Two food commodities, apples and potatoes, engineered to possess non-browning characteristic on cutting have been declared safe in the United States and Canada. These apples and potatoes do not turn brown when exposed to air. They are identical to their non-genetically engineered counterparts, except for the fact that the gene governing the browning process has been silenced using the technique of RNA interference. These apples are collectively called Arctic Apples and the potatoes will be marketed with the trade name Innate potatoes.
In 2006, two scientists, Dr. Andrew Fire and Dr. Craig C. Mellow were awarded the Nobel Prize for their discovery of the process of RNA interference or RNAi. They discovered some double stranded RNAs in the worm C. elegans that were acting in some way to turn off particular genes, and their further research led them to discover this process which they named RNAi. RNAi is a natural process in cells by which the activity of specific genes can be suppressed. This process regulates the expression of genes by switching them off, without bringing about any particular permanent genetic change. It has now been investigated and confirmed that the process of RNAi has long been regulating various biological processes such as metabolism, growth and pathogen defense in plants. RNAi is also known as gene silencing.
RNAi works by interfering with RNA molecules which carry information for protein synthesis within the cell.
Process of RNAi
In general, RNAi is triggered by double stranded RNA, which may be produced naturally in a cell or may enter the cell exogenously. An enzyme, called Dicer, cuts the long double stranded RNA into small pieces of approximately 21 nucleotides length. These small pieces could be miRNA (micro RNA; originating from endogenous long dsRNA) or siRNA (small interfering RNA; originating from exogenous sources). These RNAs then bind to the RNA-induced silencing complex (RISC). After binding, one strand of the double stranded RNA is removed, leaving the remaining strand available to bind to messenger RNA target sequences. This strand is complementary to the sequence of the target mRNA. RNA Induced Silencing Complex (RISC) cleaves mRNA or represses their translation by homology dependent mRNA degradation, which effectively silences the gene.
The use of RNAi has been extensively reported for modifying plants to enhance their nutritive value, pathogen and pest resistance, decreasing amount of unwanted metabolite production, etc.
Recently two RNAi based crops have been given regulatory approval for commercial production and sale. These are the non-browning Arctic apples and the non-browning Innate potatoes. The firms producing these crops claim that the idea behind producing the non-browning apples and potatoes is not only to improve the look of the product, but it is also intended to increase the consumption of the raw fruits along with reducing naturally occurring carcinogens (as in the case of innate potatoes). While the science behind both these products is a little complicated as both are RNAi based, in simple way it can be put as both apples and potatoes have certain genes suppressed. Both of them, though genetically modified, are grown the same way as conventional varieties. These products are likely to find a place of attraction in the fresh-cut product sales.
THE NON-BROWNING APPLES – ARCTIC APPLES
It is a common observation that once an apple is cut or bitten, it turns brown. The reason behind this browning is that when the cells of an apple fruit are damaged through a cut, bruise or bite, some enzymes, known as polyphenoloxidases (PPOs) come in contact with the polyphenolics found elsewhere in the cell. PPO catalyzes the oxidation of polyphenolics to melanins, causing browning.
In healthy apple cells, PPO is present in the plastids, while the phenolics substrates are present in the vacuoles. On cell damage, the cells loose their compartmentalization, and PPO comes in direct contact with its substrate. PPO is involved in the reaction of synthesis of quinones from diphenols. The quinones then undergo non-enzymatic condensation with lesser amounts of amino acids and proteins, to give brown pigmented polymer of lignin-like compounds.
Therefore, PPO is the key enzyme responsible for apple browning. To stop or minimize fruit browning, reducing the activities of PPO has long been considered as an effective approach. The technology of silencing the PPO genes was developed in Australia and licensed by a Canadian firm Okanagan Specialty Fruits Inc. (OSF).
