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Milestones in Chloroplast Genetic Engineering: an Environmentally Friendly Era in Biotechnology
Abstract
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.
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... Dada la preocupación por el uso de marcadores de selección cuya utilización pudiera presentar impactos en otros organismos, y teniendo en cuenta que entre los marcadores de selección más comúnmente utilizados se encuentran los de resistencia a antibióticos y herbicidas, los investigadores han tratado de buscar alternativas para el uso de marcadores ambientalmente más amigables (Daniell, 1999). Entre estos genes se encuentra el responsable de la producción de la proteína verde fluorescente (GFP) proveniente de una medusa, el cual permite seleccionar las células transformadas en cultivo in vitro, y no involucra el uso de antibióticos, de herbicidas, ni de genes de resistencia a los mismos (Daniell et al., 2002). Entre los genes de selección positiva que se están utilizando, se incluyen el que permite la utilización de sustratos específicos con el uso de genes como ßgalactosidasa (gen lacZ), el gen de la ß-glucoronidasa (gus), el de la luciferasa, y el gen de la enzima fosfomanosaisomerasa (manA) (AGBIOS, 2002). ...
... Entre los genes de selección positiva que se están utilizando, se incluyen el que permite la utilización de sustratos específicos con el uso de genes como ßgalactosidasa (gen lacZ), el gen de la ß-glucoronidasa (gus), el de la luciferasa, y el gen de la enzima fosfomanosaisomerasa (manA) (AGBIOS, 2002). Bajo este mismo contexto, se adelantan estudios relativos a la remoción de marcadores de selección por métodos de biología molecular, y a la utilización de estrategias para el uso de herencia materna citoplasmática exclusivamente, entre los que se encuentra la transferencia de genes vía cloroplastos (Daniell et al., 2002). ...
El desarrollo de técnicas de manipulación genética constituye un valioso apoyo a los sistemas de mejoramiento convencional, principalmente en aquellas situaciones en las cuales el acceso a genes de interés se encuentra limitado. La utilización de cultivos transgénicos comerciales se ha expandido en el mundo en forma acelerada desde cuando se aprobó su uso en 1994, y son numerosos los beneficios que se han determinado para el agricultor y para el ambiente. El presente artículo presenta el contexto general de la manipulación genética de cultivos, un ejemplo de desarrollos en Colombia, relativos a la transformación genética de Passiflora edulis para darle resistencia a virus y también hace referencia a aspectos de bioseguridad.
... Although the majority of genetically modified organisms are generated by the integration of transgenes in their genome, the case of photosynthetic organisms also offers the opportunity for adopting chloroplast transformation. Such organelles are remnants of endosymbiotic cyanobacteria that have gained great interest for genetic manipulation due to their homologous recombination system, facilitating the site-specific integration of foreign DNA, which avoids position effects and leads to a high copy number for the transgene; however, no silencing processes have been found in these organelles (Daniell et al., 2002;Vafaee et al., 2014). Strong specific promoters [cauliflower mosaic virus 35S (CaMV35S) promoter, a-tubulin, RBCS2, PSAD, Prrn, FCP, NIT1, and Ubi1-W] may lead to a higher transgene expression level with protein accumulations (Metting, 1996;Mayfield et al., 2003;Potvin and Zhang, 2010;Hallmann, 2015;Doron et al., 2016;Hamed, 2016;Adem et al., 2017;Zhang et al., 2017;Mutanda et al., 2020). ...
... Therefore, compared with nuclear genome manipulation, the transformation of the chloroplast genome offers various advantages, namely, uniform transgene expression and compartmentalization of the produced recombinant proteins in the chloroplast without affecting other cellular processes (Georgianna and Mayfield, 2012;Doron et al., 2016;Siddiqui et al., 2020). Achievements in chloroplast transformation have been reported in higher plants, such as tobacco, lettuce, rapeseed, potato, tomato, petunia, soybean, and cotton, and some monocots, including rice; cotton, as an efficient system for the expression of functional recombinant proteins, uses specific plastid promoterdriven and other genetic factors (Daniell et al., 2002;Verma and Daniell, 2007;Wani et al., 2015;Daniell et al., 2021). The research and development of plastid microalgal biotechnology are still a major obstacle given that such technology is well established only for the model species Chlamydomonas reinhardtii (Bateman and Purton, 2000;Rosales-Mendoza et al., 2012;Almaraz-Delgado et al., 2014;Economou et al., 2014;Munjal et al., 2014;Scranton et al., 2015;Larrea-Alvareza and Purton, 2020). ...
Microalgae constitute a diverse group of photosynthetic unicellular microorganisms that have gained immense attention for biotechnological applications. They are promising platforms for the production of high-value metabolites and biopharmaceuticals for commercial and therapeutic applications because of their physiological properties and capability to grow easily in natural and artificial environments. Although the proof-of-concept for some applications have been achieved for model species, such as Chlamydomonas reinhardtii, the genetic engineering methods for microalgae are still in their infancy. Thus, an expansion of this field is required. Chlorella vulgaris is an important algal species with a high protein content and requires focus for the development of an efficient nuclear and chloroplast transformation process. This research aimed to establish a chloroplast transformation method for the freshwater green-algae species C. vulgaris based on a specific expression vector (pCMCC, which was named after Chula Mexico Chlorella chloroplast) constructed with endogenous recombination regions, namely, 16S–trn I (left) and trn A–23S (right), and the Prrn promoter. Human basic fibroblast growth factor (bFGF) was adopted as a target biopharmaceutical to establish the chloroplast expression approach. The plasmid pCMCC:bFGF was transformed into C. vulgaris via electroporation using simple carbohydrate-based buffers, which aided in the transfer of the transgene into the chloroplast genome. Cells transformed with the pCMCC:bFGF vector were selected using kanamycin, and resistant colonies were analyzed by polymerase chain reaction and Western blotting to confirm the presence of the transgene and the recombinant bFGF, respectively. The bFGF that accumulated in the transplastomic C. vulgaris clones ranged from 0.26 to 1.42 ng/g fresh weight of biomass, and it was quantified by enzyme-linked immunosorbent assay. Therefore, the designed expression vector, in combination with an optimized electroporation protocol, constitutes a viable approach to successfully develop transplastomic lines of C. vulgaris for the potential low-cost production of biopharmaceuticals using this algal species. This study paves the way for the establishment of chloroplast biotechnology in microalgae other than the model organism C. reinhardtii.
... Transplastomic plants are obtained by the stable transformation of the plastid genome with a foreign gene via homologous recombination (Lelivelt et al., 2005). Plastid transformation is most commonly obtained by biolistic bombardment of leaves but also via polyethylene glycol (PEG) in the case of, for example, tobacco chloroplasts (Daniell et al., 2002;Golds et al., 1993). The resulting tobacco leaf explants are then regenerated and selected on a selection medium containing spectinomycin alone or in combination with streptomycin, to obtain transplastomic plants with homoplastomic chloroplast transformation in which every chloroplast carries the transgene (Obembe et al., 2011). ...
... Nevertheless, more than 100 transgenes have been successfully integrated and expressed using the plastid platform such as industrially valuable enzymes, biomaterials such as bioplastics, biopharmaceutical proteins as proinsulin (Ruhlman et al., 2007b) and somatotropin (Staub et al., 2000), antibodies, antibiotics, vaccine antigens including tuberculosis vaccine antigens (Lakshmi et al., 2013), along with transgenes that confer advantageous agronomic traits as disease or drought resistance (Verma and Daniell, 2007). Although the concept of chloroplast transformation was developed two decades ago (Daniell et al., 2002), the biotechnological potential of this production ...
... Hence, production of correctly folded and fully functional oral biopharmaceuticals would lower the total cost. Additionally, bioencapsulation of oral therapeutics within plant cell compartments like chloroplasts or plant organs like seeds provides resilience against gastric digestive enzymes and helps in absorption through microvilli of small intestine for successful delivery to the target tissues [15,16,17]. For the last 30 years, an array of biopharmaceutical production platforms ranging from plants with stable genetic modifications, to plant suspension cultures and to transient plant expression systems have been devised, scaled-up and employed for commercial scale production [18]. ...
... Though various plant tissues have been used to produce recombinant proteins like leaf and stem tissues [39,40], roots [41], fruits [42,43], tubers [44], aquatic weeds [45], hairy root culture [46], single cell culture [47,48], cell suspension culture [49,50] and transformed chloroplasts [15,17,51]. However, emergence of plant seeds as protein fication. ...
The cost-effective production of high-quality and biologically active recombinant molecules especially proteins is extremely desirable. Seed-based recombinant protein production platforms are considered as superior choice owing to lack of human/animal pathogenic organisms, lack of cold chain requirements for transportation and long-term storage, easy scalability and development of edible biopharmaceuticals in plants with objective to be used in purified or partially processed form is desirable. This review article summarizes the exceptional features of seed-based biopharming and highlights the needs of exploiting it for commercial purposes. Plant seeds offer a perfect production platform for high-value molecules of industrial as well as therapeutic nature owing to lower water contents, high protein storage capacity, weak protease activity and long-term storage ability at ambient temperature. Exploiting extraordinarily high protein accumulation potential, vaccine antigens, antibodies and other therapeutic proteins can be stored without effecting their stability and functionality up to years in seeds. Moreover, ability of direct oral consumption and post-harvest stabilizing effect of seeds offer unique feature of oral delivery of pharmaceutical proteins and vaccine antigens for immunization and disease treatment through mucosal as well as oral route.
... In 1986, tobacco became the first plant for which complete sequence of chloroplast (cp) genome (155,943 bp) was reported (Shinozaki et al. 1986). Since then, tobacco also became the most preferred model for understanding the evolutionary relationship among photosynthetic organisms and cp (plastid)-engineering or chloroplast transformation technology (CTT) for agricultural biotechnology applications (Daniell et al. 2002;. CTT brings advantages such as control of the site of gene insertion, high rates of transgene expression and protein accumulation, lack of transmission of the transgene via pollen due to the fact that plastid genes are maternally inherited and an absence of epigenetic effects (Daniell et al. 2004). ...
... CTT brings advantages such as control of the site of gene insertion, high rates of transgene expression and protein accumulation, lack of transmission of the transgene via pollen due to the fact that plastid genes are maternally inherited and an absence of epigenetic effects (Daniell et al. 2004). Improving agricultural traits such as herbicide and pathogen resistance, resistance to drought and salt tolerance, phytoremediation and molecular-pharming are all promising and potential applications of CTT (Daniell et al. 2002;Řepková 2010;Adem et al. 2017). The knowledge obtained from tobacco chloroplast genome can be easily applied to over 800 complete cp genome sequences that have been made publicly available in the National Center for Biotechnology Information (NCBI) organelle genome database (http://www.ncbi.nlm.nih. ...
This study examines the early and late age at initiation of tobacco consumption in India. It uses Global Adult Tobacco Survey (2009-10) data to this end. Bivariate and multivariate statistical techniques are used to analyze the data set. The findings reveal that the respondents living in rural areas were highly initiated in different forms of tobacco as compared with urban space. Education was one of the primary factors for initiation of tobacco use. Respondents having secondary and above education were highly initiated in late age. However, the age of tobacco initiation either early or late was significantly higher (7 per cent) among males as compared with females. Respondents belonging to the central (4.9 per cent) and northern regions (4.6 per cent) were highly initiated to tobacco as compared with other regions. Considering a decline in the initiation of tobacco consumption, local level awareness and catchy programmes play an effective role. In this, the involvement of children, women and leaders can be help to reach the goal.
... Plastid transformation has also been reported for other plants including Brassica napus [6], Lesquerella fendleri [7] and Oryza sativa [8]although problems with heteroplasmy and plant fertility have yet to be resolved. To address these problems research was focused on the use of selectable markers, the manipulation of gene expression-control elements and the development of efficient tissue-culture systems [9]. Nevertheless, the inefficient incorporation of foreign DNA into plastomes, such as occurs in Arabidopsis thaliana [10], remains to be overcome. ...
... Two hundred and thirty-seven independent kanamycin-resistant transplastomic clones from 45 bombardments (between 1 and 19 per bombardment) were recovered and 81 out of 113 tested clones showed INT-mediated attP vector insertion into the attB site. Therefore, transformation efficiencies (up to 17 per bombardment) are roughly fourfold higher than reported previously [4,8,9] (up to five per bombardment using homologous recombination and a non-lethal selection system). Nevertheless, the homologous recombination approach eliminates the introduction of unwanted plasmid sequences, which are a potential concern with INT-mediated plastid transformation. ...
