



S1: so, um, does everybody have a handout, for today? if not Aaron's got handouts f- also you'll probably need your handout, you will need your handout from, Thursday. and hopefully, the lecture'll be a little bit less painful for y'all to get through, and for me to get through than it was on Thursday. so, the point of today's lecture is to try to get into a little bit more of the nitty-gritty of plant, biotechnology. and um, it's it's such an odd, audience to try to, present this to because for some of you i know that you know the great_ all the details of the plant background and some of you know none of the details. so i'm trying to present more of an overview, of what the biotechnology process is. um, we'll start out talking a little bit about product development although not in great detail because, it just didn't seem like there was a whole lot of content that i could put there but i did try to give you some, numbers to give you a little bit of a feel for how much, plant biotechnology is going on and what impact it has on our life these days, and then, how one actually does gene transfer. we've decided on a product or in, in the case of your homework you have, an assignment to come up with a product. how do you get that gene into plants if that's your choice of how to do that particular homework assignment. and then, i'll talk about different ways in which one can get genes into plants to make a stable transformation. and then finally at the end of the lecture today i should have some time to talk about some of the very earliest, um commercial products that were, made from bi- plant biotechnology these_ there were some tomato fruit ripening, projects, and what happened to the first one and what happened to the second one. what was the, biology behind it so that you get some idea of what the, um, what they were doing at the time. so if you actually probably will need to start with your, handout from, Thursday, and about mm, i don't know, after, the phytoremediation part, there's a han- there's this page that looks kind of like this. <P :06> and again if you can't see what i'm putting on the overhead make sure you know because i know the screen up here is a little bit different than the screen that i have. basically there's two strategies when they're developing products. and you're gonna tell very s- quickly where my bias is, um, and i don't know if that's just because, that's what i'm most familiar with or if that's truly what most of plant biotechnology is doing. but there's sort of two different strategies here on the two different, pathways. the one on the left they call sort of a, more traditional, breeding pathway mutagenesis and plant breeding. and you can see molecular breeding traditional breeding, um, coming from some mutant isolation or some analytical biochemistry. this is as opposed to the m- the more, i don't know i guess modern or more technical, plant um, molecular biology, or the uh, biotechnology pathway. now the difference is in and, the whole point you've gotta, some kind of iden- a trait. we're trying to develop a trait. we're maybe making something that's more herbicide resistant or pesticide resistant or we're making, tomato fruit that are, ripe sooner or ripe later or, or have some particular trait. and one way one could do it, is to identify, basically a mutation or, some trait that you want to, um, improve upon, or exploit in some way, and traditionally they've done that by molecular breeding. breeding it back into maybe a more hybrid, uh crop or, improving vigor. and, the, more modern way would be to say okay, i know what the pathway is involved i know the gene that i'm interested in, i'm going to, engineer it, using, modern molecular biology, and cell culture, and actually, make transgenic plants then, that will yield, the product that we're looking for. so the difference the main difference in the two pathways, is the one on the right, the molecular biology one, you have to know what the, the pathway is the physiological pathway that you're dealing with. the gene that you're interested in. pathway on the left, you don't. you need to have a phenotype, you need to know what something looks like, rather than having, the full um, molecular background for this information. uh you can also see that this, i just included this from down here, this is from an article in, Trends in Biotechnology. if any of you guys really get into it it's probably a journal, that covers all different types of biotec- well it is a journal that covers all different types of biotechnology. this is an article from actually now it's five years old, but just to sorta show you how long, the development of a product might take, from start to finish, and you can see let's say we have a gene that we're very interested in, um on Thursday we're gonna talk a little bit about the development of vitamin E rich plants. and they're just beyond, this stage right now. and it's getting lots of, attention. this is where we do the filing of the patents that we're talking about this is what we, where we think we've got, this product, or potential product and so we file a lot of patents this takes one to two years. making the actual, transgenic plants can take a couple of years, before we even get into, development of the, particular variety that's of interest. regulatory approval can take quite a while. depending on, the uh, group that we're working with, i'll talk a little bit later about who you have to, get approval from. testing of the yield because as you'll see sometimes putting in a new gene, will decrease the yield of the plants and that may um be very bad in which case you might have to go back here into development a mutant discovery and doing some traditional breeding, to improve yields. and then of course testing applications and we'll talk about things that you have to worry about, when you're actually testing your products, um, and for instance, if we, decided we started out here with this really cool gene and we're trying to improve the protein content, in a particular plant but we choose a bad protein and we find out, what are we now at about seven, five to seven years down the line, that this happens to be a protein that fifty percent of the human population is allergic to, then that's a bad choice and you have to go way back at the very beginning. but even, if you don't know that it's an allergen, it's something that has to be tested for so there's a lot of, uh application testing, and product testing that goes on here. before one goes into bulking up the seed because obviously with plants you have to grow the whole crop, and, produce the grain or process the grain, or the fruit or whatever, product it is that you're looking for. this whole, process can take i think from beginning to end you can see it takes something like, eight to twelve years, from this initial great idea. so, if you're writing a grant proposal today to do, some really amazing product, then chances are, it will be done long after you get your PhD and by somebody else in the lab. or somebody else that would be in the research project at that time. the money that's involved in this uh, is really pretty amazing. some of the numbers that i'm gonna show you are, are older numbers and some of 'em, i've tried to update a little bit. um, the nonfood products, so you hafta think about this pretty broadly and it does get a little bit confusing so there's lots of nonfood products, and we'll talk about some of them here that come from genetically transformed and genetically engineered plants. the amount of money, in nineteen ninety-five, which now, i realize is quite a while ago, was about, fifteen million dollars a year. fifteen million. per year. now that was predicted, to grow in ten years so, in another, five years they're predicting it to be at greater than, three hundred and twenty million... so there's really quite a bit_ this is in the nonfood products. um, one of the reasons this is_ that we want to exploit, some of these transgenic plants, is the fact, there's a lot of, advantages we'll talk about, but, it may help us decrease our reliance on oil-derived chemicals. so lots of chemicals that're, additives in foods or, additives to plants pesticides herbicides things like that, are o- oil-derived chemicals and that's not an environmentally, good thing. um, it would help us, redirect U-S agriculture away from um, the production of su- surplus foods, if any of you come from rural backgrounds you'll realize there's been, there's not um, there's been a lotta surplus food production, um even in U-S agriculture and so that_ for instance corn, and that can be a problem because the government then spends a large amount on agriculture uh, subsidies. on, to, pays the farmers for growing foods that we don't even need, and by using engineered plants this, we can reduce some of these. um, and in fact