![]() - Click on ’Animations’ button found to the left in order to view the animation which supplements this lesson. You can also click on the animation icon within the text. - Click once on figures to see enlarged versions. - Click once on words in color to bring up their definitions. |
This first section is intended to provide a general background about what techniques are used to make nutritional changes in plants.
When genetic changes are made in crop plants, there are associated changes that occur in the plants’proteins, which in turn produce the new phenotype. These proteins arise from translation of DNA into RNA into amino acid sequences of polypeptide chains. Often, the proteins that are changed are ones that act as enzymes in the plant. These enzymes direct the synthesis of cell components that then determine the cell’s characteristics (e.g. what type of cell it is). Cells then make up tissues, organs and finally, the plant itself.
In order to make these proteins, and thereby the desired plant characteristic, several techniques are used, with the end result being addition of the desired foreign DNA into the plant.
Pathogen
One technique for introducing DNA changes into a plant involves the use of a plant pathogen, Agrobacterium tumefaciens.
In nature, this bacterium invades tissue wounds in plants(1). During this process the bacterium introduces a portion of its own DNA into the cell nuclei of the tissue it has invaded. That DNA becomes a part of the plant cell’s DNA makeup. This incorporation usually results in tumor formation within the tissue.
In the laboratory setting, the bacteria is introduced to the plant cell with its tumor causing DNA inactivated. When it invades the plant cell, it doesn’t cause tumor formation, however foreign DNA can be spliced into the bacteria’s DNA. The foreign DNA will be carried into the plant’s cell nuclei and become part of the plant’s chromosomes (1).
As part of the chromosome, promoters can then be used to express the genetic information from the foreign DNA. An important step in alteration involving nutrients is then expressing the genetic information at the correct stage of the plant’s development (e.g. leaves, flowers, fruits or seeds)(1).
![]() |
![]() |
Agrobacterium that contain a plasmid recombinant with the gene of interest are added to a solution containing callus cells | DNA, including the gene of interest, is inserted by the Agrobacterium into the nucleus of some of the callus cells where it may insert into a chromosome. |
![]() |
![]() |
Gene gun transformation begins by growing cells in tissue culture, bombarding the cells with gene coated gold particles in the gene gun, selecting out transgenic cells on selection media, and regenerating the transgenic cells into plants. | Once inside the nucleus of a cell, the genes dissolve off of the gold particle and can potentially insert into a chromosome. A major limitation of this method is that several copies of the transgene will often insert into a chromosomal position. These high copy insertion events can be detected as DNA that should not be expressed by the plant cell and the transgene copies are silenced. |
![]() |
![]() |
Proteins
Plants naturally have an incomplete amino acid composition as it relates to human amino acid requirements. This can be overcome by including foods containing the amino acids that are low in the other food, thus creating a complete source of amino acids. This technique is often used in the traditional meals from vegetarian cultures (2).
In most developed countries it is not difficult for individuals to attain complete sources of amino acids required for human health. However, in developing countries there are more challenges to overcome such as production costs, climatic restrictions on what crops can be grown, some traditional eating habits, and an overall lack of food (2).
Modifying Amino Acid Content
Research in this area has been focused on creating a complete amino acid source in plants. This is a difficult task, because plants are complex organisms. Protein expression is tissue specific, meaning that the functional requirements of each tissue dictate what proteins are manufactured there. Even within the tissue, proteins in individual cells are separated based on their function. This separation makes it essential to develop transformation systems that not only introduce the genetic information of interest, but that also work within the control mechanisms, such as tissue-specific expression (2). The introduced gene will only create the desired result when it is expressed in the correct tissue.
![]() |
![]() |
![]() |
A. Amino acids are made from simple atoms and always have an amino end and an acid end | B. Proteins or polypeptides are made by connecting amino acids together | C. The parts of the gene; the promoter and termination sequences control transcription while the coding region encodes the protein.. |
![]() |
This picture shows a purple soybean flower. |
![]() |
This picture shows a wheat spikelet. |
Research into the modification of fiber and complex carbohydrates in foods has increased. This increase has occurred due to the affects they have been found to have on lipid metabolism and diabetes control (3). The foods in this section have had their carbohydrate content modified, at least in the laboratory.
![]() |
This picture shows a grain truck filled with harvested ears of corn. |
The fatty acid composition of plants depends on climatic conditions and the genetics of the plant. These fatty acids are found as both storage oils and membrane lipids, such as glycolipids, phospholipids and lipoproteins. This section will discuss genetic changes that have been made to change the fatty acid profile in various plants.
