ArticleLiterature Review

Engineering chloroplasts: An alternative site for foreign genes, proteins, reactions and products

July 2000
Trends in Biotechnology 18(6):257-63

July 2000
18(6):257-63

Source
PubMed

Authors:

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.

OBPC SYMPOSIUM: MAIZE 2004 & BEYOND – RECENT ADVANCES IN CHLOROPLAST GENETIC ENGINEERING

Article

Full-text available

Jun 2005

The chloroplast genetic engineering approach offers a number of unique advantages, including high-level transgene expression, multi-gene engineering in a single transformation event, transgene containment via maternal inheritance, lack of gene silencing, position and pleiotropic effects and undesirable foreign DNA. Thus far, more than 40 transgenes have been stably integrated and expressed via the tobacco chloroplast genome to confer several agronomic traits and produce vaccine antigens, industrially valuable enzymes, biomaterials, and amino acids. Functionality of chloroplast-derived vaccine antigens and therapeutic proteins have been demonstrated by in vitro assays and animal studies. Oral delivery of vaccine antigens has been facilitated by hyperexpression in transgenic chloroplasts (leaves) or non-green plastids (carrots) and the availability of antibiotic-free selectable markers or the ability to excise selectable marker genes. Additionally, the presence of chaperones and enzymes within the chloroplast help to assemble complex multi-subunit proteins and correctly fold proteins containing disulfide bonds, thereby drastically reducing the costs of in vitro processing. Despite such significant progress in chloroplast transformation, this technology has not been extended to major crops. This obstacle emphasizes the need for plastid genome sequencing to increase the efficiency of transformation and conduct basic research in plastid biogenesis and function. However, highly efficient soybean, carrot, and cotton plastid transformation has been recently accomplished via somatic embryogenesis using species-specific chloroplast vectors. Recent advancements facilitate our understanding of plastid biochemistry and molecular biology. This review focuses on exciting recent developments in this field and offers directions for further research and development.

Targeting a Nuclear Anthranilate Synthase alpha-Subunit Gene to the Tobacco Plastid Genome Results in Enhanced Tryptophan Biosynthesis. Return of a Gene to Its Pre-Endosymbiotic Origin

Article

Sep 2001
PLANT PHYSIOL

Xing-Hai Zhang

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.

Regeneration and plastid transformation approaches in Arabidopsis thaliana and Rapid-Cycling Brassica rapa

Article

Full-text available

Nicotiana tabacum : PEG-Mediated Plastid Transformation

Article

Mar 2014

Stable plastid transformation in Nicotiana tabacum has been achieved by using two different methods, the biolistic method, using a particle gun, and the polyethylene glycol (PEG)-mediated transformation. PEG-mediated plastid transformation involves the treatment of isolated protoplasts (plant cells without cell wall) with PEG in the presence of DNA. We have previously shown that in Nicotiana tabacum both methods are equally efficient. The PEG-mediated transformation efficiencies range between 20 and 50 plastid transformants per experiment (10(6) viable treated protoplasts). One advantage of the PEG method is that no expensive equipment such as a particle gun is required. The only crucial points are the handling and the cultivation of protoplasts. Furthermore, markers for the selection of transformed chloroplasts are required. One of the most often used selection markers is the aadA gene which encodes for spectinomycin and streptomycin resistance. Here we describe a simplified and inexpensive protocol for the transformation of chloroplasts in Nicotiana tabacum using an optimized protoplast culture protocol.

Critical Reviews in Biotechnology Prospects of chloroplast metabolic engineering for developing nutrient-dense food crops Prospects of chloroplast metabolic engineering for developing nutrient-dense food crops

Article

Jul 2022
CRIT REV BIOTECHNOL

Addressing nutritional deficiencies in food crops through biofortification is a sustainable approach to tackling malnutrition. Biofortification is continuously being attempted through conventional breeding as well as through various plant biotechnological interventions, ranging from molecular breeding to genetic engineering and genome editing for enriching crops with various health-promoting metabolites. Genetic engineering is used for the rational incorporation of desired nutritional traits in food crops and predominantly operates through nuclear and chloro-plast genome engineering. In the recent past, chloroplast engineering has been deployed as a strategic tool to develop model plants with enhanced nutritional traits due to the various advantages it offers over nuclear genome engineering. However, this approach needs to be extended for the nutritional enhancement of major food crops. Further, this platform could be combined with strategies, such as synthetic biology, chloroplast editing, nanoparticle-mediated rapid chloroplast transformation, and horizontal gene transfer through grafting for targeting endogen-ous metabolic pathways for overproducing native nutraceuticals, production of biopharmaceuti-cals, and biosynthesis of designer nutritional compounds. This review focuses on exploring various features of chloroplast genome engineering for nutritional enhancement of food crops by enhancing the levels of existing metabolites, restoring the metabolites lost during crop domestication, and introducing novel metabolites and phytonutrients needed for a healthy daily diet. ARTICLE HISTORY

Lawrence Bogorad's Scientific Contributions and Remembrances on his 100th Birth Anniversary

Article

Full-text available

Nov 2021
PLANT BIOTECHNOL J

Kc Bansal

Lawrence Bogorad much known for his fundamental discoveries in plant molecular biology reached the pinnacle of research as a topmost international leader, occupying several key positions in his five-decade long association with plant science community. Much has been said and written about his life and his dedication to science as a pioneer in photosynthesis research. Here, we reminisce some of his key ground breaking scientific contributions, and share our experiences and appreciation for his mentorship through personal messages, as we celebrate his 100th birth anniversary that falls on August 29, 2021.

Prospects of nano- and peptide-carriers to deliver CRISPR cargos in plants to edit across and beyond central dogma

Article

Full-text available

Apr 2021

The discovery and repurposing of CRISPR/Cas have accelerated the process of precise genetic engineering to improve the yield, nutrition, and climate resilience of plants. The development of new Cas variants further empowered molecular biologists to understand and modulate the tightly regulated information flow across (DNA → RNA → protein) and beyond (epigenome and metabolome) the central dogma. However, contemporary approaches to deliver and perform CRISPR-mediated editing in plants are costly, resource-intensive, time-consuming, and have limitations such as low efficiency, tissue damage, narrow species range and restricted capacity to deliver cargo. Recent work on nano- and peptide-carriers (NC and PC) to deliver biomolecules (DNA/RNA/proteins/ribonucleoproteins) in plants exhibits the potential to address the difficulties associated with conventional techniques and further intensify the process of genetic transformation. The tunable physicochemical properties of NC and PC can be altered with acute precision to leverage their interactive capabilities displayed with biological matter, thus enabling them to traverse through the cellular barriers to deliver biomolecules. Currently, the area of NC- and PC-assisted delivery of biomolecules is in infancy and demands consolidated efforts at the interface of nanotechnology, proteomics, and plant transgenics to become a competent alternative to existing genetic transformation methods. Here, we have contextualized the advances in NC, PC, and NC–PC conjugates which makes them particularly attractive to deliver biomolecules in plants. Prominent challenges, limitations, and prospects of these carriers to deliver and perform CRISPR editing in plants are discussed. Lastly, a summary of the regulatory and safety aspects of these tiny techs is provided.

The complete chloroplast genome of greater duckweed (Spirodela polyrhiza 7498) using PacBio long reads: insights into the chloroplast evolution and transcription regulation

Article

Full-text available

Jan 2020
BMC GENOMICS

Background: Duckweeds (Lemnaceae) are aquatic plants distributed all over the world. The chloroplast genome, as an efficient solar-powered reactor, is an invaluable resource to study biodiversity and to carry foreign genes. The chloroplast genome sequencing has become routine and less expensive with the delivery of high-throughput sequencing technologies, allowing us to deeply investigate genomics and transcriptomics of duckweed organelles. Results: Here, the complete chloroplast genome of Spirodela polyrhiza 7498 (SpV2) is assembled by PacBio sequencing. The length of 168,956 bp circular genome is composed of a pair of inverted repeats of 31,844 bp, a large single copy of 91,210 bp and a small single copy of 14,058 bp. Compared to the previous version (SpV1) assembled from short reads, the integrity and quality of SpV2 are improved, especially with the retrieval of two repeated fragments in ycf2 gene. There are a number of 107 unique genes, including 78 protein-coding genes, 25 tRNA genes and 4 rRNA genes. With the evidence of full-length cDNAs generated from PacBio isoform sequencing, seven genes (ycf3, clpP, atpF, rpoC1, rpl2, rps12 and ndhA) are detected to contain type-II introns. The ndhA intron has 50% more sequence divergence than the species-barcoding marker of atpF-atpH, showing the potential power to discriminate close species. A number of 37 RNA editing sites are recognized to have cytosine (C) to uracil (U) substitutions, eight of which are newly defined including six from the intergenic regions and two from the coding sequences of rpoC2 and ndhA genes. In addition, nine operon classes are identified using transcriptomic data. It is found that the operons contain multiple subunit genes encoding the same functional complexes comprising of ATP synthase, photosynthesis system, ribosomal proteins, et.al., which could be simultaneously transcribed and coordinately translated in response to the cell stimuli. Conclusions: The understanding of the chloroplast genomics and the transcriptomics of S.polyrhiza would greatly facilitate the study of phylogenetic evolution and the application of genetically engineering duckweeds.

Transformación plastidial de maíz: Obtención de regenerantes heteroplásmicos Plastid transformation in corn: production of heteroplasmic regenerants

Thesis

Nov 2009

Susana Martin-Ortigosa

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.

Stabilization and translation of synthetic operon-derived mRNAs in chloroplasts by sequences representing PPR protein binding sites

Article

Full-text available

Feb 2018

The chloroplast is a prime target for genetic engineering in plants, offering various advantages over nuclear transformation. For example, chloroplasts allow the expression of polycistronic transcripts and thus to engineer complex metabolic pathways. Each cistron within such a longer transcript needs its own expression elements. Within the 5′-UTR, such expression elements are needed for stabilizing mRNAs and for translation of the downstream reading frame. One of the few effective expression elements used so far in transplastomic approaches is the intercistronic expression element (IEE). The IEE is derived from the psbT-psbH intergenic region and includes a target sequence of the RNA binding protein HCF107. We here show that excessive expression of the IEE can lead to specific defects of endogenous chloroplast mRNA stabilization, likely via depletion of HCF107. Key players in chloroplast transcript stabilization and translation are pentatricopeptide repeat (PPR) proteins, which are structurally related to HCF107. PPR proteins that stabilize mRNAs leave behind short RNA footprints that are indicators of their activity. We identified such sRNAs in tobacco and demonstrate that they are sufficient to stabilize and stimulate translation of mRNAs from synthetic dicistronic transgenes in chloroplasts. Thus, minimal sequence elements are generally adequate to support key steps in chloroplast gene expression, i.e. RNA stability and translation. Furthermore, our analysis expands the repertoire of available expression elements to facilitate the assembly and expression of multi-gene ensembles in the chloroplast.