Plastid transformation offers the unique advantages of high-level transgene expression and increased trans-gene containment compared with conventional trans-genic technologies. The process relies on the homologous recombination machinery of the plastid incorporating foreign DNA into the plastome, which restricts the method to species where this type of incorporation works well. However, Pal Maliga and colleagues have recently reported a novel approach for integrating foreign DNA into the plastid genome that works independently of homologous recombination. The transgenic plastid system Plastid transformation is accomplished using a multistep process in which the transformation vectors contain a selectable marker gene and passenger gene(s) flanked by plastid targeting sequences [1]. Targeting sequences enable foreign genes to be incorporated into the predetermined location through two crossover events within the homologous flanking regions. Insertion of transgenes in intergenic regions without interference with flanking genes has been successful at 14 sites throughout the plastome [2], either by biolistic DNA delivery or by polyethylene glycol treatment. Nevertheless, interference was found at one site and knockouts have been reported for 28 plastid genes. Plastid genetic engineering is emerging as an alternative new technology in plant biotechnology. Compared with conventional transgenic technologies, plastid engineering offers several potential advantages that have been extensively reviewed [1-3]. Plastid transformation has mostly been confined to Nicotiana tabacum [3] and it has taken almost ten years to extend the technology to other plant species, including Solanum tuberosum [4] and Lycopersicon esculentum [5]. Plastid transformation has also been reported for other plants including Brassica napus [6], Lesquerella fendleri [7] and Oryza sativa [8]-although problems with heteroplasmy and plant fertility have yet to be resolved. To address these problems research was focused on the use of selectable markers, the manipulation of gene expression-control elements and the development of efficient tissue-culture systems [9]. Nevertheless, the inefficient incorporation of foreign DNA into plastomes, such as occurs in Arabidopsis thaliana [10], remains to be overcome. However, incorporating transgenes into the plastome is only the first step in obtaining a genetically stable transgenic plant because several cell generations are necessary to dilute out all wild-type plastome copies [8]. Pal Maliga and colleagues recently outlined an alternative approach to incorporate foreign DNA into the plastome efficiently. The technique uses the phiC31 Streptomyces phage site-specific inte-grase (INT) to catalyze recombination to yield stable transplastomic plants [11]. Integrase-mediated recombination system Site-specific recombination systems are common in pro-karyotes and lower eukaryotes. They participate in integrating bacteriophages into host genomes. In these systems, recombinations are catalyzed by phage-encoded recombinases. These recombinases can be grouped into two types based on limited amino acid sequence homology and catalytic residues (either serine or tyrosine). The serine recombinases are referred to as the invertase or resolvase and posses a catalytic serine residue, whereas the tyrosine group members are referred to as l integrases and use a tyrosine residue during DNA cleavage. The phiC31 Streptomyces phage-encoded integrase belongs to the serine family of recombinases. Like other family members, the phiC31 phage integrase brings two recom-bination sites (the bacterial attB and phage attachment attP sites) together in a synapse and then catalyzes a concerted four-strand staggered break in the DNA sub-strates while forming transient covalent attachments with the recessed 5 0 ends [12]. One pair of half sites rotates by 1808 relative to the other to form the recombinant configuration, followed by ligation of the DNA backbone. Thus, the integration reaction generates recombinant junctions attL and attR as products. During integration, the attB and attP sites share only 3 bp sequence identity at the crossover point and 16 bp within a span of w50 bp. The crossover point for both the attachment sites is flanked by different inverted repeats (in the region of 34 bp of attB and 39 bp of attP sites) that perhaps provide binding sites for the integrase [12]. It has been observed that some integrases function autonomously whereas others catalyze the reaction with the help of DNA-binding proteins, termed accessory factors (recombination directionality factors, RDFs [13]), to influence the choice of substrates that are recombined by their cognate recombinase. The best-studied RDF is Xis, encoded by phage l, which is required in the excision reaction between attL and attR junctions to generate attP and attB as products but inhibits integrative Corresponding author: Khan, M.S. (sarwarkhan_40@hotmail.com).
... The lesser involvement of gene silencing, along with side effects, is given by this expression system. It also gives a high level of expression regarding the target gene, along with a single transformation presence compared to that of multiple genes [58]. It also includes maternal inheritance regarding the transgene, and hence there is a lesser risk for transgenic crops to get involved in the transfer of foreign genes to other crops from chloroplast expressions. ...
... The development of the Gene-Gun system for biolistic delivery of transformation vectors directly into leaf chloroplasts coupled with tissue culture methods for regeneration of transplastomic plants remains the foundation for chloroplast biotechnology (Daniell 1993;Svab et al. 1990). Transformation of chloroplasts has many intrinsic advantages over the more widely used genetic engineering of the nuclear genome (Daniell et al. 2002). With regard to the vector design, conventional chloroplast transformation methods rely on homologous recombination (HR) to integrate foreign DNA into specific sites of the plastome (Fig. 1A). ...
Key message
Novel episomal systems have the potential to accelerate plastid genetic engineering for application in plant synthetic biology.
Abstract
Plastids represent valuable subcellular compartments for genetic engineering of plants with intrinsic advantages to engineering the nucleus. The ability to perform site-specific transgene integration by homologous recombination (HR), coordination of transgene expression in operons, and high production of heterologous proteins, all make plastids an attractive target for synthetic biology. Typically, plastid engineering is performed by homologous recombination; however, episomal-replicating vectors have the potential to accelerate the design/build/test cycles for plastid engineering. By accelerating the timeline from design to validation, it will be possible to generate translational breakthroughs in fields ranging from agriculture to biopharmaceuticals. Episomal-based plastid engineering will allow precise single step metabolic engineering in plants enabling the installation of complex synthetic circuits with the ambitious goal of reaching similar efficiency and flexibility of to the state-of-the-art genetic engineering of prokaryotic systems. The prospect to design novel episomal systems for production of transplastomic marker-free plants will also improve biosafety for eventual release in agriculture.
... Regarding the controversy that has caused the transfer of genes from transgenics to other non-target crops, we can mention that the chloroplastid transformation of plants minimized horizontal and vertical gene transfer in protected crops (Daniell et al., 2002). The chloroplastid transformation of plants has had more acceptance because it has advantages such as; higher production of recombinant proteins and the transfer of modified genetic material is only through the mother (Adem et al., 2017). ...
Biotechnology is a broad interdisciplinary branch of biological sciences that consists of any technological application that uses biological systems and living organisms or their derivatives for the creation or modification of products or processes for specific uses. Within these organisms they may or may not be genetically modified. The bases of this interdisciplinary are biology, engineering, physics, chemistry, and biomedicine. Over the years the field of this science has shown relevance in pharmacology, medicine, food science, the treatment of solid, liquid and gaseous waste, industry, livestock and agriculture.
Biotechnology is present day by day in our daily lives, in the foods that we consume are sometimes supplemented with compounds, enzymes and proteins that help improve the taste, texture or general properties of food. Biotechnology has also presented great impact on the generation of raw materials to minimize the use of fossil fuels. Through different crop optimization systems and genetic manipulation, it has been possible to improve the properties of biomass in crops or biological models that are used as raw material to obtain fatty acids and alcohols.
During the year 2020, the COVID-19 pandemic was experienced worldwide, the participation of biotechnologists for the synthesis of new vaccines or establishing methodologies for rapid detection of the virus, was decisive to control the spread of the virus among the world population.
This book describes what has been the biotechnological advance in selected topics related to genetic improvement of crops, enzyme biotechnology, plant tissue culture, metabolites extraction, advances in the generation of biofuels and generation of vaccines.
The goal of this book is to emphasize the importance of biotechnology today. The editors hope that this book is to your liking and helps you in the different disciplines in which they are developed.
... Each mature leaf cell contains up to 100 chloroplasts, and each chloroplast contains ~ 100 copies of chloroplast genomic DNA. Thus, it provides a high gene copy number and leads to result in high expression of recombinant protein from integrated GOI in the chloroplast genome (Daniell et al. 2002). Chloroplast transformation is achieved by bombarding the leaf with gold particles coated with plastid DNA fragments containing the GOI and allowing the DNA to be integrated into the plastid genome via homologous recombination (Daniell et al. 1990). ...
The production of pharmaceutical compounds in plants is attracting increasing attention, as plant-based systems can be less expensive, safer, and more scalable than mammalian, yeast, bacterial, and insect cell expression systems. Here, we review the history and current status of plant-made pharmaceuticals. Producing pharmaceuticals in plants requires pairing the appropriate plant species with suitable transformation technology. Pharmaceuticals have been produced in tobacco, cereals, legumes, fruits, and vegetables via nuclear transformation, chloroplast transformation, transient expression, and transformation of suspension cell cultures. Despite this wide range of species and methods used, most such efforts have involved the nuclear transformation of tobacco. Tobacco readily generates large amounts of biomass, easily accepts foreign genes, and is amenable to stable gene expression via nuclear transformation. Although vaccines, antibodies, and therapeutic proteins have been produced in plants, such pharmaceuticals are not readily utilized by humans due to differences in glycosylation, and few such compounds have been approved due to a lack of clinical data. In addition, achieving an adequate immune response using plant-made pharmaceuticals can be difficult due to low rates of production compared to other expression systems. Various technologies have recently been developed to help overcome these limitations; however, plant systems are expected to increasingly become widely used expression systems for recombinant protein production.
... As it is said, "Let thy food be thy medicine," scientists suggest that vaccines against diseases can be produced by plants as potential source of natural recombinant vaccine. A plant-based vaccine can be produced against several diseases including dental caries, diarrhea, acquired immunodeficiency syndrome (AIDS), etc. (Daniell, Khan, & Allison, 2002 ...
The researchers are still doing efforts to develop an effective, reliable, and easily accessible vaccine candidate to protect against COVID‐19. As of the August 2020, nearly 30 conventional vaccines have been emerged in clinical trials, and more than 200 vaccines are in various development stages. Nowadays, plants are also considered as a potential source for the production of monoclonal antibodies, vaccines, drugs, immunomodulatory proteins, as well as used as bioreactors or factories for their bulk production. The scientific evidences enlighten that plants are the rich source of oral vaccines, which can be given either by eating the edible parts of plants and/or by oral administration of highly refined proteins. The use of plant‐based edible vaccines is an emerging trend as it possesses minimum or no side effects compared with synthetic vaccines. This review article gives insights into different types of vaccines, the use of edible vaccines, advantages of edible vaccines over conventional vaccines, and mechanism of action of edible vaccines. This review article also focuses on the applications of edible vaccines in wide‐range of human diseases especially against COVID‐19 with emphasis on future perspectives of the use of edible vaccines.
... Jin et al. (2012) showed the expression of agglutinin gene (pta) in leaf chloroplasts with increased broad-spectrum resistance against lepidopteran insects, aphids, and viral and bacterial pathogens. The genetically expressed osmoprotectant (yeast trehalose phosphate synthase) in tobacco plastids resulted in 20-fold higher trehalose accumulation; which resulted in plants tolerant to drought and osmotic stress (Daniell et al., 2002). ...
With the growing population, the farmers worldwide apply and depend on several
chemical approaches to meet the vegetation demand. The chemical approaches, on the
other hand, are implying a hazardous effect on both human health and the well-balanced
environment. But with the growing time, several biotechnological approaches have
been accepted for the nature of eco-friendliness and positive high attributing effect.
The biotechnological approaches with its several array of strategies vastly increased the
accuracy, and reduced the time with which it became easy to maintain proper health and
yield of the crops. Thus, the purpose of this article is to provide information regarding the
modern biotechnological approaches that can be adapted for a perfect and manageable
agricultural sector to enhance global food security.
... Hence, the gene (CsPME4) that encodes TSPME was downregulated to improve the juice quality [37]. Further, an environmentally friendly technology named, 'chloroplast transformation' is available to develop transgenic plants [38][39][40][41][42][43][44][45][46]. This technology offers several superior advantages like overexpression of transgenes up to 70% due to ...
... The advantages of chloroplast transformation over the nuclear transformation are: thousands of plastids present in photosynthetic cells of higher plants result in higher level expression (46-70% of the total soluble leaf protein) of transgene with proper folding and formation of disulfide bonds (Staub et al. 2000;Daniell and Dhingra 2002;Fernandez-San Millan et al. 2003;Lee et al. 2003;Koya et al. 2005;Oye et al. 2009;Gao et al. 2012). Transgene integrated into chloroplast DNA do not appear to undergo silencing or suffer from position effects due to their site-specific transgene integration into the chloroplast genome; scope for multi-gene engineering in a single transformation event (De Cosa et al. 2001;Jeong et al. 2004); and chloroplast genes are inherited in a strictly maternal fashion providing a natural containment method for transgenic plants, since transgene cannot be transmitted through pollen in majority of the plants (Maliga 1993;Daniell et al. 2002). ...
Production of recombinant proteins is primarily established in cultures of mammalian, insect and bacterial cells. Concurrently, concept of using plants to produce high-value pharmaceuticals such as vaccines, antibodies, and dietary proteins have received worldwide attention. Newer technologies for plant transformation such as plastid engineering, agroinfiltration, magnifection, and deconstructed viral vectors have been used to enhance the protein production in plants along with the inherent advantage of speed, scale, and cost of production in plant systems. Production of therapeutic proteins in plants has now a more pragmatic approach when several plant-produced vaccines and antibodies successfully completed Phase I clinical trials in humans and were further scheduled for regulatory approvals to manufacture clinical grade products on a large scale which are safe, efficacious, and meet the quality standards. The main thrust of this review is to summarize the data accumulated over the last two decades and recent development and achievements of the plant derived therapeutics. It also attempts to discuss different strategies employed to increase the production so as to make plants more competitive with the established production systems in this industry.
... Chloroplast transformation in sugarcane has been reported recently [3] using a vector carrying fluorescent antibiotic resistance marker gene, FALRE-S [4]. The transformed plants remained heteroplasmic after successive cycles of selection and regeneration owing to the complex anatomy and polyploidy of the plastome, though, homoplasmic clones have been successfully obtained in several plant species [5]. The sugarcane plastome is very attractive when talks about metabolic engineering where high-level expression of genes and accumulation of the product is required. ...
... Reference " Daniell et al. (2002)" was not cited anywhere in the text. Please provide in text citation or delete the reference from the reference list. ...
