9UNP0166SD_Scotty Woody and Melissa Mohn_WordPress

- Welcome to Space Place, here on South Park Street in Madison for today's PLATO Frontiers in Life Sciences presentation. We're going to hear from Scott Woody and from Melissa Mohn, who both work in the Department of Biochemistry at UW-Madison in Rick Amasino's lab. They'll be talking about how they use these rapid cycling Brassicas to figure out how plants grow. And for those of us who have a horticultural bent, also how we can grow plants better. So please join me in welcoming Melissa and Scott to PLATO Frontiers in Life Sciences. 
(applause)
 
- Thank you. Thank you all for coming today. 

It's a beautiful day out there. And I appreciate your devotion to this. I hope that we can make it worth your while. As Tom mentioned, my name is Scott Woody, and I work with Rick Amasino in the Department of Biochemistry at UW-Madison. We've been working for the last couple of years on a program that's sponsored by the Howard Hughes Medical Institute and their scholars program. The intent of the program is to improve and involve undergraduates in education. And our particular focus is on providing the tools that we think are necessary, in order to improve and augment undergrad and K-12 education in genetics. So this is a time when, you know, with gene therapy coming on the horizon, with stem cells, with personalized medicine, there will become a day very shortly when every one of us will have our own DNA sequence which will tell us something about our tendency or our likelihood of contracting certain diseases. In many ways, it's more important than ever for people to understand what is genetics. What is a gene? 

What is a genome? What's a chromosome? What is a disease that is genetically inherited? And yet, we find that particularly in K-12 settings, but even in undergraduates that the concept of genetics are not well understood. And, in fact, as I was preparing for this talk today in the current issue of the journal "Genetics," there's a study sponsored by the Genetic Society of America, which cites that, in an essay contest, evaluating the responses provided by high school students to several rather general genetics questions, and then judged for the top papers, there were misconceptions all through the essays of students who don't understand that one gene rarely controls a given trait. The students don't understand the patterns of inheritance and the ways that phenotypes are even in organisms. They don't understand what a gene is. All right? Without understanding what a gene is, students are unable to understand things like evolution. And so we're constantly faced with this whack-a-mole game, where religious concepts are trying to be introduced into the classroom under the guise of science. 

Evolution is given short shrift in the classroom. It's like the third rail of a K-12 educational curriculum. Teachers don't want to touch it. The teachers themselves are rarely well educated and well versed in genetics. And so the teaching is usually very rote and very brief. And we think that all of these things can be rectified, or at least helped, if we provide the teachers and the students with a set of tools that they can actually use to learn genetics. Now I think that you can recall from perhaps your education, the experiments of Gregor Mendel. They are usually taught in this sort of fashion. And then the students are asked to predict what the phenotype is of the resulting progeny from this. You draw out your square. 

and say this parent can produce these gametes. Say that's the male. And say this parent will serve as the female. And so you get the HET wild type and wild type. Well, that's fine and it's correct. But it goes in one ear and out of other. Right? We think that the most effective way that people learn is by doing. And we think the most effective teachers are the ones who have actually done science. So we want to provide them the tools in order to do so. 

We think that there are very simple exercises that students and teachers can do in the classroom to track patterns of inheritance, to understand the relationship between genotype and phenotype. Essentially, to recapitulate Gregor Mendel's experiments that you might recall. He worked with pea plants of varying colors and sizes, etc. So we're developing a line of Brassica rapa that is a member of a large family of plants that include bok choy, Chinese cabbage, and even turnips. You see these in your grocery store all the time. But they're morphologically very diverse. I mean, a turnip, Chinese cabbage. Quite different. Same species. This particular version is one that's known as a rapid cycling Brassica rapa. 

Some of you might be familiar with this from the Wisconsin Fast Plants program that's been run by Paul Williams and Dan Lofter out of Science House at the UW. Paul developed the Fast Plants Program about 25 years ago. And it so far, has worked to get that into the classroom where the plants serve as a model for plants, and organismal growth and development. Students can easily plant them out, watch them grow, measure them. And I've been in fourth grade classrooms doing this, and the kids just love it. They have their own plant. They name them. They do experiments on the plants, so it's usually set up teaching them the elements of the control. A controlled experiment. So you have your control, and then you have your experimental. 

