Transplastomic plant

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A transplastomic plant is a genetically modified plant in which genes are inactivated, modified or new foreign genes are inserted into the DNA of plastids like the chloroplast instead of nuclear DNA.

Contents

Currently, the majority of transplastomic plants are a result of chloroplast manipulation due to poor expression in other plastids. [1] However, the technique has been successfully applied to the chromoplasts of tomatoes. [2]

Chloroplasts in plants are thought to have originated from an engulfing event of a photosynthetic bacteria (cyanobacterial ancestor) by a eukaryote. [3] There are many advantages to chloroplast DNA manipulation because of its bacterial origin. For example, the ability to introduce multiple genes (operons) in a single step instead of many steps and the simultaneous expression of many genes with its bacterial gene expression system. [4] Other advantages include the ability to obtain organic products like proteins at a high concentration and the fact that production of these products will not be affected by epigenetic regulation. [5]

The reason for product synthesis at high concentrations is because a single plant cell can potentially carry up to 100 chloroplasts. If all these plastids are transformed, all of them can express the introduced foreign genes. [1] This is may be advantageous compared to transformation of the nucleus, because the nucleus typically contains only one or two copies of the gene. [1]

The advantages provided by chloroplast DNA manipulation has seen growing interest into this field of research and development, particularly in agricultural and pharmaceutical applications. [5] However, there are some limitations in chloroplast DNA manipulation, such as the inability to manipulate cereal crop DNA material and poor expression of foreign DNA in non- green plastids as mentioned before. [5] In addition, the lack of post- translational modification capability like glycosylation in plastids may make some human- related protein expression difficult. [6] Nevertheless, much progress has been made into plant transplastomics, for example, the production of edible vaccines for Tetanus by using a transplastomic tobacco plant. [7]

Transformation and selection procedure

Gene construct

Plant gene cassette schematic for plant transplastomics Plant Gene Cassette Schematic for Plant Transplastomics.jpg
Plant gene cassette schematic for plant transplastomics

The first requirement for transplastomic plant generation is to have a suitable gene construct that can be introduced into a plastid like a chloroplast in the form of an E. coli plasmid vector. [8] There are several key features of a suitable gene cassette including but not limited to (1) selectable marker (2) flanking sequences (3) gene of interest (4) promoter sequences (5) 5' UTR (6) 3' UTR (7) intercistronic elements. [9] The selectable marker typically tends to be an antibiotic resistant gene, which would give the plant cell the ability to tolerate being grown on antibiotic containing agar plates. [5] Flanking sequences are crucial for introduction of the gene construct at precise predetermined points of the plastid genome through homologous recombination. [4] The gene of interests introduced have many different applications and can range from pest resistance genes to vaccine antigen production. [4] Intercistronic elements (IEE) are important for facilitating high levels of gene expression if multiple genes are introduced in the form of an operon. [4] Finally, the 5' UTR and 3' UTR enhances ribosomal binding and increases transcript stability respectively. [4]

Transformation and selection

The most common method for plastid transformations is biolistics: Small gold or tungsten particles are coated with the plasmid vector and shot into young plant cells or plant embryos, penetrating multiple cell layers and into the plastid. [8] There will then be a homologous recombination event between the shot plasmid vector and the plastid's genome, hopefully resulting in a stable insertion of the gene cassette into the plastid. [8] Whilst the transformation efficiency is lower than in agrobacterial mediated transformation, which is also common in plant genetic engineering, particle bombardment is especially suitable for chloroplast transformation. Other transformation methods include the use of polyethylene glycol (PEG)- mediated transformation, which involves the removal of the plant cell wall in order to expose the "naked" plant cell to the foreign genetic material for transformation in the presence of PEG. [8] PEG- mediated transformation however, is notoriously time consuming, very technical and labor intensive as it requires the removal of the cell wall which is a key protective structural component of the plant cell. [10] Interestingly, a paper released in 2018 has described a successful plastid transformation of the chloroplast from the microalgae species N. oceanica and C. reinhardtii through electroporation. [10] Whilst no study has been attempted yet for plastid transformation of higher plants using electroporation, this could be an interesting area of study for the future.

