Course:FNH200/2012w Team10 GoldenRice

Introduction

GMO’s are currently a hot topic of debate: in a starving world, do the health benefits outweigh the potential risk? A new type of food, golden rice, has undergone extensive testing and has going through clinical trial phases to test its safety and efficacy in humans. The main issues that arise when we discuss genetically modified organisms are thus: are they safe for human consumption, will they be effective, and why do we need them in the first place ? Golden rice has been developed in response to the growing pandemic of vitamin A deficiency, which affects over half of all countries. Myths that have been cast by both sides shroud GMO's, but could a GMO, such as golden rice, be the answer for a global issue like vitamin A deficiency, or are they truly as horrendous as the anti-GMO movement claims? To dispel these facades, we are going to delve into the need for GMO’s such as golden rice, how they were created in the first place, the biotechnology required to efficiently produce these products, how golden rice is processed to ensure safety during transit and upon delivery, and finally how golden rice is compared to normal white grain rice. We will be describing the process of how golden rice is created in lab and grown in the fields.

Vitamin A Deficiency

Currently, vitamin A deficiency affects one in three children under the age of five around the world. Deficiencies cause blindness and intensify conditions like diarrhea, respiratory diseases, mumps, and measles. In addition, it allows individuals to be more susceptible to infections and diseases due to the immune and anti-oxidant function of this vitamin. However, most importantly, vitamin A deficiency claims the lives of over 700,000 children each year.

Symptoms

Since many of the symptoms of vitamin A disease are systemic, and individuals who usually suffer vitamin A deficiencies also suffer other malnutrition related diseases, retinal malfunction and blindness are key markers used to identify this deficiency. Blindness first shows up only at night as well as an increased vitamin A content to activate night-time vision. A lack of vitamin A inhibits the reformation of rhodopsin, a compound that absorbs light in your eye for visual sensations to be observed. Severe deficiency can lead to permanent damage to eyes.

Relation of Vitamin A Deficiency to Rice

Rice is a staple part of the diet of individuals in many third world countries in Southeast Asia, Africa, and South America. These countries also experience a high amount of diseases such as night-time blindness that occurs due to vitamin A deficiency. As a result, scientists have developed new rice with biotechnology called “Golden Rice” to combat the issue of blindness and death. Golden rice is a genetically modified organism that has higher provitamin A content to prevent future deficiencies. Since vitamin A is usually in vegetables and meats, typically expensive products in third world countries, “Golden Rice” will make food with excellent sources of vitamin A affordable, accessible, and culturally acceptable for individuals living in third world countries.

General Structure of Rice

Rice is composed of 4 layers:

• Husk – This is the outside uneatable protective layer of rice. It is removed during the milling process.
• Bran – This is the thin layer of skin which gives brown rice its texture. It is also removed during milling.
• Germ – This is the reproductive component of rice. It also is removed during milling.
• Endosperm – This is the inside portion of the rice grain that contains starch granules. It is not removed during the milling process.

The milling process that rice traditionally undergoes leaves rice cells with the endosperm component only. Endosperm contains photosynthetic tissue that lack two enzymes required in the biosynthesis pathway of provitamin A. As a result scientists have used a variety of techniques to ensure these enzymes are infused into the endosperm, so that rice can be an excellent source of vitamin A as well.

Enzymes missing in rice endosperm:

1. Phytoene synthase – This is an enzyme that produces phytoene, a compound required in biosynthesis pathway of vitamin A.
2. Phytoene desaturase -This is an enzyme that adds four double bonds to precursor to vitamin A.

The lack of these enzymes prevents rice endosperm from producing vitamin A.

Creation of Golden Rice

Golden Rice was created using various methods of genetic engineering on rice, specifically inserting new genes into the genetic makeup of it. The main purpose for its creation was to increase the nutritional value of rice by synthesizing micronutrients that were not present before. This process requires multiple techniques including recombination, and transformation. Recombination adds the required genes that code for the enzymes into the bacteria. Transformation is the process where genes that code for the aforementioned enzymes are incorporated into the rice endosperm. Then, the rice endosperm can express these genes to produce proteins by undergoing transcription and translation. This process produce enzymes phytoene synthase and phytoene desaturase which aids is needed for the biosynthesis of vitamin A.