Students will do things, such as feed the experimental ones colored water and asked whether they changed colors. They don't. The bad experiments are when they want to grow them on milk, which over the course of a few weeks is not such a good experiment. But nonetheless, it introduces students to the concept of measurement, observation, hypothesis testing and interpretation. They're taking data all the time and they'll plot those data out. And they feel like scientists. And they're only able to get that feeling not by being told how to do an experiment. They get that feeling by doing the experiment. And so we're providing and generating the tools necessary to do that. Now there are actually, if our interest is only to teach genetics, there are, you know, why reinvent the wheel. 

There are model systems out there, for which they are plenty of genetic resources. There's a rich history of genetics, and plays in integral role in understanding the fundamental laws of inheritance. There's vast collections of mutants. There's terrific genomic tools. The entire sequence is known. Right? Well, if you're a teacher, how are you going to maintain your stocks? Right? You're going to have to hire it out. You're going to have to have a technician that will recycle them and re-culture them periodically. 

The students themselves, I mean, fourth graders handling these? You have to etherize them in order to examine them under a microscope. When they come out of the ether, they're likely to fly away, so there goes your experiment. 
(laughter)
 Another one, baker's yeast. Terrific genetics. It's microscopic. You can see it in a colony. But as an organism, you hardly ever see students try to name their sacromyces. Right? 

And again, trying to maintain sterile technique, and have those experiments turn out right proves as quite a challenge, and even preserving the stocks between semesters. When teachers who are under tight schedule constraints don't have time to be re-culturing, to streak things out, to pour plates and get the laboratory set up. So we think that plants are an ideal genetic organism, or an ideal organism for teaching genetics in the K-12 and undergraduate classrooms. It takes minimal input to grow a plant. A little dirt, some light, some water, some seeds and you're ready to go. When the class period is over, or your semester module is over you harvest the seed, so you take whatever leftover seeds you have, and put them in a drawer until you need them again. So plants are ideal. And in fact, Paul Williams' Wisconsin Fast Plants have been ideal for growth and development. But for reasons I'll explain in a moment, they have limited utility for studies in genetics. Yes, all right. 

Let's go there. So Wisconsin Fast Plants are of the species Brassica rapa. The virtues of Wisconsin Fast Plants are rapid generation time, approximately seven weeks from seed to seed. So if you're going to teach a module, an exercise over the course of say an academic quarter, or if you want to go a couple of generations over the course of the semester, it's feasible. You can do this. They're readily accessible. And by that, I mean you cannot only get your hands on the seeds they're distributed by North Carolina or Carolina Biological Supply. And so teachers can go there and buy the accessories that they need. It's very easy to grow under minimal lighting and culture conditions, all specialized in finding very economical and simple ways for people to grow these organisms. They're also readily accessible, as you'll see, in terms of their parts. 

I'm not much of an artist, so you'll have to forgive me. You'll see the flowers are made up of whorls of organs. There's petals, stamens and carpels. The carpel makes the pistil. You'll see all this in a second. So those are all large enough to be readily manipulated, as you'll see in a second. It has the additional allure of allowing kids to, well engage in sex in the classroom. And it doesn't get any better than that. 
(laughter)
 This version of Brassica rapa is one that we call B3. 

It differs from economical or the Wisconsin Fast Plant variety in one important regard. So does anyone know what self-incompatibility means? It's a mechanism which is widely found in nature among plant species that enforces an outcrossing reproductive strategy on the plant. Now if you recall, the birds and the bees, pollen grains are deposited on the pistil. The pollen grains in a successful pollination will form an infection thread that will grow down into the pistil, into the ovule and effect fertilization. Self-incompatibility mechanisms prevent self-fertilization. And they do so through a mechanism. We don't have to go into in too much detail, but you can think of it as kind of a lock and key, sort of self-recognition mechanism. So that when pollen produced by the same plant from the anthers is deposited on the pistil, the plant has the ability to recognize that as my own pollen, and thereby prevent germination of that pollen grain, formation of an infection thread and therefore preventing fertilization. So it enforces an outcrossing reproductive strategy, maintains high levels of genetic diversity in the population, because you're always mixing. 

