Plants and other GMOs: What's out there?

Now that we've covered what genetic engineering is (and isn't) and how it's done, we get down to the real meat of the issue: the organisms themselves.

Photo credit: Ks.mini, Wikimedia Commons

This is, by necessity, a very United States-centric post.  Being located in the U.S. myself, most of my
information is centered around GMOs here; in addition, it's still U.S. companies which are pushing most of the advances in the kinds of crops consumers in the Western world see on store shelves.  That's changing gradually, as more academic and governmental institutions in places like sub-Saharan Africa and southeast Asia take charge of modifying crops important to those areas.  The center of GMO advancement still lies in the United States, though, so that has most of my focus.

According to popular belief, all the plants we eat are GMOs unless they're labeled "organic".  In actual fact, however, only about two dozen plant species have been listed as having been genetically modified, and a handful of those have gone on to gain the approval and acreage necessary to enter our food supply.  A couple of those plants provide products which are used widely in processed food, but if you pick up a random item in your supermarket's produce section, the odds are very high that it's not a GMO.

So what plants have gotten modified so far?  Here's a list from the ISAAA, an industry organization with an interest in reporting the full extent of GMO advancements:¹

• Alfalfa (Medicago sativa)
• Apple (Malus x Domestica)
• Argentine Canola (Brassica napus)
• Bean (Phaseolus vulgaris)
• Carnation (Dianthus caryophyllus)
• Chicory (Cichorium intybus)
• Cotton (Gossypium hirsutum L.)
• Creeping Bentgrass (Agrostis stolonifera)
• Eggplant (Solanum melongena)
• Eucalyptus (Eucalyptus sp.)
• Flax (Linum usitatissumum L.)
• Maize (Zea mays L.)
• Melon (Cucumis melo)
• Papaya (Carica papaya)
• Petunia (Petunia hybrida)
• Plum (Prunus domestica)
• Polish canola (Brassica rapa)
• Poplar (Populus sp.)
• Potato (Solanum tuberosum L.)
• Rice (Oryza sativa L.)
• Rose (Rosa hybrida)
• Soybean (Glycine max L.)
• Squash (Cucurbita pepo)
• Sugar Beet (Beta vulgaris)
• Sugarcane (Saccharum sp)
• Sweet pepper (Capsicum annuum)
• Tobacco (Nicotiana tabacum L.)
• Tomato (Lycopersicon esculentum)
• Wheat (Triticum aestivum)

I've put in italics the crops which don't produce food for humans.  Of the remaining 19, only the 10 in bold are currently being grown for commercial use (some others, like beans, have approval but no one grows them at this point).²  The three crops which I've underlined are maize (corn), cotton, and soybeans -- these three crops make up the vast majority of GMO acres in the United States.  Correspondingly, almost all of the yield of those three is GMO: 90% of cotton, 90% of corn, and 93% of soybeans grown in the U.S. were GMO in 2013, and the trend has been upward.³

Corn and soy are used in a lot of foods; think about how many times you see items like high fructose corn syrup and soybean oil on food labels. The current rules for the North American and European organic certification agencies exclude GMOs, but unless the ingredient is listed as organic, there's a very high probability that the ingredients derived from corn or soy are going to come from GMOs.⁴

Future crops could include bananas⁵ and oranges⁶, and cassava⁷ in Africa (where it is a significant source of food).  Wheat has been in development for some time by various sources. Several kinds of rice have been developed and even approved, but aren't currently on the market.³  And in the research world, modifications to both tobacco and garden cress (Arabidopsis thaliana) have become routine.

In addition to plants, several other organisms have been the focus of genetic modification.  A variety of salmon has recently been approved in the U.S., but it remains the only animal to have that distinction.⁸  There are a great many single-celled GMOs, however, most of which get far less press than plants and salmon.  Escherichia coli is probably the leading contender here, as a bacterium which is used to produce an enormous number of substances; the variety of E. coli being used is not the one you hear about in connection with food-poisoning reports, being harmless to humans.⁹  It shares its cousin's talent for multiplying rapidly, and it's easily modified, making it ideal for genetic work.