Polyunsaturated fatty acids
Polyunsaturated fatty acids (PUFAs) have been found to be important to human nutrition. One of the reasons for their importance is their hypocholesterolemic property related to heart disease. They are also involved in many biological processes in both structural and functional roles as parts of cell membranes, regulators of membrane permeability as well as proteins within the membrane. PUFAs are also involved in gene expression regulation, including genes that provide the information for fatty-acid synthesis, sodium-channel proteins, and cholesterol-7-alpha-hydroxylase. As a result of their part in gene expression, PUFAs impact cellular biochemical activities, transport processes involving lipid metabolism, immune responses, cold adaptation, and conditions such as carcinogenesis and cardiovascular disease (6)
Polyunsaturated fatty acids can be obtained from plant seeds, marine fish and certain mammals. It has been reported that the market for PUFAs may increase, resulting in the need for alternative sources of fatty acids (4). Genetic modifications could help provide alternative sources of these fatty acids and to provide a desirable PUFA profile to meet specific nutritional demands (6).
Trans fatty acids
Trans fatty acid consumption has been regarded as a possible risk factor for coronary heart disease. While most fatty acids are present in nature in a cis configuration (changes the shape of the molecule), trans fatty acids are created when plant oils are hydrogenated.
The concern about trans fatty acids can be overcome by the use of desaturase genes from plants (6). One example of such a gene is the Omega 6 desaturase gene from soybeans. This desaturase inserts a second double bond into oleic acid, making it polyunsaturated. Researchers have used this gene’s activity to suppress the existing gene’s activity. In other words, the addition of the second double bond is blocked. When the second double bond is not added, the result is a monounsaturated oil with no trans fatty acids created during hydrogenation (7).
Canola Oil
Canola oil developed from rapeseed was one of the first examples of nutrition concerns influencing oil seed crop developments (3). That modification was accomplished with traditional plant breeding, however now genetic methods provide a technique to make more specific modifications to plants’ fatty acid biosynthesis pathways (3). These biosynthesis pathways are controlled by multiple genes in plants, with some of the genes controlling fatty acids in specific tissues, which allows for changes that can be made in seed storage lipids (3).
Replacement of high saturated fat oils with less saturated ones is an area of research with human health implications. One example is high laurate canola oil (lauric acid is a medium chain fatty acid) created by inserting the 12:0 thioesterase gene from Umbellaria californica into canola. The benefit of the high amount of lauric acid is that the product can replace the high saturated fat tropical oils that are used in nondairy coffee whiteners and whipped toppings, as well as in confectionery products (1).
Vegetable Oil
There have been several developments used to change the fatty acid profile of vegetable oil. The first genetic modification used cyanobacterial desaturase introduction into the plant. The desaturase introduces a double bond into 16-chain and 18-chain saturated fatty acids which, if expressed in the seeds, the saturated fatty acid content of the oil could be reduced (7). The second modification uses delta 6 desaturase that introduces a double bond into linoleic acid. Linoleic acid is then converted into gamma-linoleic acid, the beneficial compound found in fish oils (7).
Deficiencies of iron, vitamins A, E and C have lead to research into increasing the amounts of these micronutrients in staple foods. It has been estimated that two billion of the world’s population are iron deficient, while 400 million of the world’s population are vitamin A deficient. Many Americans don’t consume the recommend amount of vitamin E. This section discusses these micronutrients that have been modified in food.
Vitamin E
Most of the vitamin E in the human diet comes from vegetable oils made from soybeans, maize, cottonseed and rapeseed. In these crops, alpha-tocopherol, which has the highest vitamin E activity, is found in low levels. Gamma-tocopherol, the biosynthetic precursor to alpha-tocopherol, is found in these same plants at higher levels (8). Since the precursor is available in these plants, it suggests that the synthesis of alpha-tocopherol could be increased in oil crop seeds.
The enzyme that creates the final product in the pathway, alpha-tocopherol, has been overexpressed in transgenic Arabidopsis plants. The overexpression of this gene was paired with promoters for its expression in the seed, with the result being increased tocopherol accumulation.