Every time an individual undergoes self-fertilization, heterozygosity in the gene pool or in the genome is decreased by one-half. So if you recall in diploid organisms, if you allow this to represent a gene symbol, the allele symbol. This is a homozygote. With one allele and a different allele on the homologous chromosome. It's a heterozygote. And you can have the other homozygous class as well. By limiting self-fertilization, you maintain heterozygosity in the population and genetic diversity. However, in terms of generating mutants, as we want to do in order to teach genetics, this is the problem. Right? Because what we want to be able to do is to readily provide plants to students for which you can homozygous alleles. 

So you can examine the phenotype of these homozygotes, as well as the heterozygotes. Right? Absence, or in the presence of self-incompatibility mechanisms you have to do these massive sib matings in order to do genetics. It's not impossible. But it's not at all straightforward. So several years ago, Rick Amasino, seeing the market penetration, if you will, of Paul Williams' Fast Plant program, set about to derive a line of Brassica rapa which maintains most, or all of its favorable characteristics of rapid generation time, minimal input, but eliminates the self-incompatibility mechanism. So we can propagate these lines as homozygotes, essentially. So he began a breeding program that involved three parents. Wisconsin Fast Plant, which is self-incompatible, and two other rapid cycling Brassica rapas, which just through chance have lost the self-incompatibility mechanism. Now, you would think, well why didn't you just use these? 

Well there's more to self-compatibility and self-fertility than simply losing the genetic self-incompatibility mechanism. And here's where-- In selfing individuals, there are developmental timing issues that are required for self-fertility. Recognized that the anthers have to mature, and to begin shedding pollen, at an appropriate time. And that appropriate time is when the pistil is receptive for pollen grains to land and germinate. So there's this beautiful-- In selfing organisms, it's great to watch. The timing is exquisite. And so the pistil grows up and the anthers mature, and then the pistil grows through and pollen is shed to saturation on the pistil. Right? In outcrossing individuals, often the anthers are oriented outwards. They mature at different times. 

They're relying typically on insect pollinators, rather than self-fertility. So there's more to self-fertility than loss of self-incompatibility. So Rick began a breeding program by intercrossing these three lines. This is self-compatible. Cross Fast Plants by series. Series by IMB, and Fast Plants by IMB. Grew up the resulting seeds, and then intercrossed them again mixing up the germ-- from all these lines. And then began a selecting breeding process each generation choosing the fastest plants, the most vigorous plants. Until, after nine generations, we arrive at a plant that we call B3. Ultimately, we'll give this a better name. 

Why don't you guys pull up a chair. Come around here. I'm going to put you to work. We could set up another table over here. Pull up a chair. 
(indistinct talking)
 
- There's six of them... 
(laughter)

- All right, so what we're going to do, is we're going to walk through a couple of things here. So this is the wild type. This is B3. It's about three days slower to flower than is Wisconsin Fast Plant. That is, it flowers after about 18 days after planting rather than about 15 in Fast Plant. And it's about a week later to maturity. So over all, it's about a week to ten days slower in terms of seed to seed generation time than Fast Plant. But certainly, sufficiently fast that it's useful in the classroom. This line is self-compatible and moderately self-fertile. You can see that these on the older flowers have begun to elongate due to self-pollination. 

You would never see that on a Fast Plant. The genetic self-incompatibility would prevent self-fertilization. Now, take some forceps. I'm going to ask you-- I'll show you a real quick one. So it's moderately self-fertile. But it's not fully self-fertile. That is, if you pollinate, if you hand pollinate, you'll get more seeds than you will if you don't hand pollinate. And we actually think that's a virtue, because we want students and teachers to be pollinating, right? We want them to use forceps. Or, as Paul Williams often does, bee sticks, to reach in and grab some anthers, turn them over and then deposit the pollen on the pistil. 