Substances produced with GM E. coli include:

• rBST, the hormone given to dairy cows to improve milk production.¹⁰
• Interferon, used to treat multiple sclerosis and certain types of cancer.¹¹
• Some vaccines which use isolated genetic material rather than whole cells.¹²
• Human growth hormone (hGH), used to treat dwarfism and a few other disorders.¹³

Another rapidly-multiplying organism is yeast, the same kind found in bread and beer.  It's widely used to make synthetic insulin.¹⁴

Some substances require fancy transcription and folding operations which are normally performed by human cells, and the best way to mimic that is to use something similar.  The most common stand-ins are Chinese hamster ovary (CHO) cells, which can be grown in cultures and modified to produce things which E. coli hasn't been coaxed into making.  Some things currently made with this method are follicle stimulating hormone (FSH), an important part of ovarian function and human fertility;¹⁵ human blood clotting factors, used to treat hemophiliacs and those with similar problems;¹⁶ and tissue plasminogen activator (tPA), which thins the blood in stroke victims.¹⁷  In recent years, tPA has been successfully produced in E. coli,¹⁸ which may be more common in the future -- and it's also been produced in cucumber plants, in a demonstration of the possible future of pharmacology.¹⁹

Antibodies are commonly made using various types of cells which have been modified, including bacterial, mammalian, and fungal.²⁰  Some animal vaccines are created from pathogens which have been de-fanged by removing a couple of their dangerous genes, making them harmless; these include rabies and Salmonella vaccines.²¹

The field of GMOs is both narrower and more diverse than what the popular press might lead you to believe; many important medicinal products are made using GMOs, just not the plants growing in the field that we all think about.  There has been some investigation into combining those two uses, however, creating fast-growing plants which can make drugs or hormones by the acre, ready to be purified and distributed cheaply.  That kind of application is still only found in laboratories, but it may only be a matter of time before it reaches the real world.

Now that you know which organisms are out there, it's time to dive into how they're different: what genes have been added to them, and why.  That's the next segment.  Stay tuned.

[This article is part of the series on GMOs. Jump to the first post for an overview and index.]

GMO methods

Here's where we get into the real nitty-gritty science of genetically modified organisms (GMOs): the technology used to make them.  I defined (mostly) what a GMO is in the last post; now we see just what the methods are which set them apart.

A. tumifaciens Ti plasmid, courtesy of BacMap

Gene splicing of this sort really took off some thirty years ago, when the first trials of herbicide-resistant tobacco were done in the U.S. and France.¹  Many lab experiments had been done over the previous decade, but when the tobacco field trials were done, along with the test of a spliced bacterial strain that successfully helped protect a field from frost damage,² the era of commercially viable GMOs had arrived.

Since then, the specific technologies have changed a bit, but the process remains the same. I'll lay it out, step by step.

Step 1: Identify the gene you want.

This could be a gene from a mutant of the same plant variety, a different variety, a different but related plant, a wildly different plant, or something from another kingdom entirely, such as bacteria or animals.  The more closely related it is, the easier it is to get the target plant to express that gene (read it and make a functional protein).  But there are a few tricks to get even foreign genes to work.

Gene sequencing has made selection a lot easier.  We can compare the genetics of the plant we have with those of a plant that does what we want, and pinpoint genes which might be responsible for the difference.  We have maps for what all the genes in some simple organisms do, such as some bacteria, and we can use those to find genes that look similar in other things.  And there are methods to "knock out" the genes we're interested in, to see whether the feature goes away.

So once the researchers have picked out what looks like a good candidate, the next thing is...

Step 2: Isolate the gene.

There are a bunch of little steps here, all with the same goal: get a specific tidbit of DNA into the open.  First all the DNA is pulled out of the cells in a sample, and washed clean of the remnants of cell wall, cellular machinery, and so on.  Then an enzyme or two is added to the DNA, to break it into bite-size pieces; the enzymes used have particular sequences they like to "snip" at, and one is picked which will cut as close as possible to the target gene, but leave it intact.

Since there still isn't a lot of material to work with, the bits are run through a process called polymerase chain reaction (PCR), which multiplies the target gene over and over.  Ideally, with good primers (the "hooks" used to fasten on to the piece so it can be multiplied) only that gene is amplified, and it comes out with thousands or tens of thousands of copies.  Then it's separated out from anything else in the solution, and you have your little vial of the copied gene.

Step 3: Add some extras.

Most of the time, a new gene won't get used in the plant it's put into.  Or, if it is, it won't make as much as we want of the protein it codes for.  How often a gene is used is controlled, largely, by promoters: little sequences at the beginning of the gene which tells the cell when to use it.  A plant virus known as Cauliflower mosaic virus (CaMV) came up with a couple of promoters of its own, which it added to the genes it inserted into its host during infection.  The main CaMV promoter, known as 35S, is "always on"; if the promoter is there, the gene it's attached to gets expressed all the time, and cranks out lots of orders (RNA transcripts) for its protein.³  There are other promoters used in making GMOs, including another from CaMV, but the 35S promoter is still very widely used.⁴

Once you've got your new plant, how can you tell whether your gene actually got into it?  Many insertion methods are pretty chancy.  Therefore, having some easy way to tell your gene is in there is helpful.  As a result, there are a bunch of available marker genes,⁵ which give the new plant some new, harmless ability which it's easy to test for.  They include selectable markers, such as antibiotic resistance (kanamycin is common), and reporter genes, which make some substance which can be seen or tested for chemically.  The gene for green fluorescent protein (GFP) makes a protein that glows under blacklight, and GUS creates a stain when an enzyme is used on a test cell.