Chloroplasts as Cellular Factories for the Cost-effective Production of Cellulases

Article

Full-text available

Jan 2018

Chloroplasts are vital photosynthetic organelles in plant cells that carry out several important cellular functions including synthesis of amino acids, fatty acids, and lipids and metabolism of nitrogen, starch, and Sulphur to sustain the homeostasis in plants. These organelles have got their own genome, and related genetic machinery to synthesize required proteins for various plant functions. Genetic manipulations of the chloroplast genome for different biotech applications has been of great interest due to desired features including the availability of operonal mode of gene expression, high copy number, and maternal mode of inheritance (in the most field crops). Their capacity to often express transgenes at high levels make it a cost-effective platform for the production of foreign proteins, particularly high-value targets of industrial importance, at large scale. This article reviews briefly the research work carried out to produce cellulolytic enzymes in higher plant chloroplasts. The challenges and future opportunities for the same are also discussed.

Recombinant Plant Engineering for Immunotherapeutic Production

Article

Full-text available

Dec 2017

Purpose of review: The requirement for large quantities of therapeutic proteins has fueled a great interest in the production of recombinant proteins in plant bioreactors. The vaccines and bio-therapeutic protein production in plants hold the promise of significantly lowering the cost of manufacturing life-saving drugs. This review will reflect the current status and challenges that the molecular farming platform faces becoming a strategic solution for the development of low-cost bio-therapeutics for developing countries. Recent findings: Different plant parts have been successfully identified as suitable expression systems for the commercial production of therapeutic proteins for some human and animal diseases ranging from common cold to AIDS. The processed therapeutics from such sources are devoid of any toxic components. The large-scale cultivation of these transgenic plants would be possible anywhere in the world including developing countries, which lack sophisticated drug manufacturing units. A couple of such commercially generated products have already hit the market with success. Newer methods using suitable plant viruses and recombinant gene expression systems have already been devised for producing therapeutic proteins and peptides. Summary: Plants are promising bio-factories for therapeutic protein production because of their several advantages over the other expression systems especially the advanced mechanisms for protein synthesis and post-translational modification which are very much similar to animal cells. Plant biotechnologists are much attracted to the bio-farming because of its flexibility, scalability, low manufacturing cost, as well as the lack of risk of toxic or pathogenic contamination. A number of projects on bio-farming are designed and are at various developmental stages but have not yet become available to the pharmaceutical industry. Therefore, we need further advancement in the optimization of lab protocols for up-scaling the production of such therapeutics at commercial level with a promise to offer their best clinical use.

Genetic and metabolic engineering in diatoms

Article

Full-text available

Jul 2017
PHILOS T R SOC B

Diatoms have attracted considerable attention due to their success in diverse environmental conditions, which probably is a consequence of their complex origins. Studies of their metabolism will provide insight into their adaptation capacity and are a prerequisite for metabolic engineering. Several years of investigation have led to the development of the genome engineering tools required for such studies, and a profusion of appropriate tools is now available for exploring and exploiting the metabolism of these organisms. Diatoms are highly prized in industrial biotechnology, due to both their richness in natural lipids and carotenoids and their ability to produce recombinant proteins, of considerable value in diverse markets. This review provides an overview of recent advances in genetic engineering methods for diatoms, from the development of gene expression cassettes and gene delivery methods, to cutting-edge genome-editing technologies. It also highlights the contributions of these rapid developments to both basic and applied research: they have improved our understanding of key physiological processes; and they have made it possible to modify the natural metabolism to favour the production of specific compounds or to produce new compounds for green chemistry and pharmaceutical applications. This article is part of the themed issue ‘The peculiar carbon metabolism in diatoms’.

Transgenesis and Plant Molecular Pharming

Chapter

May 2017

The use of plants as efficient biopharmaceutical factories has significantly increased in the past two decades. This is mainly due to advancements in plant biotechnology which pave the way to high-yield production of biopharmaceuticals in plants, combined with efforts made to optimize yield through upstream, downstream, and preservation strategies of recombinant proteins. The FDA’s approval to commercially release recombinant glucocerebrosidase enzyme produced in carrot cells by Protalix Biotherapeutics was the first plant-produced biopharmaceutical to be released for human consumption into the market. This is a major achievement in the field of molecular pharming. Although many other biopharmaceuticals produced in plants are in the pipeline for commercial release after undergoing various stages of clinical trials, there is room for improvement in enhancing recombinant protein yield in plants. These include exploration of innovative strategies involving genetics, genomics, epigenetics, in silico simulations and purification techniques. In this chapter, we discuss various approaches of plant biotechnology and plant genetic engineering that are being used in the molecular pharming of biopharmaceuticals.

Chloroplast Genetic Engineering: Recent Advances and Future Perspectives

Article

Full-text available

Oct 2005
CRIT REV PLANT SCI

Henry Daniell

Chloroplast genetic engineering offers a number of unique advantages, including a high-level of transgene expression, multi-gene engineering in a single transformation event, transgene containment via maternal inheritance, lack of gene silencing, position and pleiotropic effects, and undesirable foreign DNA. Thus far, over forty transgenes have been stably integrated and expressed via the tobacco chloroplast genome to confer important agronomic traits, as well as express industrially valuable biomaterials and therapeutic proteins. The hyperexpression of recombinant proteins within plastid engineered systems offers a cost effective solution for using plants as bioreactors. Additionally, the presence of chaperones and enzymes within the chloroplast help to assemble complex multi-subunit proteins and correctly fold proteins containing disulfide bonds, thereby drastically reducing the costs of in vitro processing. Oral delivery of vaccine antigens against cholera, tetanus, anthrax, plague, and canine parvovirus are made possible because of the high expression levels and antibiotic-free selection systems available in plastid transformation systems. Plastid genetic engineering also has become a powerful tool for basic research in plastid biogenesis and function. This approach has helped to unveil a wealth of information about plastid DNA replication origins, intron maturases, translation elements and proteolysis, import of proteins and several other processes. Although many successful examples of plastid engineering have set a foundation for various future applications, this technology has not been extended to many of the major crops. Highly efficient plastid transformation has been recently accomplished via somatic embryogenesis using species-specific chloroplast vectors in soybean, carrot, and cotton. Transgenic carrots were able to withstand salt concentrations that only halophytes could tolerate; more than twice the effectiveness of other engineering attempts. Recent advances in plastid engineering provide an efficient platform for the production of therapeutic proteins, vaccines, and biomaterials using an environmentally friendly approach. This review takes an in-depth look into the state of the art in plastid engineering and offers directions for further research and development.

Chloroplast-Derived Vaccine Antigens and Biopharmaceuticals: Protocols for Expression, Purification, or Oral Delivery and Functional Evaluation

Article

Full-text available

Jun 2009
Meth Mol Biol

Many vaccine antigens and biopharmaceutical proteins have been expressed at high levels via the chlo-roplast genome and their functionality has been evaluated using in vitro assays in cell cultures (i.e., macrophage lysis assay, inhibition of vesicular stomatitis virus-induced cytopathicity in baby hamster kidney cells, or inhibition of human HIV infection in TZM-BL cells) as well as protection after challenge with bacterial or viral pathogens or antitumor assays or delay the onset of insulitis in suitable animal models. Production of therapeutic proteins in chloroplasts eliminates the expensive fermentation technology. Moreover, oral delivery of chloroplast-derived therapeutic proteins eliminates expensive purification steps, cold storage, cold transportation, and delivery via sterile needles, thereby further decreasing their cost. In this chapter, we describe detailed protocols for chloroplast transformation including the construction of chloroplast transformation vectors, delivery of DNA into plant cells using particle bombardment , selection and regeneration of transformants by tissue culture, confirmation of transgene integration into the chloroplast genome and homoplasmy, evaluation of foreign gene expression, purification of foreign protein, or oral delivery via bioencapsulation, functional evaluation using in vitro and in vivo assays, and evaluation of immunity after challenge with pathogens in suitable animal models.

Transgenesis and Plant Molecular Pharming

Chapter

Feb 2017

Cloning of Flanking Regions of Plastidal Vectors in Soybean

Article

Full-text available

Mar 2016

Parviz Soleimani

Increasing Pro-Vitamin A in rice through Gene Amplification Technology

Article

Jan 2017

Three out of the eight Millennium Development Goals (MGDs); Eradication of poverty and hunger, reduction in child mortality and improving maternal health can be well addressed through food fortification initiatives. Half of the world population, particularly India relies on rice as the main food source being its main source of dietary energy. Since rice does not contain essentials vitamins like -carotene, therefore Indian population that is totally dependent on rice are at high risk of developing vitamin A deficiency. In India the burden of Vitamin A deficiency (VAD) amounts to an annual loss of 2.3 million disability-adjusted life years (DALYs). Jammu and Kashmir stands fifth after Mizoram, Goa, Assam, Manipur and West Bengal in having the highest VAD cases in the age group of 10-14 years. The prevalence of biochemical VAD among under-five years children in India is 62%, which is among the highest in the world and accounts for 330000 deaths in India alone (WHO, 2009). Combating VAD among an estimated 12 million preschool children, it is claimed that government programs to provide vitamin A supplements to the affected children are expensive and complicated. And it is asserted that conventional, non-GM methods of introducing or enhancing -carotene in other staple crops have been unsuccessful. Failure of Golden rice 1 and 2 in circumventing VAD is due to less amount of available pro-vitamin A. Assuming a -carotene to vitamin A conversion rate of 1:1, 200 grams of “Golden Rice” per day will provide enough vitamin A. Contrarily some researchers work with a conversion rate of 2:1, which implies a daily intake of 500 grams of rice to attain the FAO/ WHO figure of 400g for children between the ages of 1 and 3, thus no agreement on the amount of vitamin A that “Golden Rice 2” can in principle provide. So there is a need for the development of third generation Golden rice which produces even higher amount of beta-carotene through Gene Amplification Technology (GAT).