Alternatively, if you're just doing selfing, you can take an entire flower with its six anthers, and just tap, tap, tap, tap, tap, tap, tap, tap, tap. Right? Why don't you try it? Try it. Well, actually-- 
(inaudible)
 I think this will be fine. Does that work? If you like, you can use the magnifying glass. There it is. 

(laughter)
 Actually, as I was mentioning, Paul uses bee sticks. He orders honeybees, and you take the thorax, and you glue it to the end of a tooth pick. And then when the students in the classrooms-- Oh, they love it! You should see, you know, the teachers that I work with say, "Okay come on, buzz, buzz." And they buzz and tap, tap. But what a wonderful way to make kids think about the roll of pollinators and fertilization. 
(inaudible)
 Well, you know, it depends on the stage of the flower, the anthers sometimes are shedding vigorously sometimes not so vigorously. The nice thing is when you're going from flower to flower like that, often you're picking up more pollen, just as a bee does. 

Right? So even though you may think you're depleting the pollen on the anthers that you started with, pollen is accumulating on all these petals so you're still-- 
(inaudible)
 Yep. Well you should see it after. These flowers are probably five times of the size. 
(laughter)
 
(inaudible)

- All right. So if we came back, and you're welcome to stop by the lab, you can see that this will be extended out about that far. It will each fully fertilize, the seed pods will contain between 15 and 20 seeds. So each plant, itself, if you fertilize to saturation, pollinate to saturation, will produce about 100 to 150-200 seeds, or so. The nice thing is that each seed, because the B3 line is highly inbred, it's homozygous at all loci, effectively. So every progeny seed resulting from self-pollination will be genotypically, that is genetically, exactly the same as the parent. Often, if you self-pollinate an otherwise outcrossing line, because of the heterozygosity that exists in the genome, in the genetic background, you'll get what's known as inbreeding depression. So recall that if you have a large number of loci, which are in the heterozygous condition, where the big "A" represents the wild type, or let's say the dominant allele, dominant and recessive, does that come back to you? Typically, that refers to the functional, the normal allele. Whereas, the little "a" refers to a mutant or an allelic variant. 

Most mutations do bad things to genes. They disable gene function. But in diploid organisms, that function can be masked, that lack of function can be masked by the presence of the wild type or dominant allele. 
(inaudible)
 No. 
(inaudible)
 Well, we'll get it. We'll get there. My plants don't have eyes. But some plants have ears. 

- Tell us about that. 
(laughter)
 
- You've seen corn! 
- Oh! 
- So let's walk through this. Let's say you selfed this individual that's heterozygous at this loci. We can draw a Punnet square. Punnet square. 
- Is Mr. 

Punnet? 
- Mr. Punnet was a geneticist who developed, shortly after the turn of the century, not this century, but the prior century, at the dawn of genetics, after Mendelian genetics have been rediscovered, who helped think about, and this has become the standard way of thinking about genetics, and of understanding how crosses are going to work out. You can predict that an individual who is heterozygous is going to produce, in its gametes, one-half of the gametes will carry this dominant, the big "A" allele at a particular locus in the genome, and the other half will carry the little "a" allele. Likewise, on the female side of the mating, half of the ovules, let's say in a plant will inherit, that is will receive from the products of myosis a big "A" allele, and the other half will be little "a". You assume random combinations between pollen and ovule. And so you see that of all-- Of all of the resulting zygotes, all of the resulting progeny, one-fourth will be homozygous, big "A", big "A". One-fourth will be homozygous little "a", little "a". And one-fourth, plus one-fourth or one-half will be heterozygous just as the parents were. Imagine that the little "a", little "a" homozygous has a mutation that results in a visible phenotype. 