some of these subsidies themselves, have ranged between about, oh six and twenty-six million dollars a year. just in subsidies. that, for growing food that, is a surplus. so, um, we can change that just by changing a little bit the types of crops that are being grown. agriceuticals is this new term, maybe not so new any more... this new um, merging of agricultural biotech companies traditional agriculture companies, and, pharmaceuticals. so, mergings between like si- Ciba Geigy, and Sandoz, uh to make Novartis. Novartis is a huge no- uh agriceutical company now. and they're building, a lot of plant biotech industry and there's a huge group down, in North Carolina there's another group out in, um San Diego now where they're and they're hiring, an uh tremendous number of scientists. this merger, between to make Novartis was more than a hundred billion dollar merger. um, it's really big money, Monsanto, Dupont they're always, fighting and buyi- Dupont just, merged with Pioneer Hi-Bred, um for instance. uh Hoechst, just bought Plant Genetic Systems that was another six hundred million dollar, merger. there's just a lot of money. if we go_ this was the nonfood if we go, into more of a, a little different idea that the um, what are they called? c- uh <P :04> conventional medications i guess would be a good thing everything from aspirin to some of the most toxic cancer medications. nearly a fourth of those, have a least one active ingredient coming from plants. and in fact, f- um, something like the the World Health Organizason Organization. um, estimates that eighty percent of the world population, presently uses some kind of an herbal product, that, uh for some part of health care. and the vast majority of you probably do use, hand lotion that has an herbal product in it. or you take some of these, cold remedies that, or echinacea, or St. John's Wort um, the numbers now have have, are tremendously high that Americans alone, are expected to send spend, about, this is just in America, about six billion dollars, on some of these herbal remedies, this year. <P :04> so it's a good time to get into some of this. um this is just in the U-S. but this is actually, world wide so i wanna spend a little bit of time talking about the extent, or extant i guess the the, distribution, of biotech, around the world. on, there's been an_ that's the next page in your, this little handout that i gave you. cuz i knew you all wanted to know all of these exact numbers, but, it's because i ou- mostly because i think that some of these numbers are kinda interesting. <P :04> this is showing, just to give you some idea of, how many different crops, have been exploited, all of these different transgenic crops i mean some, most of you guys, most of you aren't plant biologists, so you would never think that they would've made, a transgenic serviceberry, i bet, in your wildest dreams. or transgenic walnut, now to be perfectly honest i haven't the foggiest what the transgenic walnut, project is. i don't know my guess is that they're trying to do, um, insecticide resistance or insect resistance, with a walnut. um, it's not too surprising they'd go after something like petunia. oil seed rape, might surprise you that's actually a, canola. um, so canola oil, hundred and eighty-one different field releases, so different projects, of this. um, kiwi, i don't have any idea what gerbera gerbera is there's only been two of those. but you can see actually there's been very, fairly large numbers, um corn, potato, potatoes uh uh, was one of the earlier crops that was easily transformed and so that was one, that we ha- there's uh been a lot of work done with. um, soybean, fifty percent, of the, soy bean and corn crop grown in the world right now is genetically modified in some way. um, and that would include most of the products then that come from soybean, and corn at this time. over a thousand fields, tests field releases that's of, about ninety-three. obviously it's probably doubled at least since then. um, let me see what else do i wanna say about that. the very first trials were with tobacco. you can see there's been a hu- over a hundred, with tobacco. the first trials was, were with tobacco in nineteen eighty-six. and that was only what fourteen years ago. so, if you think about that timeline that i gave you a seven to twelve year, um, or eight to twelve years from idea to commercialization we're just beginning to realize some of the earliest, um commercial products so we're way, way still in the log phase on this. if you look at the way, where this is done in different countries it's done all over the world in many countries you might not even think about. Zimbabwe, has uh it's own, has a single field trial. um, obviously we've got, a huge number, in the U-S. but there's, triple digits in France, in, Canada has a large number. um, Great Britain is fairly active. they've got about fifty the Netherlands, it's it's really quite a, a worldwide, technology at this point. um, the regulations of these field releases tends to focus on human safety, they also um, look at, potential risks to the environment. so the factors that they usually consider, oh the other thing i wanted to just mention, was the, regulatory commissions at this point. all of these field tests they have to satisfy various regulatory um, limits or commissions. the U-S-D-A, U-S Department of Agriculture, is what controls, m- most of these field trials. <P :04> but especially those that, are gonna focus on the U-S-D-A will especially focus on human safety. and, um, the vi- environment. <P :07> the F-D-A, Food and Drug Administration, is gonna control any of these food trials, that, affect, food products. <P :06> and the E-P-A the Environmental Protection Agency is gonna do anything, with pesticides or herbicides. <P :05> so, each field trial as as a project nears field trial they're gonna have to satisfy, often more than one of these regulatory commissions. th- this is just in this country. it's very different and in reading, some of the articles the background for this, it's very different in every country and in fact that's why some of these countries that you might not think, would have so many have a large number they tend to have, some of these countries have much lower regulatory, um limits. and so they're more likely to be, sites for early field testing. but, one project may have to satisfy regulatory limits both, in the U-S-D-A and the F-D-A because a food product is often gonna be a- of of interest for human safety. um pesticides and herbicides, are often, also gonna be, f- of interest for human safety and they're certainly gonna be, um, assessed for environmental impact. and so they often have to fit more than, or satisfy more than one of these agencies. <P :05> okay, so i wanna make a list of the factors that they consider, some of these may or may not be of some use when you're doing your homework assignment but they are things that dif- scientists depending on, the actual project, that is considered. so, none of these will be, of any surprise to you but if you were told to list 'em it would probably be a little bit hard on your own. the first of course is you have to figure out, or what has to be considered is the function of the particular gene of interest, in, the, host or the, the original donor organism... what does the gene do, in it's normal endogenous everyday location? and then, what would be the effect, of taking this, and putting it, into another plant. so the effect of the transgene, on the phenotype <P :04> of a transgenic plant <P :06> that's important right? if you take a transgene even if it's a great idea and it kills the transgenic plant it's not gonna be of any good whatsoever, downstream by the, the commercial products. a very important consideration is they look for evidence, of both toxicity to the plant, any kind of toxicity, plant to animals that are in the area to the environment, to people, and e- also allergenicity... i wish i had time to tell this great story about, uh gosh, i'm i'm not sure who that was Pioneer? uh, who developed, this great product line using a protein from Brazil nut, and got it almost ready for a commercial li- commerciality and found out that it was, highly allergenic in the human population. um, weediness... what kind of effect, does this have, on the persistence in the agricultural, habitat? so if we put a transgene, in a new plant, will it persist in that agricultural habitat? <P :08> how much does it invade, the natural habitat? <P :05> and scientists are, taking very serious looks, at some of these questions. uh, partly because these are some of the questions that have been raised and have been argued about so much in Europe. uh, what is the impact, of these transgenes? on nontargeted organisms? <P :05> what are we doing inadvertently? unintentional, unintended effects. what is the