Plants are unique in that their cells contain three genomes: in the nucleus, mitochondria, and chloroplasts. Advances in recombinant DNA technology allow the manipulation of all three genomes. Insertion of foreign genes in the nucleus results in low gene expression, random integration of the transgenes, and gene silencing due to epigenetic interactions. Introducing transgenes into the chloroplast genome offers several advantages that include extraordinary gene expression levels (>70% of total soluble proteins), uniform gene expression due to non-Mendelian inheritance, and site-specific integration of the transgene at a chosen location on the chloroplast genome. The most attractive feature of developing transgenic field crops perhaps lies in the fact that chloroplast DNA is not carried in the pollen, particularly in field crops. The inheritance of chloroplasts in angiosperms is predominantly maternal, thereby providing a natural transgene containment system. Despite the huge potential, chloroplast transformation has not been extended to field crops, particularly cotton. There is only one study reporting the development of transgenic cotton expressing a kanamycin selectable marker. The possible reasons that currently hinder the application of this technology to cotton include prolonged and inefficient tissue culture-based protocols for recovering transplastomic plants and the limitation to develop transgenic plants from nongreen plastids through somatic embryogenesis. Efforts are underway to extend chloroplast transformation in agronomically important crops. In this chapter, we highlight the potential of chloroplast transformation technology for developing transgenic cotton for sustainable cotton production.
... whereas PCR is more sensitive with 0.01% limit of detection. Transcriptional or translational gene silencing is a natural phenomenon which is routinely observed in nuclear transformation work (Daniell et al., 2002;Huntzinger et al., 2011). Moreover, low expression or gene silencing could be avoided by using more effective regulatory elements (promoters and terminators) or by the introduction of synthetic ht gene in other cellular compartments like plastome, which facilitates pre-determined and precise insertions of transgene(s) with high level of expression (Nazir et al., 2012). ...
Herbicides are the chemicals used to kill unwanted plants or weeds which can compete with main crop for light, water and other useful nutrients. Two types of herbicides i.e. selective and non-selective are being used but the later one is more efficient and it cannot differentiate between the crops and weeds. Hence, development of herbicide tolerant (ht) plant is an ultimate solution to cope with the challenge of non-selective ones. Nucleotide sequence of ht bacterial gene encoding 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase was modified according to crop codon usage. The synthetic herbicide tolerant gene was cloned in a gateway compatible plant expression vector and introduced into an Agrobacterium strain LBA4404. Tissue culture independent in-planta transformation system was used to integrate the transgene into cotton plants at three different stages of flower after pollination. Flower shedding was the main problem observed during inoculation. Maximum inoculated bolls were developed at 16 hours after pollination and plantlets were screened using different doses of roundup spray having glyphosate as an active ingredient. Successful transgene integration was verified using PCR and Southern blot analysis in plants surviving at @ 600 mL/acre roundup herbicide. Whereas on higher dose of herbicide transgenic plant did not manage to survive. These results showed that the cotton is successfully transformed with ht gene but the expression of transgene is low or it may have been gone in silent mode. Gene silencing is a natural phenomenon which could be avoided by using more efficient regulatory elements or by introducing the transgene into other genetic compartments of plant. In addition, development of more transformation events could result in transgenic cotton plants with higher transgene expression.
... One of the major bottlenecks in developing stable plastid transformation in monocots has been their regeneration from non-green embryonic cells, containing undifferentiated plastids. Other barriers in developing homoplasmic transplastomic plants, particularly of rice, might be the low level of marker gene expression in non-green plastids in embryogenic cells because of a low genome copy number and low rates of protein synthesis [13]. ...
We report here plastid transformation in sugarcane using biolistic transformation and embryogenesis-based regeneration approaches. Somatic embryos were developed from unfurled leaf sections, containing preprogrammed progenitor cells, to recover transformation events on antibiotic-containing regeneration medium. After developing a proficient regeneration system, the FLARES (fluorescent antibiotic resistance enzyme, spectinomycin and streptomycin) expression cassette that carries species-specific homologous sequence tails was used to transform plastids and track gene transmission and expression in sugarcane. Plants regenerated from streptomycin-resistant and genetically confirmed shoots were subjected to visual detection of the fluorescent enzyme using a fluorescent stereomicroscope, after genetic confirmation. The resultant heteroplasmic shoots remained to segregate on streptomycin-containing MS medium, referring to the unique pattern of division and sorting of cells in C4 monocotyledonous compared to C3 monocotyledonous and dicotyledonous plants since in sugarcane bundle sheath and mesophyll, cells are distinct and sort independently after division. Hence, the transformation of either mesophyll or bundle sheath cells will develop heteroplasmic transgenic plants, suggesting the transformation of both types of cells. Whilst developed transgenic sugarcane plants are heteroplasmic, and selection-based regeneration protocol envisaging the role of division and sorting of cells in the purification of transplastomic demands further improvement, the study has established many parameters that may open up exciting possibilities to express genes of agricultural or pharmaceutical importance in sugarcane.
... Recently, there has been a negative public perception against GM O crops due to the documented transfer of transgenes to wild relatives of a few p lant species. Maternal inheritance of a transgene, and thus effective elimination of t ransgene escape via pollen, is possible if a t ransgene is integrated into the chloroplast genome [35]. Not surprisingly, therefore, plastids have recently become a popular target for exploration in genetic engineering. ...
For as long as man has been cultivating crops and raising animals, there have been modifications of the genomes of these plants and animals. The p rogress made in current genetic technology allows for modify ing the genome very precisely, one gene at a time. Research has made possible targeted changes in varieties of plants that have enabled man to increase both yields and the quality of these crops. Crops developed through genetic engineering are common ly known as transgenic crops or genetically modified (GM) crops. This review will detail benefits of genetic modifications in crop production, and describe additional concepts in crop biotechnology that will make more d irect contributions to food quality, environmental benefits, pharmaceutical production, and non-food crops.
... This has 76 been done through " genome editing" whereby designer nucleases primarily effect the way a 77 protien is regulated within a plant based system. (16) 78 79 Plant model selection is based on several factors, such as an ease of development, techniques 80 available for the genetic transformation, high scale production of therapeutic agents, organ 81 harvesting and processing of PMPs . An example is that of tobacco plant which is the plant of 82 choice for green matter synthesis as it witholds high biomass production rate. ...
... As about 10,000 plastid DNAs are present in each plant cell, useful extra proteins generally accumulate at higher levels in plastid, rather than nuclear transformants (Bock 2007;Daniell et al. 2009;Maliga 2003;Scotti et al. 2012). It is thought that homologous recombination of plastid DNA occurs only infrequently in a few plastids that receive a transformation vector by particle bombardment (Daniell et al. 2002;Maliga and Bock 2011). Therefore, untransformed wild-type plastid DNAs normally remain in antibiotic-resistant transformants that are regenerated in the primary selection step (Ye et al. 2003). ...
Plastid transformants form biofactories that are able to produce extra proteins in plastids when they are in a homoplasmic state. To date, plastid transformation has been reported in about twenty plant species; however, the production of homoplasmic plastid transformants is not always successful or easy. Heteroplasmic plants that contain wild-type plastids produce fewer target proteins and do not always successfully transfer transgenes to progeny. In order to promote the generation of homoplasmic plants, we developed a novel system using barnase–barster to eliminate wild-type plastids from heteroplasmic cells systematically. In this system, a chemically inducible cytotoxic barnase under a plastid transit signal was introduced into nuclear DNA and barster, which inhibits barnase, was integrated into plastid DNA with the primary selection markers aminoglycoside 3′-adenylyltransferase (aadA) and green fluorescence protein (GFP) gene. As expected, the expression of the plastid barnase was lethal to cells as seen in leaf segments, but barster expression in plastids rescued them. We then investigated the regeneration frequency of homoplasmic shoots from heteroplasmic leaf segments with or without barnase expression. The regeneration frequency of homoplasmic-like shoots expressing barnase–barster system was higher than that of shoots not expressing this. We expect that the application of this novel strategy for transformation of plastids will be supportive to generate homoplasmic plastid transformants in other plant species.
... Plastid transformation in higher plants has been established in the recent past to engineer many agronomic traits including insect and pathogen resistance, herbicide resistance, abiotic stress tolerance, increased photosynthesis and also production of edible crops engineered to produce 'biopharmaceuticals' (Daniell et al, 2002;Koop et al, 2007;Maliga and Block, 2011;Verma and Daniell, 2007;Wang et al., 2009]. ...
The world population is increasing at a very rapid pace. Therefore, to fulfill the need of ever increasing population, there is urgent need to enhance the food production. Although total arable land, which has reached its maximal utilization, may even decrease. Moreover, climate change is another major threat to global food security. However, complex traits i.e. crop productivity is difficult to manipulate and also time consuming if we go through conventional breeding methods for any change in genetic makeup. The availability of a very limited gene pool has led to the exploitation of plasma genes in generating superior crop varieties in terms of production and productivity. Plant mitochondrial genome is relevant to the common phenomenon of cytoplasmic male sterility (CMS), as in past also, many hybrid varieties were developed using CMS technique. Organelles transformation has opened many options for crop improvement, biomolecules, production of enzymes having applications in many industries, pharmaceuticals, and nutraceuticals etc. Organelle transformation have advantage in the sense that genetic information are maternally inherited and also can be used as an additional safeguard that can eliminate the risk 22 Biotechnology in Plant Protection and Crop Improvement of trans-gene escape by pollen release. On the other hand, from the literature study it is clear that transformation of chloroplast genome is not an easy tool and needs more interventions to manipulate of chloroplast genome.
... In such cases, organic synthesis requires many steps to obtain target metabolites from simple substrates, constituting a great hurdle for industrial largescale synthesis of plant-specific secondary metabolites (Bruggink et al., 2003;Laine, 2017). On the other hand, microbial fermentation which is another method for synthesising metabolites enables the supply of stable plant-specific secondary metabolites without an environmental impact (Daniell et al., 2002;Velasco et al., 2000). ...
Plant-biosynthesised secondary metabolites are unique sources of pharmaceuticals, food additives, and flavourings, among other industrial uses. However, industrial production of these metabolites is difficult because of their structural complexity, dangerousness and unfriendliness to natural environment, so the development of new methods to synthesise them is required. In this study, we developed a novel approach to identifying alternative bacterial enzyme to produce plant-biosynthesised secondary metabolites. Based on the similarity of enzymatic reactions, we searched for candidate bacterial genes encoding enzymes that could potentially replace the enzymes in plant-specific secondary metabolism reactions that are contained in the KEGG database (enzyme re-positioning). As a result, we discovered candidate bacterial alternative enzyme genes for 447 plant-specific secondary metabolic reaction. To validate our approach, we focused on the ability of an enzyme from Streptomyces coelicolor strain A3(2) strain to convert valencene to the grapefruit metabolite nootkatone, and confirmed its enzymatic activity by gas chromatography-mass spectrometry. This enzyme re-positioning approach may offer an entirely new way of screening enzymes that cannot be achieved by most of other conventional methods, and it is applicable to various other metabolites and may enable microbial production of compounds that are currently difficult to produce industrially.
... One obstacle to developing plastid transformation of commercial plants has been that their plant regeneration occurs from non-green embryonic cells (containing proplastids), rather than from leaf cells with functional chloroplasts [22]. Chloroplast transformation is potentially an ecologically viable alternative for plant genetic engineering. ...
We highlight the importance of the mixed genetic approaches (classical and currents) to improve the social perception related to the GMOs acceptance. We pointed out that CRISPR/Cas9 events could carry DNA variability/rearrangements related to somaclonal variations or epigenetic changes that are independent from the editing per se. The transformation of single cells, followed by plant regeneration, is used to generate modified plants, transgenic or genome editing (CRISPR/Cas9). The incidence of undesirable somaclonal variations and/or epigenetic changes that might have occurred during in vitro multiplication and regeneration processes, must be carefully analyzed in replicates in field trials. One remarkable challenge is related to the time lapse that selects the modified elite genotypes, because these strategies may spend a variable amount of time before the results are commercialized, where in all the cases it should be take into account the genotype x environment interactions. Furthermore, this combination of techniques can create an encouraging bridge between the public opinion and the community of geneticists who are concerned with plant genetic improvement. In this context, either transgenesis or genomic editing strategies become complementary modern tools to facing the challenges of plant genetic improvement. Their applications will depend on case-by-case analysis, and when
... The improved transformation efficiency and foreign gene expression can be achieved through utilization of endogenous flanking sequences and regulatory elements (Birch-Machin, Newell, Hibberd, & Gray, 2004;Maliga, 2004;Tangphatsornruang, Birch-Machin, Newell, & Gray, 2010). Transformation of chloroplast genome offers a number of advantages over nuclear transformation including a high level of transgene expression, polycistronic transcription, lack of gene silencing or positional effect and transgene containment (Bock, 2007;Daniell, Khan, & Allison, 2002;Maliga, 2002Maliga, , 2004. Tangphatsornruang et al. (2011) reported the complete chloroplast genome sequence of rubber tree as being 161,191 bp in length including a pair of inverted repeats of 26,810 bp separated by a small single copy region of 18,362 bp and a large single copy region of 89,209 bp. ...
