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Unit 13: Genetically Modified Organisms
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David L. Dornbos, Jr., PhD

Dornbos is the Global Head of Production Research at Syngenta Seeds.

What is Bt?

Bt stands for Bacillus thuringiensis, and it is a fairly ubiquitous soil-borne bacteria.

When was Bt first isolated?

People first learned about Bacillus thuringiensis around 1901. It was found to be the culprit of something that was killing silkworms, and so it was a problem to those who were trying to grow silk.

What was done with that knowledge?

They understand that they had a problem. I don't know if they ever resolved the problem and reduced it. But eventually people learned that the same organism, Bt, controlled other insect species in the same family of insects, the Lepidoptera.

And in the 1920's through the '30s the first applications were developed of this insecticidal mix in Europe, primarily in France and probably some other countries as well. They applied these insecticides primarily to vegetable crops and they found that they could control some of the insects that were causing them problems, reducing aerial spraying.

What makes Bt so effective?

Well, I don't know why Bacillus thuringiensis has this special quality to it. In fact, what it has is a gene that codes for a protein, and this protein, at the appropriate pH, which is associated with the guts of certain insects-those in the lepidopteran family-will crystallize. There are many different types of Bt's and as they crystallize, they attach to specific receptors in insect stomachs or guts. And when they attach to those receptors, they essentially paralyze the insect's gut; they block it, and prevent it from feeding further. As a result those insects are killed.

The Bt protein is very specific. It controls only certain species within the class. It won't even protect or control all of the Lepidoptera insects, and it does so with varying degrees of efficacy.

What is the pyramid-shaped structure in the Bt micrograph?

That's actually a picture of the Bt crystal.

Why is it shaped like that?

The crystalline structure is somehow shaped to fit in a receptor found in the insect itself. And when it does that, it occludes the gut, so the insect basically has to eat a small amount of the material that contains Bt, whether it's the Bt in the form of an insecticidal spray that an organic farmer would use or it's the Bt inside of a genetically engineered corn plant. The insect has to consume a certain amount of this Bt protein to basically block all of the receptors or at least a large proportion of the receptors in its gut. When it does that, the gut becomes occluded, the insect has to stop feeding, and within 24 to 48 hours the insect will then die if it's sensitive.

Does the insect have to eat a whole lot to be affected?

No, it doesn't have to eat a whole lot. If you look at the surface of a corn leaf that, a corn borer larva, for example, has eaten, it leaves just a very small divot. If you were out in the field, you probably would never find it. You could find it in the lab when you feed them small pieces of leaf so it just leaves just a small little divot, about the size of a pinprick.

Does the European corn borer feeding hurt the corn plant?

No, the feeding damage by the European corn borer on corn really causes no economic damage at all. The real issues arise when the corn borer larva grows larger and tunnels into the midribs of the leaves or into the stalk. That's where the economic damage occurs.

Is there a Bt protein variant that works against other insects?

There is a type of Bt protein that is still in development that controls the corn root worm. And the corn root worm is in a completely different family than the European corn borer. The European corn borer is in the Family Lepidopteron. The corn root worm is in the coleopteran family.

What is a Lepidopteron insect?

A lepidopteron insect is a family of insects that is commonly known as the butterfly family. And so the European corn borer is one example of the lepidopteron insect; the Monarch butterfly is another; the black swallow-tail would be a third.

So, each strain of Bt works against a different family of insects?

There are thousands of different types of Bt proteins, and as they form crystals in the guts of insects, each different protein forms a slightly different type of crystalline structure, and those different Bt's then are selective for different types of insects.

So, for example, you have in one case the Bt that is currently used in corn to control the European corn borer, but a different Bt to control the corn root worm, which is a Coleopteran insect. It's a different family of insect altogether.

Is that why Bt is so effective?

That is definitely one of the strong benefits for Bt. It's very important in any kind of insect control management program to have the target be as specific as possible.

Is Bt what organic farmers use?

One of the tools that organic farmers do have is the use of Bt, and they'll use it as either a liquid formulation or sometimes simply as a dried powder. They'll primarily use it with vegetables in the cabbage family. And it does a very nice job of controlling the cabbage looper, which those who have had gardens in the past know that these are the small green worms that can eat holes through the leaves and become very voracious and prevent the flower for the vegetable from even making any kind of a fruit that we can consume.

So it's very effective. It's been available for use since 1961. It is actually considered to be the safest insecticide on the marketplace. It's the only one with a reentry time of zero, which means you can apply this insecticide and then eat the vegetable almost at the same moment.

What are the drawbacks of Bt?