These markers are typically tacked onto the desired gene before packing the whole thing up, and they are present in the mature modified plant.  That said, they're pretty useless in the field, and unlikely to have any effect on the plant's survival or the animals who eat it.  There's been more of a question about the effect of the promoters, but I'll go into that in a later segment.

Finally, we're ready to...

Step 4: Get the gene into the plant.

There are a bunch of ways to do this.⁶  The earliest method for introducing genes into plants used a bacteria called Agrobacterium tumefaciens, better known as the cause of crown gall (if you grow roses, you might have heard of it).  A.t. has a loop of genetic material which can cut into the DNA of just about any non-grassy plant, and insert its cargo of a handful of genes.  Scientists took that carrier loop, known as a plasmid, and deleted the native A.t. genes; then they could splice in anything they wanted.  All they had to do then was to breed up a bunch of these bacteria, with their new genetic cargo, and let them infect some cells from the target plant.  It worked pretty well, as methods go, and it was the means used to create the first generation of genetically modified plants.

Beyond Agrobacterium, there are some viruses that can also carry genes when they infect a plant, and viruses or other bacteria which can modify bacterial GMOs.

When a plant cell is stripped of its cell wall, so that all it has is a cell membrane similar to those of our own cells, a small electric shock can open up holes in the membrane long enough to slip some DNA in.  If it's done right, that DNA is taken into the cell and treated as if it belonged.  Specific chemicals can have the same effect of temporarily punching little holes in the cell membrane to allow new genes in.  These methods, electroporation and chemical poration, are much more successful than Agrobacterium at modifying grassy plants.  Vacuum infiltration, using vacuum to open up cell walls enough to infuse genetic material, has a similar end result.

Microinjection uses a small needle to inject DNA into plant cells.

Finally, the most recent and most widely applied category of modification is bioballistics.  The DNA cargo is loaded onto tiny "bullets" made of tungsten or gold, which are then fired at the plant like a shotgun.  Some of those bullets penetrate cells and drop their cargo, which is then taken up.  It sounds very haphazard and inefficient, but it works surprisingly well, and has been used to create many of the newer GMOs.  It helps that this "gene gun" works on grassy plants, such as rice and corn, which can be hard to modify by other means.

Step 5: Make a new plant.

Now that you've got the gene into the plant... but wait, it's only in a handful of cells at this point.  The next thing is to make whole plants, where every cell is modified.

In some cases, like bioballistics, the embryo of a seed can be the part modified, so it's pretty simple to grow it up into a whole plant.  With Agrobacterium modification, chemical poration, or electroporation, it takes some work, because the cells that have been modified are random tissue cells.  Fortunately, plants have the ability to grow whole new individuals from any single cell, if the conditions are right.  The procedure to do that is called micropropagation, or tissue culture, and it's used for other purposes such as creating disease-free plants from virus-infested parents.⁷  With time and care, the individual modified cells are coaxed into making roots and shoots, and finally they're indistinguishable from those grown from seed.  It's more difficult with grassy plants, though many of the problems have been solved over the years.

At this point, the plants can be test-grown in the greenhouse to make sure they grow properly and have all the desired traits.  In many cases with crop plants, the last step is...

Step 6: Breed a new variety.

Plants which are easy to modify aren't always the ones that offer the best performance as a crop.  So, once it's clear that the target gene (the transgene) has successfully made it into new plants, those modified plants are bred with a high-yielding commercial variety, using traditional methods.  The offspring are examined to make sure they still have the transgene, and several generations down the road, the new variety is stable and ready for general use.


So there you have it.  The process of creating modified plants has brought up a bunch of questions (separate from those raised by the genes themselves), including some about the safety of the promoters, how genetically stable the new plants are, and whether randomly-inserted genes might disrupt the plant in such a way that it would become harmful to eat.  I'll address those issues all together in a later segment, under "Concerns".

Next up: which plants, and which genes, have been thrown into the GMO pool so far.

[This article is part of the series on GMOs. Jump to the first post for an overview and index.]

GMOs defined

When talking about genetically modified organisms (GMOs), it helps to start by defining them.  To that end, let's see what definitions the various authorities use.

Steffen Dietzel, courtesy of Wikimedia Commons

The Random House dictionary definition:
[A]n organism or microorganism whose genetic material has been altered by means of genetic engineering.