likelihood... and the consequences so if it does happen, that the transgene would move, to other organisms or of movement. <P :08> to other plants, that's primarily by, cross pollination... and then, i realize this is gonna go. the last one that, would be just probably below the line there of the board, are some, less, defined maybe ethical considerations <P :05> about the acceptability for example of putting antibiotic resistance markers, in food products. now, there's an article, they comment, that came out, in a journal called Trends in Plant Science. <P :06> that addresses some of these concerns... very directly, for G-M crops. G-M being genetically, modified, plants. um, this is in December, of ninety-nine so, what, just over a month ago. volume, four number twelve, page four-sixty-seven, to sixty-nine or so. uh it's just a couple of pages. it's just an interesting article for those of you who, are a little bit interested in this. you don't really care about the plant part but are a little bit interested about, some of this controversy surrounding, the genetically modified crops. what is the public's perception, what is the scientific response to it? and i'd go into it but, it's not, it's not that interesting to me which is unfortunate because uh as a scientist i can refute most of the, complaints and the comments, uh but that's sort of not my purpose in this part of the lecture. if you're interested in that that is here. so for instance, what are they doing about antibiotic resistance in plants? and is that really a problem? if you eat a plant that has an antibiotic resistance marker are you gonna become resistant to that antibiotic? or is your intestinal flora? the simple answer is no you're not, and no your intestinal flora is not either, and it explains all about it in here. um it does tend to be, it's rude to say hysteria, um and it's also rude to say ignorance but it's a little bit of both. and, this is actually just a nice very general comment, and commentary of showing some of the studies that are being done. what it also does is it's very honest about saying you know scientists didn't really think about, invasiveness in natural habitats, what are these transgenes doing? we didn't really think, when they first started developing these, are we gonna be impacting nontarget organisms, and so it does, a nice job of saying now what they're looking at and what they're finding in some of these studies. so if you happen to be interested in some of this stuff that's more there, but i didn't do much with it, so. a little bit more on the extent of biotechnology, um turns out that actually China, is the country that has the, largest areas of transgenic crops. in fact, um in this, one article that i looked at they predicted that by the year two thousand, seventy percent of the total tobacco being cultivated in China, would be transgenic. that tends to be mostly transgenic, tobacco that's um, has, a gene that, confers resistance to cucumber mosaic virus. now, most people don't care i mean, most of you guys don't really care what we do to plants to engineer, insect resistance or herbicide resistance. it only came when we started changing the food that people started to care what was happening in the plant world and gene- genetically modified crops. and so these things have come a long way before they got any public attention. but, a very large percentage this is seventy percent of tobacco, i told you fifty percent of the corn and the soybean in the, world that's being grown is genetically modified, mostly for, against in this case this is a cucumber mosaic virus tobacco mosaic virus most of these viruses are, um, we've engineered resistance against some of these major problems. um, in fact, in tobacco, the transgenes, have uh increased the yield of the tobacco five to seven percent so it's pretty hard to tell, farmers especially in some of these, more developing countries, i'm sorry we can give you a plant, that now is resistant to it's major pest, has a five to seven percent higher yield but you can't use it. so i mean there's, that, there is nothing wrong, with the plant itself. it allows them to decrease the amount, of insecticide, they put on their crop. that's a good thing right? we're applying fewer chemicals then to the crops. um, anyway so this is, many of these plants are now, completely commercialized at this point. um, let's see. i think i've said all of that. uh, the examples, um, i'm gonna go through a whole bunch of examples. the next page in your handout is just a little bit more information. <P :09> millions of hectares, um, this gives you all the information for all the countries besides China, they don't even have China on here. let's see if we put China in here, on this same table, uh, in, the year two thousand so these are basically from nineteen ninety-eight so a couple years old now. the prediction was that, for China, in the year two thousand, that about fourteen million, hectares, in China alone, would be, transgenic, and this is for trans- fourteen million hectares so it continues down from here, of transgenic tobacco, only... so, the point i'm just trying to make here is that here we're saying, twenty-eight million hectares in the world, total, would be transgenic and yet just in China, or at least in this group, with China alone fourteen million of 'em of the tobacco that's being cultivated was transgenic at that point. what's maybe more interesting is just some of these examples, of the, plant characteristics that have been, modified, the vast majority of what's been done, has been done with the insect resistance, viral resistance, and herbicide, tolerance. so, and most people that are not, plant biotechnologists couldn't care less about most of this work. and so it really hasn't received much attention. some of these down here i'll talk about these because they're of more interest to, maybe a general population. and, um they're some of the more interesting, um, strategies. yeah? 
S2: are there any examples of, uh pests who have developed, ways of getting around the, resistance? 
S1: yeah, sure, that's one of the big, problems or one of the big concerns and that's one of the, the, one of the biggest concerns is this, this bis- uh, the B-T toxin. and, by um, pu- using this as a way to prevent infection by this particular insect, one concern was that the insects would become resistant to the toxin. that was being used to kill them off. and, this article actually i thought this was a quite interesting article, and it is a problem. so that has been a concern. if we start trying to, change these insects will we just get insects that are resistant and then, then you've got the same problem all over again. and one thing that it talks about is that, some of that's a dose, response. and one of the problems that they have is that, c- currently the transgene is usually expressed at fairly low levels in the plant, and so it may not be a (hike) so when you have_ it's just like with antibiotics, you're more likely to develop resistance if you're ha- you're being exposed to a toxin or an antibiotic at a low level. one way, what they're finding now is that, if they express these transgenes, in chloroplast, they can produce tremendously high levels of the toxin, that will actually knock off the insects, before they're able to develop resistance so, the short answer is yes they are, having to be concerned about, uh uh, insect-developed resistance. the other part is that they're also now trying to deal with ways of getting around it. and, because in a chloroplast you could make many copies of the same gene, more than at the, on the nuclear level. um, they're f- they're using this as one way to get around it. they're actually using, expressing these genes in the plastids, the chloroplast especially of these plants is a way to get around a lot of problems that are, i had listed over here. but you have to read the article for all those details. um, okay so, three things that you sort of have to think about ar- the ones we're gonna talk about o- the ones we're gonna talk about in case, people really care we're gonna talk a little bit about delayed ripening, we're gonna talk a little bit about, the phytase content, we're gonna talk some about starc- uh starch metabolism. don't know if we'll talk any about that. glyphosate if anybody cares that's Roundup, Roundup is uh, pretty common, uh, herbicide, that was developed by Monsanto. um, i think those are kind of the main ones that we'll talk about. but when one's, looking at, a gene say for delayed ripening that's one of the examples we're gonna look at in a little bit... have to have, make sure that this, introduced enzyme, whatever the introduced enzyme, has affinities for a substrate, so that it can compete with an endogenous enzyme if the plant already makes one enzyme why do we need to put in a transgene? so