Bt is primarily used by organic farmers for farming vegetables, which is a fairly high value cropping system. In the case of large-volume corn production it would very expensive to try to spread this material over a wide acreage.

The other issue with it is that it is prone to breaking down fairly rapidly under sun exposure-this protein is literally put on the surface of the plant. So the shelf life of effectiveness on the plant than is relatively short.

So do you have to keep applying it?

Yes, and in the case of vegetable crops, that's okay. It's a high value enough of a crop that they can do that. But in the case of corn, which is not necessarily a high value crop, it's a commodity crop, reapplication would be prohibitively expensive. In fact, it's often the cost of applying Bt-to rent an airplane or to drive a highboy over top of the corn when it's big-that gets to be very, very expensive.

When did the idea of genetically engineering crops to contain Bt first arise?

I think the idea probably started to develop in the late 1970's and the early '80s as the biotechnology tools themselves started to develop. These biotechnology tools are fairly sophisticated, and they developed over a long period of time-10, 15, 20 years.

So I don't know exactly when the idea popped into somebody's head, but somebody came up with the idea that we have a compound that we know is very safe and it has some serious limitations for use in production agriculture situation, and they thought, hey, maybe this is a slam-dunk, ready-made case of applying a biotechnology combination of tools to get this thing to work in a corn plant-again, at a very specific targeted pest.

What is recombinant DNA?

Recombinant DNA stands for simply the recombining of DNA or deoxyribonucleic acid, the so-called building blocks of life.

And recombining DNA or reorganizing it has been done for a long, long time. A conventional corn breeder or soybean breeder will make crosses between two different lines, and in doing that they will recombine hundreds of thousands of genes at a time, and it's very much a hit-or-miss adventure. They'll take what they want to be one parent that has a certain desired trait and they'll take another parent that has many desired traits. They'll combine them and then they'll select the progeny from that cross, and they'll select only those few individuals that have both traits that they were interested in.

What is different about recombination, cross-pollination and selective breeding?

Recombining DNA transcends both science forms. So in the case of genetic engineering we're recombining DNA but in a very targeted sense. You're taking a very specific gene from perhaps a soil bacterium like Bacillus thuringiensis and you're combining into the corn genome so it's still recombinant DNA but now you're crossing the lines of two different species of organisms.

What is the first step in creating recombinant DNA?

The first step is to really identify the gene of interest, and through the long history of Bacillus thuringiensis with silkworms and European corn borers, scientists knew that there was some component in the DNA that produced a trait that had a very desirable end result. Somehow, through a process of working with DNA extracts from Bacillus thuringiensis, they were able to isolate out the single gene that cause the end results.

What is the next step?

Well, the next step is to figure out how to get the gene from a bacterium into the genome of a corn plant and make it active at a high enough expression level that it will control the insect when the insect feeds on it. Early on, there were two strategies that were discussed, whether we should take a high-dose strategy or a low-dose strategy. And for various reasons, including resistance management control methods, we definitely went after what we called a high-expression strategy. In other words, we wanted the corn plant to produce a lot of Bt protein.

And so to make the corn plant be able to sense, recognize, and treat as its own a gene from the soil bacteria, there needed to be several pieces added to the gene. One that was used initially was an antibiotic, and the reason for attaching an antibiotic gene to the European corn borer controlling gene was so that you could sense whether or not you were actually moving the gene of interest to where you want it to be, and expressing it.

The other part that you need is a series of promoters and controllers so that when the gene is in the genome of the corn plant, it gets turned on. The third part that they needed was another type of a marker, and we use as a second marker an herbicide-resistant gene, which is a gene that confers resistance to Liberty Herbicide.

Is that what a multi-construct gene is?

That is the name for it. It's a construct. It actually has a series of genes in sequence that are led by a promoter. Then you go through a process of trying to take this construct, replicate it, because you have to have thousands or maybe even millions of copies of this gene, and then somehow you have to get it into the corn cell. Once you've got a possibility of getting the gene into a corn cell in a position where it could be expressed, and having the promoter and the marker genes in there, it gives you some insight of whether or not it's working without having to grow full size plants and go to the field with it, which is a very expensive and time consuming process.

How do you get a gene construct into a plant cell?

There are several ways of getting a gene construct into a corn plant. There are certain organisms that will carry pieces of DNA from one spot to another, and one of those is a plasmid. So you can create your construct, expose it to a plasmid, and the plasmid will readily take this DNA in as part of itself and the plasmid can cross a cell wall into the corn genome and will transfer the DNA into the corn genome. That's one way.

