Background
Nutritional Importance of Omega-3 Fatty Acids and Docosahexaenoic Acid
Of a large number of fatty acids of high biological value to the human body, the most notable are omega-3 long-chain polyunsaturated fatty acids. This is the main component of fish oil, which, when consumed by humans, has a positive effect on the construction properties of cell membranes and blood vessels. It should be recognised that these molecules alone cannot be synthesized in the body, and consequently, the person has to eat them with food to maintain a full diet. One of the most important representatives of polyunsaturated fatty acids is docosahexaenoic acid (Echeverría et al., 2017). An adequate amount of these molecules in the diet positively correlates with the formation of brain and eye structures of the embryo, as well as postpartum functional development of the child.
Despite the severe study of polyunsaturated fatty acids, among researchers, there is still an established opinion about the daily norm, which is necessary for human consumption. Omega-3 fatty acids (2019) indicates a daily requirement of 1.6 g of acids for men and 1.1 g for women. Of this amount, 250 mg of the substance should be docosahexaenoic acid (Tressou et al., 2019). In this context, a study of the sources that supply the most significant amount of DHA to the body draws attention: according to Omega-3 fatty acids (2019), this is Atlantic salmon. At the same time, no docosahexaenoic acid is found in the composition of natural vegetable oils, although its sufficient amount could solve the problem of creating a complete diet. The increased emphasis on omega-3 fatty polyunsaturated acids is becoming a defining goal for nutraceuticals in the coming decades (Panse and Phalke, 2016). For this reason, special attention should be given to genetic modifications of existing plant materials to produce foods rich in fatty acids, and in particular DHA.
Status of Knowledge on Genetic Modifications of Vegetable Oils
The definition of the crop used as an object of genetic manipulation is of primary importance in the search for alternatives to fatty acid production. It should be admitted that the prevailing scientific literature is devoted to the modification of Canola, a plant with low levels of omega-9 fatty acids. In the context of the importance of genetic manipulations with Canola, it should be noted that vegetable oils derived from this crop have high nutritional and commercial value, with the potential to reduce serum lipid deposits (Ghobadi et al., 2019). On the other hand, the technical grade of such oil has found full application as a biofuel to replace traditional counterparts in diesel engines (Ge, Yoon, and Choi, 2017). Vegetable oil from Canola is incredibly essential for humans, and therefore, its modifications are justified.
Traditionally, the authors use similar technologies to insert genes of interest into the Canola genome. In supporting document 1 (2017), the authors of the study proposed a transformation transfer of a cassette of seven genes isolated from the genome of yeast and microscopic algae. These genes were responsible for the synthesis of proteins involved in the production and storage of omega-3 polyunsaturated fatty acids. The genetic engineering method used by the authors was the transformation of plasmid pJP3416_GA7-ModB into the host organism. Interestingly, biochemical analysis and establishment of the affinity of amino acid residual sequences did not reveal similarity with toxic forms of proteins. This fact unambiguously postulates the safety of the modified Canola for human eating. On the other hand, a critical remark is the fact that the thermally unstable proteins produced by the plant were found in negligible quantities, which is especially relevant in the context of the task at hand.
Meanwhile, it should be recognised that the granting of the food safety status is preceded by several rigorous procedures and examinations. Napier, Olsen, and Tocher (2019) have successfully demonstrated that genetically modified crop lines can be authorized by regulatory authorities because toxic properties are not traditionally detected. In other words, modified plants have the potential to replace existing animal counterparts that produce omega-3 fatty acids.
In the context of the task, special attention should be paid to the lack of works on genetic modification of the sunflower genome (Helianthus annuus). Expanded search by keywords in the most popular electronic databases does not allow finding any significant works in which this plant would be manipulated to produce the described proteins. It is fair to note that the task of creating laboratory versions of the wild sunflower with the modified genome is still found in several works, although the ultimate goal of the researchers is different from that applicable to this work. Thus, Jacob, Sujatha, and Varaprasad (2017) showed the possibility of introducing a gene resistant to the toxic effects of herbicides in the locus Ahasl1. On the other hand, a modification of sunflower to create a hyperaccumulator of heavy metals sorbed from the soil was also shown to be technically feasible (Saxena et al., 2020). These examples allow us to conclude that the practice of genetic manipulation of the plant genome is generally known among researchers, although it is not common.