Biotechnology

"Biotechnology is an umbrella term that covers a broad spectrum of tools and techniques, ranging from fermentation (bread, wine, cheese) to plant and animal breeding, cell and tissue culture, antibiotic production and genetic engineering. The traits of every organism are encoded in its genetic material (DNA or RNA) which is organized into individual units called genes. Genetic modification is achieved by changing the code or organization of the genetic material of an organism. This includes, but is not limited to, moving a gene or genes from one organism to another (this is commonly called genetic engineering)"

Due to development of sophisticated molecular techniques, the knowledge of biosynthetic pathways has grown significantly. ‘All organisms share a good proportion of their genes and metabolic functions, making it possible to draw conclusions from one species to another and to do research on model organisms with short life cycles while taking advantage of an exhaustive genetic infrastructure already available’.

Since macronutrients in plants are unevenly distributed not only between plant species but also plant tissues, leaves often contain all macronutrients. For rice endosperm to meet human nutritional requirements, it is necessary to modify so that the endosperm accumulates enough vitamin A to meet the nutritional needs of those living in poverty who use rice primarily to nourish themselves.

Golden rice can not be created through classical breeding. Beta-carotene is only present in the non-edible part of the rice and is not found in the endosperm. This lead bioscientists to embark on the ‘bold idea of reintroducing the biochemical pathway leading to beta-carotene, the most important pro-vitamin A, into rice but putting it under control of a promoter that's specific to endosperm’.

This process requires multiple techniques including recombination, and transformation. Recombination adds the required genes that code for the enzymes into the bacteria. Transformation is the process where genes that code for the aforementioned enzymes are incorporated into the rice endosperm. Then, the rice endosperm can express these genes to produce proteins by undergoing transcription and translation. This process produce enzymes phytoene synthase and phytoene desaturase which aids is needed for the biosynthesis of vitamin A.

Make Recombinant DNA

The plasmids of bacteria Agrobacterium tumefaciens have genes encoded for enzymes required for vitamin A synthesis

1. Isolate DNA- Circular pieces of DNA called plasmids from A. tumefaciens are isolated. This is done by the use of detergents, or by using lysozyme to break down the cell walls. DNA is then precipitated (taken) out of the solution by the use of a centrifuge.
2. Restrict DNA - Pieces of this plasmid are cut using restriction enzymes (endonucleases) that cleave DNA at certain positions. Each strand of DNA will have ends where the new DNA added can bind to.
3. Add new DNA - The new DNA that code for Phytoene synthase, Phytoene desaturase are added into the solution and incubation occurs. Pieces of the new DNA will bind to the plasmid at the free ends specific (complementary) to it. Since this is a random process, not all plasmids will have this new DNA added to the plasmid.
4. Optional Step - This optional step most often is used by scientists but is not required. These new plasmids are inserted back into the A. tumefaciens.

The end result of this process of recombination results in bacteria A. tumefaciens to have the genes that are not present in the rice endosperm. If the optional step is not completed, scientists have free plasmids with the genes required for vitamin A synthesis.

Transformation

Plasmids of bacteria A. tumefaciens must integrate into the host DNA

Soil bacterium Agrobacterium tumefaciens is used to produce many genetically modified organisms as they infect plant cells and causes tumors. When this bacteria infects wounded plant cells, it disturbs the normal healing process by creating a cancerous growth instead of a protective layer to cover the wound. This tumor can grow in a tissue culture and prolifterate without hormones in the culture medium. To produce golden rice, these A. tumefaciens bacteria have plasmids that have the phytoene synthase, phytoene desaturase, and Lycopene -cyclase genes incoded in it to infect rice cells.