Another way is by using another bacterium called the Agrobacterium tumefaciens and this is again an organism that was studied a long time ago for a long period of time. For some reason, this particular bacterium is very receptive to taking up foreign DNA, absorbing it as its own, going into the cell of another organism and depositing or leaving chunks of DNA behind.

The third way is ballistics, and it uses a 22-caliber shell and, in this case, in the early days it was packed with very small gold dust that was coated with the gene construct and was literally discharged into callus tissue from a corn plant. And it was literally just shot into cells, hoping that some DNA sticks in the right spot.

Are there problems with ballistics or the gene gun method?

There are some technical problems, and there are what we call IP problems. The technical problem is it's a hit-and-miss adventure, so you have to have a very good marker-screening method because you can shoot, if you will, the callus tissue of many different corn plants or many different extracts of corn plants in a very short period of time.

So typically what they would do is have lab technicians grow corn calli, pretty much continuously, and then they would let those calli sit in a growth chamber for a period of hours, a couple of days, and then they would spray herbicide on top of the Petri dish. And the calli that had the gene construct that went into a cell and was expressing would not be killed by the herbicide, and that would be the minority of cases. Certainly most of the calli did not take up the DNA. You know you did not get the gene into the right spot and have it expressed when those calli were very quickly killed by the herbicide. Of course one of the genes in the construct was a herbicide resistance gene, and the antibiotic was used for the same thing, so you could spray the calli with either a herbicide or an antibiotic, and that way you knew if the calli had a cell left in it, at least at one cell in it, that it was expressing the marker gene. You still don't know for sure if it's expressing the Bt gene because these insertions using the ballistics are often considered to be messy.

How do you get foreign genes onto plasmids-via restriction enzymes?

There are probably dozens of different types of restriction enzymes, and different restriction enzymes will cleave DNA between prescribed base pairs. So the restriction enzymes are a way of getting down to smaller pieces, more manageable pieces of DNA, and even single genes.

With Bt, did you use a plasmid to get it into the corn genome?

Different Bt types of corn that are out in the marketplace utilize different ways of getting the Bt gene into the plant in the first place. In the case of the first Bt event, which was "event 176," created by Ciba Seeds, the gene was inserted into the corn plant using a ballistics method.

How does the totipotency of plants aid in genetic engineering?

The fact that plant cells are totipotent, which means that they have the potential to express all genes, does make it easy to produce a callus tissue from a few cells, and then you can insert the desired gene construct into that callus and you can, through the manipulation of sequences or combinations of hormones and growth regulators, produce a full-size and viable and reproductive plant from that single callus tissue. And with plants that's certainly an advantage.

What are marker genes?

Marker genes are simply genes that allow you to determine if the desired gene has been inserted or has been assumed into a position into the target plant in such a way that it's expressed.

So an example of a marker gene could be an antibiotic, it could be an herbicide, and so the marker simply tells you if the construct insertion went well or not. If the marker gene is active, it tells you that there's a good chance that the gene targeted to be put in there-in this case Bt-is there. It does not tell you for sure that it is there but it tells you that there is a good chance that it is.

What are the genes used in making Bt corn?

The early versions of Bt corn contained a couple of marker genes; Event 176 contained some promoter actives and two marker genes. There was a gene that made it resistant to a particular antibiotic, and there was a gene that made it resistant to a particular herbicide. There was also the Bt gene itself. And then there were probably a couple more codons to indicate that it was the end of that gene sequence, so when the plants going through the process of replicating protein from the DNA it knows where to stop and start.

Those were the early days. Now, in the more common and the more modern Bt events, we do not use antibiotics. Antibiotics have been completely taken out of the recipe since the first couple years.

Why?

Antibiotics are very convenient to screen the plants we get in the lab. But some of the people who potentially consume corn products of a GMO nature are opposed to having antibiotics active in the plant or being part of the construct, and the concern is that through the process of continually exposing nature to the antibiotic that whatever it was an antibody to might become resistant to it. So it's a matter of simply frequency of exposure.

We still need to use markers, but the markers that we use are for the traits and a very common area would be herbicide sensitivity.

What were the challenges in creating Bt corn?

There were a lot of issues along the way. It was nearly a 10- to 15-year process. There were issues from a regulatory point of view. A lot of work that needed to be done to confer enough information so that it would be generally understood to be safe to the environment, that it would be safe to the consumer, that it was not going to cause any unwanted effects. It had to be demonstrated that it was effective in controlling the target pest that we were out after in the first place, so there were a whole series of science issues or technical issues.

The tools of biotechnology we're actually developing as scientists-we're trying to develop the end product, and so by focusing on trying to build effective Bt corn, that actually drove the development of many of the tools of science to get us there.