Or let's say knocks out the ability of the plant to do photosynthesis. That's not a good thing. So these individuals will be lost in the population. They'll be highly selected against. But when you try to self an ordinarily outcrossing individual, there are say, 25,000 genes in the plant genome, 50% of which exist ordinarily in the heterozygous condition. Now you self, and you bring together all those homozygous conditions that were masked, but are suddenly unmasked in the homozygous. And so you start to see what's known as inbreeding depression. These cumulative effect of homozygosing these deleterious, or harmful mutant alleles. Okay? Through the selective breeding program, Rick arrived at a version of Brassica rapa, which is genetically uniform. 

It's homozygous at all loci. And it's evident that the loci, the collections of alleles that it has, are ones that allow for a perfectly functioning plant. So generation after generation after generation, you recapitulate the same phenotype that you start with, without suffering inbreeding depression. This also simplifies the process of generating mutants. That's what I'm going to talk about now. Our goal is to generate a set of tools so students can look at the transmission of genetically determined traits through successive generations. Can they derive the laws through their own experiments, the laws of inheritance, just as Gregor Mendel did back in the late 1800s? We think they can. We think it's not beyond them. But first we have to generate the mutants. 

Beginning about last year, we undertook a mutagenesis. Once we arrived at a plant that we thought, well, you know, this is pretty good. It's pretty vigorous. It looks just like Paul William's Fast Plant, which is useful for us, because Paul's plants are in classrooms across the country and across the world. They're used in, you know, over 88 countries in the world. So if teachers who might be hesitant, and it's going to be a tough sell to get them to take on any new sort of tool, they're going to say well, that's Fast Plant, I know how to grow that. It gives them confidence. It lowers the activation energy, if you will, to getting them to considering using it. So we arrived at this plant. It's self-compatible. 

It's reasonably self-fertile. Now the task is to generate a large collection of mutants. We need to induce changes in the DNA sequence, which will effect the gene. When that gene, that mutant gene is made homozygous, we hope to observe visible phenotypes. A change in the appearance of the plant that's genetically determined. We do this by a process called mutagenesis. By exposing seeds to a chemical called EMS, ethyl methanesulfonate. What EMS does is it acts as an alkylating agent, so it changes Gs, which you might recall ordinarily pair complementarily with C. But by attaching an alkyl group, to the G changes its base pairing property, so that upon replication-- Upon DNA replication, this alkylated G will pair like A. And so, direct insertion of a T on the opposite strand. 

C still pairs like C, so it will pair with G. Now we go one more round of DNA replication, and we have this T calling for an A. So, we've effectively changed this G-C based pair that's embedded, and let's say it's in a coding region of a gene. We changed this G-C to an A-T based pair. We've induced a mutation. And the question is, does that result in a phenotype? Well in the plant that you're growing immediately after EMS treatment, we have among-- There's 10 chromosomes in rapa. Each genome at the level of EMS that we're using will suffer about 500 to 750 G-C to A-T base pair changes. Scattered at random through the genome. Remember, this is a diploid organism, so it has two copies of each gene. 

We're saying that we've induced a G-C to A-T mutation at this site. It's homolog, that is, the other copy of that locus is unlikely to be affected in that same experiment. So, this plant actually grows, after you plant the seed, as a heterozygote. Right? It has the wild type copy here. It has an altered copy on the other chromosome. So now we have to go through that selfing of the heterozygote. We self-pollinate those plants to generate what's called the M2, the second mutagenized generation. Because we selfed it, we've given those gametes an opportunity to combine in the homozygous condition. When we plant those seeds out, we inspect the plants and ask, is there any mutant phenotypes. 

Have we induced a change in the genome which results in a visible change in phenotype or appearance of the plant? So with the help of a team of undergraduates, including Melissa and about ten others. It varies from time to time, we worked last summer to do successive rounds of mutagenesis. We generated around 5,000 to 7,500 mutagenized families. Recognized that if these grew from mutagenized seeds, and you hand pollinated each of these flowers, at maturity, you would harvest a family of seeds. Right? 
- How long does it take it to seed? 
- About three weeks. Three to four weeks after pollination. So you generate a family of seeds each derived from the same plant. 