1. Wounds are created on the rice cells in the solution, making them protoplasts. Protoplasts are plant cells without their cell walls, allowing for easier penetration.
2. A. tumefacians attach themselves to the wound site. Portions of the plasmid are cut out by rice enzymes and enter the rice cell. These cut pieces of DNA enter the nucleus and integrate into the plant chromosome in a random manor. Currently, scientists cannot direct exactly where the plasmid portion will be integrated into the DNA.
3. The genes transferred include the tumor-causing genes. These carry information to make auxin and cytokine, which are hormones that signal cells to become cancerous. Cancerous cells simply multiply continuously without any regulation. In additions, compounds called opines are produced by infected plant cells. Opines are only produced by infected plant cells, so it can be used as a marker to ensure that these plants cells are infected.
4. These infected rice cells will rapidly divide and produce daughter cells that will have the genes for vitamin A synthesis.

If the scientist decided not to incorporate the plasmids back into the bacteria, they will use the biolistic transformation method. This involves the coating of DNA with micro-sized metal particles, and bombarding them with a particle gun into intact cells at high velocities. A fraction of these plant cells will incorporate portions of the DNA into its genome, which can then be isolated to have rice cells with the Phytoene synthase and phytoene desaturase genes.

Transcription and Translation

DNA transcription and translation take place so vitamin A can be synthesized. Now these rice cells are genetically modified organisms, as scientists have used biotechnology to modify the qualities of the rice. These rice cells now have all DNA required to produce vitamin A by itself. The endosperm will now produce the two originally missing enzymes, phytoene synthase and phytoene desaturase that will catalyze chemical reactions in order to produce provitamin A.

Processing

Harvesting and milling

Genetically modified Golden rice is grown, collected, and processed in the same process as white rice. The rice undergoes four core processes: harvesting, drying, milling, and storage. Harvesting occurs after rice plants have matured and ripened to produce four to five tillers each with a flowering head contains rice granules. Once harvesting is complete, the un-milled rice is given the name ‘paddy rice’. Paddy rice is delivered to a storage facility where they are cleaned and dried. Cleaning occurs to remove any debris such as grass, stones, and leaves are removed.

Drying

Paddy rice is then dried to maintain the quality of the rice by in-store drying method or the sun-drying method. The in-store drying method involves uses a storage compartment with controlled heat, air moisture, and wind conditions. Although it produces greater rice quality, it requires a facility and has longer drying times. The sun drying method simply uses heat produced from the sun. Although the sun drying method is inexpensive, it can reduce yields as farmers do not have control over the moisture content and prevalent pests. Delays in drying can cause vitamin A content to reduce due to heat respiration and oxidative damage. The paddy rice are also monitored to maintain optimal temperature and moisture levels so the rice does not go rancid and to avoid conditions for optimal pest growth. Inadequate monitoring can reduce the quality, quantity, and palatability of the Golden rice, which will in turn result in more individuals being vitamin A deficient.

Milling

The drying process is followed by the milling process which uses machines to remove the husk layer of the rice. This leaves behind brown rice with the bran, germ, and endosperm layers still intact. This variety of rice has higher content of vitamins and minerals as the bran and germ layers are nutrient rich. The brown rice can be packaged and sold to the public, or it can be further processed. It is typically processed further as the germ and bran layers contain many nutrients and oils that can under oxidation and make the rice rancid. Further milling can remove both the germ and bran layers to leave behind polished rice with only the inner-endosperm layer remaining. If this rice has undergone transformation with the encoded genes for vitamin A biosynthesis, it will be called Golden rice. Otherwise this further milled rice will be known as white rice.

Polishing

After milling, rice is passed through rice polishing machines to make the surface smooth and to give rice its typical shine. The colour of rice changes from its slightly brown-yellow tint to a standardized glowing white colour. This gives rice its aesthetic appeal and it allows for a better grade.

Sorting

Rice undergoes quality control and discoloured, damaged, immature, and off-shaped rice is removed in this stage by the use of machines.

Packing

Rice is packaged into various sized containers and either placed into storage or loaded onto transportation vehicles to deliver rice to food distributors.