we have to make sure that the introde- introduced, enzyme, will bind to whatever it's substrate is in the transgenic plant, it had to be, it's important that we look at, the amount of the protein that's being produced the product that's being produced in the plant. that's a little bit what he was asking and if, or my answer to what he was asking is if we're, producing enough of the toxin for instance to actually kill off the insects that are eating on the plant. uh, and also the localization the compartmentalization, it's important that the product, is produced in the appropriate compartment within the cell. so that it's completely functional. most of the examples that i'll talk about and most of the examples you would think of, do happen to be cytosolic proteins. uh in the case, of, Roundup and glyphosate in fact the enzyme that they're making they had to make sure they targeted that, to the chloroplast. because that was where it's most active. and so, it had to make sure that the compartmentalization, the final localization, was correct. also within the tissue you have to make sure that it's expressed in the appropriate tissue, it doesn't do any good to have an insect toxin being expressed in the root if the insects are chewing up the leaves. so, tissue expression, has to be appropriate and so you'd wanna think about that when you're designing your product. okay so some of the, examples let's see, one more page here. couple more pages. couple more pages in your handout just look at um, giving you just a little bit more information this one talks about some very specific examples, who's actually developing them, and at what state they were at. now this is an older article so all of these that are actually pending, have all been authorized at this point or else completely dropped from the program. um, this is the, this one the tomato delayed fruit ripening. we'll be talking about this in a minute. this is the Flavr-Savr, tomato. uh, D-NAP's Group this is the Endless Summer, tomato. <P :07> all of these companies, still exist in one form or another they may or may not be called exactly this, uh, but they are all still doing, various forms of plant biotechnology. um <P :04> oh yeah this was just the last then, of the, examples they're sort of odd examples... amelistris- we're gonna talk about each of these very briefly, amylose-free starch cyclodextrins and trehalose. these are, several compounds that have been, um, expressed in plants. so in this this what we're listing here is how, plants have been exploited as bioreactors. some of you think of growing up micro-organisms and getting them to produce large amounts of things. plants have been exploited to produce large amounts of trehalose cyclodextrins we'll talk about that in a second and why we might wanna even do that or why that might want to be done. um, Thursday i'll talk a little bit about, using, plants to express something like hirudin which is uh, an anti- a blood anticoagulant, and so being able to make very large amounts of this pharmaceutical, uh using, plants to express large amounts of antigens to be able to work as natural vaccines. um, phytase we're gonna talk about that a little bit today. this is uh, uh ra- currently this is an additive in animal feed, and, so, it's yet another chemical that's brough- bought and fed to, certain, livestock or in this case boiler(sic) chickens, but, if it can be, actually engineered into the feed, then it can provide, the um nutrition that they need without having to do more additives. and we'll talk about that a little bit too. think those are the main ones. that we'll talk about so. okay. <P :10> figure one this is just my prop so that i can talk about a few of these different examples because most of you and certainly i hadn't heard of many of these things before i started preparing these kinds of lectures. amylose-free starch. so, that's this guy over here. amylose-free starch is something that they're trying to, uh that has been developed, and expressed in plants, and they do it by antisense expression we're gonna talk about antisex- sense expression in a little bit um, by antises- sense, expressing, some of the genes involved in the synthesis of this guy, this is important because microwave-ready foods, um, need, a lot of amylose-free starch. i know that is gonna thrill Aaron to no end. um, because, it um, when you microwave foods that have this stuff in it it makes a clear p- p- paste and it doesn't deteriorate when you microwave it. and so now when we're developing all of these foods that are microwavable, they want more of this kind of starch in your food because it doesn't like coagulate in to a cloudy mess when you, microwave it. so you want to look for this in your food i guess. cyclodextrins, are another very common, product that they're engineering now, using plants as bioreactors, metabolically engineering various_ this is, these are just a whole bunch of examples of carbohydrate metabolism. ways they're altering carbohydrate metabolism in plants. make more cyclodextrins, most of you probably don't remember and i don't blame you but i actually talked about cyclodextrins on, um, last Thursday, those were one of the, uh soil additives that i talked about, that you could put in the soil, and it would um, free up some of the metals, for tak- being taken up into the plant. this is not actually why. the main reason it's used, um, it turns out these cyclodextrin are really, like cylindrical molecules cylindrical structures, they're carbohydrates. and, they carry, what they call guest molecules. so, they're used in pharmaceutical delivery, drug delivery because these are um, carbohydrates they're very hydrophobic, um, they're used for flavor and odor enhancement. so that your food, smells better and tastes better. um they're used, to remove undesirable compounds like caffeine, so, this is one of these products because it's this, cylindrical structure, and it can carry guest molecules away. these guest molecules include undesirable molecules like, caffeine. um that some people think they don't, want in their food. trehalose, this guy over here, this one is i guess important. um, it's currently made from yeast. so, a lotta these were products that were not just sort of found in plants and we decided this was a good idea. these often come from other sources. trehalose, is made in yeast you can get yeast to overproduce it. it costs two hundred bucks a kilogram... to make trehalose. plants would make this much less expensively and the reason that we would want to make trehalose, is that, this is an additive that's added to foods. food additive. and it improves, this is gonna, be good for Aaron too, processed food and dried food, and it makes it, fresher, makes it smell fresher. and they taste fresher. i don't know how you can add a chemical that, this uh this is the chemical that gives freshness taste to your food. but, anyway that's what it does. um and so it'd be much less expensive to be able to produce it in plants than to have to isolate it out of the yeast, that it's currently being used in. phytase is down here. this is just some, results. currently, phytase is, is a feed phosphate. so this is a source of phosphate, that's fed to the little broiler chickens, the big broiler chickens whatever, so, if you feed, if you, use this as an additive, the reason to use it as an additive for the broiler chickens is to get them to grow bigger, you want bigger broiler chickens because that's what people buy. but y- you have to buy it as a chemical, an alternative is to take this fungus, and actually isolate the protein out of it. you can make it grow as much but it's much more difficult. and so what some, group, is doing, is actually using, is has e- has engineered, <COUGH> sorry. tobacco seeds, that will express, phytase. so it expresses the gene for phytase this is a fungal gene, it expresses the gene actually in the tobacco seeds, the tobacco seeds can be added to the chicken feed the chickens, eat these seeds, you don't have to add any chemical you don't have to isolate it from the fungus, and you can get the same level of growth, from the broiler chickens that you can get, by doing all the extra work. this is also good, because, if you could feed it to them, in their seeds, it decreases the amount that they excrete in their manure and if you grew, broiler chickens apparently that's an important consideration. i knew you were worried about that. <SS LAUGH> um, let's see, packaging them is, in these kinds of seeds is very safe, and stable. and nobody gets hurt. it's very good. um, don't have to purify it. don't have to do all these big isolation things to get it out of the fungus. you basically just let the plant, produce the enzyme. it's a very good thing for broiler chickens. okay. now, i have just a couple of other short examples that i wanna give you. and we'll