Chloroplast Biotechnology in Higher Plants: Expressing Antimicrobial Genes in the Plastid Genome (original) (raw)
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Chloroplast Genomics and Genetic Engineering for Crop Improvement
Agricultural Research, 2012
Chloroplast genome sequence information is crucial for understanding the evolutionary relationship among photosynthetic organisms and in chloroplast (plastid) genetic engineering for agricultural biotechnology applications. Plastid transformation technology in crop plants offers numerous advantages over nuclear transformation, including high transgene expression, multiple transgene stacking through operon transfer to plastid genome, lack of epigenetic gene silencing and transgene containment due to maternal inheritance of plastids. More importantly, this technology permits expression of native bacterial genes at much higher level than the levels achievable in nucleus. However, only a handful of crops are amenable to routine plastid transformation due to technical difficulties. The plastid transformation in plants necessitates development of species-specific transgene delivery vector, which ideally should consist of homologous recombination sequences and endogenous plastid regulatory elements for efficient transgene integration and stable protein expression. However, inadequate plastid genome sequence information in majority of agriculturally important species has limited the development of transplastomic crops with desired traits. The recent advancement in high-throughput genome sequencing has resulted in the availability of complete plastid genome sequences in more than 230 photosynthetic organisms, including more than 130 higher plants. The availability of genome sequence data of more crop plants will offer an opportunity to construct species-specific plastid vectors, thus provide a newer platform for efficient plastid genetic engineering with a variety of agronomic applications, including high insect and pathogen resistance, herbicide resistance, tolerance to drought, salt and cold stresses, cytoplasmic male sterility, metabolic pathway engineering, production of antigens, biopharmaceuticals and bio-fuels. However, the major challenges ahead are to develop and implement this novel toolkit efficiently in most major crops for desirable agronomic applications.
Breakthrough in chloroplast genetic engineering of agronomically important crops
Trends in Biotechnology, 2005
Chloroplast genetic engineering offers several unique advantages, including high-level transgene expression, multi-gene engineering in a single transformation event and transgene containment by maternal inheritance, as well as a lack of gene silencing, position and pleiotropic effects and undesirable foreign DNA. More than 40 transgenes have been stably integrated and expressed using the tobacco chloroplast genome to confer desired agronomic traits or express high levels of vaccine antigens and biopharmaceuticals. Despite such significant progress, this technology has not been extended to major crops. However, highly efficient soybean, carrot and cotton plastid transformation has recently been accomplished through somatic embryogenesis using speciesspecific chloroplast vectors. This review focuses on recent exciting developments in this field and offers directions for further research and development.
PLANT PHYSIOLOGY, 2001
The antimicrobial peptide MSI-99, an analog of magainin 2, was expressed via the chloroplast genome to obtain high levels of expression in transgenic tobacco (Nicotiana tabacum var. Petit Havana) plants. Polymerase chain reaction products and Southern blots confirmed integration of MSI-99 into the chloroplast genome and achievement of homoplasmy, whereas northern blots confirmed transcription. Contrary to previous predictions, accumulation of MSI-99 in transgenic chloroplasts did not affect normal growth and development of the transgenic plants. This may be due to differences in the lipid composition of plastid membranes compared with the membranes of susceptible target microbes. In vitro assays with protein extracts from T 1 and T 2 plants confirmed that MSI-99 was expressed at high levels to provide 88% (T 1 ) and 96% (T 2 ) inhibition of growth against Pseudomonas syringae pv tabaci, a major plant pathogen. When germinated in the absence of spectinomycin selection, leaf extracts from T 2 generation plants showed 96% inhibition of growth against P. syringae pv tabaci. In addition, leaf extracts from transgenic plants (T 1 ) inhibited the growth of pregerminated spores of three fungal species, Aspergillus flavus, Fusarium moniliforme, and Verticillium dahliae, by more than 95% compared with non-transformed control plant extracts. In planta assays with the bacterial pathogen P. syringae pv tabaci resulted in areas of necrosis around the point of inoculation in control leaves, whereas transformed leaves showed no signs of necrosis, demonstrating high-dose release of the peptide at the site of infection by chloroplast lysis. In planta assays with the fungal pathogen, Colletotrichum destructivum, showed necrotic anthracnose lesions in non-transformed control leaves, whereas transformed leaves showed no lesions. Genetically engineering crop plants for disease resistance via the chloroplast genome instead of the nuclear genome is desirable to achieve high levels of expression and to prevent pollen-mediated escape of transgenes. ; fax 407-384 -2062.