The commercial production of high quality natural rubber (NR) solely depends on Hevea brasiliensis Muell. Arg, (Para rubber tree) and accounts for >98% of total production worldwide. NR with its unique properties is an essential commodity for the automobile industry and its synthetic counterparts are in no way substitute to it. The rubber tree genome is very complex and plays an important role in delivering the unique properties of Hevea. But a lack of knowledge on the molecular mechanisms of rubber biosynthesis, disease resistance, etc., in elite clones of rubber still persists. Marker-assisted selection and transgenic techniques were proved to be advantageous in improving the breeding efficiency for latex yield, disease resistance, etc. The suppression subtractive hybridization (SSH), in the form of subtracted cDNA libraries and microarrays, can assist in searching the functions of expressed genes (candidate gene approach). Expressed sequence tags (ESTs) related to various metabolic aspects are well utilized to create EST banks that broadly represent the genes expressed in one tissue, such as latex cells, that assists in the study of gene function and regulation. Transcriptome analysis and gene mapping have been accomplished in Hevea at various stages. However, a selection criterion to delineate high yielding genotypes at the juvenile stage has not been accomplished so far. This is the main pit fall for rubber breeding apart from stock–scion interactions leading to yield differences among a clonally multiplied population. At least four draft genome sequences have been published on Hevea rubber, and all give different genome size and contig lengths—a comprehensive and acceptable genomic map remains unfulfilled. The progress made in molecular markers, latex biosynthesis genes, transcriptome analysis, chloroplast and mitochondrial DNA diversity, paternity identification through Breeding without Breeding (BwB), stimulated latex production and its molecular intricacies, molecular biology of tapping panel dryness, genomics for changed climates and genome mapping are discussed in this review. These information can be utilized to improvise the molecular breeding programs of Hevea in future.
... Development in metabolic engineering allowed use of different plants such as tobacco & p. patens as production hosts for artemisinin. Furthermore, among all three methods discussed above in-plant production methods are more cost effective and environmentally friendly (Daniell et al. 2002) but these methods are commercially not available yet. In future, these two methods are seems to be promising because multigene transformation, targeting cellular compartments and transcriptions can increase level of production. ...
Malaria is a deadly disease caused by a parasite and spread among people by mosquito bites. In humans, ‘P. Falciparum’ is identified as the most common species of parasite that causes malaria. The world is constantly fighting with Malaria problem, about 219 million malaria cases found in the year 2017. According to WHO’s 2018 report (World Health Organisation), most of the malaria cases are from African and Asian region. Five countries carry nearly half of the malaria burden in the world namely, Nigeria (25%), Democratic Republic Congo (11%), Mozambique (5%), India (4%) and Uganda (4%) (WHO 2018).
Malaria is causing a huge social and economic burden on the world, about 456,000 child deaths occur due to malaria each year, which means 50 children deaths per hour (UNICEF 2019). The world is constantly looking for new options to eradicate malaria from society, in 2016, about 600 million dollars were invested in R&D on Malaria (WHO 2018).
There are many medicines available to treat malaria, but it is still difficult to make it available in all regions of the developing countries such as Africa and India. Researchers are looking for a cheaper and easy to available option for treating Malaria, one of which is the development of antimalarial drugs from plants like ‘Artemisia Annua’. This plant produces an antimalarial drug called ‘Artemisinin’ it can be derived by using semisynthetic process (White 1997).
The traditional way to produce artemisinin is by cultivating a large amount of ‘A. Annua’, (Namuli et al. 2018) revealed that putting efforts in plant growth regulators, fertilisers, high yielding strains and variation in growth condition of A.Annua plants can increase the artemisinin content. Still, it is difficult to fulfil increasing artemisinin demand in the world. The annual demand for artemisinin was about 1.6 billion treatments and forecast to grow to about 1.7 billion treatments by 2021 (WHO 2018). In this essay, we will discuss various approaches used to increase the production of antimalarial drug ‘Artemisinin’.
... Una de las limitaciones de la transformación plastidial en general, no sólo de monocotiledóneas, es la transformación de especies vegetales que se propaguen in vitro por embriogénesis somática. La regeneración a partir de células que contienen proplastidios afecta al bajo nivel de expresión del gen marcador de selección, ya que los proplastidios tienen pocas copias del plastoma y ratios bajos de síntesis de proteínas (Daniell et al., 2002). ...
Maize is one of the most important crops for the nutritional intake of humans, being such via direct utilization as food or through livestock breeding. The constant growth of human population, the increasing meat consumption in emerging economy countries, the use of grain for biofuel production or the climate change are demanding an increase in the productivity of this crop. Plant biotechnology offers diverse procedures to achieve this objective, being plastid transformation one of them. This technique brings important advantages to plant transformation such as high expression level of the gene of interest or prevent the transgene transmission through pollen to other crops due to the maternal inheritance of plastids. Achieving plastid transformation of maize would allow the transfer of those advantages to this important crop.
We have developed diverse in vitro culture procedures and worked with different maize genotypes to select the best candidates to be plastidially transformed. The best protocol tested has been the regeneration through somatic embryogenesis induced from immature embryos. We also obtained a green calli culture procedure using the peptide hormone -PSK, designed a second round of regeneration on selective media using mature somatic embryos and a protocol for culturing callus cell aggregates on liquid media as an alternative to cell suspensions.
The three selection schemes tested were antibiotic based: the gene aadA gene and streptomycin, the nptII gene and kanamycin and the Aph(4) gene and hygromycin selection. Those genes were cloned in four different plastid transformation vectors which introduced the transgenes into the following regions of the inverted repeat regions of the plastome: 16SRNAr-trnV-ORF85/ORF58 or 16SRNAr-trnI/trnA-23SRNAr. A total of 54 transformation experiments were made with 12 different types of materials and 1738 maize regenerants were obtained, 7 of which were transplastomic regenerants with a high level of heteroplasmy. Six of them were obtained in streptomycin selection and 1 with hygromycin.
It was also designed an alternative selection scheme which was based on the complementation of 3 nuclear photosynthetic albino mutants: csr1-1, crs1-2 y crs2-2. We obtained embryogenic calli from those mutants cultured in light or dark conditions from immature embryos. The mutated gene was introduced in a plastid transformation vector which introduces the gene into the 16SRNAr-trnI/trnA-23SRNAr recombination zone. No regenerants were obtained from 6 transformation experiments.
... Further, presence of chaperonin proteins in plastids [23] helps proteins to be biologically active. Precisely describing the technology, a gene of interest under the control of a promoter and a terminator along with another gene that confers resistance to an antibiotic is cloned between the DNA fragments from the plastome -used as left and right borders -to target the enclaved genes into the plastome through two homologous recombination events [24,25]. The antibiotic gene is used to select and screen the recipient plants. ...
... This design is to facilitate the integration of transgenes through homologous recombination. In most circumstances, the adenyltransferase gene aadA is included between the homologous regions, which allows for selection with spectinomycin and streptomycin, the most widely used antibiotics for plastome engineering [111] . Any other genes to be inserted, such as fluorescent markers, are adjacent, and often oriented in the opposite direction to aadA. ...
Plastids are subcellular organelles which perform photosynthesis in plants and algae. They are descended from free-living photosynthetic bacteria which were engulfed by early eukaryotes ~1.5 billion years ago. Nearly all plastids retain a small genome separate from the nuclear genome of the plants in which they reside, which is termed the plastome (plastid genome). This genome encodes many of the proteins required for photosynthesis, as well as bacteria-like transcriptional and translational machinery. Biotechnological techniques for making precise modifications to the plastome have been available for three decades, nearly as long as the equivalent techniques for the plant nuclear genome. However, while nuclear genetic engineering quickly moved from a laboratory novelty to a tool used for the improvement of crop plants planted on over a billion acres, plastome engineering remains largely confined to research use. The primary reasons for this lack of application are the species restrictions and technical difficulty of the transformation process, which until recently was only possible in a few species.
In 2007, a protocol for plastid transformation was reported for Marchantia polymorpha, a thalloid liverwort classically used as a model species. Marchantia offers rapid generation time, small size, simple genetics, and asexual reproduction by means of gemmae, small disks of tissue which provide a powerful platform for live-tissue microscopy. In this thesis, tools for Marchantia plastid transformation are systematically improved by the generation of the first plastome sequence assembly for a widely used laboratory strain of Marchantia, optimisation of the transformation protocol itself, and a comparison of the in vivo activity of plastid regulatory elements through a fluorescent marker and quantitative microscopy. The highly conserved nature of the land plant plastome suggests the improvements to plastid transformation developed in Marchantia will translate to other species.
... Moreover, the chloroplast DNA sequences offers adequate information to study the phylogenetics and phylogeography of angiosperms at lower taxonomic levels [8]. In past decades, there have been great advances in our understanding of chloroplasts [22][23][24][25], in terms of their origin, structure, evolution, forward and reverse genetics, and genetic engineering. Moreover, the emergence of second-generation sequencing technology means that there is now less demand for research into chloroplast genomics [26,27]. ...
We sequenced and analyzed the complete chloroplast genome of Aster tataricus (family Asteraceae), a Chinese herb used medicinally to relieve coughs and reduce sputum. The A. tataricus chloroplast genome was 152,992 bp in size, and harbored a pair of inverted repeat regions (IRa and IRb, each 24,850 bp) divided into a large single-copy (LSC, 84,698 bp) and a small single-copy (SSC, 18,250 bp) region. Our annotation revealed that the A. tataricus chloroplast genome contained 115 genes, including 81 protein-coding genes, 4 ribosomal RNA genes, and 30 transfer RNA genes. In addition, 70 simple sequence repeats (SSRs) were detected in the A. tataricus chloroplast genome, including mononucleotides (36), dinucleotides (1), trinucleotides (23), tetranucleotides (1), pentanucleotides (8), and hexanucleotides (1). Comparative chloroplast genome analysis of three Aster species indicated that a higher similarity was preserved in the IR regions than in the LSC and SSC regions, and that the differences in the degree of preservation were slighter between A. tataricus and A. altaicus than between A. tataricus and A. spathulifolius. Phylogenetic analysis revealed that A. tataricus was more closely related to A. altaicus than to A. spathulifolius. Our findings offer valuable information for future research on Aster species identification and selective breeding.
Styphnolobium japonicum is a significant resource of ornamental and medicinal plants. In this study, we employed high-throughput sequencing to assemble nine chloroplast genomes of S. japonicum. We compared and reconstructed the phylogenetic relationships of these genomes, along with three publicly available chloroplast genomes. Our results showed that the length of the 12 S. japonicum chloroplast genomes ranged from 158,613 bp to 158,837 bp, all containing 129 unique functional genes. The genetic diversity within S. japonicum chloroplast genomes was relatively low, with π = 0.00029, Theta-W = 0.00028, and an indel frequency of 0.62 indels/1 kb. Among the four regions, the SSC region exhibited the highest genetic diversity and indel frequency, while the IR region had the lowest. Non-coding regions displayed greater genetic variation compared to coding regions, with a few highly variable regions identified. The phylogenetic tree constructed revealed that the major cultivars of S. japonicum originated from two genetic ‘sources. S. japonicum ‘JinhuaiJ2’ had an independent origin and showed close relatedness to S. japonicum var. violacea, S. japonicum var. japonicum, and S. japonicum f. oligophylla. On the other hand, other major cultivars shared a common genetic origin and were closely related to S. japonicum f. pendula. This study highlights the variability of chloroplast genomes within S. japonicum and provides insights into the genetic origins of major cultivars and their relationships with different varieties and forma.
Avian influenza is a disease of poultry that causes economic losses to poultry farms. Conventional vaccine production requires chicken embryos for virus proliferation with time-consuming and also affected by egg supply during the avian influenza virus outbreak. There are several advantages of plant cells as bio-factories for vaccine production, including low cost, low risk of contamination, and even edible. In this study, we chose carrot (Daucus carota) as a bioreactor to produce hemagglutinin (HA), an avian influenza virus antigen protein. HA gene linked with heat-labile enterotoxin B subunit (LTB) gene whose product plays the role of adjuvant protein was codon optimized with codon usage bias of carrot for high-level expression. The native and codon-optimized LTB–HA were transformed into carrots by Agrobacterium-mediated transformation. T-DNA insertion of two version constructs was confirmed by polymerase chain reaction. Integration of codon-optimized LTB–HA gene in all transgenic carrot lines was identified as a single copy by Southern blot analysis. LTB–HA protein expressed in carrot cells was determined through enzyme-linked immunoassay. Codon-optimized LTB–HA gene showed a better expression level than the native LTB–HA gene in transgenic carrots. The highest expression level of LTB–HA in codon-optimized LTB–HA-transformed carrot taproot reached up to 0.0108% of total soluble protein. This study is the first report about expression of HA antigen of H5N2 subtype AIV in a horticultural crop. Our transgenic carrots have successfully produced HA protein which shows great potential to become an edible vaccine against avian influenza virus.
Tobacco is one of the important commercial crops in the world and is cultivated in more than 120 countries. Various biotic stresses viz. pests, diseases and parasitic weeds infect tobacco from seedling stage to leaf harvest and during post-harvest leaf management there-by severely affecting its leaf yield and quality. Development and deployment of host plant resistance is a sustainable option to minimize these losses. A number of varieties resistant to major biotic stresses viz. TMV, back shank, brown spot, blue mold, powdery mildew, root-knot nematodes, cater pillar, aphid etc. infecting tobacco were developed through conventional breeding. However, lack of reliable sources of resistance, narrow genetic variability, natural barriers of crossing among existing species, longer period required for developing stable homogeneous lines, undesirable associations between the resistant gene and yield and quality contributing characters either due to pleiotropic effects of the resistance gene per se, or due to linkage drag effects caused by the presence of deleterious genes linked to resistant gene, laborious process of screening/phenotyping segregating generations, etc. are slowing down the progress in developing tobacco varieties resistant to biotic stresses through traditional breeding. These limitations can be successfully overcome through molecular breeding and genome designing strategies. In this chapter, the current knowledge about genetic resources, the status of utilization of molecular markers in germplasm characterization and development of molecular maps, identified linked markers and quantitative trait loci (QTLs) to various biotic stresses, omics resources characterized, resistant genes cloned, accessible genomic resource databases etc. were summarized for their effective utilization in designing tobacco genome for higher yields and biotic stress resistance. In addition, advances in Marker-assisted selection (MAS) strategies, gene editing technologies and other genome designing strategies, and their possible utilization in designing tobacco genotypes for biotic stress resistance were also discussed.KeywordsTobacco
Nicotiana
DiseasesPestsStressResistanceGenetic mapsDiversityGenome sequencingMarkersGenomic resourcesQTLGenome designingMASGene editingCloningDatabases
p>Any novel transgenic crop requires rigorous testing prior to commercialisation using an ecological risk assessment framework. The aim of this study is to help inform this process using the parasitoid Cotesia marginiventris, the host Spodoptera littoralis, purified Cry1Ab insecticidal protein and Bt176 transgenic maize.
