, coupled to hyper-expression of vaccine antigens, and the use of plant-derived antibiotic-free selectable markers, augur well for oral delivery of edible vaccines and biopharmaceuticals that are currently beyond the reach of those who need them most.Chloroplast transformation is an environmentally friendly approach to plant genetic engineering that minimizes out-crossing of transgenes to related weeds or crops [1,2] and reduces the potential toxicity of transgenic pollen to non-target insects [3]. Because the plastid genome is highly polyploid, transformation of chloroplasts permits the introduction of thousands of copies of foreign genes per plant cell, and generates extraordinarily high levels of foreign protein [3]. Chloroplast transformation vectors use two targeting sequences that flank the foreign genes and insert them, through homologous recombination, at a precise, predetermined location in the organelle genome (Fig. 1). This results in uniform transgene expression among transgenic lines and eliminates the 'position effect' often observed in nuclear transgenic plants. Gene silencing, frequently observed in nuclear transgenic plants, has not been observed in genetically engineered chloroplasts. The ability to express foreign proteins at high levels in chloroplasts and chromoplasts, and to engineer foreign genes without the use of antibiotic resistant genes [4,5],make this compartment ideal for the development of edible vaccines [6]. Moreover, the ability of chloroplasts to form disulfide bonds and to fold human proteins has opened the door to high-level production of biopharmaceuticals in plants [7]. Furthermore, foreign proteins observed to be toxic in the cytosol are non-toxic when accumulated within transgenic chloroplasts [6,8]. Chloroplast and nuclear genetic engineering are compared in Table 1.

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“…Examples are those that used npt II, hpt and bar to develop the Wrst generation of transgenic crops regardless of tissue systems (Haldrup et al 1998a;Miki and McHugh 2004), confers antibiotic resistance along with the gene of interest. Once transgenic plants are produced these genes serves no useful purpose, but may cause weediness and endanger natural ecosystems (Daniell et al 2001b).…”

Section: Introductionmentioning

confidence: 99%

Plant native tryptophan synthase beta 1 gene is a non-antibiotic selection marker for plant transformation

Hsiao

Sanjaya

et al. 2006

Planta

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Gene transformation is an integral tool for plant genetic engineering. All antibiotic resistant genes currently employed are of bacterial origin and their presence in the field is undesirable. Therefore, we developed a novel and efficient plant native non-antibiotic selection system for the selection of transgenic plants in the model system Arabidopsis. This new system is based on the enhanced expression of Arabidopsis tryptophan synthase beta 1 (AtTSB1) and the use of 5-methyl-tryptophan (5MT, a tryptophan [Trp] analog) and/or CdCl2 as selection agent(s). We successfully integrated an expression cassette containing an AtT-SB1 cDNA driven by a cauliflower mosaic virus 35S promoter into Arabidopsis by floral dip transformation. Transgenic plants were efficiently selected on MS medium supplemented with 75 microM 5MT or 300 microM CdCl2 devoid of antibiotics. TSB1 selection was as efficient as the conventional hygromycin selection system. Northern blot analysis of transgenic plants selected by 5MT and CdCl2 revealed increased TSB1 mRNA transcript whereas uneven transcript levels of hygromycin phosphotransferase II (hpt) (control) was observed. Gas chromatography-mass spectrometry revealed 10-15 fold greater free Trp content in AtT-SB1 transgenic plants than in wild-type plants grown with or without 5MT or CdCl2. Taken together, the TSB1 system provides a novel selection system distinct from conventional antibiotic selection systems.

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Antibiotic-free chloroplast genetic engineering – an environmentally friendly approach

Cited by 35 publications

References 15 publications

Isolation of a rice regeneration quantitative trait loci gene and its application to transformation systems

Isolation of a rice regeneration quantitative trait loci gene and its application to transformation systems

Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology

Plant native tryptophan synthase beta 1 gene is a non-antibiotic selection marker for plant transformation

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