Vitamin A
Rice
Rice grains do not naturally contain beta-carotene, the precursor to vitamin A. However, they do contain a substance, geranylgeranyl pyrophosphate that can be converted to beta-carotene. This conversion requires four enzymes in the biosynthetic pathway. The four genes for these enzymes have been isolated from daffodils and bacteria. These genes were then transferred into rice via Agrobacterium mediated transformation (9). The result of this transfer was several transgenic plants that had all four genes expressed, with a visible yellow endosperm that indicates the presence of carotenoids (9). A meal-sized portion of the transgenic rice contains enough vitamin A to meet the daily requirements for the vitamin (10).
Tomatoes
Research into carotenoid biosynthesis in tomatoes has lead to knowledge about genes such as Psy1, Pds, beta-Lcy and Epsilon-Lcy. Psy1 and Pds, which control lycopene synthesis, are up-regulated during the ripening process, while beta-Lcy and Epsilon-Lcy, which control lycopene cyclisation, are down-regulated. The result of this regulation is beta-carotene (the major dietary precursor of vitamin A) levels in ripe tomatoes less than 15% of the total carotenoids present.
Beta-carotene has been increased in tomatoes with overexpression of Arabidopsis beta-Lcy cDNA in the plant, which is fused to the Pds promoter. The Pds promoter, as mentioned before, is up-regulated during ripening. The result was increased beta-carotene in the tomato (11). The level of beta-carotene was 6 mg (the equivalent of the vitamin A RDA).
Iron
Rice
The accumulation of iron and ferritin in seeds occurs naturally. The seed ferritin is a target trait since it is a natural source of iron for development in plants and animals, it is already a normal seed component, it appears to be under accessible genetic control, and it is bioavailable (12).
The gene encoding the plant iron storage protein, phytoferritin, was cloned. Researchers hope that this gene can be used via overexpression in cereal crops to fortify them with bioavailable iron (8).
Another technique to fortify rice has been the transfer of the soybean ferritin gene via Agrobactrium-mediated transformation (9). A promoter for seed storage protein was used as a reference to express the soybean gene in the endosperm of the rice. The result was a threefold increase in iron content compared to untransformed controls (9).
The elimination of phytate, a sugar-like molecule that binds dietary iron, has been another focus of improving iron content in rice. A fungal gene for phytase, an enzyme that breaks down phytate, has been introduced into rice. The result of this addition was an increase in the bioavailability of the iron (9).
Vitamin C
Fruits and vegetables supply approximately 91% of the vitamin C in the U.S. food supply (3). The substantial variation of the amount of vitamin C in crops suggests that there is a potential for genetic modification.
Tomatoes
Currently, tomatoes with increased vitamin C content have been developed with traditional cross breeding between cultivated and wild species. However, poor yield often co-exists with the increased vitamin C. Until the interaction is better understood, modification of vitamin C in tomatoes has been discontinued (3).
Flavonoids
Flavonoids have been defined as polyphenolic compounds found in plants. These compounds are involved in plant growth and development. There has also been research that suggests flavonoids are beneficial to human health, possibly due to their antioxidant properties (5). Currently, over 400 individual flavonoids have been discovered.
The benefits include protection against chronic diseases, such as cardiovascular disease, that can be obtained by consuming foods containing these compounds. Their antioxidant properties towards free radicals have potential in anticancer activity, such as antiproliferation and promotion of differentiation and apoptosis (13).
There is some difficulty in obtaining good sources of flavonoids since many food sources have only small amounts in their edible parts. Foods may also contain flavonoids with suboptimal antioxidant characteristics, making them less than ideal sources (5).
Genetic engineering could play a role in improving these sources. Research in this area has lead to identification of enzymes needed to synthesize flavonoids, genes that encode for these enzymes, and genes that regulate the production of flavonoids. These developments puts researchers closer to being able to upregulate the flavonoid biosynthesis as well as to direct synthesis of desirable flavonoids in crop plants (5).
Isoflavonoids
Isoflavonoids are a class of flavonoids found mainly in legumes. They have been found to have oestrogenic and anticancer activity. This leads to the possibility of using isoflavones to treat or prevent hormone-related disorders.
The gene that encodes the main enzyme in isoflavonoid formation, cytochrome P450 mono-oxygenase isoflavone synthase (IFS) has been cloned. When this gene was expressed in plants, such as tobacco and maize, more isoflavonoids were found in the plant where the general flavonoid pathway was already active (5).
Experiments have also been done with non-legume plants. A transfer of the IFS gene found in soybeans to Arabidopsis (a flowering plant) resulted in conversion of narigenin to an isoflavone, genistein, a phytoestrogen of high medical interest (13). Success in studies such as this give researchers hope that they will be able to increase isoflavonoid production in crops such as tomato, maize, wheat or rice (5).