Under direct exposure in injecting trials, parasitoid tibia length and sex ratio showed no significant difference between C. marginiventris injected with CrylAb or distilled water. Mean pupation was significantly longer in Cry1Ab infected treatment (9.5 days) when compared to the injected control group (9.1 days). C. marginiventris raised on S. littoralis hosts ingesting the toxin at 100μg g<sup>-1</sup> Cry1Ab showed a significantly extended pupation time (11.1 days) when compared to controls (9.5 days), mean tibia length was also significantly shorter at this concentration. The negative effects on pupation time and tibia length were therefore only measurable at 5-10 times plant expression levels. ELISA analysis confirmed that S. littoralis hosts ingested high amounts of Cry1Ab whilst feeding on both plant tissue and artificial diet treatments. However, whilst concentrations of Cry1Ab were found in S. littoralis larvae, frass and parasitoid cocoons none was detectable in parasitoid adults. Therefore the toxin is not accumulating up the food chain and appears to be secreted by C. marginiventris juveniles prior to adult emergence.
Further laboratory feeding experiments were carried out to establish the host mediated effects of Cry1Ab on C. marginiventris attack rate, host preference, parasitism success, emergence success and tibia length when reared on S. littoralis hosts ingesting 200, 20, 0μg g<sup>-1</sup> Cry1Ab, Bt176 transgenic maize or its near isoline. Host preference of adult parasitoids was also assessed to establish if adults showed any avoidance behaviour towards hosts reared on Bt176 maize, no avoidance behaviour was found. There was no significant difference between attack rate, proportion of attacks resulting in cocoon formation or proportion of adults emerging between the treatments. Tibia length was significantly shorter in the Bt176, 200 and 20μg g<sup>-1</sup> CrylAb treatments when compared to the near isoline and control artificial diet treatments. However, there was a trend towards reduced parasitism and emergence success following exposure to the Cry1Ab toxin. C. marginiventris larvae were exposed to the toxin via S. littoralis hosts at 100μg g<sup>-1</sup> over two generations. Following two generations of exposure to the toxin pupation time was significantly longer (10.4 days) when compared to controls (10.0 days). Neither tibia length, parasitism or emergence success were significantly different following exposure over two generations.</p
Chloroplasts are one of the most studied organelles of the plant in terms of their evolution, ultrastructure, genomic organization, and biochemistry, specifically in relation to the different photosynthetic pathways in the plants. The very sustenance of life on earth depends on these subcellular organelles. Chloroplasts are of endosymbiotic origin and in spite of having its own genome independent of the nuclear genome, over 95% of chloroplast proteins are encoded by nuclear genes. We discuss the various carbon-assimilating pathways in plants with specific reference to the different types of C4 pathways. In this chapter, we discuss the endosymbiotic origins of chloroplast with specific reference to protein transport and chloroplastic tRNA, which are a family of highly conserved genes through the various events that occurred during the evolution of chloroplasts. We also discuss on the method of transgene introduction through chloroplast genome engineering which can be used to achieve tolerance to various climate related threats and evolution of climate ready cultivars.
Edible vaccines hold great promise as a cost-effective, easy-to-administer, easy-to-store, fail-safe and socioculturally readily acceptable vaccine delivery system, especially for the poor developing countries. It involves introduction of selected desired genes into plants and then inducing these altered plants to manufacture the encoded proteins. Introduced as a concept about a decade ago, it has become a reality today. A variety of delivery systems have been developed. Initially thought to be useful only for preventing infectious diseases, it has also found application in prevention of autoimmune diseases, birth control, cancer therapy, etc. Edible vaccines are currently being developed for a number of human and animal diseases. There is growing acceptance of transgenic crops in both industrial and developing countries. Resistance to genetically modified foods may affect the future of edible vaccines. They have passed the major hurdles in the path of an emerging vaccine technology. Various technical obstacles, regulatory and non-scientific challenges, though all seem surmountable, need to be overcome. This review attempts to discuss the current status and future of this new preventive modality.
The sections in this article are
Introduction
Historical Aspects
Unique Features of Chloroplast Genetic Engineering
Maternal Inheritance and Gene Containment
Crop Species Stably Transformed via the Plastid Genome
Agronomic Traits Conferred via the Plastid Genome
Transgenic Plastids as Bioreactors
Biomaterials, Enzymes, and Amino Acids
Conclusions
Acknowledgements
The public has working notions of heredity that permit functionally adequate judgment in most matters, but that differ from the perspective of molecular genetics in significant ways. Non-specialists also have limited knowledge of plant and animal breeding prior to the advent of recombinant DNA methods for gene transfer. Concerns about the “deficit model” of science communication notwithstanding (see Chap. 12), a discussion of key terms (genes, GMOs, mutations, and biotechnology itself), reveals the ethical significance of this gap between expert and lay understanding of genetics. The chapter also functions as an overview and introduction to agrifood biotechnology for non-specialists. Past errors, globalization and growing environmental consciousness create a need for greater vigilance and ethical awareness in the discovery, design and implementation of technological innovations. The interpretive disconnect between experts and the public becomes significant because it frustrates effective public oversight, ethical reflection and democratic participation in the development of biotechnology.
With distribution of genetic materials and advance molecular characteristics,
the chloroplast is prokaryotic compartments within the eukaryotic plants that
have turned into a crucial source for the genetic engineering and transplastomic
plants are becoming more popular means of agricultural development with elevated
crop yield. To address global agricultural problems, genetic modification of crop
plants is a rapid and promising solution to adapt the environment-friendly and well-controlled
farming system. The transplastomic plant with high accumulation of foreign
proteins (up to 45–46% TSP) and stable transgene expression with gene
containment can be a unique choice for the agricultural innovation of coming centuries.
Although the transplastomic plants still facing encumber to ensure the full
potential exploitation and expansion as an economical means, the removal of hardness
and obstacles of this technology and commercialization can contribute for the
sustainable development of future agriculture. In this book chapter, we intend to
recapitulate the up to date development and achievement of transplastomic plant
including gene transfer procedures in plastid genomes, regulable expression of plastid
transgenes, plant trait improvement by foreign gene expression, biopharmaceuticals
production, engineering of metabolic pathways in plant, study of transformation
mediated RNA editing technologies, bio-safety issues and public concerns on transplastomic plants and overall beneficial aspects. We believe that the utilization of
transplastomic plants will ensure an eco-
Plant genetic engineering has become an inevitable tool in the molecular breeding of crops. Significant progress has been made in the generation of novel plastid transformation vectors and optimized transformation protocols. There are several advantages of plastid genome engineering over conventional nuclear transformation. Some of the advantages include multigene engineering by expression of biosynthetic pathway genes as operons, extremely high-level expression of protein accumulation, lack of transgene silencing, etc. Transgene containment owing to maternal inheritance is another important advantage of plastid genome engineering. Chloroplast genome modification usually results in alteration of several thousand plastid genome copies in a cell. Several therapeutic proteins, edible vaccines, antimicrobial peptides, and industrially important enzymes have been successfully expressed in chloroplasts so far. Here, we critically recapitulate the latest developments in plastid genome engineering. Latest advancements in plastid genome sequencing are briefed. In addition, advancement of extending the toolbox for plastid engineering for selected applications in the area of molecular farming and production of industrially important enzyme is briefed.
An imbalance in oxygen supply to cardiac tissues or formation of thrombus leads to deleterious results like pulmonary embolism, coronary heart disease and acute cardiac failure. The formation of thrombus requires clinical encounter with fibrinolytic agents including streptokinase, urokinase or tissue plasminogen activator. Irrespective to urokinase and tissue plasminogen activator, streptokinase is still a significant agent in treatment of cardiovascular diseases. Streptokinase, being so economical, has an important value in treating cardiac diseases in developing countries. This review paper will provide the maximum information to enlighten all the pros and cons of streptokinase up till now. It has been concluded that recent advances in structural/synthetic biology improved SK with enhanced half-life and least antigenicity. Such enzyme preparations would be the best thrombolytic agents.
The 2.5 Å structure of the cytochrome b6f complex provides a basis for control of the rate‐limiting electron transfer step of oxygenic photosynthesis associated with the plastoquinol/quinone exchange pathway. Here, a structural change was made at a site containing two proline residues which border the intra‐cytochrome pathway for plastoquinol/quinone exchange. The proline side chains confer a larger aperture for passage of plastoquinol/quinone. Change of these prolines to alanine in the cyanobacterium Synechococcus sp. PCC 7002 results in attenuation of this rate‐limiting step, observed by a two‐fold reduction in the rate of cell growth, O2 evolution, and plastoquinol‐mediated reduction of cytochrome f. This study demonstrates modification by site‐directed mutagenesis of photosynthetic energy transduction based on rational application of information in the atomic structure. This article is protected by copyright. All rights reserved.
The effect of salt stress on ethanol endurance of yeast cells was studied. Cells grown under increased NaCl concentrations were more ethanol tolerant than controls. The increase in trehalose content under hyper-saline conditions has been suggested to allow cells to withstand higher ethanolic conditions. There seems to be an overlap between osmotolerance and ethanol endurance in Saccharomyces cerevisiae.
Transient expression of β-glucuronidase (GUS) in different cellular compartments following biolistic delivery of chloroplast or nuclear expression vectors into wheat leaves or calli, derived from anther culture or immature embryos, is reported here. When pB1121, the nuclear GUS vector, was used to bombard wheat cells, the β-glucuronidase product, an insoluble indigo dye, was observed evenly throughout the cytosol. But, when the chloroplast expression vector pHD203-GUS was used for bombardments, the indigo dye (GUS product) was subcellularly localized within the chloroplasts of wheat cells. The observation of GUS expression in albino plastids, when anther culture derived albino leaves were bombarded with the chloroplast expression vector pHD203-GUS, suggests the presence of a functional protein synthetic machinery in these organelles. GUS expression was also observed in regenerable calli derived from wheat immature embryos bombarded with pHD203-GUS. Leaves or calli bombarded with pUC19, as negative controls, did not show any GUS expression. These results constitute the first demonstration of foreign gene expression in chloroplasts of a monocot and that a dicot chloroplast promoter functions in a monocot chloroplast.
Bioelastic protein-based polymers (PBP) have several medical (prevention of post-surgical adhesions) and non-medical (biodegradable
plastic) applications. This study compares expression levels of PBP genes (synthetic) integrated into the nuclear genome or
the large single-copy (LSC) or inverted repeat (IR) region of the chloroplast genome in transgenic tobacco plants. Polymer
transcripts accumulated up to 100-fold higher in the IR plants than in those of nuclear transgenic plants. Integration of
foreign genes into all of the chloroplast genomes (homoplasmy) and higher levels of polymer transcripts were observed only
in the IR and not in LSC transgenic plants. Expression of the polymer protein was further confirmed by Western blot analysis.
Several environmental problems related to plant genetic engineering may prohibit advancement of this technology and prevent
realization of its full potential. One such common concern is the demonstrated escape of foreign genes through pollen dispersal
from transgenic crop plants to their weedy relatives, creating super weeds or causing gene pollution among other crops or
toxicity of transgenic pollen to nontarget insects. The high rates of gene flow from crops to wild relatives (as high as 38%
in sunflower and 50% in strawberries) are certainly a serious concern. Maternal inheritance of the herbicide resistance gene
via chloroplast genetic engineering has been shown to be a practical solution to these problems. Another common concern is
the suboptimal production of Bacillus thuringiensis (Bt) insecticidal protein or reliance on a single (or similar) B.t. protein in commercial transgenic crops, resulting in
B.t. resistance among target pests. Clearly, different insecticidal proteins should be produced in lethal quantities to decrease
the development of resistance. Such hyperexpression of a novel B.t. protein in chloroplasts has resulted in 100% mortality
of insects that are up to 40 000-fold resistant to other B.t. proteins. Yet another concern is the presence of antibiotic
resistance genes in transgenic plants that could inactivate oral doses of the antibiotic or be transferred to pathogenic microbes
in the GI tract or in soil, rendering them resistant to treatment with such antibiotics. Cotransformation and elimination
of antibiotic resistant genes from transgenic plants using transposable elements via breeding are promising new approaches.
Genetic engineering efforts have also addressed yet another concern, i.e., the accumulation and persistence of plastics in
our environment by production of biodegradable plastics. Recent approaches and accomplishments in addressing these environmental
concerns via chloroplast genetic engineering are discussed in this review.