In order to persist and be stably maintained in the cell, a plasmid DNA molecule must contain an origin of replication, which allows it to be replicated in the cell independently of the chromosome. When foreign DNA is first introduced to the plant tissue, not all chloroplasts will have successfully integrated the introduced genetic material. [5] There will be a mixture of normal and transformed chloroplast within the plant cells. This mix of normal and transformed chloroplasts are defined to be "heteroplasmic" chloroplast population. [5] Stable gene expression of the introduced gene requires a "homoplasmic" population of transformed chloroplasts in the plant cells, where all the chloroplasts in the plant cell has successfully integrated the foreign genetic material. [5] Typically, homoplasmicity can be achieved and identified through multiple rounds of selection on antibiotics. [5] This is where the transformed plant tissue are grown repeatedly on agar plates that contain antibiotics like spectinomycin. [5] Only plant cells that have successfully integrated the gene cassette as shown above will be able to express the antibiotic resistance selectable marker and therefore grow normally on agar plates containing antibiotics. [5] Plant tissue that do not grow normally will have a bleached appearance as the spectinomycin antibiotic inhibits the ribosomes in the plastids of the plant cell, thereby preventing maintenance of the chloroplast [5] However, as heteroplasmic population of chloroplasts may still be able to grow on agar plates effectively, many rounds of antibiotic selection and regrowth are required to cultivate a plant tissue that is homoplasmic and stable. [5] Generation of homoplasmic plant tissue is considered to be a major difficulty in transplastomics and incredibly time consuming. [8]

Example of a tomato plant grafting for agricultural purposes Tomato graft union.jpg
Example of a tomato plant grafting for agricultural purposes

Grafting

Some plant species such as Nicotiana tabacum are more receptive to transplastomics compared to members of the same genus such as Nicotiana glauca and Nicotiana benthamiana. [11] An experiment conducted in 2012 highlighted the possibility of facilitating transplastomics for difficult plant species using grafting. Grafting occurs when two different plants are joined together and continue to grow, this technique has been widely employed in agricultural applications and can even occur naturally in the wild. [12] A transplastomic N. tabacum plant was engineered to have spectinomycin resistance and GFP fluorescence. [11] Whilst the nuclear transgenic plants N. benthamiana and N. glauca were engineered to have kanamycin antibiotic resistance and YFP fluorescence. [11] The transplastomic plant and the nuclear transgenic plants were then grafted unto each other and the grafted tissues were then analysed. [11] Fluorescence microscopy and antibiotic selection on agar plates with both kanamycin and spectinomycin revealed that the grafted plant tissue had both transplastomics and nuclear transgene DNA material. [11] This was further confirmed through PCR analysis. [11] This study highlighted that plastids like the chloroplast are able to pass between cells across graft junctions and result in the transfer of genetic material between two different plant cell lines. [11] This finding is significant as it provides an alternative pathway for generation of transplastomic plants for species that are not as easily transformed using our current experimental methodology as seen above. [11]

Optimizing transgene expression

Inducible expression systems such as theoriboswitches and pentatricopeptide repeat proteins have been widely studied in an effort to control and modulate expression of transgene products in transplastomic plants. [13] One big advantage in using inducible expression systems is to optimize concentration of transgene protein production. [13] For example, young plants need to devote energy and resources into growth and development to become mature plants. [13] Constitutive expression of the transgene would therefore be detrimental for plant growth and development, as it takes away valuable energy and resources to express the foreign gene construct instead. [13] This would result in a poorly developed transplastomic plant with low product yield. [13] Inducible expression expression of the transgene would overcome this limitation and allow the plant to mature fully like a normal wildtype plant before it is induced chemically to begin production of the transgene which can then be harvested. [13]

Biological containment and agricultural coexistence

Nicotiana tobacco plant Patch of Tobacco (Nicotiana tabacum ) in a field in Intercourse, Pennsylvania..jpg
Nicotiana tobacco plant

Genetically modified plants must be safe for the environment and suitable for coexistence with conventional and organic crops. A major hurdle for traditional nuclear genetically modified crops is posed by the potential outcrossing of the transgene via pollen movement. Initially it was thought that, plastid transformation, which yields transplastomic plants in which the pollen does not contain the transgene, not only increases biosafety, but also facilitates the coexistence of genetically modified, conventional and organic agriculture. Therefore, developing such crops was a major goal of research projects such as Co-Extra and Transcontainer.