Plant Molecular Pharming

Chapter

Oct 2016

The use of plants as efficient biopharmaceutical factories has significantly increased in the past two decades. This is mainly due to advancements in plant biotechnology which pave the way to high-yield production of biopharmaceuticals in plants in addition to the efforts made in optimizing the yield through upstream, downstream, and preservation strategies of recombinant proteins. The FDA’s approval to commercially release recombinant glucocerebrosidase enzyme produced in carrot cells by Protalix Biotherapeutics was the first plant-produced biopharmaceutical to be released for human consumption into the market. This is a major achievement in the field of molecular pharming. Although many other biopharmaceuticals produced in plants are in the pipeline for commercial release after undergoing various stages of clinical trials, there is still room for improvement in enhancing recombinant protein yield in plants by exploring some innovative strategies involving in genetics, genomics, epigenetics, in silico, and purification techniques. In this chapter, we discuss various approaches of plant biotechnology and plant genetic engineering that are being used in the molecular pharming of biopharmaceuticals.

Compartmentalized Metabolic Engineering for Artemisinin Biosynthesis and Effective Malaria Treatment by Oral Delivery of Plant Cells

Article

Full-text available

Oct 2016
MOL PLANT

Artemisinin is highly effective against drug-resistant malarial parasites, which affects nearly half of the global population and kills >500 000 people each year. The primary cost of artemisinin is the very expensive process used to extract and purify the drug from Artemisia annua. Elimination of this apparently unnecessary step will make this potent antimalarial drug affordable to the global population living in endemic regions. Here we reported the oral delivery of a non-protein drug artemisinin biosynthesized (∼0.8 mg/g dry weight) at clinically meaningful levels in tobacco by engineering two metabolic pathways targeted to three different cellular compartments (chloroplast, nucleus, and mitochondria). The doubly transgenic lines showed a three-fold enhancement of isopentenyl pyrophosphate, and targeting AACPR, DBR2, and CYP71AV1 to chloroplasts resulted in higher expression and an efficient photo-oxidation of dihydroartemisinic acid to artemisinin. Partially purified extracts from the leaves of transgenic tobacco plants inhibited in vitro growth progression of Plasmodium falciparum-infected red blood cells. Oral feeding of whole intact plant cells bioencapsulating the artemisinin reduced the parasitemia levels in challenged mice in comparison with commercial drug. Such novel synergistic approaches should facilitate low-cost production and delivery of artemisinin and other drugs through metabolic engineering of edible plants.

Transgenesis and Plant Molecular Pharming

Chapter

Oct 2016

Abstract The use of plants as efficient biopharmaceutical factories has significantly increased in the past two decades. This is mainly due to advancements in plant biotechnology which pave the way to high-yield production of biopharmaceuticals in plants in addition the efforts made in optimising the yield through upstream, downstream and preservation strategies of recombinant proteins. The FDA’s approval to commercially release recombinant glucocerebrosidase enzyme produced in carrot cells by Protalix Biotherapeutics was the first plant produced biopharmaceutical to be released for human consumption into the market. This is a major achievement in the field of molecular pharming. Although many other biopharmaceuticals produced in plants are in the pipeline for commercial release after undergoing various stages of clinical trials, there is still room for improvement in enhancing recombinant protein yield in plants by exploring some innovative strategies involving in genetics, genomics, epigenetics, in silico and purification techniques. In this chapter, we discuss various approaches of plant biotechnology and plant genetic engineering that are being used in the molecular pharming of biopharmaceuticals.

Optimization of Cellulase Production from Paenibacillus jamilae BRC 15-1

Article

Dec 2015

In this study was selected the cellulolytic microorganism and investigated optimum condition of cellulase production for the cellulosic bioethanol production. A bacterial strain Paenibacillus jamilae BRC15-1, was isolated from soil of domestic reclaimed land. For optimizing cellulase production from the selected strain, various culture parameters were investigated such as culture medium, pH (pH 4~10), temperature (25{\sim}50^{\circ}C) and culture time (2~72 h). As a result, P. jamilae BRC15-1 efficiently produced cellulase from cellulosic biomass under following conditions: 24 h of culture time (pH 7, 40^{\circ}C) in manufactured media of CMC (carboxymethyl cellulose) with peptone. Optimum saccharifying condition of crude enzyme produced from P. jamilae BRC15-1 was identified on pH 6 and 40^{\circ}C of reaction temperature, respectively. This crude enzyme from P. jamilae BRC15-1 was used for saccharification of pretreated sweet sorghum (Sorghum bicolor var. dulciusculum Ohwi) bagasse under the optimal condition. Finally, pretreated sweet sorghum bagasse including 0.1 g of glucan was saccharified by crude enzyme of P. jamilae BRC15-1 into 2.75 mg glucose, 0.79 mg xylose and 1.12 mg arabinose.

Potential of Chloroplast Genome in Plant Breeding

Article

Full-text available

Sep 2010

Jana Repková

Chloroplast engineering (or chloroplast transformation technology, CTT) is a strategy consisting of inserting a transgene into the chloroplast genome of a plant instead of its nuclear genome. 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. Tobacco remains the species most amenable to CTT to date, although chloroplast genetic engineering has also been achieved successfully in crops such as maize, tomato, cotton, potato, rice and sugar beets. Improving agricultural traits such as herbicide and pathogen resistance, resistance to drought, salt tolerance and phytoremediation potential are all promising applications. Molecular pharming is another area of chloroplast engineering with high potential; the production of a wide range of products such as vaccine antigens, pharmaceutical proteins (human somatotropin, human serum albumin, human interferon, monoclonal antibodies) and industrial proteins (avidin, beta casein, liquid crystal polymers, xylanase, anthranilate synthase) is economically beneficial in comparison with bacterial cultivation or animal cell cultures. This review summarises the current status of CCT and its potential economic impact from the viewpoint of high levels of transgene expression and high accumulation of foreign proteins.

Synthesis of Recombinant Products in the Chloroplast

Chapter

Sep 2014

For the engineering of new traits in plants and algae, modification of the plastid genome offers significant advantages. These include transgene integration by homologous recombination, high levels of expression, multigene engineering in polycistronic units, and reduced transmission through pollen. Numerous tools are available for DNA delivery, selection of transformants, removal of undesirable markers and efficient expression of single or multiple transgenes. The list of plants and algae with successful plastid transformation is increasing and includes some crop plants. Its further extension to agronomically relevant species and appropriate cultivars, together with advances in synthetic biology, will be key to the future development of plastids as green factories for the production of high-value metabolites or proteins.

Analysis of paternal plastid inheritance in Arabidopsis thaliana and its impact on biosafety of gene-modified plants

Article

Full-text available

Mar 2011

Ashraf Elsayed

Plastid-encoded genes are maternally inherited in most plant species. Transgenes located on the plastid genome are thus within a natural confinement system, preventing their distribution via pollen. However, a low-frequency leakage of plastids via pollen seems to be universal in plants. Here we report that a very low-level paternal inheritance in Arabidopsis thaliana occurs under field conditions. As pollen donor an Arabidopsis accession (Ler-Ely) was used, which carried a plastid-localized atrazine resistance due to a point mutation in the psbA gene. The frequency of pollen transmission into F1 plants, based on their ability to express the atrazine resistance was 1.9 9 10-5. We extended our analysis to another cruciferous species, the world-wide cultivated crop Brassica napus. First, we isolated a fertile and stable plastid transformant (T36) in a commercial cultivar of B. napus (cv Drakkar). In T36 the aadA and the bar genes were integrated in the inverted repeat region of the B. napus plastid DNA following particle bombardment of hypocotyl segments. Southern blot analysis confirmed transgene integration and homoplasmy of plastid DNA. Line T36 expressed Basta resistance from the inserted bar gene and this trait was used to estimate the frequency of pollen transmission into F1 plants. Afrequency of\2.6 9 10-5 was determined in the greenhouse. Taken together, our data show a very low rate of paternal plastid transmission in Brassicacea. Moreover, the establishment of plastid transformation in B. napus facilitates a safe use of this important crop plant for plant biotechnology.

Molecular Farming of Plants and Animals for Human and Veterinary Medicine

Chapter

Full-text available

Jan 2002

Tobacco is readily amenable to genetic engineering and has many desirable agronomic attributes, like high biomass yield and high soluble protein levels that are essential for crops used to produce recombinant proteins. It is a non-food crop, making containment in an agricultural setting feasible. Most production systems are based on the accumulation of proteins in leaves, eliminating the need for flowering and pollen production. These attributes make tobacco an ideal bioreactor for the large-scale production of biopharmaceutical recombinant proteins. As a consequence, a wide variety of the recombinant proteins, from simple peptides to complicated multimeric molecules like hemoglobin or secretory antibodies, have been produced successfully in tobacco. Many of these proteins have therapeutic or industrial uses.

Transplastomic plants for innovations in agriculture. A review

Article

Full-text available

Jun 2015
AGRON SUSTAIN DEV

Food production has to be significantly increased in order to feed the fast growing global population estimated to be 9.1 billion by 2050. The Green Revolution and the development of advanced plant breeding tools have led to a significant increase in agricultural production since the 1960s. However, hundreds of millions of humans are still undernourished, while the area of total arable land is close to its maximum utilization and may even decrease due to climate change, urbanization, and pollution. All these issues necessitate a second Green Revolution, in which biotechnological engineering of economically and nutritionally important traits should be critically and carefully considered. Since the early 1990s, possible applications of plastid transformation in higher plants have been constantly developed. These represent viable alternatives to existing nuclear transgenic technologies, especially due to the better transgene containment of transplastomic plants. Here, we present an overview of plastid engineering techniques and their applications to improve crop quality and productivity under adverse growth conditions. These applications include (1) transplastomic plants producing insecticidal, antibacterial, and antifungal compounds. These plants are therefore resistant to pests and require less pesticides. (2) Transplastomic plants resistant to cold, drought, salt, chemical, and oxidative stress. Some pollution tolerant plants could even be used for phytoremediation. (3) Transplastomic plants having higher productivity as a result of improved photosynthesis. (4) Transplastomic plants with enhanced mineral, micronutrient, and macronutrient contents. We also evaluate field trials, biosafety issues, and public concerns on transplastomic plants. Nevertheless, the transplastomic technology is still unavailable for most staple crops, including cereals. Transplastomic plants have not been commercialized so far, but if this crop limitation were overcome, they could contribute to sustainable development in agriculture

Complementation of the Nuclear Antisense rbcS-Induced Photosynthesis Deficiency by Introducing an rbcS Gene into the Tobacco Plastid Genome

Article

Nov 2002

Xing-Hai Zhang

;The small subunit (SS) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a nuclear gene-encoded protein that is imported into chloroplasts where it assembles with the large subunit (LS) after removal of the transit peptide to form Rubisco. We have explored the possibility that the severe deficiency in photosynthesis exhibited in nuclear transgenic tobacco (line 5) expressing antisense rbcS coding DNA that results in low SS and Rubisco protein content [Rodermel et al. (1988) Cell 55: 673] could be complemented by introducing a copy of the rbcS gene into its plastid genome through chloroplast transformation. Two independent lines of transplastomic plants were generated, in which the tobacco rbcS coding sequence, either with or without the transit sequence, was site-specifically integrated into the plastid genome. We found that compared with the antisense plants, expression of the plastid rbcS gene in the transplastomic plants resulted in very high mRNA abundance but no increased accumulation of the SS and Rubisco protein or improvement in plant growth and photosynthesis. Therefore, there is a limitation in efficient translation of the rbcS mRNA in the plastid or an incorrect processing and modification of the plastid-synthesized SS protein that might cause its rapid degradation.