The development of Bt corn drove technology?

The European corn borer causes in excess of a billion dollars worth of damage in the United States alone every year. Farmers in many cases would experience corn borer damage, and they couldn't do anything about it, or if they were going to try to do something about it, many farmers did spray more conventional insecticidal sprays.

I guess it was first of all difficult to know when was the optimum time to apply and then, secondly, to apply in such a way that those sprays would be effective. So there was a lot of money in this interest. There was a big market and so there were a number of companies that invested heavily in the development of biotechnology tools, specifically to try as quickly as possible bring to market something that worked to provide a control alternative for this pest.

Once plant has the gene, how and where is it expressed?

Once you have transformed corn plant and you've gotten the gene construct into the plant, the next question is to understand if it is expressed, and if so, how.

Typically, this involves first doing a tissue test in the lab. In the greenhouse you produce a small plant and you can take a leaf disc and you can expose this little leaf disc with a control that you believe does not have Bt in it to European corn borer larva and very quickly you'll see if the corn plant is expressing Bt. Those larva will die within 24 to 48 hours so you have a very quick screen that way.

Depending on the construct you've developed, the Bt protein, or any protein that you've put into a plant, it could be expressed in different plant parts. There are promoters that will turn on the gene, if you will, in specifically roots and maybe not elsewhere in the plant. And there are promoters that will turn on the gene in all plant parts.

The earliest event which is, again, event 176, turned on the gene in leaf tissues, kind of anything green on a plant and the tassel, including pollen, but not the silks. And different expression patterns and the potential use of different expression patterns have advantages and disadvantages. If the goal is to control the corn root worm with a different kind of Bt, and the larva that feed on corn feed on the roots, it would make sense to express that protein heavily in the roots to knock out that feeding point, because that's where the damage is caused.

Different strains of Bt need different promoters?

There are not a huge number of different types of Bt's today on the commercial market. There are probably five or six, but each one is somewhat different. And it is very, very important for the farmer to pay close attention to what the differences are.

I got an interesting story or feedback on some of the early days again in '98 and '99. There were two different types of Bt available in the market. They were both called Yield Guard. But one contained resistance to an herbicide; the other one did not. And so farmers who bought the Yield Guard that did not contain the herbicide resistance sometimes misunderstood what they had and sprayed a field with the herbicide and promptly killed their entire corn crop.

What are the challenges ahead with Bt?

One of the benefits of Bt is of course is that it's very selective. It controls the European corn borer and it may suppress corn ear worm and fall armyworm, but that's it. But that's also a disadvantage. Farmers have the need to control many other insect pests. So, yes, there is an effort to produce other types of Bt's or other types of GMOs, if you will, that control other problematic insects in corn.

All at one time?

All at one time.

So selectivity is good and bad?

The current situation is that there are two types of corn hybrids out there. There is a type of Bt corn that will probably be marketed very, very soon in the U.S. that controls the corn root worm, but that's a different kind of Bt completely than the kind that controls the European corn borer.

The problem from a farmer's perspective is that maybe he's got corn root worm and European corn borer in the same field, so to have two different control methods in two different hybrids doesn't really help him much. The goal would be to have one hybrid so that a farmer could plant a field that contains the ability to control all the lepidopteron species-European corn borer, fall armyworm, corn ear worm and corn root worm. And that would be a package, a genetically modified organism, if you will, a hybrid that could control really all of the major insect pests in the U. S. Midwest in one hybrid.

How close are we to doing that?

Well, through the continued improvement in the genetic engineering tools and the ability to put more genes together and to have a more complicated construct and to get it into a corn plant in such a way that it works, that technology is definitely improving, so we're getting closer to being successful in doing that but we're still probably looking at 2005-2006.

Are we seeing less insecticide spraying with Bt corn?

I don't think we know yet if the advent of Bt corn has reduced overall spraying or not. One thing the use of Bt corn has done is it's definitely made farmers much more aware of how much yield they were losing to this particular insect pest and it's given them a new tool to help control this pest. But there are issues from a farmer's perspective on whether or not to use Bt corn.

One of the biggest issues is simply the cost: seed that contains Bt is more expensive than seed that does not contain Bt. And the farmer does not know at planting time if he's going to have a significant outbreak of European corn borer in a particular field or not. In some areas of the corn belt, there is greater likelihood that there is going to be an outbreak than others.

And so the farmer is left with an economic decision and so in some cases farmers, yeah, they decide not to use Bt corn. In 2002, roughly 34% of the corn planted in the U.S. was Bt corn. But that leaves 66% that was probably not Bt corn, and they are going to need to use the more conventional scouting methods for European corn borer.