Packaging and regulations

Golden Rice is not a commercially available product in Canada. Thus there are no regulation or packaging details available currently. However, for Golden Rice to be exported to a country, a risk assessment is to be done by the receiving country according to the Cartagena Protocol on Biosafety.

“Cartagena Protocol is an international agreement which aims to ensure safe handling, transport and use of living modified organisms resulting from modern biotechnology that may have adverse effects on biological diversity, taking also into account risks to human health.” The requirements are the following:

1. Name and identity of the GMO (event number, OECD unique identifier), including domestic classification, if any, of the Biosafety level of the GMO in the exporting country.
2. Risk Assessment Classification
3. Information on the recipient or parental organism
4. Information on the donor organisms
5. Description of the nucleic acid or the modification introduced, the vector and technique used and the resulting characteristics of the GMO
6. Information on the location, geographic, climatic, and environmental characteristics of the receiving environment, including any centres of origin or biological diversity
7. Intended use of the GMO or derived products
8. Quantity or volume of the GMO to be transferred
9. A risk assessment report
10. Suggested methods for the safe handling and storage, transport, use, including packaging, labelling, documentation, disposal and contingency procedures where appropriate, as well as the detection and identification and their specificity, sensitivity and reliability
11. The regulatory status of the GMO within the country of export, including any restrictions, and if the GMO has been banned in the country of export the reasons for the ban
12. Results and purpose of any notification by the exporter to other countries regarding the GMO to be transferred

Comparison Between White Rice and Golden Rice

Golden and ordinary rice have major differences. To begin with, Golden rice is Gold in colour. That’s because Golden rice is rich in Beta-carotene, that our body turns into Vitamin A.

Besides containing high levels of Vitamin A, GM rice also contains very high levels of Iron and Zinc. 'Normal rice has 10 parts per million. The new GM rice has been modified such that it has an additional 110% iron at 21 parts per million. This benefit would impact mainly third-world countries’.

Also, surprisingly in terms of pesticides, GM rice uses and emits less to the environment. ‘The quantity and cost of pesticides applied to conventional rice is 8 to 10 times as high as that applied to the insect-resistant genetically modified rice. In short, use of the genetically modified rice enables the farmers to reduce pesticide use by 15 pounds per acre, an 80% reduction compared with pesticide use by farmers producing normal rice’.

Similar to regular white rice, Golden rice is grown, collected, and packaged the same way. As previously stated, both types of rice undergo the same four core processes: harvesting, drying, milling, and storage.

It is worthwhile to know that Golden Rice is still in its experimental phase due to its possible hazards. Where white rice is readily available in our markets, Golden rice has yet to pass the regulatory authorities. The Golden rice project has faced a lot of scepticism from regulatory boards and pressure groups, such as The Green Peace.

Public Opinion

Video

http://www.youtube.com/watch?v=PsnZkT-GbQ0

Conclusion

Golden rice is a GMO, that has been developed in response to the rising vitamin A deficiency epidemic. It is grown in the same way as rice, has been in testing for over 10 years, and was created by splicing different genes into the rice through a myriad of processes to regulate the natural vitamin A production, giving it a golden color. It undergoes the same processes as rice before being packaged and is still not being distributed throughout the world. Albeit being in testing for so long, and having no issues arising from golden rice use, it is still stuck in the processes of being accepted. This is a huge issue that many GMO's are currently facing, and hopefully will be overcome in the near future. Golden rice has the ability to prevent a significant level of deaths, but it has been held back due to possible health concerns. Importation has been so heavily regulated that actually getting one approved is a daunting task, preventing people from receiving a possibly life saving food.

Possible Exam Question

Which of the following biotechnological techniques are used in the formation of golden rice?

• a) transfection and transformation
• b) transformation and recombination
• c) transfection and recombination
• d) recombination only
• e) transformation only
• f) transfection only

Correct answer: b)

• List the four steps of rice processing. (Harvesting, drying, milling, packaging)