Towards Engineering Dark-Operative Chlorophyll Synthesis Pathways in Transgenic Plastids

Chapter

Mar 2013

Muhammad Sarwar Khan

Biotecnología Vegetal (español)

Book

Full-text available

Apr 2005

Prefacio La ciencia es una construcción humana que busca entender cómo está hecho el Universo y su acervo es, sin duda, patrimonio de la humanidad. Si cada uno, dentro de sus respectivos campos, tornara accesible parte de ese patrimonio, se estaría dando a cualquier comunidad la oportunidad de avanzar con seriedad y eficacia por el camino del progreso y lo más importante, con el fiel sustento de una estructura con buenos cimientos. Dentro de cualquier sistema educacional, la Universidad es el vértice de la pirámide del conocimiento y sus alumnos, en las más variadas disciplinas, atraviesan dentro de ella todas las dimensiones en un juego espacio-tiempo que finalmente les garantiza una posición privilegiada dentro de una sociedad. Pero visto esto desde un punto de vista menos individualista, existe en cada uno de nosotros una responsabilidad y compromiso dentro de un contexto histórico continuo. En cada momento el país espera con paciencia y serenidad la aplicación actualizada y eficiente de esos conocimientos por parte de esos estudiantes, quienes en último término constituyen la esperanza para la construcción de un proyecto-político más tangible. Al dar nuestra contribución en este libro, estamos difundiendo y facilitando conocimientos que, de otra manera, no son de fácil alcance para muchos. Por eso la razón de este libro de biotecnología, asunto éste bastante debatido en el escenario internacional y nacional, especialmente en relación con la transgenia. En sus páginas, los lectores encontrarán contenidos escritos de una manera didáctica, fluida y precisa sobre el estado del arte actual de la biotecnología de plantas. Queremos dejar claro que nuestros lectores, no será el público en general, sino estudiantes y profesionales del área de plantas que usarán esta obra como auxilio o consulta. Las personas que participan de este esfuerzo editorial, son especialistas dentro de sus respectivos campos y viven o conocen la realidad de nuestra Sudamérica. Esto es, en cierta forma, una garantía de pertinencia en la información entregada, la que esperamos disminuya en alguna medida la creciente distancia que existe en el uso de este conocimiento científico en el país. En cuanto al potencial biotecnológico de plantas, el libro en su primera parte aborda aspectos y potencial del cultivo in vitro de células, tejidos y diferentes órganos vegetales. Se presentan primeramente aspectos históricos y del potencial referido al cultivo de tejidos in vitro. Se desarrollan a continuación los, medios de cultivo, nutrientes esenciales, addenda y diversos tipos reguladores de crecimiento requeridos para la inducción de procesos específicos y requerimientos de laboratorio. La segunda parte del libro está vinculada al potencial morfogénico de células, tejidos y su capacidad regenerativa conducente a la rediferenciación a plantas, de acuerdo a sus propiedades, origen y respuestas obtenidas in vitro. Esta ruta se inicia con el establecimiento del explante, enfatiza en el requerimiento de asepsia, problemas de oxidación fenólica, la morfogénesis inducida y la aclimatación del material en terreno. A continuación se trata sobre aplicaciones como también de eventuales limitaciones del sistema, aparte de costos por opción de estas biotecnologías. La tercera parte del libro nos introduce a la revolución vivida durante los años ´70, la tecnología del DNA recombinante. Aquí se presentan nociones básicas de ésta y algunas de sus herramientas más comunes, las que a juicio de los autores, deben formar parte del léxico de cualquiera de nuestros estudiantes para su trabajo en el laboratorio. Esto dará paso a una de los productos más controversiales de estas nuevas tecnología, la transformación genética de plantas. De ella se muestran sus bases y experimentos. Una vez establecida esta línea base, se muestran las nuevas tendencias en esta técnica, como son la prescindencia de genes de resistencia a antibióticos como reporteros o la transformación de organelos, como forma de evitar el flujo génico desde estos cultivos a los convencionales. La parte cuarta y final, nos presenta de forma resumida, la gran gama de herramientas moleculares que se utilizan tanto en el mejoramiento vegetal como en la caracterización varietal de especies. Estos marcadores moleculares, fueron la modesta base para el comienzo de la utilización de sistemas automatizados de estudios de los genomas. De esta forma, aquí se incluyen y describen utilidades y desventajas de los marcadores moleculares, para finalizar con un vistazo simplificado de la compleja ruta hacia el conocimiento del funcionamiento de los genomas bajo circunstancias específicas, lo que se conoce como genómica funcional. Se incluyen además, a manera de guías simplificadas, tablas con formulaciones de medios nutritivos, reguladores de crecimiento, metodología básica sobre transformación y de marcadores moleculares. La semilla está lanzada. Vamos a esperar como el ambiente la recibe. Los Autores

Biotecnología Vegetal (texto básico biotecnología en español)

Book

Full-text available

Apr 2005

Construction of Novel Chloroplast Expression Vector and Development of an Efficient Transformation System for the Diatom Phaeodactylum tricornutum

Article

Full-text available

Apr 2014
MAR BIOTECHNOL

Plastids are ideal subcellular hosts for the expression of transgenes and have been successfully used for the production of different biopolymers, therapeutic proteins and industrial enzymes. Phaeodactylum tricornutum is a widely used aquatic feed species. In this study, we focused on developing a high-efficiency plastid expression system for P. tricornutum. In the plastid transformation vector, the site selected for integration was the transcriptionally active intergenic region present between the trnI and trnA genes, located in the IR (inverted repeat) regions of the plastid genome. Initially, a CAT reporter gene (encoding chloramphenicol acetyltransferase) was integrated at this site in the plastid genome. The expression of CAT in the transformed microalgae conferred resistance to the antibiotic chloramphenicol, which enabled growth in the selection media. Overall, the plastid transformation efficiency was found to be approximately one transplastomic colony per 1,000 microalgae cells. Subsequently, a heterologous gene expression cassette for high-level expression of the target gene was created and cloned between the homologous recombination elements. A TA cloning strategy based on the designed XcmI-XcmI sites could conveniently clone the heterologous gene. An eGFP (green fluorescent protein) reporter gene was used to test the expression level in the plastid system. The relatively high-level expression of eGFP without codon optimisation in stably transformed microalgae was determined to account for 0.12 % of the total soluble protein. Thus, this study presents the first and convenient plastid gene expression system for diatoms and represents an interesting tool to study diatom plastids. Electronic supplementary material The online version of this article (doi:10.1007/s10126-014-9570-3) contains supplementary material, which is available to authorized users.

Identification and utilization of cleistogamy gene cl7(t) in rice (Oryza sativa L.)

Article

Full-text available

Mar 2014
J EXP BOT

Gene transformation is an important method for improvement of plants into elite varieties. However, the possibility of gene flow between genetically modified (GM) crops and similar species is a serious public issue that may potentially endanger ecological stability. Cleistogamy is expected to be an ideal genetic tool for preventing transgene propagation from GM crops. A rice mutant, cl7(t), was created by ethyl methanesulfonate mutagenesis. The mutant exhibited cleistogamy, and had closed spikelets, reduced plant height, and altered morphology of the leaves, panicle, and seeds. Anatomical investigations revealed that the cl7(t) mutant contained more vascular bundles and thicker stems than the wild type, which increased the mechanical strength of its internodes, and anti-lodging ability. Further studies demonstrated that the force required to open the lemma and palea was higher in the cl7(t) mutant, and there was weak swelling ability in the lodicules, which leads to cleistogamy. Allelic analyses and complementation tests indicated that cl7(t) was a novel allele of dep2, a mutant that was previously reported to have similar panicle morphology. Sequence analysis showed that cl7(t) had a single nucleotide substitution (C to A) in the third exon that leads to a Ser substitution with a stop codon, giving a truncated DEP2 protein. Quantitative RT-PCR and in situ hybridization tests demonstrated that there was lower CL7(t) expression level in the spikelets and weaker CL7(t) signals in the lodicules of the cl7(t) mutant compared with wild type, which implies that CL7(t) might participate in the development of lodicules. To improve the agronomic traits of cl7(t) to fit the needs of field production, the cl7(t) mutant was crossed with an intermediate-type rice variety named Guanghui102, which bears some important agronomic traits, including increased grain numbers and high rate of seed setting. Through multi-generational pedigree selection, cleistogamy lines with improved economic traits were obtained, which can be used for the selection of ecologically safe GM rice varieties.

Production and characterisation of transgenic cauliflower plants containing abnormal chloroplasts

Article

Dec 2013
SCI HORTIC-AMSTERDAM

Plant Edible Vaccines: A Revolution in Vaccination

Chapter

Jul 2013

Abstract Plants have been used as a source for many pharmaceutical since long. However, utilization of plant systems for production of edible vaccines has been a comparatively recent phenomenon. There are several potential advantages of plant derived vaccines over other conventional systems of vaccine production such as mammalian or avian cell culture. The cost of vaccines is one factor preventing further use of vaccination, leaving hundreds of thousands of children susceptible to preventable diseases. Especially for developing world this novel technique proved to be a boon for its low cost of production, convenient administration, easy storage and negligible chances of infection whereas the conventional system of vaccine production limits the applicability of vaccines in many parts of the world. These vaccines are prepared by introducing selected desired genes into plants and inducing these genetically modified plants to manufacture the encoded proteins. Transgenic plants may provide an ideal expression system, in which transgenic plant material can be fed directly as oral dose of recombinant vaccines. Expression of vaccines in plant tissue eliminates the risk of contamination with animal pathogen, provides a heat stable environment and enables oral delivery thus eliminating infection related hazards. Identification of transgenic material, containment of the transgenes and control of recombinant protein may be potential problems for large scale production of vaccines in plants. Factors like scaling up production as well as distribution and handling of transgenic plant material must comprise the future consideration in this field.