Evolving levels of resistance in insects to the bioinsecticide Bacillus thuringiensis (Bt) can be dramatically reduced through the genetic engineering of chloroplasts in plants. When transgenic tobacco leaves expressing Cry2Aa2 protoxin in chloroplasts were fed to susceptible, Cry1A-resistant (20,000- to 40,000-fold) and Cry2Aa2-resistant (330- to 393-fold) tobacco budworm Heliothis virescens, cotton bollworm Helicoverpa zea, and the beet armyworm Spodoptera exigua, 100% mortality was observed against all insect species and strains. Cry2Aa2 was chosen for this study because of its toxicity to many economically important insect pests, relatively low levels of cross-resistance against Cry1A-resistant insects, and its expression as a protoxin instead of a toxin because of its relatively small size (65 kDa). Southern blot analysis confirmed stable integration of cry2Aa2 into all of the chloroplast genomes (5,000–10,000 copies per cell) of transgenic plants. Transformed tobacco leaves expressed Cry2Aa2 protoxin at levels between 2% and 3% of total soluble protein, 20- to 30-fold higher levels than current commercial nuclear transgenic plants. These results suggest that plants expressing high levels of a nonhomologous Bt protein should be able to overcome or at the very least, significantly delay, broad spectrum Bt-resistance development in the field.
Expression of chloramphenicol acetyltransferase (cat) by suitable vectors in chloroplasts of cultured tobacco cells, delivered by high-velocity microprojectiles, is reported here. Several chloroplast expression vectors containing bacterial cat genes, placed under the control of either psbA promoter region from pea (pHD series) or rbcL promoter region from maize (pAC series) have been used in this study. In addition, chloroplast expression vectors containing replicon fragments from pea, tobacco, or maize chloroplast DNA have also been tested for efficiency and duration of cat expression in chloroplasts of tobacco cells. Cultured NT1 tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 (negative control), 35S-CAT (nuclear expression vector), pHD312 (repliconless chloroplast expression vector), and pHD407, pACp18, and pACp19 (chloroplast expression vectors with replicon). Sonic extracts of cells bombarded with pUC118 showed no detectable cat activity in the autoradiograms. Nuclear expression of cat reached two-thirds of the maximal 48 hr after bombardment and the maximal at 72 hr. Cells bombarded with chloroplast expression vectors showed a low level of expression until 48 hr of incubation. A dramatic increase in the expression of cat was observed 24 hr after the addition of fresh medium to cultured cells in samples bombarded with pHD407; the repliconless vector pHD312 showed about 50% of this maximal activity. The expression of nuclear cat and the repliconless chloroplast vector decreased after 72 hr, but a high level of chloroplast cat expression was maintained in cells bombarded with pHD407. Organelle-specific expression of cat in appropriate compartments was checked by introducing various plasmid constructions into tobacco protoplasts by electroporation. Although the nuclear expression vector 35S-CAT showed expression of cat, no activity was observed with any chloroplast vectors.
The uptake and expression by plastids isolated from dark-grown cucumber cotyledons (etioplasts) of two pUC derivatives, pCS75 and pUC9-CM, respectively carrying genes for the large small subunits of ribulose bisphosphate carboxylase/oxygenase of Anacystis nidulans or chloramphenicol acetyltransferase, is reported. Untreated etioplasts take up only 3% as much DNA as that taken up by EDTA-washed etioplasts after 2 hr of incubation with nick-translated [32P]-pCS75. The presence or absence of light does not affect DNA uptake, binding, or breakdown by etioplasts. Calcium or magnesium ions inhibit DNA uptake by 86% but enhance binding (23-200%) and breakdown (163-235%) of donor DNA by EDTA-treated etioplasts. Uncouplers that abolish membrane potential (delta psi), transmembrane proton gradient (delta pH), or both do not affect DNA uptake, binding, or breakdown by etioplast. However, both DNA uptake and binding are severely inhibited by ATP. Presumably this results from the hydrolysis of ATP, because the poorly hydrolyzable analog adenyl-5'-yl imidodiphosphate does not inhibit the uptake or binding of DNA by etioplasts. beta-Lactamase specified by the ampicillin resistance gene of pCS75 can be detected only in EDTA-treated etioplasts that have been incubated with the plasmid pCS75. After the incubation of EDTA-treated etioplasts with pCS75, immunoprecipitation using antiserum to the small subunit of ribulose bisphosphate carboxylase/oxygenase from A. nidulans reveals the synthesis of small subunits; these are smaller by 2 kDa than the cucumber small subunit encoded by the nuclear genome. Treatment of etioplasts with 10 mM EDTA shows a 10-min duration to be optimal for the expression of chloramphenicol acetyltransferase encoded by pUC9-CM. A progressive increase in the expression of this enzyme is observed with an increase in the concentration of pUC9-CM in the DNA uptake medium. The plasmid-dependent incorporation of [35S]methionine by EDTA-treated organelles declines markedly during cotyledon greening in vivo.
Glyphosate is a potent herbicide. It works by competitive inhibition of the enzyme 5-enol-pyruvyl shikimate-3-phosphate synthase (EPSPS), which catalyzes an essential step in the aromatic amino acid biosynthetic pathway. We report the genetic engineering of herbicide resistance by stable integration of the petunia EPSPS gene into the tobacco chloroplast genome using the tobacco or universal vector. Southern blot analysis confirms stable integration of the EPSPS gene into all of the chloroplast genomes (5000-10,000 copies per cell) of transgenic plants. Seeds obtained after the first self-cross of transgenic plants germinated and grew normally in the presence of the selectable marker, whereas the control seedlings were bleached. While control plants were extremely sensitive to glyphosate, transgenic plants survived sprays of high concentrations of glyphosate. Chloroplast transformation provides containment of foreign genes because plastid transgenes are not transmitted by pollen. The escape of foreign genes via pollen is a serious environmental concern in nuclear transgenic plants because of the high rates of gene flow from crops to wild weedy relatives.
Pollen-mediated movement of transgenes from transplastomic oilseed rape (Brassica napus) into wild relatives will be avoided if chloroplasts are maternally transmitted. We assess the probability of chloroplast exchange between conventional oilseed rape and wild Brassica rapa to model the future behavior of transplastomic cultivars. Primers specific to cpDNA were used to demonstrate maternal inheritance of chloroplasts in 47 natural hybrids between cultivated B. napus and wild B. rapa. We conclude that there will be no or negligible pollen-mediated chloroplast dispersal from oilseed rape. Transgene introgression could still occur in mixed populations, however, if B. napus acted as the recurrent female parent. Rate of transfer would then depend on the abundance of mixed populations, their persistence as mixtures, and hybridization frequency within stands. A low incidence of sympatry (0.6-0.7%) between wild B. rapa and cultivated B. napus along the river Thames, UK, in 1997 and 1998, suggests mixed stands will form only rarely. Eighteen feral populations of B. napus also showed a strong tendency toward rapid decline in plant number, seed return, and ultimately, extinction within 3 years. Conversely, hybrid production is significant in mixed stands, and the absence of control practices means that oilseed rape will have slightly greater persistence. We infer that some introgression from transplastomic B. napus into B. rapa is inevitable in mixed populations even though such populations will occur infrequently and will tend to lose B. napus plants relatively quickly. Chloroplast exchange will be extremely rare and scattered.
A galinstan expansion femtosyringe enables femtoliter to attoliter samples to be introduced into prokaryotes and subcellular compartments of eukaryotes. The method uses heat-induced expansion of galinstan (a liquid metal alloy of gallium, indium, and tin) within a glass syringe to expel samples through a tip diameter of about 0.1 microm. The narrow tip inflicts less damage than conventional capillaries, and the heat-induced expansion of the galinstan allows fine control over the rate of injection. We demonstrate injection of Lucifer Yellow and Lucifer Yellow-dextran conjugates into cyanobacteria, and into nuclei and chloroplasts of higher organisms. Injection of a plasmid containing the bla gene into the cyanobacterium Phormidium laminosum resulted in transformed ampicillin-resistant cultures. Green fluorescent protein was expressed in attached leaves of tobacco and Vicia faba following injection of DNA containing its gene into individual chloroplasts.
Rice (Oryza sativa), a major staple food, is usually milled to remove the oil-rich aleurone layer that turns rancid upon storage, especially
in tropical areas. The remaining edible part of rice grains, the endosperm, lacks several essential nutrients, such as provitamin
A. Thus, predominant rice consumption promotes vitamin A deficiency, a serious public health problem in at least 26 countries,
including highly populated areas of Asia, Africa, and Latin America. Recombinant DNA technology was used to improve its nutritional
value in this respect. A combination of transgenes enabled biosynthesis of provitamin A in the endosperm.
Transgenic plants have become attractive systems for production of human therapeutic proteins because of the reduced risk of mammalian viral contaminants, the ability to do large scale-up at low cost, and the low maintenance requirements. Here we report a feasibility study for production of a human therapeutic protein through transplastomic transformation technology, which has the additional advantage of increased biological containment by apparent elimination of the transmission of transgenes through pollen. We show that chloroplasts can express a secretory protein, human somatotropin, in a soluble, biologically active, disulfide-bonded form. High concentrations of recombinant protein accumulation are observed (>7% total soluble protein), more than 300-fold higher than a similar gene expressed using a nuclear transgenic approach. The plastid-expressed somatotropin is nearly devoid of complex post-translational modifications, effectively increasing the amount of usable recombinant protein. We also describe approaches to obtain a somatotropin with a non-methionine N terminus, similar to the native human protein. The results indicate that chloroplasts are a highly efficient vehicle for the potential production of pharmaceutical proteins in plants.
In nuclear transgenic plants, expression of multiple genes requires introduction of individual genes and time-consuming subsequent backcrosses to reconstitute multi-subunit proteins or pathways, a problem that is compounded by variable expression levels. In order to accomplish expression of multiple genes in a single transformation event, we have introduced several genes into the chromoplast genome. We confirmed stable integration of the cry2Aa2 operon by PCR and Southern blot analyses in T(0) and T(1) transgenic plants. Foreign protein accumulated at 45.3% of the total soluble protein in mature leaves and remained stable even in old bleached leaves (46.1%), thereby increasing the efficacy and safety of transgenic plants throughout the growing season. This represents the highest level of foreign gene expression reported in transgenic plants to date. Insects that are normally difficult to control (10-day old cotton bollworm, beet armyworm) were killed 100% after consuming transgenic leaves. Electron micrographs showed the presence of the insecticidal protein folded into cuboidal crystals. Formation of crystals of foreign proteins (due to hyperexpression and folding by the putative chaperonin, ORF 2) provides a simple method of purification by centrifugation and enhances stability by protection from cellular proteases. Demonstration of expression of an operon in transgenic plants paves the way to engineering new pathways in plants in a single transformation event.
The objective of this study was to determine if mRNA sequences downstream of the translation initiation codon are important for translation of plastid mRNAs. We have employed a transgenic approach, measuring accumulation of the neomycin phosphotransferase (NPTII) reporter enzyme translationally fused with 14 N-terminal amino acids encoded in the rbcL or atpB plastid genes. NPTII accumulation from wild-type and mutant rbcL and atpB segments was compared. We report that silent mutations in the rbcL segment reduced NPTII accumulation 35-fold. In contrast, mutations in the atpB mRNA reduced NPTII accumulation only moderately from approximately 7% (w/w) to approximately 4% (w/w) of the total soluble cellular protein, indicating that the importance of sequences downstream of the translation initiation codon are dependent on the individual mRNA. Information provided here will facilitate transgene design for high-level expression of recombinant proteins in chloroplasts by translational fusion with the N-terminal segment of highly expressed plastid genes or by introduction of silent mutations in the N-terminal part of the coding region.
Phosphinothricin (PPT) is the active component of a family of environmentally safe, nonselective herbicides. Resistance to PPT in transgenic crops has been reported by nuclear expression of a bar transgene encoding phosphinothricin acetyltransferase, a detoxifying enzyme. We report here expression of a bacterial bar gene (b-bar1) in tobacco (Nicotiana tabacum cv Petit Havana) plastids that confers field-level tolerance to Liberty, an herbicide containing PPT. We also describe a second bacterial bar gene (b-bar2) and a codon-optimized synthetic bar (s-bar) gene with significantly elevated levels of expression in plastids (>7% of total soluble cellular protein). Although these genes are expressed at a high level, direct selection thus far did not yield transplastomic clones, indicating that subcellular localization rather than the absolute amount of the enzyme is critical for direct selection of transgenic clones. The codon-modified s-bar gene is poorly expressed in Escherichia coli, a common enteric bacterium, due to differences in codon use. We propose to use codon usage differences as a precautionary measure to prevent expression of marker genes in the unlikely event of horizontal gene transfer from plastids to bacteria. Localization of the bar gene in the plastid genome is an attractive alternative to incorporation in the nuclear genome since there is no transmission of plastid-encoded genes via pollen.
Chloroplast genetic engineering offers several advantages over nuclear transformation including high levels of gene expression and gene containment. However, a consequence of placing a transgene in the chloroplast genome is that the antibiotic resistance genes used as selectable markers are highly amplified. Engineering genetically modified (GM) crops without the use of antibiotic resistance genes should eliminate the potential risk of their transfer to the environment or gut microbes. Therefore, the betaine aldehyde dehydrogenase (BADH) gene from spinach was used in this study as a selectable marker. The selection process involves conversion of toxic betaine aldehyde (BA) by the chloroplast BADH enzyme to non-toxic glycine betaine, which also serves as an osmoprotectant. Chloroplast transformation efficiency was 25-fold higher in BA selection than with spectinomycin. In addition, rapid regeneration was obtained. Transgenic shoots appeared within 12 days in 80% of leaf disks (up to 23 shoots per disk) under BA selection compared to 45 days in 15% of disks (1 or 2 shoots per disk) under spectinomycin selection. Southern blots confirmed stable integration of foreign genes into all of the chloroplast genomes (approximately 10,000 copies per cell) resulting in homoplasmy. Transgenic tobacco plants showed 15- to 18-fold higher BADH activity at different developmental stages than untransformed controls. Transgenic plants were morphologically indistinguishable from untransformed plants and the introduced trait was inherited stably in the subsequent generation. This is the first report of genetic engineering of the higher plant chloroplast genome without the use of antibiotic selection. The use of naturally occurring genes in spinach for selection, in addition to gene containment, should ease public concerns regarding GM crops.