However, a study conducted on the tobacco plant in 2007 has disproved this theory. Led by Ralph Bock from the Max Planck Institute of Molecular Plant Physiology in Germany, researchers studied genetically modified tobacco in which the transgene was integrated in chloroplasts. [14] A transplastomic tobacco plant generated through chloroplast mediated transformation was bred with plants that were male sterile with an untouched chloroplast. [14] The transplastomic plants were engineered to have resistance to the antibiotic spectinomycin and engineered to produce a green fluorescent protein molecule (GFP). [14] Therefore, it was hypothesized that any offspring produced by from these two lines of tobacco plant should not be able to grow on spectinomycin or be fluorescent, as the genetic material in the chloroplast should not be able to transfer via pollen. [14] However, it was found that some of the seeds were resistant to the antibiotic and could germinate on spectinomycin agar plates. [14] Calculations showed that 1 out of every million pollen grains contained plastid genetic material, which would be significant in an agricultural farm setting. [14] Because tobacco has a strong tendency towards self-fertilisation, the reliability of transplastomic plants is assumed to be even higher under field conditions. Therefore, the researchers believe that only one in 100,000,000 GM tobacco plants actually would transmit the transgene via pollen. Such values are more than satisfactory to ensure coexistence. However, for GM crops used in the production of pharmaceuticals, or in other cases in which absolutely no outcrossing is permitted, the researchers recommend the combination of chloroplast transformation with other biological containment methods, such as cytoplasmic male sterility or transgene mitigation strategies. This study showed that whilst transplastomic plants do not have absolute gene containment, the level of containment is extremely high and would allow for coexistence of conventional and genetically modified agricultural crops. [14]

There are public concerns regarding a possible transmission of antibiotic resistant genes to unwanted targets including bacteria and weeds. [15] As a result of this, technologies have been developed to remove the selectable antibiotic resistance gene marker. One such technology that has been implemented is the Cre/lox system, where the nuclear encoded Cre recombinase can be placed under control of an inducible promoter to remove the antibiotic resistant gene once homoplasmicity has been achieved from the transformation process. [16]

Potato beetle larvae Potato beetle larvae.jpg
Potato beetle larvae

Examples and the future

A recent example of transplastomics in agricultural applications was conferring potato plants protection against the Colorado potato beetle. [17] This beetle is dubbed a "super-pest" internationally because it has gained resistance against many insecticides and are extremely voracious feeders. [17] The beetle is estimated to cause up to 1.4 million USD in crop damages annually in Michigan alone. [18] A study conducted in 2015 by Zhang utilized transplastomics to introduce double stranded RNA producing transgenes into the plastid genome. [17] The double stranded RNA confers protection to the transgenic potato plant via a RNA interference methodology, where consumption of the plant tissue by the potato beetle would result in silencing of key genes required by the beetle for survival. [17] There was a high level of protection conferred, the leaves of the transplastomic potato plant were mostly unconsumed when exposed to the adult beetles and larvae. [17] The investigation also revealed an 83% killing efficacy for larvae that consumed the leaves of the transplastomic plant. [17] This study highlights that as pests gain resistance to traditional chemical insecticides, the use of transplastomics to deliver RNAI- mediated crop protection strategies could become increasingly viable in the future. [17]

Another notable transplastomics based approach is the production of artemisinic acid through transplastomic tobacco plants which is the precursor molecule that can be used to produce artemisinin. [19] Artemisinin- based combination therapy is the preferred and recommended treatment of choice by the WHO (World Health Organization) against malaria. [19] Artemisinin is naturally derived from the plant Artemisia annua , however, only low concentrations of artemisinin in the plant can be harvested naturally and there is currently an insufficient supply for the global demand. [19] A study conducted in 2016 led by Fuentes, managed to introduce the artemisininic acid production pathway into the chloroplast of N. tabacum through a biolistics approach before using their novel synthetic biology tool COSTREL (combinatorial supertransformation of transplastomic recipient lines) to generate a transplastomic N. tabacum plant that had a very high arteminisin acid yield. [20] This study illustrates the potential benefits of transplastomics for bio-pharmaceutical applications in the future.