RECENT TRENDS OF INNOVATIONS IN CHEMICAL AND BIOLOGICAL SCIENCES VOLUME -I

Book

Full-text available

Aug 2022

Chemical sciences and Biological science play an important role in the evolutionary concept of the living world. This book Recent Trends Innovation Chemical and Biological Science: An Approach towards Qualitative and Quantitative Studies and Applications is a considerable effort taken by different authors in the discipline to provide new methodologies of research, its applications, and practical inducements of chemical sciences and Biological Science. The various themes in the book such as application of biological organisms, ethnomedicinal used in different human disorder, biological activity of Indian medicinal plants, Ethnobotanical study ,Ecofriendly energy, Transplastomic plants, Role of Sacred Groves in Biodiversity Conservation, Medicinal property rich plants comphora and Different traditional parts in India its application. It covers topic from environment science like effect of toxic chemical on environment. Also covered point from pharmacognosy like as the pharmacological property of Euphorbiaceae. It cover topic like phytochemistry biochemistry and active ingredients Indian medicinal plants .From chemical science subject like organic and inorganic and as well as applied chemistry included such Their Biological Activities It also cover there under medicinal and computational chemistry This book acts as an intermediary manual between Chemical sciences with other disciplines paving a way for ideas to new research in the respective arena. The experiments described in the boom chapters are such as should be performed by everyone beginning the study of chemistry, and would also serve as an excellent introduction to a course of qualitative and quantitative analysis. All scientists, academicians, researchers, and students working in the fields of chemistry, biology, physics, materials science, and engineering, among other fields, will find this book quite valuable. This book with valuable book chapters from eminent scientists, academicians, and researchers will surely be a part of almost information for the coming new research taken by the researchers in the field of chemical sciences and other disciplines in the future.

Chloroplast Genetic Engineering for Enhanced Agronomic Traits and Expression of Proteins for Medical/Industrial Applications

Chapter

Apr 2018

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

Chloroplast Engineering and Production of Biopharmaceuticals

Chapter

Nov 2009

Kathleen Hefferon

Engineering Plant Biochemical Pathways for Improved Nutritional Quality

Chapter

Aug 2002

Genetic Engineering Technologies

Chapter

Jul 2004

Genetic engineering of crop plants represents a major milestone in modern agricultural science. The advent of recombinant DNA technology in the early 1970s and the subsequent development of DNA transfer techniques provided exciting opportunities for plant scientists to insert foreign genes from both prokaryotic and eukaryotic organisms into the genome of crop plants and achieve transgene expression. Technological advancements in plant tissue culture techniques facilitated introduction of foreign genes into the plant genome to produce transgenic plants. Transgenic plants expressing novel traits now are being widely cultivated for their improved yield, quality, and other value-added characteristics. It should be noted, however, that in most instances genetic engineering techniques provide only an alternative approach to conventional breeding programs. In crop improvement, conventional breeding and hybrid seed production are the mainstay in ongoing efforts directed toward varietal development (Morandini and Salamini, 2003). Nonetheless, modern genetic engineering technologies offer several unique advantages over conventional hybridization approaches. For example, in vitro DNA transfer techniques permit introduction of genes and other genetic elements among sexually unrelated organisms, thereby bypassing biological barriers. Such genetic manipulation can be accomplished using a large quantity of plant materials in a relatively small space with a year-round artificially controlled growth environment. Hence, use of genetic engineering techniques complements and expedites conventional breeding programs by increasing diversity of genetic resources, enhancing efficiency and reducing length of time needed to introgress desirable traits into existing elite crop varieties. Genetic engineering also allows utilization of exotic genes for development of transgenic plants to produce proteins with novel nutritive, pharmaceutical, agrichemical, and industrial characteristics (Fischer and Emans, 2000).

Biohydrogen Production: A Volume in the Encyclopedia of Sustainability Science and Technology, Second Edition

Chapter

Jan 2019

Tobacco ribosomal DNA spacer element stimulates amplification and expression of heterologous genes

Article

Full-text available

Jan 2000

Engineering the Chloroplast Genome to Confer Stress Tolerance and Production of Pharmaceutical Proteins

Chapter

Jan 2002

Henry Daniell

The life-supporting metabolic process of photosynthesis is carried out in endosymbiotic organelles, the chloroplasts. Scientific endeavors over the last decade have enabled us to successfully engineer the chloroplast genome. This singular development has opened new avenues for agricultural biotechnology and has contributed immensely to our understanding of the basic genetic mechanisms operative within the organelle. The chloroplast genome has been engineered to express agronomically important traits such as disease-resistance, drought-tolerance, herbicide-resistance, insect resistance and production of antibodies, biopharmaceuticals and edible vaccines. The ability to hyper-express prokaryotic and eukaryotic proteins without the drawbacks of gene silencing or position effects, combined with the advantage of gene containment due to plastid maternal inheritance, make chloroplast transformation technology both useful and environmentally-friendly. The recent use of a plant-derived selectable marker for chloroplast transformation is an important development that should help allay public fears about GM foods. In addition, the extension of chloroplast transformation technology to edible plant species such as potato and tomato, coupled with the hyper-expression of antigens, opens up the possibility of oral delivery of vaccines and other biopharmaceuticals. This review focuses on some of these recent accomplishments and their impact.

Algae as a promising resource for biofuel industry: facts and challenges: Algae as a valuable chance for biofuels

Article

Dec 2016
INT J ENERG RES

Energy is the main driving force of society today that should be handled as a whole starting from production to consumption. With the rapid increase in the energy necessity, alternative methods and sources are becoming a crucial topic that should be scientifically highlighted with all their pros and cons. Especially the problems related to the fossil sources of energy triggered the search on the renewable alternatives like algae. In order to reach the desired amounts of energy with the satisfactory quality and quantity, understanding the algae as a living thing with the biological mechanism and existing production technologies are the key points to have a projection for commercialization. In this regard, technical facts and challenges on algal biofuel production should be evaluated. Keeping in mind the specifications and possible advantages related to their taxonomy, algae can serve as a promising source to reduce fossil fuel consumption. With the progress in the modern technology, reaching an effective production process will be possible, and this will help the algal biofuels to prove their maturity as a sustainable source for future. Within this context, the aim of this review is to point out the crucial technical challenges about algal fuels comprising both the macroalgae and microalgae as a reliable source of renewable energy. Copyright

Significance of Cytoplasmic DNA in Plant Breeding

Chapter

Jun 2010

Introduction Some Basic Information on DNA in the Cytoplasm of Plants Agronomic Traits Influenced by Cytoplasmic Factors Breeding Using Cytoplasmic Factors Conclusion Literature Cited

Structural and Functional Genomics of Symbiotic Arbuscular Mycorrhizal Fungi

Chapter

Jan 2004

The absorbing organs (roots, rhizomes) of nearly all terrestrial plant families host an intimate symbiotic association, called a mycorrhiza, with specialized functional groups of soil fungi. The most common type of root symbiosis is the arbuscular mycorrhiza where soil fungi interact with a tremendous diversity of plant species, including many forest trees and agricultural, horticultural, and fruit crops (Gianinazzi et al., 2002). The fungi involved are very ancient microorganisms compared to other true fungi. Fossil data and molecular phylogenetic analyses indicate that their origin dates back to the Ordovician-Devonian era some 460 to 400 million years ago (Remy et al., 1994; Redecker et al., 2000), coinciding with land colonization by early plants. Since then, arbuscular mycorrhizal (AM) fungi have become an integral part and key components of most terrestrial ecosystems (Smith and Read, 1997). Their ability to enhance plant resistance to biotic and abiotic stresses makes them potentially powerful biotools for low-input agriculture (Gianinazzi et al., 2002), and it is believed that their biodiversity can influence plant community structure in natural ecosystems (van der Heijden et al., 1998). More than 100 species of AM fungi have been described, and many of them are held in international culture collections (IBG/BEG, 1993; INVAM, 1996). Their taxonomical status has been a matter of debate because of their asexual nature and the difficulty to affiliate them closely to existing fungal groups. Until recently, they were organized into six genera, distributed in four families, and grouped into a unique order, Glomales (Zygomycota) which comprises Gigaspora and Scutellospora (Gigasporaceae), belonging to the suborder Gigasporineae, and Glomus, Sclerocystis (Glomaceae), Acaulospora, and Entrophospora (Acaulosporaceae), clustered in the Glomineae (Morton and Benny, 1990). However, a revised classification is presently being considered which places them in a new phylum, the Glomeromycota with four new orders (Glomerales, Archeaosporales, Paraglomales, and Diversisiporales) (Schüßler et al., 2001). For clarification, the generic term “AM fungi” will therefore be used above the species level throughout this chapter.

Environmentally Friendly Approaches in Biotechnology: Engineering the Chloroplast Genome to Confer Stress Tolerance

Article

Aug 2002

Henry Daniell

Chapter Ten Production of cellulases in plants for biomass conversion

Article

Dec 2001
Recent Adv Phytochemistry

Kathleen J. Danna

Plant biomass, the most abundant renewable resource on earth, is a potential source of fermentable sugars for the production of clean-burning, alternative transportation fuels such as ethanol. Bioconversion of plant biomass to fermentable glucose involves the enzymatic hydrolysis of cellulose, a major polysaccharide constituent of the plant cell wall. Enzymes from thermophilic organisms are particularly suited for this application because they are thermostable, resistant to protease, and tolerant toward other stresses. Commercially available microbial cellulases are prohibitively expensive for bioethanol processes. Manufacturing heterologous cellulases in crop-plant bioreactors could significantly reduce costs associated with enzyme production and could offer a potentially high-volume alternative to traditional methods. Because transgenic plants that produce large amounts of bacterial glucanases (up to 26% of total soluble leaf protein) exhibit normal growth and development, this approach offers much promise.

The dynamic history of plastid genomes in the Campanulaceae sensu lato is unique among angiosperms

Article

Jul 2014

Eric B. Knox

Significance Photosynthesis in plants occurs in the chloroplast, which is one developmental form of the eukaryotic plastid that evolved endosymbiotically from a cyanobacterium. During their algal ancestry, most plastid genes were transferred to the nucleus or otherwise lost, and genome architecture and organization stabilized prior to the origin of land plants. The plastid genome in some groups of plants subsequently became dynamic, which prompts the question: Why? This study shows that the extensive rearrangements in Campanulaceae plastid genomes include dozens of newly inserted protein-coding genes that likely originated from the nucleus. Better understanding of this unique evolutionary potential of Campanulaceae plastids to acquire new DNA may help bioengineers incorporate genes into plastids of other plants.