Anthranilate synthase (AS), the control enzyme of the tryptophan (Trp) biosynthetic pathway, is encoded by nuclear genes, but is transported into the plastids. A tobacco (Nicotiana tabacum) cDNA (ASA2) encoding a feedback-insensitive tobacco AS α-subunit was transformed into two different sites of the tobacco plastid genome through site-specific insertion to obtain transplastomic plants with normal phenotype and fertility. A high and uniform level of ASA2 mRNA was observed in the transplastomic plants but not in the wild type. Although the plants with the transgene insertion atndhF-trnL only expressed one size of theASA2 mRNA, the plants with the transgene incorporated into the region between accD and open reading frame (ORF) 184 exhibited two species of mRNA, apparently due to readthrough. The transplastomic plants exhibited a higher level of AS α-subunit protein and AS enzyme activity that was less sensitive to Trp-feedback inhibition, leading to greatly increased free Trp levels in leaves and total Trp levels in seeds. Resistance to an AS inhibitor, 5-methyl-Trp, was found during seed germination and in suspension cultures of the transplastomic plants. The resistance to the selection agent spectinomycin and to 5-methyl-Trp was transmitted maternally. These results demonstrate the feasibility of modifying the biosynthetic pathways of important metabolites through transformation of the plastid genome by relocating a native gene from the nucleus to the plastid genome. Very high and uniform levels of gene expression can be observed in different lines, probably due to the identical insertion sites, in contrast to nuclear transformation where random insertions occur.
We describe here the development of a reproducible plastid transformation system for potato and regeneration of plants with uniformly transformed plastids. Two distinct tobacco-specific plastid vectors, pZS197 (Prrn/aadA/TpsbA) and pMON30125 (Prrn/GFP/ Trps16::PpsbA/aadA/TpsbA), designed for integration into the large single copy and inverted repeat regions of the plastid genome, respectively, were bombarded into leaf explants of potato line FL1607. A total of three transgenic lines were selected out of 46 plates bombarded with pZS197 and three transgenic lines out of 104 plates were obtained with pMON30125. Development of a high frequency leaf-based regeneration system, a stringent selection scheme and optimization of biolistic transformation protocol were critical for recovery of plastid transformants. Plastidexpressed green fluorescent protein was used as a visual marker for identification of plastid transformants at the early stage of selection and shoot regeneration. The establishment of a plastid transformation system in potato, which has several advantages over routinely used nuclear transformation, offers new possibilities for genetic improvement of this crop.
The effect of salt stress on ethanol endurance of yeast cells was studied. Cells grown under increased NaCl concentrations were more ethanol tolerant than controls. The increase in trehalose content under hyper-saline conditions has been suggested to allow cells to withstand higher ethanolic conditions. There seems to be an overlap between osmotolerance and ethanol endurance in Saccharomyces cerevisiae.
The green fluorescent protein gene (gfp) is a widely used reporter in both animals and plants. Fusions between the plastidrrnpromoter or theEscherichia coli trcpromoter and thegfpcoding region have been delivered to chloroplasts using gold or tungsten microprojectiles, and fluorescence from GFP was visible in individual tobacco chloroplasts and in the abnormally large chloroplasts of thearc6 mutant ofArabidopsis thaliana2–4 days after bombardment. The fusion of thegfpcoding region to the bacterialtrcpromoter demonstrated that a bacterial promoter is active in chloroplastsin vivo. GFP was also detectable in amyloplasts of potato tubers and in chromoplasts of marigold petals, carrot roots and pepper fruits 4 days after bombardment. This demonstrates that GFP can be used as a reporter for transient gene expression in chloroplasts and in non-photosynthetic plastids in a range of higher plants.
Certain higher plants synthesize and accumulate glycine betaine, a compound with osmoprotectant properties. Biosynthesis of glycine betaine proceeds via the pathway choline betaine aldehyde glycine betaine. Plants such as tobacco (Nicotiana tabacum L.) which do not accumulate glycine betaine lack the enzymes catalyzing both reactions. As a step towards engineering glycine betaine accumulation into a non-accumulator, spinach and sugar beet complementary-DNA sequences encoding the second enzyme of glycine-betaine synthesis (betaine aldehyde dehydrogenase, BADH, EC 1.2.1.8) were expressed in tobacco. Despite the absence of a typical transit peptide, BADH was targeted to the chloroplast in leaves of transgenic plants. Levels of extractable BADH were comparable to those in spinach and sugar beet, and the molecular weight, isoenzyme profile and K
m for betaine aldehyde of the BADH enzymes from transgenic plants were the same as for native spinach or sugar beet BADH. Transgenic plants converted supplied betaine aldehyde to glycine betaine at high rates, demonstrating that they were able to transport betaine aldehyde across both the plasma membrane and the chloroplast envelope. The glycine betaine produced in this way was not further metabolized and reached concentrations similar to those in plants which accumulate glycine betaine naturally. Betaine aldehyde was toxic to non-transformed tobacco tissues whereas transgenic tissues were resistant due to detoxification of betaine aldehyde to glycine betaine. Betaine aldehyded ehydrogenase is therefore of interest as a potential selectable marker, as well as in the metabolic engineering of osmoprotectant biosynthesis.
Chloroplast genetic engineering offers several advantages over nuclear genetic engineering, including gene containment and hyperexpression. However, introducing thousands of copies of transgenes into the chloroplast genome amplifies the antibiotic resistance genes. Two recent articles report different and novel strategies to either remove antibiotic resistance genes or select chloroplast transformants without using these genes. This should eliminate their potential transfer to microorganisms or plants and ease public concerns about genetically modified crops.
The conversion of genetic information stored in DNA into a protein product proceeds through the obligatory intermediate of messenger RNA. The steady-state level of an mRNA is determined by its relative synthesis and degradation rates, i.e., an interplay between transcriptional regulation and control of RNA stability. When the biological status of an organism requires that a gene product’s abundance varies as a function of developmental stage, environmental factors or intracellular signals, increased or decreased RNA stability can be the determining factor. RNA stability and processing have long been known as important regulatory points in chloroplast gene expression. Here we summarize current knowledge and prospects relevant to these processes, emphasizing biochemical data. The extensive literature on nuclear mutations affecting chloroplast RNA metabolism is reviewed in another article in this volume (Barkan and Goldschmidt-Clermont, this issue).
Over the past decade, scientific advances in molecular biology and immunology have improved understanding of many diseases and led to the development of novel strategies for vaccination. The development of plants expressing vaccine antigens is a particularly promising approach. Plant-derived antigenic proteins have delayed or prevented the onset of disease in animals and have proven to be safe and functional in human clinical trials. Future areas of research should further characterize the induction of the mucosal immune system and appropriate crop species for delivery of animal and human vaccines.
Plastid transformation in higher plants is accomplished through a gradual process, during which all the 300-10,000 plastid genome copies are uniformly altered. Antibiotic resistance genes incorporated in the plastid genome facilitate maintenance of transplastomes during this process. Given the high number of plastid genome copies in a cell, transformation unavoidably yields chimeric tissues, which requires the identification of transplastomic cells in order to regenerate plants. In the chimeric tissue, however, antibiotic resistance is not cell autonomous: transplastomic and wild-type sectors both have a resistant phenotype because of phenotypic masking by the transgenic cells. We report a system of marker genes for plastid transformation, termed FLARE-S, which is obtained by translationally fusing aminoglycoside 3"-adenyltransferase with the Aequorea victoria green fluorescent protein. 3"-adenyltransferase (FLARE-S) confers resistance to both spectinomycin and streptomycin. The utility of FLARE-S is shown by tracking segregation of individual transformed and wild-type plastids in tobacco and rice plants after bombardment with FLARE-S vector DNA and selection for spectinomycin and streptomycin resistance, respectively. This method facilitates the extension of plastid transformation to nongreen plastids in embryogenic cells of cereal crops.
Expression vectors for Chlamydomonas reinhardtii chloroplast transformation have been constructed with transcription and translation signals from chloroplast genes. The bacterial
aadA sequence, coding for aminoglycoside 3″ adenyl transferase, was inserted in these vectors and introduced into the C. reinhardtii chloroplast by particle gun transformation. The stable tranagenic expression of this foreign protein in the chloroplast confers
spectinomycin and streptomycin resistance to the transformed cells. This new marker can be used as a reporter of gene expression,
and as a portable selectable cassette for chloroplast reverse genetics. Targetted gene disruption mutants of loci required
for photosynthesis, tscA and psaC, were thus obtained. A gene disruption of an unidentified open reading frame, ORF472, remained heteroplasmic, suggesting
that it has a vital function.
Four transgenic Nicotiana tabacum plants were generated that expressed a murine monoclonal antibody kappa chain, a hybrid immunoglobulin A-G heavy chain, a murine joining chain, and a rabbit secretory component, respectively. Successive sexual crosses between these plants and filial recombinants resulted in plants that expressed all four protein chains simultaneously. These chains were assembled into a functional, high molecular weight secretory immunoglobulin that recognized the native streptococcal antigen I/II cell surface adhesion molecule. In plants, single cells are able to assemble secretory antibodies, whereas two different cell types are required in mammals. Transgenic plants may be suitable for large-scale production of recombinant secretory immunoglobulin A for passive mucosal immunotherapy. Plant cells also possess the requisite mechanisms for assembly and expression of other complex recombinant protein molecules.
Magainins are a family of linear, amphipathic, cationic antimicrobial peptides, 21 to 27 residues in length, found in the skin of Xenopus laevis. They kill microbial targets through disruption of membrane permeability. They exhibit selectivity, on the basis of their affinity for membranes which contain accessible acidic phospholipids, a property characterizing the cytoplasmic membranes of many species of bacteria. Magainins are broad-spectrum antimicrobial agents exhibiting cidal activity against Gram-negative and Gram-positive bacteria, fungi and protozoa. In addition these peptides lyse many types of murine and human cancer cells at concentrations 5-10-fold lower than normal human cells. Because of their selectivity, broad spectrum, low degree of bacterial resistance and ease of chemical synthesis, magainins are being developed as human therapeutic agents. The most advanced candidate is MSI-78, a 22-residue magainin analogue. This peptide is currently in human Phase IIb/III clinical trials in studies intended to evaluate its efficacy as a topical agent for the treatment of impetigo. Preclinical studies have demonstrated that analogues of magainin exhibit activity in vivo against malignant melanoma and ovarian cancer cells in mouse models. Intravenous administration of several magainin analogues has been shown to treat effectively systemic Escherichia coli infections in the mouse.
In the bacterium Alcaligenes eutrophus, three genes encode the enzymes necessary to catalyze the synthesis of poly[(R)-(-)-3-hydroxybutyrate] (PHB) from acetyl-CoA. In order to target these enzymes into the plastids of higher plants, the genes were modified by addition of DNA fragments encoding a pea chloroplast transit peptide, a constitutive plant promoter, and a poly(A) addition sequence. Each of the modified bacterial genes was introduced into Arabidopsis thaliana by Agrobacterium-mediated transformation, and plants containing all three genes were obtained by sexual crosses. These plants accumulated PHB up to 14% of the dry weight as 0.2- to 0.7-micron granules within plastids. In contrast to earlier experiments in which expression of the PHB biosynthetic pathway in the cytoplasm led to a deleterious effect on growth, expression of the PHB biosynthetic pathway in plastids had no obvious effect on the growth or fertility of the transgenic plants and resulted in a 100-fold increase in the amount of PHB that accumulated. We conclude that there does not appear to be any biological barrier to high-level production of PHB in higher plants. The high level of PHB accumulation also suggests that the synthesis of plastid acetyl-CoA is regulated by a mechanism which responds to metabolic demand.
We report on a novel chimeric gene that confers kanamycin resistance on tobacco plastids. The kan gene from the bacterial transposon Tn5, encoding neomycin phosphotransferase (NPTII), was placed under control of plastid expression signals and cloned between rbcL and ORF512 plastid gene sequences to target the insertion of the chimeric gene into the plastid genome. Transforming plasmid pTNH32 DNA was introduced into tobacco leaves by the biolistic procedure, and plastid transformants were selected by their resistance to 50 micrograms/ml of kanamycin monosulfate. The regenerated plants uniformly transmitted the transplastome to the maternal progeny. Resistant clones resulting from incorporation of the chimeric gene into the nuclear genome were also obtained. However, most of these could be eliminated by screening for resistance to high levels of kanamycin (500 micrograms/ml). Incorporation of kan into the plastid genome led to its amplification to a high copy number, about 10,000 per leaf cell, and accumulation of NPTII to about 1% of total cellular protein.
We report here a 100-fold increased frequency of plastid transformation in tobacco by selection for a chimeric aadA gene encoding aminoglycoside 3"-adenylyltransferase, as compared with that obtained with mutant 16S rRNA genes. Expression of aadA confers resistance to spectinomycin and streptomycin. In transforming plasmid pZS197, a chimeric aadA is cloned between rbcL and open reading frame ORF512 plastid gene sequences. Selection was for spectinomycin resistance after biolistic delivery of pZS197 DNA into leaf cells. DNA gel-blot analysis confirmed incorporation of the chimeric aadA gene into the plastid genome by two homologous recombination events via the flanking plastid gene sequences. The chimeric gene became homoplasmic in the recipient cells and is uniformly transmitted to the maternal seed progeny. The ability to transform routinely plastids of land plants opens the way to manipulate the process of photosynthesis and to incorporate novel genes into the plastid genome of crops.