Despite transplastomics being non- viable for non green plastids at the moment, plant transplastomics work done on the chloroplast genome has proved extremely valuable. [4] The applications for chloroplast transformation includes and is not limited to agriculture, bio-fuel and bio-pharmaceuticals. [4] This is because of a few factors, which include ease of multiple transgene expression in the form of operons and high copy number expression. [4] The study of transplastomics still remains a work in progress. More research and development is still required to improve other areas such as transplastomics in non- green plastids, inability to transform cereal crops through transplastomics and a way to circumvent the lack of glycosylation capability in the chloroplast. [4] Further improvements in this field of study will only give us a potential robust biotechnological route in many applications important in our day to day lives.

Related Research Articles

<span class="mw-page-title-main">Chloroplast</span> Plant organelle that conducts photosynthesis

A chloroplast is a type of membrane-bound organelle known as a plastid that conducts photosynthesis mostly in plant and algal cells. The photosynthetic pigment chlorophyll captures the energy from sunlight, converts it, and stores it in the energy-storage molecules ATP and NADPH while freeing oxygen from water in the cells. The ATP and NADPH is then used to make organic molecules from carbon dioxide in a process known as the Calvin cycle. Chloroplasts carry out a number of other functions, including fatty acid synthesis, amino acid synthesis, and the immune response in plants. The number of chloroplasts per cell varies from one, in unicellular algae, up to 100 in plants like Arabidopsis and wheat.

<span class="mw-page-title-main">Genetically modified organism</span> Organisms whose genetic material has been altered using genetic engineering methods

A genetically modified organism (GMO) is any organism whose genetic material has been altered using genetic engineering techniques. The exact definition of a genetically modified organism and what constitutes genetic engineering varies, with the most common being an organism altered in a way that "does not occur naturally by mating and/or natural recombination". A wide variety of organisms have been genetically modified (GM), from animals to plants and microorganisms. Genes have been transferred within the same species, across species, and even across kingdoms. New genes can be introduced, or endogenous genes can be enhanced, altered, or knocked out.

<span class="mw-page-title-main">Plastid</span> Plant cell organelles that perform photosynthesis and store starch

The plastid is a membrane-bound organelle found in the cells of plants, algae, and some other eukaryotic organisms. They are considered to be intracellular endosymbiotic cyanobacteria. Examples include chloroplasts, chromoplasts, and leucoplasts.

<span class="mw-page-title-main">Horizontal gene transfer</span> Type of nonhereditary genetic change

Horizontal gene transfer (HGT) or lateral gene transfer (LGT) is the movement of genetic material between unicellular and/or multicellular organisms other than by the ("vertical") transmission of DNA from parent to offspring (reproduction). HGT is an important factor in the evolution of many organisms. HGT is influencing scientific understanding of higher order evolution while more significantly shifting perspectives on bacterial evolution.

<i>Agrobacterium</i> Genus of bacteria

Agrobacterium is a genus of Gram-negative bacteria established by H. J. Conn that uses horizontal gene transfer to cause tumors in plants. Agrobacterium tumefaciens is the most commonly studied species in this genus. Agrobacterium is well known for its ability to transfer DNA between itself and plants, and for this reason it has become an important tool for genetic engineering.

<span class="mw-page-title-main">Gene gun</span> Device used in genetic engineering

In genetic engineering, a gene gun or biolistic particle delivery system is a device used to deliver exogenous DNA (transgenes), RNA, or protein to cells. By coating particles of a heavy metal with a gene of interest and firing these micro-projectiles into cells using mechanical force, an integration of desired genetic information can be introduced into desired cells. The technique involved with such micro-projectile delivery of DNA is often referred to as biolistics, short for "biological ballistics".

Pharming, a portmanteau of "farming" and "pharmaceutical", refers to the use of genetic engineering to insert genes that code for useful pharmaceuticals into host animals or plants that would otherwise not express those genes, thus creating a genetically modified organism (GMO). Pharming is also known as molecular farming, molecular pharming or biopharming.