Nif gene transfer and expression in chloroplasts: Prospects and problems

Article

Full-text available

Jan 1997

The engineering of plants capable of fixing their own nitrogen is an extremely complex task, requiring the co-ordinated and regulated expression of 16 nif genes in an appropriate cellular location. We suggest that plastids may provide a favourable environment for nif gene expression provided that the nitrogenase enzyme can be protected from oxygen damage. Using the non-heterocystous cyanobacteria as a model, we argue that photosynthesis could be temporally separated from nitrogen fixation in chloroplasts by restricting nitrogenase synthesis to the dark period. We report preliminary data on the introduction and expression of one of nitrogenase components, the Fe protein, in transgenic tobacco and Chlamydomonas reinhardtii. Finally we discuss potential avenues for further research in this area and the prospects for achieving the ultimate goal of expressing active nitrogenase in cereal crops such as rice.

Targeted Manipulation of Maize Genes in vivo Using Chimeric RNA/DNA Oligonucleotides

Article

Full-text available

Aug 1999

Site-specific heritable mutations in maize genes were engineered by introducing chimeric RNA/DNA oligonucleotides. Two independent targets within the endogenous maize acetohydroxyacid synthase gene sequence were modified in a site-specific fashion, thereby conferring resistance to either imidazolinone or sulfonylurea herbicides. Similarly, an engineered green fluorescence protein transgene was site-specifically modified in vivo. Expression of the introduced inactive green fluorescence protein was restored, and plants containing the modified transgene were regenerated. Progeny analysis indicated Mendelian transmission of the converted transgene. The efficiency of gene conversion mediated by chimeric oligonucleotides in maize was estimated as 10−4, which is 1–3 orders of magnitude higher than frequencies reported for gene targeting by homologous recombination in plants. The heritable changes in maize genes engineered by this approach create opportunities for basic studies of plant gene function and agricultural trait manipulation and also provide a system for studying mismatch repair mechanisms in maize.

Transient Foreign Gene Expression in Chloroplasts of Cultured Tobacco Cells After Biolistic Delivery of Chloroplast Vectors

Article

Full-text available

Feb 1990

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.

Evidence for variation in the quantity of DNA among plastids of Acetabularia

Article

Full-text available

Feb 1970
J CELL BIOL

The DNA content of individual plastids of the giant unicellular algae Acetabularia mediterranea, and Polyphysa cliftoni was studied. Four methods were used for localizing DNA: acridine orange staining, radioautography following actinomycin D-(3)H treatment, electron microscopy of thin tissue sections, and electron microscopy of osomotically disrupted plastids. With each method, DNA was readily detected in 20-35% of plastids, but no DNA was observed in the remaining 65-80%. The results further showed that in those plastids with detectable DNA the amount of DNA present was variable. The sensitivity and reliability of the localization methods are discussed, and the possible implications of these findings are considered.

Exchange of Protein Molecules Through Connections Between Higher Plant Plastids

Article

Full-text available

Jul 1997

Individual plastids of vascular plants have generally been considered to be discrete autonomous entities that do not directly communicate with each other. However, in transgenic plants in which the plastid stroma was labeled with green fluorescent protein (GFP), thin tubular projections emanated from individual plastids and sometimes connected to other plastids. Flow of GFP between interconnected plastids could be observed when a single plastid or an interconnecting plastid tubule was photobleached and the loss of green fluorescence by both plastids was seen. These tubules allow the exchange of molecules within an interplastid communication system, which may facilitate the coordination of plastid activities.

Containment of herbicide resistance through genetic engineering of the chloroplast genome

Article

Full-text available

May 1998

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.

Chloroplast Division in Higher Plants Requires Members of Two Functionally Divergent Gene Families with Homology to Bacterial ftsZ

Article

Full-text available

Jan 1999

The division of plastids is critical for viability in photosynthetic eukaryotes, but the mechanisms associated with this process are still poorly understood. We previously identified a nuclear gene from Arabidopsis encoding a chloroplast-localized homolog of the bacterial cell division protein FtsZ, an essential cytoskeletal component of the prokaryotic cell division apparatus. Here, we report the identification of a second nuclear-encoded FtsZ-type protein from Arabidopsis that does not contain a chloroplast targeting sequence or other obvious sorting signals and is not imported into isolated chloroplasts, which strongly suggests that it is localized in the cytosol. We further demonstrate using antisense technology that inhibiting expression of either Arabidopsis FtsZ gene (AtFtsZ1-1 or AtFtsZ2-1) in transgenic plants reduces the number of chloroplasts in mature leaf cells from 100 to one, indicating that both genes are essential for division of higher plant chloroplasts but that each plays a distinct role in the process. Analysis of currently available plant FtsZ sequences further suggests that two functionally divergent FtsZ gene families encoding differentially localized products participate in chloroplast division. Our results provide evidence that both chloroplastic and cytosolic forms of FtsZ are involved in chloroplast division in higher plants and imply that important differences exist between chloroplasts and prokaryotes with regard to the roles played by FtsZ proteins in the division process.

A Tool for Functional Plant Genomics: Chimeric RNA/DNA Oligonucleotides Cause in vivo Gene-Specific Mutations

Article

Full-text available

Aug 1999

Self-complementary chimeric oligonucleotides (COs) composed of DNA and modified RNA residues were evaluated as a means to (i) create stable, site-specific base substitutions in a nuclear gene and (ii) introduce a frameshift in a nuclear transgene in plant cells. To demonstrate the creation of allele-specific mutations in a member of a gene family, COs were designed to target the codon for Pro-196 of SuRA, a tobacco acetolactate synthase (ALS) gene. An amino acid substitution at Pro-196 of ALS confers a herbicide-resistance phenotype that can be used as a selectable marker in plant cells. COs were designed to contain a 25-nt homology domain comprised of a five-deoxyribonucleotide region (harboring a single base mismatch to the native ALS sequence) flanked by regions each composed of 10 ribonucleotides. After recovery of herbicide-resistant tobacco cells on selective medium, DNA sequence analyses identified base conversions in the ALS gene at the codon for Pro-196. To demonstrate a site-specific insertion of a single base into a targeted gene, COs were used to restore expression of an inactive green fluorescent protein transgene that had been designed to contain a single base deletion. Recovery of fluorescent cells confirmed the deletion correction. Our results demonstrate the application of a technology to modify individual genetic loci by catalyzing either a base substitution or a base addition to specific nuclear genes; this approach should have great utility in the area of plant functional genomics.

A galinstan expansion femtosyringe for microinjection of eukaryotic organelles and prokaryotes

Article

Full-text available

Oct 1999

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.

Engineering the Provitamin A (??-Carotene) Biosynthetic Pathway into (Carotenoid-Free) Rice Endosperm

Article

Full-text available

Feb 2000

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.

Genetic Transformation of Wheat Mediated by Agrobacterium tumefaciens

Article

Full-text available

Dec 1997

A rapid Agrobacterium tumefaciens-mediated transformation system for wheat was developed using freshly isolated immature embryos, precultured immature embryos, and embryogenic calli as explants. The explants were inoculated with a disarmed A. tumefaciens strain C58 (ABI) harboring the binary vector pMON18365 containing the [beta]-glucuronidase gene with an intron, and a selectable marker, the neomycin phosphotransferase II gene. Various factors were found to influence the transfer-DNA delivery efficiency, such as explant tissue and surfactants present in the inoculation medium. The inoculated immature embryos or embryogenic calli were selected on G418-containing media. Transgenic plants were regenerated from all three types of explants. The total time required from inoculation to the establishment of plants in soil was 2.5 to 3 months. So far, more than 100 transgenic events have been produced. Almost all transformants were morphologically normal. Stable integration, expression, and inheritance of the transgenes were confirmed by molecular and genetic analysis. One to five copies of the transgene were integrated into the wheat genome without rearrangement. Approximately 35% of the transgenic plants received a single copy of the transgenes based on Southern analysis of 26 events. Transgenes in T1 progeny segregated in a Mendelian fashion in most of the transgenic plants.

Transformation of Maize Cells and Regeneration of Fertile Transgenic Plants

Article

Full-text available

Aug 1990
PLANT CELL

A reproducible system for the generation of fertile, transgenic maize plants has been developed. Cells from embryogenic maize suspension cultures were transformed with the bacterial gene bar using microprojectile bombardment. Transformed calli were selected from the suspension cultures using the herbicide bialaphos. Integration of bar and activity of the enzyme phosphinothricin acetyltransferase (PAT) encoded by bar were confirmed in all bialaphos-resistant callus lines. Fertile transformed maize plants (R0) were regenerated, and of 53 progeny (R1) tested, 29 had PAT activity. All PAT-positive progeny analyzed contained bar. Localized application of herbicide to leaves of bar-transformed R0 and R1 plants resulted in no necrosis, confirming functional activity of PAT in the transgenic plants. Cotransformation experiments were performed using a mixture of two plasmids, one encoding PAT and one containing the nonselected gene encoding [beta]-glucuronidase. R0 plants regenerated from co-transformed callus expressed both genes. These results describe and confirm the development of a system for introduction of DNA into maize.

Perturbation of chloroplast DNA amounts and chloroplast gene transmission in Chlamydomonas by 5-fluorodeoxyuridine

Article

Full-text available

Nov 1977

5-Fluorodeoxyuridine selectively decreases the rate of chloroplast DNA replication in Chlamydomonas resulting after several generations of growth in equilibrium levels as low as one-seventh of normal. When the maternal parent is treated prior to mating, the decrease of chloroplast DNA appears to perturb the normal maternal transmission of chloroplast genes, dramatically increasing the proportion of exceptional zygotes transmitting chloroplast genes from the paternal parent.

Discovery of the T-DNA of Agrobacterium Tumefaciens

Chapter

Feb 1998

Milton P. Gordon

The Discovery of Tissue Culture

Chapter

Dec 1998

Zhi-hong Xu

The Chloroplast, Its Genome and Possibilities for Genetically Manipulating Plants

Article

Jan 1979

Lawrence Bogorad

We can imagine altering the genetic potential of an organism by deleting or introducing structural genes. We can visualize altering patterns of gene expression by manipulating regulatory DNA sequences. Exercise of either of these two genetic engineering approaches will reveal important biological principles and may well lead to important applications.

Plant Tissue Culture

Article

Jan 1969

G. G. HENSHAW

Plant Tissue Culture and Plant Morphogenesis By R. G. Butenko. Translated from the Russian. Pp. iv + 291. (Israel Program for Scientific Translations: Jerusalem. Distributed in the UK by H. A. Humphrey: London, October 1968.) 105s.