talk about the transgenic plants. sweet potatoes, this guy at, Tuskegee University, has um, engineered sweet potatoes. sweet potatoes are an important crop in certain parts of the country. and what he's done with the sweet potatoes is increase protein content. essential amino acids most of you have heard of essential amino acids and you know that they're essential because that's what they're called, they're called essential because we can't make them, we have to eat the amino acids, so what this guy has done, is approve the in- improved the nutritional value of sweet potatoes, i wrote sweat potatoes sweet potatoes, by um, impro- increasing the amount of the essential amino acids, that these sweet potatoes produce. and they, he's improved, and increased the um, protein content in these guys. about two and a half to five fold increase. um, there's a very slight increase in the overall yield of the sweet potatoes. so somehow increasing the proteins made them very good. now he has just tested them on hamsters, and he found that hamsters, fed on these sweet potatoes for twenty-eight days, weighed fifty-six percent more than hamsters that were not fed these, transgenic sweet potatoes that were fed, control sweet potatoes for twenty-eight days. so, this guy has, begun at least the process of testing his, product on a nonhuman, subject at this point. the other example that, i think is somewhat interesting, because i had never considered it, is astaxanthin, <P :05> astaxanthin, i have no idea if that's the, appropriate pronounce, pronunciation, is a pigment. it's a caratinoid pigment. it's a natural pigment. it's found in toba- it's been, engineered, it's production has been engineered in tobacco. this, little guy, costs, it's a pigment, twenty-six hundred bucks a kilogram. currently, on the open market. what one would want this for is to tint your flowers. it's a nice little reddish orange color. also, farm-raised shrimp, you know all those shrimps you buy in the grocery store. farm-raised salmon. nobody wants to buy you know if they're pale right? so they add, astaxanthin to these guys so they make 'em nice and orange. and in fact, i've never seen this but, when i taught this last year somebody had seen it and and, nodded, that you can actually feed this stuff to chickens, if you feed this stuff to chickens it makes their, the yolk, a brilliant orange. why you want this vibrant orange in your egg yolk i don't know but somebody wants that so. <SS LAUGH> astaxanthin is_ this is the, um, currently extracted at this price, they get it from sea shells. or they synthesize it chemically so organic, syn- chemists or whatever have figured out a way to do this. basically, the way this guy who's who's um, engineered this in plants what he does, is he has found_ so beta-carotene, i'll do it up here. beta-carotene is a natural pigment, in plants, and what he did was he found, an enzyme, from, green algae, it just happens to be called, ketolase, which is about as useless general a term for an enzyme as you can imagine. and this, particular enzyme, is what was engineered into these tobacco, it makes a product, called, canthaxanthin. canthaxanthin, can serve as a, substrate for a number of plant enzymes. natural plant enzymes then, that can, convert, canthaxanthin, into astaxanthin, and make it a lot easier, to, um extract it. this stuff actually, will accumulate then in the nectaries in the flower. so, you can go by and just collect all these little flowers and get, the astaxanthin outta there and, feed it to the chickens and there you have, vibrant orange, egg yolks. so, i'm not sure that this is what i would spend the rest of my life working on, when it is something that somebody has done, to do, with plant biotechnology. now, why would you choose this besides money? um, microbial systems one could use, could engineer perhaps a microbial system to produce astaxanthin, or to produce trehalose or something like that. one of the problems with microbial systems tend to be from, prokaryotic systems they don't do post-translational modifications that are often necessary, and so, that, limited capacity for some of those modifications make them of limited use in many cases. often bacterial fermentation large-scale can be very expensive. often very expensive to, um isolate the aggregates many of you have tried to overexpress proteins and bacteria, and know that all you get are big fat aggregates and those aggregates aren't terribly useful, and they can spend a lotta money trying to solubilize those aggregates. transgenic animals are another possibility if you wanted to get, astaxanthin produced in sp- specific transgenic animals, um there tends to be more public concerns when we start dealing with transgenic, animals more ethical concerns. so, many, people are, avoiding_ i suppose there'd be a lot of ethical concerns if you wanted to use animals simply to, produce astaxanthin to feed to your chickens. um, plants tend to have lower upstream costs. it doesn't cost all that much actually to hire a bunch of plant biotechnologists to figure out how to do, something like this. the, costs downstream tend to be a little bit higher, so that's the actual growing of the crop and doing the, production of the grain or the fruit whatever the lower, the downstream products are. um, of course, it's important to ma- to design, your plant, uh system correctly. um and that's something that we're gonna talk about now for the rest of today. and then, on to Thursday. it's important that you have appropriate, high-level expression... if you want to make a product use a plant as a bioreactor you wanna make sure it's expressed at high levels, you're gonna have to make sure that you use the appropriate promoters, appropriate, leader sequences for, the expression of the gene. you hafta optimize codon usage plants tend to have a slightly different codon usage, than animals and microbes. hafta make sure that the, messenger R-N-A destabilizing sequences have been removed because, in many cases a pl- a, a messenger R-N-A that's stable in one system may not be stable in another system. sometimes it's important to use, other systems besides tobacco although tobacco worked here. tobacco was not useful for this guy down in Tuskegee because most people don't run around eating raw tobacco. and he wanted, to use a crop, that could be used in developing countries. so where he was improving the protein content. tobacco's not a good, choice for this guy that's developing edible vaccines for the exact same reason. but, tobacco's a very good place to do, use as a bioreactor to be able to, produce a large amount material that you wanna isolate. so your ch- choice of species is gonna be important. um, that's, important to make sure it goes to the right compartment, because if it's only, has to function in the chloroplast you have to make sure its got the targeting signal for that. um, and of course it has to be able to fold or, uh into the appropriate, ma- uh, structure. the final, post translational modifications all have to occur that was why we didn't wanna use, microbial systems so we need to make sure that it will occur in the plant, system. um, that's, probably enough on that. so that's sort of a, big general introduction, to il- and a lot of sort of small examples of ways in which plant biotechnology has been used. um, may or may not be some of the more important ways but at least, they do illustrate some of the variety, of plant biotechnology at this point. i wanna move on then, to, how one, actually does, gene transfer, and regenerate plants. what would you do? or how would you do this? assuming that you've now decided on, what product it is you want to overproduce in plants, or what, trait you're trying to capitalise on. um, basically, one starts with, a cloned gene of interest... this is assuming that you've done a lot of biology already and you know exactly what gene you wanna work for, or work with. some gene of interest that's gonna do your trait, that you're interested in. um, it's important then to have stable introduction of that gene... into some part of the plant. so a stable introduction of the foreign D-N-A... this can be accomplished by several different transformation techniques. transformation is the term for how you get a gene into an organism. the two that i wanna talk about are Agrobacterium-mediated transformations. <P :06> and, um, more of a vectorless, gene transfer. <P :08> particle bombardment is an example that we'll talk about in a minute electroporation. assuming you've gotten stable integration and it has to be stable that means that it doesn't go away if you go home for the night. it stays, introduced into this organism, into the plant... then you need to be able to regenerate, the whole plant... intact. this is not trivial this is ta- depending on the species that you're dealing with this has