<span class="mw-page-title-main">Genetically modified crops</span> Plants used in agriculture

Genetically modified crops are plants used in agriculture, the DNA of which has been modified using genetic engineering methods. Plant genomes can be engineered by physical methods or by use of Agrobacterium for the delivery of sequences hosted in T-DNA binary vectors. In most cases, the aim is to introduce a new trait to the plant which does not occur naturally in the species. Examples in food crops include resistance to certain pests, diseases, environmental conditions, reduction of spoilage, resistance to chemical treatments, or improving the nutrient profile of the crop. Examples in non-food crops include production of pharmaceutical agents, biofuels, and other industrially useful goods, as well as for bioremediation.

A transgene is a gene that has been transferred naturally, or by any of a number of genetic engineering techniques, from one organism to another. The introduction of a transgene, in a process known as transgenesis, has the potential to change the phenotype of an organism. Transgene describes a segment of DNA containing a gene sequence that has been isolated from one organism and is introduced into a different organism. This non-native segment of DNA may either retain the ability to produce RNA or protein in the transgenic organism or alter the normal function of the transgenic organism's genetic code. In general, the DNA is incorporated into the organism's germ line. For example, in higher vertebrates this can be accomplished by injecting the foreign DNA into the nucleus of a fertilized ovum. This technique is routinely used to introduce human disease genes or other genes of interest into strains of laboratory mice to study the function or pathology involved with that particular gene.

<span class="mw-page-title-main">Gene delivery</span> Introduction of foreign genetic material into host cells

Gene delivery is the process of introducing foreign genetic material, such as DNA or RNA, into host cells. Gene delivery must reach the genome of the host cell to induce gene expression. Successful gene delivery requires the foreign gene delivery to remain stable within the host cell and can either integrate into the genome or replicate independently of it. This requires foreign DNA to be synthesized as part of a vector, which is designed to enter the desired host cell and deliver the transgene to that cell's genome. Vectors utilized as the method for gene delivery can be divided into two categories, recombinant viruses and synthetic vectors.

<span class="mw-page-title-main">Gene targeting</span> Genetic technique that uses homologous recombination to change an endogenous gene

Gene targeting is a biotechnological tool used to change the DNA sequence of an organism. It is based on the natural DNA-repair mechanism of Homology Directed Repair (HDR), including Homologous Recombination. Gene targeting can be used to make a range of sizes of DNA edits, from larger DNA edits such as inserting entire new genes into an organism, through to much smaller changes to the existing DNA such as a single base-pair change. Gene targeting relies on the presence of a repair template to introduce the user-defined edits to the DNA. The user will design the repair template to contain the desired edit, flanked by DNA sequence corresponding (homologous) to the region of DNA that the user wants to edit; hence the edit is targeted to a particular genomic region. In this way Gene Targeting is distinct from natural homology-directed repair, during which the ‘natural’ DNA repair template of the sister chromatid is used to repair broken DNA. The alteration of DNA sequence in an organism can be useful in both a research context – for example to understand the biological role of a gene – and in biotechnology, for example to alter the traits of an organism.

Plant transformation vectors are plasmids that have been specifically designed to facilitate the generation of transgenic plants. The most commonly used plant transformation vectors are termed binary vectors because of their ability to replicate in both E. coli, a common lab bacterium and Agrobacterium tumefaciens, a bacterium used to insert the recombinant (customized) DNA into plants. Plant Transformation vectors contain three key elements;

In molecular cloning, a vector is any particle used as a vehicle to artificially carry a foreign nucleic sequence – usually DNA – into another cell, where it can be replicated and/or expressed. A vector containing foreign DNA is termed recombinant DNA. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Of these, the most commonly used vectors are plasmids. Common to all engineered vectors are an origin of replication, a multicloning site, and a selectable marker.

<span class="mw-page-title-main">Plant genetics</span> Study of genes and heredity in plants

Plant genetics is the study of genes, genetic variation, and heredity specifically in plants. It is generally considered a field of biology and botany, but intersects frequently with many other life sciences and is strongly linked with the study of information systems. Plant genetics is similar in many ways to animal genetics but differs in a few key areas.