An improved, helium-driven biolistic device

Article

Jan 1991

Chloroplast Division in Higher Plants Requires Members of Two Functionally Divergent Gene Families with Homology to Bacterial f tsZ

Article

Dec 1998

Katherine W. Osteryoung

Evidence that inducible C4-type photosynthesis is a chloroplastic CO2-concentrating mechanism in Hydrilla, a submersed monocot

Article

Feb 1997

Hydrilla verticillata (L.f.) Royle exhibits an inducible C4-type photosynthetic cycle, but lacks Kranz anatomy. Leaves in the C4-type state (but not C3-type) contained up to 5-fold higher internal dissolved inorganic carbon (DIC) concentrations than the medium, indicating that they possessed a CO2-concentrating mechanism (CCM). Several lines of evidence indicated that the chloroplast was the likely site of CO2 generation. From C4-type leaf [DIC] measurements, the estimated chloroplastic free [CO2] was 400 mmol m–3. This gave a calculated 2% O2 inhibition of photosynthesis, which was identical to the measured value, and provided independent evidence that the estimated [CO2] was close to the true value. A homogeneous distribution of DIC in the C4-type leaf could not account for such a high [CO2], or the resultant low O2 inhibition. For C3-type leaves the estimated chloroplastic [CO2] was only 7 mmol m–3, which gave high, and similar, calculated and measured O2 inhibition values of 22 and 26%, respectively. The CCM did not appear to be located at the plasma membrane, as it operated at low and high pH, indicating that it was independent of use of HCO3– from the medium. Also, both C3- and C4-type Hydrilla leaves showed pH polarity in the light, with abaxial and adaxial boundary layer values of about pH 4·0 and 10·5, respectively. Thus, pH polarity was not a direct component of the CCM, though it probably improved access to HCO3. Additionally, iodoacetamide and methyl viologen greatly reduced abaxial acidification, but not the steady-state CCM. Inhibitor studies suggested that the CCM required photosynthetically generated ATP, but Calvin cycle activity was not essential. Both leaf types accumulated DIC in the dark by an ATP-requiring process, possibly respiration, and C4-type leaves fixed CO2 at 11·8% of the light rate. The operation of a CCM to minimize photorespiration, and the ability to recapture respiratory CO2 at night, would conserve DIC in a densely vegetated lake environment where daytime [CO2] is severely limiting, while [O2] and temperatures are high.

Stable chloroplast transformation in potato: Use of green fluorescent protein as a plastid marker

Article

Jul 1999

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.

New genes boost rice nutrients

Article

Jan 1999

T. Gura

Transient expression of green fluorescent protein in various plastid types following microprojectile bombardment

Article

Dec 1998
PLANT J

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.

A specific increase in chloroplast gene mutations following growth of Chlamydomonas in 5-fluorodeoxyuridine

Article

Oct 1979

Growth of Chlamydomonas in the thymidine analog 5-fluorodeoxyuridine (FdUrd) leads to a reduction in the amount of chloroplast DNA and also alters the pattern of chloroplast gene transmission in crosses (Wurtz et al., 1977). We have now found that growth of Chlamydomonas in FdUrd also increases at least 10 to 20 fold the frequency of cells expressing antibiotic resistant or non-photosynthetic mutations in the chloroplast genome with no concomitant increase in nuclear gene mutations with similar phenotypes. Clearly this effect is not locus specific since the non-photosynthetic chloroplast mutations thus far isolated comprise 9 recombinationally separate loci in the chloroplast genome (Shepherd et al., 1977, 1979). Only with the use of FdUrd has isolation of this important class of non-photosynthetic mutations been possible. The efficiency of recovery of chloroplast gene mutations rises as FdUrd concentration increases from 0.1 to 1.0 mM. At higher concentrations of FdUrd, growth rates and mutant yields are reduced. We propose the analog increases the yield of chloroplast mutations by a two-step process in which mutations are first induced as a result of thymidine starvation and then become expressed because the chloroplast DNA has been greatly reduced in ploidy and possibly damaged. FdUrd has its maximal effect on both recovery of chloroplast gene mutations and chloroplast gene transmission in crosses after cells grown in the presence of the analog for several generations remain at stationary phase for about 24 h. These observations suggest that chloroplast DNA metabolism is very active in non-dividing stationary phase cells of Chlamydomonas.

Development of the particle inflow gun for DNA delivery to plant cells

Article

Jan 1992

A simple and inexpensive particle bombardment device was constructed for delivery of DNA to plant cells. The Particle Inflow Gun (PIG) is based on acceleration of DNA-coated tungsten particles using pressurized helium in combination with a partial vacuum. The particles are accelerated directly in a helium stream rather than being supported by a macrocarrier. Bombardment parameters were partially optimized using transient expression assays of a -glucuronidase gene in maize embryogenic suspension culture and cowpea leaf tissues. High levels of transient expression of the -glucuronidase gene were obtained following bombardment of embryogenic suspension cultures of corn and soybean, and leaf tissue of cowpea. Stable transformation of embryogenic tissue of soybean has also been obtained using this bombardment apparatus.

Plastid transformation in Arabidopsis thaliana

Article

Nov 1998

Plastid transformation is reported in Arabidopsis thaliana following biolistic delivery of transforming DNA into leaf cells. Transforming plasmid pGS31A carries a spectinomycin resistance (aadA) gene flanked by plastid DNA sequences to target its insertion between trnV and the rps12/7 operon. Integration of aadA by two homologous recombination events via the flanking ptDNA sequences and selective amplification of the transplastomes on spectinomycin medium yielded resistant cell lines and regenerated plants in which the plastid genome copies have been uniformly altered. The efficiency of plastid transformation was low: 2 in 201 bombarded leaf samples. None of the 98 plants regenerated from the two lines were fertile.

Why don't plants fix nitrogen?

Article

Nov 1984
TRENDS BIOTECHNOL

The process of biological nitrogen fixation is restricted to a small number of prokaryotic organisms and the majority of the world's crop plants depend on chemical fertilizers to achieve optimum yields. Need this always be the case or have recent advances in the genetic analysis of nitrogen fixation made the construction of nitrogen-fixing plants a realistic goal?

Fluorescent antibiotic resistance marker for tracking plastid transformation in higher plants

Article

Oct 1999

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.

Inheritance and Expression of Chimeric Genes in the Progeny of Transgenic Maize Plants

Article

Oct 1990
Bio Tech

We obtained transgenic maize plants by using high-velocity microprojectiles to transfer genes into embryongenic cells. Two selectable genes were used to confer resistance to either chlorsulfuron or phosphinothricin, and genes encoding either E. coli beta-glucuronidase or firefly luciferase were used as markers to provide convenient assays for transformation. When regenerated without selection, only two of the eight transformed embryogenic calli obtained produced transgenic maize plants. With selection, transgenic plants were obtained from three of the other eight calli. One of the two initial lines produced 15 fertile transgenic plants. The progeny of these plants contained and expressed the foreign genes. Luciferase expression could be visualized, in the presence of added luciferin, by overlaying leaf sections with color film.

High-velocity microprojectiles for delivering nucleic acids into living cells. 1987.

Article

Feb 1992
Biotechnology

We report here a novel phenomenon, namely that nucleic acids can be delivered into plant cells using high-velocity microprojec-tiles. This research was conducted in the hope of circumventing some of the inherent limitations of existing methods for delivering DNA into plant cells1-6. After being accelerated, small tungsten particles (microprojectiles) pierce cell walls and membranes and enter intact plant cells without killing them. Microprojectiles were used to carry RNA or DNA into epidermal tissue of onion and these molecules were subsequently expressed genetically. This approach can therefore be used to study the transient expression of foreign genes in an intact tissue. It remains to be shown that smaller cell types, as are found in regenerable plant tissues, can be stably transformed by this method. If this proves possible, it would appear to provide a broadly applicable transformation mechanism capable of circumventing the host-range restrictions of Agrobacterium tumefaciens1, and the regeneration problems of protoplast transformation2-5.

Studies on Chlamydomonas Chloroplast Transformation: Foreign DNA Can Be Stably Maintained in the Chromosome

Article

Feb 1989

As shown originally by Boynton and co-workers (Boynton, J.E., Gillham, N.W., Harris, E.H., Hosler, J.P., Johnson, A.M., Jones, A.R., Randolph-Anderson, B.L., Robertson, D., Klein, T.M., Shark, K.B., and Sanford, J.C. [1988]. Science 240, 1534-1538), a nonphotosynthetic, acetate-requiring mutant strain of Chlamydomonas reinhardtii with a 2.5-kilobase pair deletion in the chloroplast Bam 10 restriction fragment region that removes the 3' half of the atpB gene and a portion of one inverted repeat can be transformed to photosynthetic competency following bombardment with microprojectiles coated with wild-type Bam 10 DNA. We have found that assorted other circular plasmids, single-strand DNA circles, or linear, duplex DNA molecules containing the wild-type atpB gene can also complement the same mutant. DNA gel blot hybridization analysis of all such transformants indicates that the complementing DNA has integrated into the chromosome at the atpB locus and suggests that a copy-correction mechanism operating between the inverted repeats maintains sequence identity in this region. Sequences from the intact inverted repeat may be recruited to restore the incomplete copy when exogenous DNA with only a portion of the deleted sequence is introduced. Furthermore, a foreign, unselected-for, chimeric gene flanked by chloroplast DNA sequences can be integrated and maintained stably in the chloroplast chromosome. The bacterial neomycin phosphotransferase structural gene fused to the maize chloroplast promoter for the large subunit gene of ribulose-1,5-biphosphate carboxylase (rbcL) has been integrated into the inverted repeat region of the Bam10 restriction fragment. RNA transcripts that hybridize to the introduced foreign gene have been identified.

Chloroplast Transformation in Chlamydomonas with High Velocity Microprojectiles

Article

Jul 1988

Bombardment of three mutants of the chloroplast atpB gene of Chlamydomonas reinhardtii with high-velocity tungsten microprojectiles that were coated with cloned chloroplast DNA carrying the wild-type gene permanently restored the photosynthetic capacity of the algae. In most transformants of one of the mutants, a fragment with a 2.5-kilobase deletion was restored to normal size by a homologous replacement event; in about 25 percent of the transformants the restored restriction fragment was 50 to 100 base pairs smaller or larger than that of wild type. About one-fourth of the transformants of this mutant contained unintegrated donor plasmid when first examined. This plasmid persisted in four different transformants after 65 cell generations of continuous liquid culture but was lost from all transformants maintained on plates of selective medium. The restored wild-type atpB gene remains in all transformants as an integral part of the chloroplast genome and is expressed and inherited normally.