Several approaches have been used in the past to manipulate genes in chloroplasts—that is, in the generation of chloroplast mutants, protoplast fusion, organelle inactivation, and chloroplast recombination. Reports of the introduction of chloroplasts into albino protoplasts and the observation of variegated progeny with the transfer of only two chloroplasts open up the possibilities of introduction of transformed chloroplasts into recipient protoplasts. Transient expression of foreign genes in chloroplasts of tobacco, sugar beet, and wheat cells, and in leaves or calli has been observed. It is clear that chloroplast promoters are interchangeable among monocots and dicots; the cat gene driven by the maize rbcL promoter functions in tobacco chloroplasts and the uidA gene driven by the pea psbA promoter functions in wheat chloroplasts. In addition, chloroplast vectors introduced into isolated protoplasts by the electroporation or polyethylene glycol (PEG)-mediated DNA uptake do not express foreign genes in the nuclear compartment.
The bacterial gene aadA is an important and widely used selectable marker for manipulation of the chloroplast genome through biolistic transformation. Because no other such marker is available, two strategies for recycling of the aadA cassette have been developed. One utilizes homologous recombination between two direct repeats flanking the aadA cassette to allow its loss under non-selective growth conditions. A second strategy is to perform co-transformation with a plasmid containing a modified, non-essential chloroplast gene and another plasmid in which the aadA cassette disrupts a chloroplast gene known to be essential for survival. Under selective growth conditions the first mutation can be transferred to all chloroplast DNA copies whereas the aadA insertion remains heteroplasmic. Loss of the selectable marker can be achieved subsequently by growing the cells on non-selective media. In both cases it is possible to reuse the aadA cassette for the stepwise disruption or mutagenesis of any gene in the same strain.
A new vector, pFaadAII, for transformation of plastids of Nicotiana tabacum L. has been developed. It harbours a chimeric gene consisting of the aadA coding region from Escherichia coli, the 16S rDNA promoter from tobacco combined with a synthetic ribosome-binding site, a 500-bp fragment containing the 3' untranslated transcript region (UTR) of the Chlamydomonas rbcL gene and 3.75-kb (5') and 0.95-kb (3') tobacco plastome sequences allowing for targeting the foreign sequences to the intergenic region between the rpl32 and trnL genes of the tobacco plastome. The vector thus targets foreign sequences to the small single-copy region of the plastome, which has so far not been modified by transformation. Leaf protoplasts of Nicotiana tabacum L. were treated with polyethylene glycol (PEG) in the presence of the vector. The protocol for PEG treatment aiming at plastome transformation was optimized. Cell lines were cultured in the presence of spectinomycin and streptomycin using a novel and efficient protoplast culture and selection system. Regenerants were characterized by polymerase chain reaction (PCR) analysis, Southern hybridization and reciprocal crossing. The transformation procedure is described in detail and parameters influencing its efficiency are presented. Special effort is placed on analyzing suitable selection conditions. Only a proportion of the cell lines with a resistant phenotype could be confirmed by molecular analysis and/or reciprocal crossings to represent plastome transformants. Integration of the plastome specific aadA cassette into the nuclear genome accounted for a fraction of the resistant cell lines. Still, as many as 20-40 plastome transformants can be expected from the treatment of 10(6) protoplasts. Therefore, the improved protocol for PEG-mediated plastome transformation in combination with the new aadA-vector supplies a simple, reproducible and cost-efficient alternative to the biolistic procedure.
The enzyme cytosine deaminase (CD) encoded by codA catalyzes deamination of cytosine to uracil. CD is present in prokaryotes and in many eukaryotic micro-organisms, but is absent in higher plants. 5-fluorocytosine (5FC) is metabolized in CD-expressing cells, causing cellular death. A chimeric codA has been introduced into the tobacco plastid genome and 5FC was used to select against tissue culture cells and seedlings expressing CD. This negative selection scheme will be useful in identifying nuclear genes which control plastid gene expression in higher plants.
The Bacillus thuringiensis (Bt) crystal toxins are safe biological insecticides, but have short persistance and are poorly effective against pests that feed inside plant tissues. Production of effective levels of these proteins in plants has required resynthesis of the genes encoding them. We report that amplification of an unmodified crylA(c) coding sequence in chloroplasts up to approximately 10,000 copies per cell resulted in the accumulation of an unprecedented 3-5% of the soluble protein in tobacco leaves as protoxin. The plants were extremely toxic to larvae of Heliothis virescens, Helicoverpa zea, and Spodoptera exigua. Since the plastid transgenes are not transmitted by pollen, this report has implications for containment of Bt genes in crop plants. Furthermore, accumulation of insecticidal protein at a high level will facilitate improvement in the management of Bt resistant insect populations.
To compare the differential effects of cry2A operon orf2 (29-kDa protein gene) and Cry11A operon orf3 (20-kDa protein gene) on Cry2A synthesis and inclusion formation, we expressed the cry2A gene along with either the 29-kDa gene, 20-kDa gene, or both genes. Constructs containing 20-kDa, in the presence or absence of 29-kDa, produced more Cry2A than constructs which lacked this gene. Cry2A synthesis was also higher when the 29-kDa gene was included with 20-kDa in the construct. However, even in the presence of increased Cry2A synthesis facilitated by the 20-kDa gene, typical Cry2A crystals did not form if the 29-kDa gene was not included in the construct. These results suggest that the 29-kDa and 20-kDa proteins have different functions, with the 20-kDa protein acting like a molecular chaperone to enhance net Cry2A synthesis, and the 29-kDa protein likely serving as a template for the stabilization of Cry2A molecules and their organization into the rectangular inclusion characteristic of wild-type Cry2A crystals.
Genes encoding critical steps in the synthesis of osmoprotectant compounds are now being expressed in transgenic plants. These plants generally accumulate low levels of osmoprotectants and have increased stress tolerance. The next priority is therefore to engineer greater osmoprotectant synthesis without detriment to the rest of metabolism. This will require manipulation of multiple genes, guided by thorough analysis of metabolite fluxes and pool sizes.
5' and 3' untranslated regions (UTRs) of plastid RNAs act as regulatory elements for post-transcriptional control of gene expression. Polyethylene glycol-mediated plastid transformation with UTR-GUS reporter gene fusions was used to study the function of the psbA, rbcL and rpl32 UTRs in vivo. All gene fusions were expressed from the same promoter, i.e. the promoter of the 16S-rRNA gene, such that variations in RNA and protein levels would be due to the involved UTR elements alone. Transgenic tobacco lines containing different combinations of UTRs showed fivefold variation in the uidA-mRNA level (RNA stability) and approximately 100-fold differences in GUS activity, a measure of translation activity. The rbcL 5'-UTR conferred greater mRNA stability than the psbA 5'-UTR on uidA transcripts. In contrast, the psbA 5'-UTR enhanced translation of GUS to a much greater extent compared to the rbcL 5'-UTR. The psbA 5'-UTR also mediated light-induced activation of translation which was not observed with other constructs. Deletion mutagenesis of an unanalysed terminal sequence element of the psbA 5'-UTR resulted in a twofold drop in uidA-mRNA level and a fourfold decrease in translation efficiency. Exchange of 3'-UTRs results in up to fivefold changes of mRNA levels and does not significantly influence translation efficiency. The mechanical impacts of these results on plastid translation regulation are discussed.
Plant genetic engineering via the nucleus is a mature technology that has been used very productively for research and commercial biotechnology. By contrast, the ability to introduce foreign genes at specific locations on a chloroplast's chromosome has been acquired relatively recently. Certain limitations of nuclear genome transformation methods might be overcome by the site-specific introduction of genes into plastid chromosomes. In addition, plastids, mitochondria and other subcellular organelles might provide more favorable environments than the nuclear-cytoplasmic compartment for certain biochemical reactions and for accumulating large amounts of some gene and enzyme products.
The discovery that chloroplasts have semi-autonomous genetic systems has led to many insights into the biogenesis of these organelles and their evolution from free-living photosynthetic bacteria. Recent developments of our understanding of the molecular mechanisms of translation in chloroplasts suggest selective pressures that have maintained the 100-200 genes of the ancestral endosymbiont in chloroplast genomes. The ability to introduce modified genes into chloroplast genomes by homologous recombination and the recent development of an in vitro chloroplast translation system have been exploited for analyses of the cis-acting requirements for chloroplast translation. Trans-acting translational factors have been identified by genetic and biochemical approaches. Several studies have suggested that chloroplast mRNAs are translated in association with membranes.
Removal of antibiotic resistance genes from genetically modified (GM) crops removes the risk of their transfer to the environment or gut microbes. Integration of foreign genes into plastid DNA enhances containment in crops that inherit their plastids maternally. Efficient plastid transformation requires the aadA marker gene, which confers resistance to the antibiotics spectinomycin and streptomycin. We have exploited plastid DNA recombination and cytoplasmic sorting to remove aadA from transplastomic tobacco plants. A 4.9 kbp insert, composed of aadA flanked by bar and uidA genes, was integrated into plastid DNA and selected to remove wild-type plastid genomes. The bar gene confers tolerance to the herbicide glufosinate despite being GC-rich. Excision of aadA and uidA mediated by two 174 bp direct repeats generated aadA-free T(0) transplastomic plants containing the bar gene. Removal of aadA and bar by three 418 bp direct repeats allowed the isolation of marker-free T(2) plants containing a plastid-located uidA reporter gene.
Escherichia coli mRNA translation is facilitated by sequences upstream and downstream of the initiation codon, called Shine–Dalgarno (SD)
and downstream box (DB) sequences, respectively. In E.coli enhancing the complementarity between the DB sequences and the 16S rRNA penultimate stem resulted in increased protein accumulation
without a significant affect on mRNA stability. The objective of this study was to test whether enhancing the complementarity
of plastid mRNAs downstream of the AUG (downstream sequence or DS) with the 16S rRNA penultimate stem (anti-DS or ADS region)
enhances protein accumulation. The test system was the tobacco plastid rRNA operon promoter fused with the E.coli phage T7 gene 10 (T7g10) 5′-untranslated region (5′-UTR) and DB region. Translation efficiency was tested by measuring neomycin
phosphotransferase (NPTII) accumulation in tobacco chloroplasts. We report here that the phage T7g10 5′-UTR and DB region
promotes accumulation of NPTII up to ∼16% of total soluble leaf protein (TSP). Enhanced mRNA stability and an improved NPTII
yield (∼23% of TSP) was obtained from a construct in which the T7g10 5′-UTR was linked with the NPTII coding region via a
NheI site. However, replacing the T7g10 DB region with the plastid DS sequence reduced NPTII and mRNA levels to 0.16 and 28%,
respectively. Reduced NPTII accumulation is in part due to accelerated mRNA turnover.
RNA maturation and modulation of RNA stability play important roles in chloroplast gene expression. In vitro and in vivo studies have shown that both the 5'- and 3'-untranslated regions (UTRs) contain sequence and structural elements that guide these processes, and interact with specific proteins. We have previously characterized the spinach chloroplast petD 3'-UTR in detail by in vitro approaches. This stem-loop forming sequence is a weak terminator but is required for RNA maturation and also exhibits sequence-specific protein binding. To test petD 3'-UTR function in vivo, tobacco chloroplast transformants were generated containing uidA reporter genes flanked by variants of the petD 3'-UTR, including one which does not form an RNA-protein complex in vitro, and one which lacks a stem-loop structure. Analysis of uidA mRNA indicated that a stable secondary structure is required to accumulate a discrete mRNA, and that changes in the 3'-UTR sequence which affect protein binding in vitro can also affect RNA metabolism in vivo. The 3'-UTR also influenced beta-glucuronidase protein accumulation, but not in proportion to RNA levels. These results raise the possibility that in tobacco chloroplasts, the 3'-UTR may influence translational yield.
Plastid transformation (transplastomic) technology has several potential advantages for biotechnological applications including the use of unmodified prokaryotic genes for engineering, potential high-level gene expression and gene containment due to maternal inheritance in most crop plants. However, the efficacy of a plastid-encoded trait may change depending on plastid number and tissue type. We report a feasibility study in tobacco plastids to achieve high-level herbicide resistance in both vegetative tissues and reproductive organs. We chose to test glyphosate resistance via over-expression in plastids of tolerant forms of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Immunological, enzymatic and whole-plant assays were used to prove the efficacy of three different prokaryotic (Achromobacter, Agrobacterium and Bacillus) EPSPS genes. Using the Agrobacterium strain CP4 EPSPS as a model we identified translational control sequences that direct a 10,000-fold range of protein accumulation (to >10% total soluble protein in leaves). Plastid-expressed EPSPS could provide very high levels of glyphosate resistance, although levels of resistance in vegetative and reproductive tissues differed depending on EPSPS accumulation levels, and correlated to the plastid abundance in these tissues. Paradoxically, higher levels of plastid-expressed EPSPS protein accumulation were apparently required for efficacy than from a similar nuclear-encoded gene. Nevertheless, the demonstration of high-level glyphosate tolerance in vegetative and reproductive organs using transplastomic technology provides a necessary step for transfer of this technology to other crop species.
The use of plants for medicinal purposes dates back thousands of years but genetic engineering of plants to produce desired biopharmaceuticals is much more recent. As the demand for biopharmaceuticals is expected to increase, it would be wise to ensure that they will be available in significantly larger amounts, on a cost-effective basis. Currently, the cost of biopharmaceuticals limits their availability. Plant-derived biopharmaceuticals are cheap to produce and store, easy to scale up for mass production, and safer than those derived from animals. Here, we discuss recent developments in this field and possible environmental concerns.