<span class="mw-page-title-main">Genetically modified tomato</span>

A genetically modified tomato, or transgenic tomato, is a tomato that has had its genes modified, using genetic engineering. The first trial genetically modified food was a tomato engineered to have a longer shelf life, which was on the market briefly beginning on May 21, 1994. The first direct consumption tomato was approved in Japan in 2021. Primary work is focused on developing tomatoes with new traits like increased resistance to pests or environmental stresses. Other projects aim to enrich tomatoes with substances that may offer health benefits or be more nutritious. As well as aiming to produce novel crops, scientists produce genetically modified tomatoes to understand the function of genes naturally present in tomatoes.

<span class="mw-page-title-main">History of genetic engineering</span>

Genetic engineering is the science of manipulating genetic material of an organism. The first artificial genetic modification accomplished using biotechnology was transgenesis, the process of transferring genes from one organism to another, first accomplished by Herbert Boyer and Stanley Cohen in 1973. It was the result of a series of advancements in techniques that allowed the direct modification of the genome. Important advances included the discovery of restriction enzymes and DNA ligases, the ability to design plasmids and technologies like polymerase chain reaction and sequencing. Transformation of the DNA into a host organism was accomplished with the invention of biolistics, Agrobacterium-mediated recombination and microinjection. The first genetically modified animal was a mouse created in 1974 by Rudolf Jaenisch. In 1976 the technology was commercialised, with the advent of genetically modified bacteria that produced somatostatin, followed by insulin in 1978. In 1983 an antibiotic resistant gene was inserted into tobacco, leading to the first genetically engineered plant. Advances followed that allowed scientists to manipulate and add genes to a variety of different organisms and induce a range of different effects. Plants were first commercialized with virus resistant tobacco released in China in 1992. The first genetically modified food was the Flavr Savr tomato marketed in 1994. By 2010, 29 countries had planted commercialized biotech crops. In 2000 a paper published in Science introduced golden rice, the first food developed with increased nutrient value.

<span class="mw-page-title-main">Genetic engineering techniques</span> Methods used to change the DNA of organisms

Genetic engineering techniques allow the modification of animal and plant genomes. Techniques have been devised to insert, delete, and modify DNA at multiple levels, ranging from a specific base pair in a specific gene to entire genes. There are a number of steps that are followed before a genetically modified organism (GMO) is created. Genetic engineers must first choose what gene they wish to insert, modify, or delete. The gene must then be isolated and incorporated, along with other genetic elements, into a suitable vector. This vector is then used to insert the gene into the host genome, creating a transgenic or edited organism.

An edible vaccine is a food, typically plants, that produce vitamins, proteins or other nourishment that act as a vaccine against a certain disease. Once the plant, fruit, or plant derived product is ingested orally, it stimulates the immune system. Specifically, it stimulates both the mucosal and humoral immune systems. Edible vaccines are genetically modified crops that contain antigens for specific diseases. Edible vaccines offer many benefits over traditional vaccines, due to their lower manufacturing cost and a lack of negative side effects. However, there are limitations as edible vaccines are still new and developing. Further research will need to be done before they are ready for widespread human consumption. Edible vaccines are currently being developed for measles, cholera, foot and mouth disease, Hepatitis B and Hepatitis C.

A plastid is a membrane-bound organelle found in plants, algae and other eukaryotic organisms that contribute to the production of pigment molecules. Most plastids are photosynthetic, thus leading to color production and energy storage or production. There are many types of plastids in plants alone, but all plastids can be separated based on the number of times they have undergone endosymbiotic events. Currently there are three types of plastids; primary, secondary and tertiary. Endosymbiosis is reputed to have led to the evolution of eukaryotic organisms today, although the timeline is highly debated.

<span class="mw-page-title-main">Pal Maliga</span> A plant molecular biologist

Pal Maliga is a plant molecular biologist. He is Distinguished Professor of Plant Biology and Laboratory Director at the Waksman Institute of Microbiology, Rutgers University. He is known for developing the technology of chloroplast genome engineering in land plants and its applications in basic science and biotechnology.

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