Arctic apples do not show signs of browning
Scientists at Okanagan Speciality Fruits developed these non-browning apples using the technique of RNA interference to block the activity of PPO. They used gene silencing to develop a new type of apple in which a gene from the original apple was deleted. They named these apples as Arctic Apples. Two varieties of apple, namely, Granny Smith and Golden Delicious, have been modified so far using this technique.
Strategy to develop non browning apples
Genetic engineering using Agrobacterium based transformation was used to achieve this goal. First and foremost, the genes encoding PPO in apple were identified. Scientists were able to identify ten such genes of their interest. They categorized these genes into four groups. One gene from each group was used for further developments.
DNA fragments were then produced from each of the four genes and combined into a single gene. They named it as PGAS, taking the first letter of each of the gene selected (PPO2, GPO3, APO5 and pSR7). This transgene was incorporated into a vector for further apple transformation. The hybrid gene was placed under the control of the cauliflower mosaic virus 35s promoter (PCAMV35s) and nopaline synthase terminator (TNOS). Apple cells were transformed with bacterium Agrobacterium tumefaciens carrying vector GEN-03, and then transgenic plants with the expected transgene PGAS selected. The transformed plants were analyzed for reduced or non-browning characteristics in the fruit. On analysis the PPO activity was found to be significantly reduced in these apples.
NON-BROWNING POTATOES – INNATE
The potato that does not brown on cutting was developed by J. R. Simplot Company. They used RNAi to silence 4 different proteins. Scientists at this company engineered a popular potato variety Russet Burbank to contain late blight resistance, low acrylamide potential, reduced black spot, and lower reducing sugars. These potatoes have resistance to the serious disease late blight, caused by fungus Phytophthora infestans. In addition, three potato quality issues were addressed: (1) the presence of high amounts of reducing sugars which cause brown spots on accumulation (2) enzymatic browning (3) presence of high level of non-essential free amino acid asparagine which gets oxidized to form carcinogenic compound acrylamide upon baking or frying. Enzymatic browning occurs due to PPO activity as is the case in apples. In potatoes, browning also occurs non-enzymatically because of the partial degradation of starch into sucrose and fructose. Also, these reducing sugars react with amino acids on heating to produce a variety of desired compounds contributing to flavor, but also produce acrylamide which is harmful on consumption.
Scientists at Simplot silenced the invertase gene in potato tubers to reduce the conversion of sucrose to reducing sugars, allowing for storing potatoes at colder temperatures. The company believes that the overall yield improvement lowered fungicide use because of late blight resistance would be beneficial due to lesser harmful impact on the environment due to reduced fungicide use. They also found that low asparagine and low reducing sugars resulted in greater than 70% reduction in acrylamide even after extended cold storage, addressing this potential health risk for consumers and the food industry.
Strategy used
The innate potatoes were developed using Agrobacterium mediated transformation technique. First the plants were transformed to reduce the expression of the gene asparagine synthetase (to reduce amount of asparagine) and PPO (to reduce polyphenol oxidase). These transformed plants were then again transformed with genes to induce late blight resistance and silence invertase. The transformed potatoes were compared for their composition to untransformed ones and were found to show food safety equivalence with the untransformed potatoes.
REGULATORY APPROVAL
Both, Arctic apples and Innate potatoes, have been granted approval for commercial sale in the United States. The Innate potatoes were approved by the United States Department of Agriculture (USDA), Food and Drug Administration (FDA) and Animal and Plant Health Inspection Service (APHIS). Three potato varieties, Ranger Russet, Russet Burbank and Atlantic Potatoes, are known by the trade name Innate. The Arctic apples were approved for sale in the United States by USDA and FDA and in Canada by Canadian Food Inspection Agency (CFIA) and Health Canada.
Now that RNAi based foods are coming to the market, it can be concluded that this kind of genetic modification is new in its kind, because these are not a traits aimed at farmers, but directly at consumers. The earlier commercialized biotech crops were aimed to improve pest resistance or herbicide resistance in crops standing in the field, but these traits will be directly visualized by the commodity consumers.