Well, as they've learned how much yield that they're losing, they're much more aggressive in doing the scouting and then potentially spraying for European corn borer. So on those acres where Bt corn was planted, there are certainly likely much less insecticides applied, but there may be other acres that wouldn't have been applied earlier that are applied now.

What are the problems with conventional insect management?

The tools of scouting and the use of insecticides were available long before Bt corn came along. You have to look for European corn borer eggs and larva, depending on whether you're trying to control or trying to estimate the amount of economic yield loss you're going to have.

The primary problem with conventional spraying is to know when to spray. Depending on where you live in the corn-belt, you may have one, two or more than three generations of corn borer in a single growing season. And so when you're scouting for first generation European corn borer, you're typically looking at small corn plants that have a whorl, and you can look for larvae feeding in the whorl. If you can time the pesticide application well to when those young larvae are feeding on leaves in the whorl, insecticidal control can be fairly effective. If you miss it and you're slightly late and the larva has already tunneled into the cornstalk, it's too late. You can't get insecticide to where the larva is, and so therefore you can spray but you're losing your money and you're putting insecticide where it doesn't need to be.

The second generation and the third generations are much more difficult. Now you have a full-grown corn plant, and basically what you've got to scout for are the small egg masses on the underside of the leaf, and there is only a period of about a few days or less where those larva hatch and they're on the surface of the leaf in such a position that insecticide would get to them and control them, after which they quickly go down the leaf sheath and wrap tightly around the stalk-insecticide can't get to them there-and then they tunnel into the midribs or into the stalk where you can't get to them. So the window of application on second generation ECB is very narrow and very difficult to hit.

How does Bt corn prevent crop loss?

Bt corn is very effective in preventing crop loss associated with damage caused by European corn borer. This particular technology trait is very, very effective against the ECB so the control rate is 99.9% plus. There are other disease pests that farmers can lose yield to, so European corn borer is just one of the concerns that they have to be aware of.

Are you concerned about insect resistance to Bt?

Another concern of the use of this kind of technology is the potential that European corn borers could become resistant to the Bt gene or protein that's been put into the corn plant. And Bt corn is a very nice resource and one that we certainly do not need to squander. And so through a process of collaboration between industry and university scientists particularly, a method was developed by which farmers could plant blocks of corn, and 80-85% of the corn can be Bt, but 20% of the corn needs to be non-Bt.

The whole concept of Insect-Resistant Management and planning these blocks of corn are to reduce the likelihood that resistance would develop. There's very little doubt that if you did not have an IRM strategy that the European corn borer would become resistant. It's only a matter of when and the use of an IRM strategy is to try to slow that down.

How does Syngenta make sure farmers adhere to the IRM strategy?

We work with farmers to make sure that they understand what an Insect-Resistant Management program is and why it is used. We try to help them understand the bigger picture, that we would like to have Bt corn be viable and available 10, 15, 20 years down the road and this is part of your insurance policy to make sure that's likely to happen.

So part of it is education. Part of it is regulatory. We do keep track of the counties in which we sell Bt and non-Bt corn, and in which volumes, and we report back to the EPA on a regular basis. And then the EPA monitors it as well, and obviously the intent is to keep a county growing 80% or less Bt corn.

How do you respond to charges of environmental organizations like Greenpeace?

I think it's clear from looking at yield responses, looking at corn plant health that Bt corn does provide a very significant financial benefit to the farmer. It's not a standalone. It is one tool that a farmer has available in their arsenal to control a very problematic insect pest.

So I think the jury is very, very clear that there is a significant advantage to having Bt out there. At the same time, I certainly agree that as much as we have the right to explain our point of view that they should have the right to explain their point of view and to make that perfectly clear as well.

Will this type of genetic engineering replace conventional breeding?

No, I do not believe it can ever replace conventional breeding. It has deflected some of the resources from conventional breeding, but again, these are very selective traits. They only hit one or two target pests or problems at a time, and a conventional corn breeder when they breed they're selecting against hundreds of thousands of traits at a single time. And so there's no way that biotechnology, which is kind of the rifled approach, can replace more of the shotgun approach of conventional breeding.

Are GMO's good or bad?

You cannot make that kind of distinction. Genetic engineering has all the potential to be good and it has all the potential to be misused, and that comes back to ethics, having a good moral founding, and if you have that kind of background or that kind of a base, there's a huge potential of what you can use biotechnology for-and it's not just plants. It's plants, it's animals, it's medicine. You can do a lot to the benefit of mankind with the proper focusing of some very good tools.


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