This phenomenon cannot seem justified, since vegetable oil isolated from sunflower is one of the most popular liquid oils. According to Regitano et al. (2016), this type of oil is generally rich in fatty acids, but of unsaturated oils, the highest content is characteristic of omega-6. This means that traditional, natural oils have almost no omega-3 fatty acids. Along with the high popularity of the product, its genetic modification to introduce the gene cassette responsible for the production of polyunsaturated fatty acid DHA is justified.
Natural Breeding of Sunflower and Sunflower Oil
Sunflower is an annual plant, which has a relatively large height and nutritional value of seeds. With the formation of the first shoots begins the formation of the generative organs of the plant. During flowering, which lasts about two weeks, cross-pollination of the flowers takes place, culminating in the development of a seed fruit. Gradually, the seed forms a hard, dark coat, and the overall moisture content of the fruit is noticeably reduced. Depending on the variety, oil fatty acids, mostly oleic and linoleic acids, are synthesized and stored inside the cells. The harvested crop goes to the oil production workshops, where a liquid essence is obtained by pressing from the nuclei of the fruit.
It should be noted that sunflower oils have ambiguous value to the body. To date, the scientific community has formed two points of view on the correctness of daily intake of this type of oil (Health benefits, 2019). Ongoing disputes among researchers have led to the fact that many myths have emerged in public about the danger of sunflower oil for people of all ages. On the one hand, researchers argue that the abundance of omega-6 fatty acids negatively affects the metabolic balance of the body. Although it is an essential fatty acid, omega-6 can cause inflammation in the body (Jandacek, 2017). Moreover, carcinogenic products of oil decomposition may be released if food preparation is impaired. Nevertheless, the low content of trans fats along with a small amount of saturated fatty acids makes sunflower oil useful. The main advantage of this type of oil is the reduction of bad cholesterol, along with an increase in good cholesterol. It should be admitted that research in this area has been carried out for a long time, so enough material has been accumulated to confirm the positive qualities of sufficient sunflower oil consumption. This, in turn, led to the FDA officially recognizing sunflower oil as safe for health and acceptable for retail sales (FDA completes the review, 2018). In other words, the choice of sunflower as an object of future genetic manipulation is justified.
Data on Docosahexaenoic Acid
It should be noted that docosahexaenoic acid is an essential representative of omega-3 fatty unsaturated acids, the absence of which negatively affects the functionality of the human body. This type of molecule is not produced within the body itself, and hence, only external intake is possible (Calder, 2016). However, except for some microscopic algae, docosahexaenoic acid is practically absent in vegetable fats but is widely represented in animals: salmon, herring.
The biological value of acid is difficult to overestimate: in the absence of DHA in the human body in the supply, quantities observed functional pathology of the development and growth of tissue and organic structures. It is well known that insufficient acid intake in early childhood can lead to long-term obesity (Foster et al., 2017). There is also research on the development of pathologies in adult patients. According to Dawczynski et al. (2018), with sufficient daily intake, DHA can have a stimulating effect on the body’s anti-inflammatory processes. In other words, docosahexaenoic acid has critical biological properties for humans, so ignoring this product is unacceptable. In vegetable sunflower oils, almost zero content of this acid can be found (Fat composition, 2016). At the same time, Colombo et al. (2018), indicates that the gene responsible for acid metabolism may be introduced into plant feedstocks. Based on the above, the desire of researchers to intensify the artificial production of DHA can be understood. Taking into account the lack of any reliable scientific information about the genetic modification of sunflower to produce a plant capable of producing DHA, it should be noted that this work is highly unique and innovative.