Merriam Webster doesn't have a definition of GMO, but defines genetic engineering:
[T]he group of applied techniques of genetics and biotechnology used to cut up and join together genetic material and especially DNA from one or more species of organism and to introduce the result into an organism in order to change one or more of its characteristics.  (Take a deep breath after that one.)

The World Health Organization's contribution:
Genetically modified organisms (GMOs) can be defined as organisms in which the genetic material (DNA) has been altered in a way that does not occur naturally. The technology is often called "modern biotechnology" or "gene technology", sometimes also "recombinant DNA technology" or "genetic engineering". It allows selected individual genes to be transferred from one organism into another, also between non-related species.

The European Union's legal definition:
An organism is "genetically modified", if its genetic material has been changed in a way that does not occur under natural conditions through cross-breeding or natural recombination.¹

Finally, the US Food and Drug Administration's regulatory definition:
In the case of foods, genetically engineered plant foods are produced from crops whose genetic makeup has been altered through a process called recombinant DNA, or gene splicing, to give the plant desired traits. Genetically engineered foods are also known as biotech, bioengineered, and genetically modified, although "genetically modified" can also refer to foods from plants altered through methods such as conventional breeding.

So there you go -- clear as mud.  However, the general consensus is that genetically modified organisms are plants (or animals) altered by individual-gene splicing methods which have been developed in the last 30 years.  The term "GMO" generally refers to organisms deliberately modified by viruses, bacteria, "gene gun", injection, electrical shock, or chemical poration, to carry a specific set of new genes that they didn't have before.  It doesn't usually include mutation (by radiation or chemicals), so-called "wide crosses" where distant cousins are bred together, various hybrids, or cloning plants from a single cell or growing point.

Even before the advent of real gene splicing, we were messing with our food to a serious degree.  Many people point to corn, which was bred up from a grassy weed, as an example of how we've always manipulated food plants.²  What I think of, however, are the more recent developments of ruby red grapefruit and seedless watermelons.  "Star Ruby", the first of the seedless red grapefruits, was the result of treating grapefruit seeds with radiation.³  Seedless watermelon plants are hybrids, made by crossing a regular watermelon with one which has been treated with a chemical to double its chromosomes; the child of that cross has three of each chromosome, which short-circuits its seed production.⁴  It would have taken hundreds or thousands of years to breed these plants by traditional methods, though it is theoretically possible.  Both mutations and doubled chromosomes happen in nature.

GMOs, by contrast, couldn't be made using old-world methods at all.  One of the definitions of species is whether two individuals can successfully breed; a plant (corn) and a bacterium (Bacillus thuringiensis) are too far apart to cross.  We have enough trouble breeding together cousins like wheat and rye, and only succeeded with the aid of the chemical colchicine.⁵  So a crop like Bt corn, which has genes from Bacillus thuringiensis inserted into it to repel pests, could only come out of the modern methods I mentioned above.

Now that we have some idea of the result, the next step is to look at those methods.  That will be the next post.

[This article is part of the series on GMOs.  Jump to the first post for an overview and index.]

Investigation: Genetically Modified Organisms

The purpose of this blog is to take the products of science, especially those which we encounter in everyday life, and explain the actual facts of them in plain English.  To that end, I can't think of a more appropriate, or more timely, topic to discuss than genetically modified organisms (GMOs).

Public domain image, courtesy of USDA ARS.

When I started this blog, I had an eye on doing a series on GMOs.  Recently, I've joined an effort by a
national non-profit organization to help its members understand agricultural issues, and GMO crop plants are part of that.  So it's time to explain them, in all their complexity: what they actually are, how they're created, what plants we're looking at, testing, regulation, health and environmental concerns, the whole shebang.

There's so much emotional investment in this topic -- nothing hits closer to home than food, water, or shelter -- that it's particularly important to drill down to real facts, on all sides, and answer as many of the nagging questions as possible.  These articles will be, to the best of my ability, impartial.  All that this series assumes is that major governments are not out to poison their people, the scientific process and community are (on the whole) to be trusted, and that there really are hard, solid facts to build conclusions from.  If you're willing to grant those points, let's go ahead with a rational investigation.

The Series
As each piece is completed, I will post a link to it here; bookmark this post to serve as an index.  More will be added as I complete the initial research and figure out how to group upcoming topics.

Definitions
Methods (and a peek into genetics)
Plants and other organisms
Genes
Testing and regulation
Market share, availability, and restrictions
Concerns
Potential benefits and drawbacks
Afterword

I have a full segment planned on specific questions and concerns, especially about health and environmental risks, where I will address them in Q&A format.  If you have a specific concern that you'd like me to address, please tell me, and I'll add it to the list if it isn't already there.

Thanks for reading, and stay tuned.