Here are some common questions about diet and cancer, including phytonutrients from the American Cancer Society.
Flavonols
Another class of flavonoids, flavonols, has been found to be good antioxidants and protective against cardiovascular disease. Tomatoes naturally contain this compound in the peel of the fruit in the form of quercetin. The peels also contain another flavonoid, chalcone narichalcone. Research data has suggested that flavonol biosynthesis in the peel is limited in part by low levels of gene expression of chalcone isomerase (CHI). Increasing synthesis of the CHI in the tomato was accomplished by using the petunia CHI gene to develop transgenic tomatoes. The result was a large increase in the amount of quercetin (70-fold) and flavonol levels within the tomato comparable to onions, which are naturally high in flavonols (5).
Here is some information about phytonutrients and cardiovascular disease from the American Heart Association.
Evaluation of transgenic plants has also included a study of levels of inherent toxic and antinutritive substances. This has been done since genetic modification has the potential to affect expression of genes not directly involved in the nutritional (or other type) of change being made (14).
Antinutrients have been defined as substances that inhibit or block important pathways in metabolism, especially digestion (14). These substances reduce utilization of nutrients by the body, such as proteins, vitamins and minerals. The result is a decrease in the body’s ability to use the nutrients, even though they are present in the food.
Secondary, or unintentional, effects on the plant can occur when genetic material is introduced. With the transfer of one gene, multiple changes in the plant can result. Some of these effects could include increased synthesis activity of already present biochemical metabolism pathways, augmented synthesis caused by increased activation of other genes, decreased production of catabolic enzymes, or reduced degradation of substances (14).
The possibility of these effects being present in genetically engineered foods has lead to review of several products (14). The table below outlines food crops that have been tested for increased levels of antinutrients following genetic modification. The net result was a lack of significantly increased antinutrient levels in these foods, however it is important to recognize that these substances could exist and we need to be aware of them.
Rapeseed oil
Naturally occurring antinutrients
1. A Golden Bowl of Rice. Nature Biotechnology. 1999;17(9):831.
2. Chakraborty S, Chakraborty N, Datta A. Increased nutritive value of transgenic potato by expressing a nonallergenic seed albumin gene from Amaranthus hypochondriacus. Proceedings of the National Academy of Sciences of the United States of America. 2000;97(7): 3724-3729.
3. Day PR. Genetic modification of proteins in food. Critical Reviews in Food Science and Nutrition. 1996;36(suppl):S49-67.
4. Farnham MW, Simon PW, Stommel JR. Improved phytonutrient content through plant genetic improvement. Nutrition Reviews. 1999:57(9);S19-26.
5. Forkmann G, Martens S. Metabolic engineering and applications of flavonoids. Current opinion in biotechnology. 2001:12(2);155-160.
6. Gill I, Valivety R. Polyunsaturated fatty acids, Part 1: Occurrence, biological activities and applications. Trends in Biotechnology. 1997: 15(10);401-409.
7. Hefford MA. Engineering nutritious proteins. Biotechnology and Genetic Engineering. 1997;14:191-210.
8. Khush GS. Challenges for meeting the global food and nutrient needs in the new millennium. The Proceedings of the Nutrition Society. 2001;60(1):15-26.
9. Kinney AJ. Designer oils for better nutrition. Nature Biotechnology. 1996;14(8):946.
10. Kochian LV, Garvin DF. Agricultural approaches to improving phytonutrient content in plants: an overview. Nutrition Reviews. 1999:57(9):S13-18.
11. Novak WK, Haslberger AG. Substantial equivalence of antinutrients and inherent plant toxins in genetically modified novel foods. Food and Chemical Toxicology. 2000:38(6);473-483.
12. Rosati C, Aquilani R, Dharmapuri S. Metabolic engineering of beta-carotene and lycopene content in tomato fruit. The Plant Journal: for cell and molecular biology. 2000;24(3):413-419.
13. Ruf S, Hermann M, Berger IJ. Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit. Nature Biotechnology. 2001;19(9): 870-875.
14. Theil EC, Burton JW, Beard JL. A sustainable solution for dietary iron deficiency through plant biotechnology and breeding to increase seed ferritin control. European Journal of Clinical Nutrition. 1997;51(supp 4):S28-31.
15. Van der Meer IM, Boxy AG, Bosch D. Plant-based raw material: Improved food quality for better nutrition via plant genomics. Current Opinion in Biotechnology. 2001;12(5):488-492).