Significance and Innovation
Docosahexaenoic acid, as one of the most important representatives of omega-3 fatty acids, is an integral part of a complete human diet. It has been shown that a sufficient amount of the substance consumed daily has a positive effect on the development of tissue and organic structures in the body. On the contrary, if the daily dose is comparatively less than the established norm, that is 250 mg, a person may have pathological changes in the brain structures and general development. These provisions justify the increased demand for this product and taking into account the uniqueness of its sources of origin, it was necessary to develop alternative production options (Betancor et al., 2017). The literature review demonstrated the lack of scientific papers on genetic modifications of sunflower to introduce the genes responsible for the synthesis and stock of DHA. This work is incredibly significant and promising since along with public value, it aims to develop and study the mechanisms of sunflower modification by genes isolated from microalgae and yeast.
Currently, many researchers in the field of genetic engineering are turning to the mechanism of vector transformation to transfer genes from the carrier to the host cell. This is a very convenient approach, but its implementation requires time for plasmid recombination and careful control of the survival of competent cells. At the same time, there is an alternative method of multiple genes introduction into the host genome: gene gun. It is worth admitting that this is a rather reliable approach, which allows injecting into the cytoplasm of cells several genes deposited on a heavy metal micro-particle. Moreover, this method of genetic engineering is well suited for the modification of plant forms, so this paper was chosen so unique for this problem. Summing up the innovation of this project, it should be repeated: genetic modification is carried out on a rare plant for existing research using a method that is not very common in the subject papers. In other words, the implementation of this experiment will allow for solving simultaneously two problems related both to the choice of the biological object of study and instrumental methods.
Aims of the Project
The search for alternatives to the production of docosahexaenoic acid is crucial to public health issues. Understanding whether genetically modified sunflower will produce enough omega-3 fatty acids for daily consumption will broaden the horizons for future research. Based on the results of this study, it will be possible to analyze the significance of sunflower varieties for DHA production statistically and to identify in more detail the external and internal factors affecting the synthesis.
Aims
Conduct the literature review of case studies and determine the degree of uniqueness.
- Create a cassette of donor genes isolated from microscopic algae and yeast.
- Using the gene gun method, introduce seven genes of interest into the host cell, make sure that they are accretive.
- Growing the first experimental sunflower line.
- Carry out PCR-analysis, microchipping to detect the presence of transgenes in the sunflower genome.
- Carrying out selective crossbreeding to create generations F1 and F2, guaranteed to have transgenes.
- Stimulation of self-pollination of descendants to obtain diploid individuals.
- Carrying out examination and quality control of products.
- Production of stably multiplying plant genetically modified crops on the consumer market.
Methodology
This proposal is driven by the studies of Supporting document 1 (2017), Napier, Olsen, and Tocher (2017), and Ursin (2003), which detailed that inclusion of DHA synthesis genes in terrestrial plant cells is possible. However, the technology and the biological object of the study have been changed to study the previously unexplored properties of GMO sunflower. In the proposed project, the first stage of the experiment is the isolation of seven different genes from host cells using restriction (DNA restriction and electrophoresis, 2019). Four representatives of the seaweed and two representatives of the yeast will be placed on a Petri dish under sterile conditions and, after identification of the genes of interest, complementary restriction with enzymes will be performed.
With the help of integration enzymes (REMI), the isolated genes are inserted into single-chain gene cassettes, after which the seven-genes cassette is embedded in the plasmid of the vector using a ligase enzymatic solution. Then, a recombinant plasmid is applied to a Wolfram or Gold particle, and the callus of undifferentiated sunflower cells grown on the gel is bombarded (Hanson, Taylor, and Wernick, 2016). In the initial stages of the study, it is crucial to perform regular screening to eliminate defective or incompetent cells. Subsequently, the callus is transplanted into Murashige and Skoog medium and actively supplied with growth hormones to provoke the development of the whole plant. The resulting genetically modified sample is crossed with an alternative wild form, and at the F1 stage PCR research, microchipping, and genome blots of offspring are performed. Detection of the cassette sought allows for continuing breeding crosses up to F2, when it is necessary to conduct another genomic examination. Haploid specimens of F2 are crossed by self-pollination to obtain a diploid specimen. This line of organisms requires careful testing not only for the presence of genes of interest but also for overall toxicity and potential health risks to the consumer.
Reference List
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