taken many, uh scientists many many years. one can now regenerate intact, uh plants from many species but not all of them. and then, assuming one can regenerate, a transgenic plant, it's important that your, gene that you're interested in, is expressed. so the expression of, the introduced gene... now these are the things, that one has to figure out. okay what am i going to, how am i gonna determine if the gene is expressed? what am i gonna assay? what is likely to go wrong? can we generate the plant? have i chosen an appropriate species? um, at this point obviously tobacco, soybean, corn, rice, most of the standard ones can be easil- relatively easily transformed. maybe not as easily as you might, expect but, it's not too bad. so essentially, one has to desid- design, an appropriate, um, transgenic construct. we've got a cloned gene, depending on, how we're gonna put it into the plant we design a construct, most of you are familiar with this and i'm actually not gonna give any specific examples. in a plasma based, transgenic, um strategy, you have some kind of a promoter <P :04> that promoter, is sort of the on-off switch for the expression of your gene of interest. this is your gene of interest. astaxanthin production perhaps, the trehalose production, the enzyme that's responsible for that maybe you're trying to produce that. the promoter the choice of promoter can be very important. it can be of plant origin. it might be of viral origin it might be of bacterial origin. and you might have to look up some of the papers that i've listed when you're doing your homework to figure out what kind of a promoter you want. you have to make sure that you know whether you want to provide, constitutive expression. do you want this gene on at all times? do you want that plant making your toxin? or making, the gene to make trehalose at all times? if you have a relatively innocuous, gene product it may be fine. or do you want it to have an induced expression? if you want contis- constitutive expression, put it under the control of a promoter that's on all the time that's completely on. do you want it expressed in all tissues? if you have something like you're increasing protein content well you probably want that in all tissues and you certainly want it in the tissues where you're gonna be eating it. but, you may not want it in all tissues. a toxin you may not want in all tissues. do you want environmentally inducible expression? a heat, shock, promoter for instance. that will only come out, under certain conditions. monocots are another problem but, i won't talk about that. specific monocot promoters for, for things in monocots. you also need to have and this is where, the antibiotic resistant often comes in, some kind of a, a genetic marker. what's gonna be your marker? how are you gonna know, if your, transformation has been successful? how will you know if this plasmid, has been transferred, into this new plant? you need, some kind of a genetic marker. antibiotic resistance is one that's most common and has gotten the most public information. herbicide resistance is another possibility. you might be able to use a marker for herbicide resistance. grow up all of your transgenic plants on a plate, apply that herbicide, only those that have been transformed are gonna live and everybody else is gonna die. and then you can take those transgenic plants and go from there. you have to be able to have some way to identify your transgene. there's what the point is there. okay. so, what i've assumed here is that you're gonna introduce your your D-N-A into the plant, using a plasmid. a plasmid just refers to this ci- circular piece of D-N-A. you can also introduce, the D-N-A in the plants as naked D-N-A. so this is a plasmid... there's your gene of interest. you're being very specific about how it's going to be expressed, where it's gonna be expressed. but you might, want to simply, use the naked D-N-A, for your gene of interest. and put that into plants. <P :04> now, the first page on the handout from today... believe it or not <P :07> should show you... this figure. which i don't find terribly useful but some people might... these are different um, choices, one has to make when designing transgenic plants. what exactly are you gonna do? what are you gonna transform? the most traditional way and the way that i, when i've made my transgenic plants in the past, is basically this pathway... that's the most usual or traditional way. you take a tissue explant that means to take some part of the plant, and cut it out. you can make callus, callus is is sort of like cauliflower, it's what it looks like. it's nongreen it's completely undifferentiated and, under certain hormonal conditions you can get, this callus. you can find callus on here i think other places. and then add, here's where you're adding the D-N-A. so these blobs, are actually adding your transgene, make up a whole new plant. um, we've used root pieces cut up little roots, transfo- transform those. most people now use vacuum infiltration and actually take the, uh a very young plant and vacuum infiltrate so cause vacuum to uh, get the D-N-A sucked up through the le- the fruits there. um, hormones are involved in most of these steps. um, not a lot to say here. this_ the open circlers(sic) are Agro-mediated transformations. the closed circles, so this is using Agro Agro is a, bacterium that we'll talk about in a second. this is using, particle gun, bombardment this is where you use naked D-N-A bombardment <P :05> this all happens because plants have this, relatively cool um, principle or, characteristic that they're totipotent. what that means is that any single cell of a plant, at any time during its development, can be dedifferentiated. and then under certain, hormones will redifferentiate into a full plant. unlike you and i, because at full development, it is very difficult to take a single adult cell, dedifferentiate that into, an undifferentiated cell and regenerate a whole organism. plants however, that can be done and that's the basis for all of these regeneration and regenerative processes. we basically take some kind of a plant tissue, under hormonal, conditions you could make a callus, and then regenerate a plant from that. um <P :04> that's probably good enough. the hard thing, about doing these transformations either using bacteria, or using a particle gun is you've got this piece of D-N-A, you've gotta get it through the cell wall, into the plant as a whole. so, if we've got a cell. (i'll put this one up.) a plant cell has a relatively, hard cell wall. and you're trying to get that, piece of D-N-A somehow, through the cell wall not only, across the cell wall and the cell membrane, but actually into the nucleus of the cell, where it then will be expressed. and so, that can be pretty difficult. um, one way to do it is through this po- particle gun bombardment. um, another way and i'll talk about that in just a second another way is, using high voltage electroporation. and, el- making the cells open up long enough to get the D-N-A in. particle gun bombardment is a lot like what it sounds. you take a little bit of naked D-N-A, and you, precipitate on to like gold particles or tungsten particles. and you literally shoot it into the plant cell. it's a, it's like a gun, it's not exactly a gun but it's like a gun. and they used to use gunpowder to shoot, the D-N-A into the cells. um, now they tend to use more like uh, a helium, tank, to to shoot it in. the trick and i- s- it is a bit of a trick is to, get just the right amount of Tungsten or, gunpow- gunpowder, that's got your D-N-A coated on it, so that it gets into the cell and it doesn't just go right through the tissue. so, it's important, to_ you have all these variables to test but, because this D-N-A, and this is the D-N-A out here, coming into the cell, and you think of just shooting a gun at a cell you know what are the chances you're actually gonna land in the cell without going all the way through? and so there's a lotta work that's been done, to try to do that. this actually th- using helium um, works, better than using gunpowder now um, and most of the companies that sell these guns these, these uh bombardment systems use helium. um, this has been very helpful in developing, monocot transformations for in- example. um let's see. one advantage is you can shoot the D-N-A into almost any tissue. you really only need a little bit of tissue so, like if you t- if you imagine taking a petri dish and that's what they do, you can take, an embryo for instance, and shoot the D-N-A into an embryo. i mean D-N-A's not very big right? you just have to get it into a single cell. but you can take little pieces of leaves, and shoot up a whole, plateful of that, and then, allow it to express and become a callus and then grow up a whole plant from that. it's been really helpful, like