DNA Supercoiling Affects in vitro Transcription of Two Maize Chloroplast Genes Differently

Article

Sep 1985

Two adjacent, divergently transcribed, developmentally regulated genes of the maize chloroplast chromosome have different superhelical density/transcriptional activation profiles when transcribed in vitro by the homologous DNA-dependent RNA polymerase. Promoter-specific transcription of the gene for the beta and epsilon subunits of coupling factor 1 (cf1BE) increases and plateaus from templates of increasing negative superhelicity, while transcription of the gene for ribulose bisphosphate carboxylase large subunit (rcL) rises and then falls. Maximal transcription from the two promoters occurs at different template negative superhelical densities and transcription of the two genes is stimulated to different degrees. The different superhelicity profiles alter the molar ratios of the two transcripts over an order of magnitude. Changes in DNA conformation represent one possible mechanism for the differential regulation of the genes.

Large plasmid in Agrobacterium tumefaciens essential for crown gall-inducing ability

Article

Dec 1974

THE gram-negative bacterium Agrobacterium tumefaciens induces crown gall tumours in many, mostly dicotyledonous, plants. Zaenen et al.1 demonstrated the presence of one or more large plasmids in a number of crown gall-inducing Agrobacterium strains belonging to seven different Agrobacterium groups. They were not able to find such plasmids in eight non-pathogenic Agrobacterium strains belonging to four of the same groups2,3. They therefore formulated the hypothesis that the genetic information for the tumour-inducing principle4 in crown gall-inducing Agrobacterium strains is carried by one or several large plasmids.

Chloroplast transformation in plants: polyethylene glycol (PEG) treatment of protoplasts is an alternative to biolistic delivery systems

Article

Jun 1993

Nicotiana plumbaginifolia protoplasts were directly transformed by PEG treatment with a cloned 16S rRNA gene isolated from a double antibiotic-resistant Nicotiana tabacum plastid mutant. Putative plastid transformants were selected in cell culture by their spectinomycin resistance and identified by their unselected streptomycin resistance. Alternatively, cell lines were selected in the presence of both antibiotics. The cell line (and its regenerated plants) selected solely for spectinomycin resistance demonstrated an extensive segregation of streptomycin resistance in subsequent tests, while the double-selected line showed stable resistance for both antibiotics. The resistance markers were inherited maternally. In the putative plastid transformants the origin of the resistance mutations was identified by the absence of an AatII site, missing in the donor N. tabacum plastid gene (spectinomycin resistance site) but present in that of wild-type N. plumbaginifolia, and a sequence analysis of the particular nucleotide changes in both resistance sites. Restriction enzyme analysis of total plastid DNA (ptDNA), and the recloning and full sequencing of the fragment introduced, investigated in one of the plastid transformants, showed no DNA rearrangements accompanied with the integration process. Sequence analysis indicated a targeted, homologous integration of the DNA fragment introduced but an unexpectedly complete homology of the parental ptDNA sequences in this region prevented the location of borders. Although the frequency of plastid transformant colonies (2 x 10(-5)) should still be improved, this method for stable chloroplast DNA transformation is comparable with or more efficient than the particle bombardment techniques.

Red/Far-Red and Blue Light-Responsive Regions of Maize rbcS-m3 are Active in Bundle Sheath and Mesophyll Cells, Respectively

Article

Jan 1996

Leaves of the C4 plant maize have two major types of photosynthetic cells: a ring of five large bundle sheath cells (BSC) surrounds each vascular bundle and smaller mesophyll cells (MC) lie between the cylinders of bundle sheath cells. The enzyme ribulose bisphosphate carboxylase/oxygenase is encoded by nuclear rbcS and chloroplast rbcL genes. It is not present in MC but is abundant in adjacent BSC of green leaves. As reported previously, the separate regions of rbcS-m3, which are required for stimulating transcription of the gene in BSC and for suppressing expression of reporter genes in MC, were identified by an in situ expression assay; expression was not suppressed in MC until after leaves of dark-grown seedlings had been illuminated for 24 h. Now we have found that transient expression of rbcS-m3 reporter genes is stimulated in BSC via a red/far-red reversible phytochrome photoperception and signal transduction system but that blue light is required for suppressing rbcS-m3 reporter gene expression in MC. Blue light is also required for the suppression system to develop in MC. Thus, the maize gene rbcS-m3 contains certain sequences that are responsive to a phytochrome photoperception and signal transduction system and other regions that respond to a UVA/blue light photoperception and signal transduction system. Various models of "coaction" of plant photoreceptors have been advanced; these observations show the basis for one type of coaction.

Integration of foreign sequences into the tobacco plastome via polyethylene glycol-mediated protoplast transformation

Article

Feb 1996

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.

Transformation of rice mediated by Agrobacterium tumefaciens

Article

Oct 1997

Agrobacterium tumefaciens has been routinely utilized in gene transfer to dicotyledonous plants, but monocotyledonous plants including important cereals were thought to be recalcitrant to this technology as they were outside the host range of crown gall. Various challenges to infect monocotyledons including rice with Agrobacterium had been made in many laboratories, but the results were not conclusive until recently. Efficient transformation protocols mediated by Agrobacterium were reported for rice in 1994 and 1996. A key point in the protocols was the fact that tissues consisting of actively dividing, embryonic cells, such as immature embryos and calli induced from scutella, were co-cultivated with Agrobacterium in the presence of acetosyringonc, which is a potent inducer of the virulence genes. It is now clear that Agrobacterium is capable of transferring DNA to monocotyledons if tissues containing 'competent' cells are infected. The studies of transformation of rice suggested that numerous factors including genotype of plants, types and ages of tissues inoculated, kind of vectors, strains of Agrobacterium, selection marker genes and selective agents, and various conditions of tissue culture, are of critical importance. Advantages of the Agrobacterium-mediated transformation in rice, like on dicotyledons, include the transfer of pieces of DNA with defined ends with minimal rearrangements, the transfer of relatively large segments of DNA, the integration of small numbers of copies of genes into plant chromosomes, and high quality and fertility of transgenic plants. Delivery of foreign DNA to rice plants via A. tumefaciens is a routine technique in a growing number of laboratories. This technique will allow the genetic improvement of diverse varieties of rice, as well as studies of many aspects of the molecular biology of rice.

High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens

Article

Jul 1996

Transformants of maize inbred A188 were efficiently produced from immature embryos cocultivated with Agrobacterium tumefaciens that carried "super-binary" vectors. Frequencies of transformation (independent transgenic plants/embryos) were between 5% and 30%. Almost all transformants were normal in morphology, and more than 70% were fertile. Stable integration, expression, and inheritance of the transgenes were confirmed by molecular and genetic analysis. Between one and three copies of the transgenes were integrated with little rearrangement, and the boundaries of T-DNA were similar to those in transgenic dicotyledons and rice. F1 hybrids between A188 and five other inbreds were transformed at low frequencies.

Amplification of a Chimeric Bacillus Gene in Chloroplasts Leads to an Extraordinary Level of an Insecticidal Protein in Tobacco

Article

May 1995
Bio Tech

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.

Co-ordinated expression of multiple enzymes in different subcellular compartments in plants

Article

Nov 1998

A gene expression system designed for coordinated expression of multiple genes in plants and their targeting to specified subcellular locations was tested. A series of genes encoding polyproteins containing the tobacco vein mottling virus (TVMV) Nla proteinase along with two other reporter genes (those encoding the Escherichia coli acetate kinase (ACK) and Tn9 chloramphenicol, acetyl transferase (CAT) enzymes) were assembled. The respective coding sequences of these genes were separated by a TVMV Nla proteinase recognition sequence. In addition, in some instances, chloroplast targeting information (a transit peptide (TP) from a pea rbcS gene) was incorporated into the polyprotein. We found that the Nla proteinase can be used to express, as individual polypeptides, the ACK and CAT proteins, and that these proteins retain enzymatic activity. Polyproteins with the structure TP-Nla-ACK-CAT or TP-ACK-CAT-Nla failed to yield chloroplast-localized ACK and CAT proteins, although the latter did give rise to a chloroplast-localized ACK-CAT polyprotein. These results indicate that the Nla proteinase acts in cis more rapidly than transport of proteins into the chloroplast, but that chloroplast localization can take place before complete processing of the polyprotein. Polyproteins with the structures ACK-Nla-TP-CAT and TP-ACK-Nla-TP-CAT yielded appropriately processed and targeted ACK and CAT. Our results show that subcellular localization signals can be effectively recognized in the context of a polyprotein, and they suggest an appropriate strategy for simultaneous engineering of multiple subcellular compartments in plants.

Biotechnology. New genes boost rice nutrients

Article

Sep 1999

Trisha Gura

BIOTECHNOLOGYST. LOUIS-- The latest high-tech version of rice, described here last week at the 16th International Botanical Congress, has been genetically engineered to contain b-carotene, the precursor to vitamin A, as well as a healthy dose of iron. This achievement is a Herculean feat of gene transfer. Unlike other genetically engineered crops, which contain only one or two foreign genes, the new rice strain carries a total of seven foreign genes from two separate pathways. But beyond that, it's also a major leap on a more humanitarian front: It may offer improved nutrition for the billions of people in developing nations who depend on rice as a staple food.

Stable Transformation of Plastids in Higher Plants

Article

Dec 1990

Stable genetic transformation of the plastid genome is reported in a higher plant, Nicotiana tabacum. Plastid transformation was obtained after bombardment of leaves with tungsten particles coated with pZS148 plasmid DNA. Plasmid pZS148 (9.6 kilobases) contains a 3.7-kilobase plastid DNA fragment encoding the 16S rRNA. In the 16S rRNA-encoding DNA (rDNA) a spectinomycin resistance mutation is flanked on the 5' side by a streptomycin resistance mutation and on the 3' side by a Pst I site generated by ligating an oligonucleotide in the intergenic region. Transgenic lines were selected by spectinomycin resistance and distinguished from spontaneous mutants by the flanking, cotransformed streptomycin resistance and Pst I markers. Regenerated plants are homoplasmic for the spectinomycin resistance and the Pst I markers and heteroplasmic for the unselected streptomycin resistance trait. Transgenic plastid traits are transmitted to the seed progeny. The transgenic plastid genomes are products of a multistep process, involving DNA recombination, copy correction, and sorting out of plastid DNA copies.

Transient expression of green fluorescent protein in various plastid types following microprojectile bombardment.

Jan 1998
627-632

Hibbard J.M.

Engineering chloroplasts: An alternative site for foreign genes, proteins, reactions and products

Abstract

No full-text available

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