A protocol for expression of foreign genes in chloroplasts
Nature Protocols, 2008
Several major costs associated with the production of biopharmaceuticals or vaccines in fermentation-based systems could be minimized by using plant chloroplasts as bioreactors, which facilitates rapid scale-up. Oral delivery of chloroplast-derived therapeutic proteins through plant cells eliminates expensive purification steps, low temperature storage, transportation and sterile injections for their delivery. Chloroplast transformation technology (CTT) has also been successfully used to engineer valuable agronomic traits and for the production of industrial enzymes and biomaterials. Here, we provide a detailed protocol for the construction of chloroplast expression and integration vectors, selection and regeneration of transformants, evaluation of transgene integration and inheritance, confirmation of transgene expression and extraction, and quantitation and purification of foreign proteins. Integration of appropriate transgenes into chloroplast genomes and the resulting high levels of functional protein expression can be achieved in B6 months in lettuce and tobacco. CTT is eco-friendly because transgenes are maternally inherited in most crop plants.
OBPC Symposium: Maize 2004 & beyond—Recent advances in chloroplast genetic engineering
2005
The chloroplast genetic engineering approach offers a number of unique advantages, including high-level transgene expression, multi-gene engineering in a single transformation event, transgene containment via maternal inheritance, lack of gene silencing, position and pleiotropic effects and undesirable foreign DNA. Thus far, more than 40 transgenes have been stably integrated and expressed via the tobacco chloroplast genome to confer several agronomic traits and produce vaccine antigens, industrially valuable enzymes, biomaterials, and amino acids. Functionality of chloroplastderived vaccine antigens and therapeutic proteins have been demonstrated by in vitro assays and animal studies. Oral delivery of vaccine antigens has been facilitated by hyperexpression in transgenic chloroplasts (leaves) or non-green plastids (carrots) and the availability of antibiotic-free selectable markers or the ability to excise selectable marker genes. Additionally, the presence of chaperones and enzymes within the chloroplast help to assemble complex multi-subunit proteins and correctly fold proteins containing disulfide bonds, thereby drastically reducing the costs of in vitro processing. Despite such significant progress in chloroplast transformation, this technology has not been extended to major crops. This obstacle emphasizes the need for plastid genome sequencing to increase the efficiency of transformation and conduct basic research in plastid biogenesis and function. However, highly efficient soybean, carrot, and cotton plastid transformation has been recently accomplished via somatic embryogenesis using species-specific chloroplast vectors. Recent advancements facilitate our understanding of plastid biochemistry and molecular biology. This review focuses on exciting recent developments in this field and offers directions for further research and development.
Technical Advances in Chloroplast Biotechnology
Transgenic Crops [Working Title], 2019
Chloroplasts are highly organized cellular organelles after master organelle nucleus. They not only play a central role in photosynthesis but are also involved in several crucial cellular activities. Advancements in molecular biology and transgenic technology have further groomed importance of the organelle, and they are the most ideal ones for the expression of transgene. No doubt, limitations are there, but still research is advancing to resolve those. Certain valuable traits have been engineered for improved agronomic performance of crop plants. Industrial enzymes and therapeutic proteins have been expressed using plastid transformation system. Synthetic biology has been explored to play a key role in engineering metabolic pathways. Further, producing dsRNA in a plant's chloroplast rather than in its cellular cytoplasm is more effective way to address desired traits. In this chapter, we highlight technological advancements in chloroplast biotechnology and its implication to develop biosafe engineered plants.
Recent achievements obtained by chloroplast transformation
Plant Methods, 2017
Chloroplasts play a great role for sustained wellbeing of life on the planet. They have the power and raw materials that can be used as sophisticated biological factories. They are rich in energy as they have lots of pigment-protein complexes capable of collecting sunlight, in sugar produced by photosynthesis and in minerals imported from the plant cell. Chloroplast genome transformation offers multiple advantages over nuclear genome which among others, include: integration of the transgene via homologus recombination that enables to eliminate gene silencing and position effect, higher level of transgene expression resulting into higher accumulations of foreign proteins, and significant reduction in environmental dispersion of the transgene due to maternal inheritance which helps to minimize the major critic of plant genetic engineering. Chloroplast genetic engineering has made fruit full progresses in the development of plants resistance to various stresses, phytoremediation of toxic metals, and production of vaccine antigens, biopharmaceuticals, biofuels, biomaterials and industrial enzymes. Although successful results have been achieved, there are still difficulties impeding full potential exploitation and expansion of chloroplast transformation technology to economical plants. These include, lack of species specific regulatory sequences, problem of selection and shoot regeneration, and massive expression of foreign genes resulting in phenotypic alterations of transplastomic plants. The aim of this review is to critically recapitulate the latest development of chloroplast transformation with special focus on the different traits of economic interest.