with growing up certain, um monocot tissues. not so great for dicots for, a number of other reasons. dicot systems they tend, we tend to be using the Agrobacterium-based systems. um, i think the next page on your handout kind of just summarizes an example, of using Agrobacterium-based system <P :04> basically, what this thing summarizes is just an old example this is one way they were using, E-P-S-P synthase this is one of the genes involved, in the glyphosate resistance that's uh, Roundup tolerance. and what they did was they took the gene out of a plant, put it, onto a plasmid, so now they have, the bacterial plasmid, with the plant gene on it. put that bacterial plasmid with the plant gene on it, back into, the Agrobacterium. Agrobacterium was a natural soil bacteria that causes coun- crown gall tumors on plants, cu- i- normally, if you go out in nature, you'll find galls tumors on plants and it's caused by a bacterium, this is one of the bacteria that does it. so when we engineer, this bacteria now it can express, the gene that we're interested in, put it back into a plant cell. the way that one gets Agrobacterium into a plant cell i'll show you in just a second, allow those cells to grow up, and then you can regenerate whole plants, that are transgenic that express, the gene in this case the E-P-S-P gene. and th- and in that case those plants are now, confer a a glyphosate resistant or Roundup resistance that, the nontransgenic plants would not be. and so they're, able to withstand spraying, with glyphosate. um, so in this case they're able to select, the, the cells, that have taken up the plant gene, because they can put, the herbicide on these little plates, and then grow up whole plants that're herbicide resistant. okay. the, details although, you certainly don't need to know them but some people are somewhat interested in, how one exactly gets the D-N-A from the bacterial cell, into the plant cell. so i have a little picture of that here. and it's really kinda cool. it happens from wounding right? you gotta imagine these roots are growing around in the soil, and they're gonna get wounded, at some point. when the, plant gets wounded, it puts out some of these wound signals. and so it actually s- puts out an exudate. the exudate itself, attracts the bacterium, so this is the bacterial cell here. and Agrobacterium does grow naturally, in, in the soil. so here's your, bacterium, here's a root. that's been wounded over here, putting out this signal, maybe you wanna put the whole root over there. when it puts out this signal, this happens to be, a sugar receptor, that's on the surface of the bacterium, and it causes the expression, of a whole system of genes. the vir genes the virulence genes you can kind of, it sounds sort of like an infection just to have virulence. this is the, D-N-A that's in the Agrobacterium, anyway so when this wounding happens you're inducing the production, of a whole bunch of genes, it in it um, one of the sets of genes, or one of the plasmids that is expressed in this bacteria, is the T-I plasmid. it's a tumor, inducing, plasmid. that's why it's called T-I... and, so this is the actual plasmid where we would be putting our foreign D-N-A. where we would be putting our gene of interest, is on that T-I plasmid. the T-I plasmid, expresses then what's called, the T-D-N-A... the T-D-N-A is the D-N-A that's going to be transferred. <P :05> okay so your gene that you're expressing that foreign gene is on the plasmid. when that plasmid is expressed when those genes are expressed it's part of the T-D-N-A. some of the other genes that the Agrobacterium are expressing at that time, will coat the D, T-D-N-A so your T-D-N-A is now in here, it's being coated by these vir genes, these vir genes now come over here and interact with some more vir genes and, you can kind of imagine, this whole snake now here's your T-D-N-A inside, it's got the foreign gene you're interested in, is actually now being inserted into the plant cell. so the vir genes_ there's a whole buncha different ones. are the ones that're actually responsible for transferring, the D-N-A, into this other cell. just because we've gotten into the other cell, now remember i said we had to get into the nucleus of the other cell. and that's where these Vir-E-two and Vir-D-two they're kinda cool because they actually have, nuclear localization signals on these proteins. so these genes have, made protein, in the bacterium, they're coating the transfer D-N-A, the D-N-A that's being transferred into the plant cell, your, foreign D-N-A now is coated with some bacterial proteins that have nuclear localization signals, which allows it to go through the nuclear pore complex. that's what the N-P-C is. and, you can integrate now, your f- the foreign gene, or the, T-D-N-A, into the plant's cell. what they're showing here is, under normal conditions, what's carried on that transferred D-N-A, makes genes for these opines. these are actually nutrients for the Agrobacterium i mean why would ena- in nature why would a plant do this? and it's because, or why would the bacterium do this? they actually then get their own nutrition, the plant comes here, makes the nutrition to feed the bacterium. it's these plant hormones that're produced by the soil bacterium, that result in tumor production. so this is where the tumor part would come from. <P :04> this is, nutrients, for the bacterium. <P :05> now in most of the vectors that you would use in the lab, you don't actually make these, either of these because we're using fake bac- in a way sort of fake bacterium we've us- we're using engineered bacteria. so that instead of, making, the hormones to make tumors, which wouldn't be very good for us trying to make plants, with tumors, and we don't really care if we feed the Agrobacterium, the genes that we're expressing the gene that we're integrating into the plant cell is our foreign gene of interest. so that's how one gets, a tr- a, a gene expressed, in another plant cell. alright relatively quickly, i only have about five minutes that'll be almost enough time. i wanna go over at least a little bit of this tomato fruit ripening stuff. <P :06> so, this was the first type of um transgenic plant products that actually reached the market, and became available commercially. um, i know that Flavr-Savr is not being commercially marketed anymore i don't know about Endless Summer i, honestly, don't pay any attention to it. it wouldn't bother me at all to eat Endless Summer tomatoes. fruit ripening has been a problem some of you i know are from, California and are probably very well aware of problems with growing tomatoes. let me just tell you a little bit about it and this is the next page of your, handout so you don't have to write any of this down. and i don't have to write any down i can read it to you. basically, tomato fruit ripening involves all kind of physiological changes and people have been studying this for, many many years. what happens? how do tomatoes get ripe? well obviously color changes. think of a tomato, a nonripe tomato is green, you know it's ripe when it's red. there's a lotta pigmentation changes that're going on. the texture changes. an unripe tomato is hard a ripe tomato, is soft. so there's a lotta cell wall and structural changes that are going on. and of course, the flavor and the aroma of the tomato change when it goes from unripe to ripe. so they kn- understand, a lot about the physiology in all of this. they know that the hormone ethylene, is what induces fruit ripening, and that as fruit ripens it produces ethylene and so it's kind of a cycle so the ethylene, produces ripening, as it ripens then, you need more ethylene, and so this is the cycle that, the hormone that's responsible for it. well that problem is if any of you ever, have grown stuff in your, garden you know that ripened fruit, gets soft, and it's really easily damaged when you harvest it. when you try to process it and if you think about doing this at a commercial level this could be a real problem, for those of us who wish we had fresh tomatoes out here in Michigan right now, would like for them to be able to ship fresh tomatoes from California out here and it's not easy at this point in, this season. so one of the solutions was to harvest the fruit, when they were immature, and then, as they're being transported in the truck, to give 'em ethylene, and allow, ripening to occur then. sounds okay you can a ri- you can harvest a green, fruit that's still pretty hard. it's gonna be, um not so much damaged give it some ethylene. the problem is the fact, that if we're harvesting these, prematurely, they really don't taste so much like vine-ripened fruit. they're easier to harvest but they don't taste like it so they're, the solution the biotech solution was so, okay let's just inhibit, the ripening-related genes. and like i said they'd been studying this for a number of years, the next page in your handout goes through what some of those ripening, related genes are. <P :06> this shows the ethylene pathway, of tomato fruit ripening, lot of people spent a lot of years studying this. starts out with methionine which is just at the top of the page you've got it on your handout, and you can see there's a number of enzymes that i've marked in green here. polygalacturonase A-C-C synthase A-C-C deaminase A-C-C oxidase, these are just um, all related to this particular, component in the pathway. we know that downstream, as you make ethylene, you need all these enzymes to make ethylene. ethylene is the hormone of fruit ripening. and that that leads to, flavor and aroma production that you get during ripening cell wall softening that you get during ripening and the appropriate pigment synthesis that you get during ripening. so what they exploited when they made, these transgenic tomatoes was two things. the first thing they decided to do, was lef- s- was to... um, prevent production, of polygalacturonase, P-G. because if, if they could do that, then they wouldn't get, the softening of the walls. and, the other thing then, that and that was what resulted in the Flavr-Savr. what, the other thing they decided to do was to prevent ripening in the first place and that was what happened with, Endless Summer. so let me just talk about those a little bit more. let's see... so one of the, the ways they did this, was by, what's called antisense R-N-A... what antisense R-N-A is is that, taking, a single-stranded R-N-A for the gene in this case making polygalacturonase, taking and turning it around so antisense making it ne- n- the other direction. so i just took my gene for polygalacturonase, i turned it around, and i make antisense R-N-A most of you know that ant- that R-N-A is normally single-stranded. and if you take, a sense R-N-A and an antisense R-N-A, they make a duplex. this duplex R-N-A. and, that's how they get, they use antisense technology and they're actually doing this now in a lot of drug-based therapies so, many of you may have heard of this. to make duplex R-N-A, and duplex R-N-A or this antisense R-N-A, then inhibits the production of a gene. they think that the main reason for this is that the R-N-A duplex is degraded. that double-stranded R-N-A in a cell is recognized as foreign R-N-A. there's other possibilities of what's happening. it may be not, getting R-N-A splicing, it may be that you're not getting good transport to the cytoplasm and it may be that you're not getting, translation so. the whole point and the reason i present this is just for, some of you if you've never heard of antisense R-N-A, you can get some idea of what it is and it's basically taking the gene, turning it around, expressing it in the plant so that it binds up all of the single stranded, endogenous R-N-A, and prevents expression or, final product production. what they did then, was, to make the Flavr-Savr, was to use antisense of, the polygalacturonase gene. that next page in your handout, almost the last page in your handout, shows a couple of things. the very top is just about what i was just telling you only a little bit differently. the Yang cycle refers to, the Yang just because of Shang Fa Yang who was this, really amazing scientist who studied ethylene production for many many years he retired, he was at U-C Davis. but, here's A-C-C synthase. this is the um, this is one of the genes that they, <COUGH> sorry, that they used antisense to prevent production of ethylene A-C-C deaminase A-C-C oxidase, polygalacturonase was produced. this is down here, if we don't, produce as much of that, then we wouldn't get the f- the the um, the problems with the wall. and what the bottom here shows, is you have to just look at basically this is activity, versus R-N-A, but both this is, the test. if they add one antisense gene, they get neither activity or two antisense genes, nor messenger R-N-A production for paly- polygalacturonase. this is just the proof. control, a nontransgene, control nontransgene, control nontransgene and you can tell that, if the antisense is being expressed, they weren't getting the production. it sounded like a really great idea, that, they could, you know d- decrease production of this, um, and they found out that they could do this. you can see, or you can imagine, that if we just, affect this we're not gonna change color. we're not gonna affect any of these other genes and so you would get a beautiful red tomato. it would have, it would be almost like it was ripe enough, but it would have, not very much degradation, because of the, there wouldn't be any polygalacturonase, and so that was a really good thing that, Calgene was the company in California they marketed it as Flavr-Savr, but they found out that, by inhibiting the rotting, that um, it was able to, uh, um ripen on the vine, and they found out that they had to take it, it didn't work. they had to take it out of the ma- off the market, because, conventional tomato picking machines, um, damaged the fruits. and so it didn't work at all. it was a great idea, but it didn't work in the end. when they got it to, commercial production, and the, the way they were, picking tomatoes at the time, didn't work, and so they had to go back. and that was when they went back and, um, did, the other kind they got, they used antisense on A-C-C synthase, this is where, they came up with what's called Endless Summer... so they transgenic_ they used transgenic plants that expressed, an antisense gene for A-C-C synthase, and, then that would prevent production of ethylene, and they could use then ethylene to actually trigger they could f- they could then later control, when they wanted to trigger, um, ethylene production in fruit ripening and so that was, that actually worked for them, the last page of your handout is just, a little piece from a brochure that D-NAP put out, D-NAP is the name of the company, that developed, Endless Summer tomatoes. and it tells a little bit about it. and that at the bottom it tells a little bit about, D-NAP's technology. this was a little three fold brochure that they put out. and they actually had an order form i didn't, include the order form it's very old now. and it was extremely expensive this was when they first came out, a a f- actually a few years ago. and i don't_ does anybody even know if they're still selling these things? e- i don't know do you know? no? i don't know. so, that's just that was the first set, of transgenic plants that came to commercial production. um, so what i wanna do then on Thursday, is talk a little bit more about metabolic engineering and um, some m- biomedical, purd- products that were produced, um the edible vaccines, and um this blood clo- this antiblood clotter hirudin that's made on, um, oil bodies actually. 
<P :13> 
S3: so what was problem with with picking them before they're ripe? 
S1: problem was they got, um soft. so that they, they got softer than they wanted them to. and so, they weren't they just, they split. and they didn't expect that. <S3 LAUGH> i don't know why. 
S3: i guess, i don't know. it just seems, to me that like picking them early wouldn't be that different than, shutting off the A-C-C synthase 
S1: well the picking you mean oh why don't they now. the problem with picking early that's what they do now. i mean that's what you're getting in [S3: yeah ] in the stores now. and people, think it tastes gross so, so, 
S3: well can't, can't they, expose them ethylene then after that? and if it do- that doesn't (xx) 
S1: if you can stop ethylene production under, so that way you would have to make it so it's not a normal, [S3: so you just want to stop the ethylene until you get, ] a normal pathway. that's right so that you can control it. [S3: okay ] so that's what the Endless Summer did was stop it so that they could, control when they, actually ripened. otherwise they'd probably all ripen when they hit Wyoming or soemthing before they got all the way out here. 
S3: they'd have to like have some sort of ethylene, sequestering (xx) 
S1: well they ha- you know the tru- they have the trucks the trucks are specialized for ethylene production. [S3: oh yeah ] and they have like little compartments that, spray out i suppose it's like a little gassing truck or something. 
S4: i heard that they used a, (bis toxins) to make sure they're flavored the right way. they made like, this, great tomato but then they used this
S1: but they used the wrong thing as their starting material. 
S4: right they used the, these packing [S1: well that, yeah so that, you know ] tomatoes to start with
S1: maybe that was part of the problem then too, yeah. <P :04> so more than you ever wanted to know about, plant biotechnology right? 
S4: you know what? 
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