But each plant carries a constellation of mutations, 500 to 750 mutations, that is different than the ones that occurred in this, that is different than the ones that occurred in this, and is different than the ones that occurred in this. So you're doing the experiment over and over and over again. Like I said 5,000-7,500 M2 mutagenized generations families were generated. Then we've been planting them out. We just look at them, and ask is one of these things not like the other. And so, I just want to show you some of the mutants that we've recovered so far. I hope you can see this. We can pass them around, if you like. This is wild type B3. A little younger than those plants. 

This is a class of mutants we call the high mutants. Planted at the same date. But you'll notice-- Actually, you tell me, what's the difference? 
(inaudible)
 The overall height is different. In fact, at earlier stages, there is even a more noticeable phenotype. That is this region of the stem from the germinated seed to these cotyledons, the embryonic leaves, they're called the hypocotyls. And it's much extended in these class of mutants. In this isolet that goes on and that over extension of the stem segments is repeated with each successive node. So whereas there's not much distance between the cotyledons and the true leaves on the stem of the wild type plant, you see there's additional extension in this mutant. 

Right? 
- Is that very different from, I can't tell from here really. This seems like a fair amount of distance. 
- Well if you look at-- These will elongate further. So we always have to grow the wild type beside the mutant, and that's the only legitimate comparison you can make. Because there's variation in the wild type phenotype, as well. But then you look for gross abnormalities. We had the virtue in our screen, our only criteria for an "interesting mutant" is it has to have a phenotype, an appearance that no fourth grader could miss. Right? If it's going to be useful in the classroom, there can't be ambiguity in the phenotype. 

They have to be able to score the mutant and distinguish it from the wild type. So beyond that, anything goes. Now here's another mutant that we've isolated. And I think that you will agree that this seems to be somewhat defective in its ability to deploy the photosynthetic machinery. It's pale green. Now we're in the early stages. We don't know what the gene product is that's affected in these mutants. We don't know the genetic map position yet, but we do know that if you self-fertilize these plants, and grow them out, that you'll see the mutant phenotype again in the next generation. So the change is caused by a stable genetic change. And finally, and I want you to see this one up close. 

I'll bring it over in a second. What's wrong with this plant? 
- Those aren't fully complete flowers. 
- There's no petals. 
- That's what I mean. 
- Right. Petals aren't essential for fertilization. They might be out in nature, where they may be useful for attracting pollinators. But for our purposes, it doesn't interfere with fertilization, it doesn't interfere with seed production. Can you see? 

- Yes. 
(inaudible)
 
- That's a wild type. 
- Oh, that's a wild type. 
- Good eye, good eye! But see, it's just that sort of question, you know, when students look at it, and they say wait a minute what's wrong with this. We think that they can solve that problem. They can answer that question, you know, we would say, "I don't know. What do you think it is?" 

- Oh, god, yes. I'm glad you didn't. 
(laughter)
 
- All right. These are the sorts of mutants. We now have a collection of approximately 250 mutant lines that fall into about 30 different phenotypic classes. They include these without petals. They include a variety of pigmentation mutants from fully pale green plants to plants which form fully normal cotyledons, darkly pigmented green cotyledons, but then successive leaves are pale green. We have variegated mutants, where the leaves will form, for instance, a very sharp distinctions in portions of the leaves that have albino tissue. 

No chloroplasts, no chlorophyll adjacent to fully deep green wild type regions of the leaf. And that's interesting, because often those are cytoplasmically chloroplast determined. And the mode of inheritance of chloroplast mutations, or the chloroplast genome, is different than the nuclear genome. So what we're going to do is we're going to develop curricular modules, exercises in genetics experiments that teachers and students can use that will allow them to explore the principles of genetics. And I'm going to ask you guys to do just that. We'll start with the wild type. Melissa, could you hand out the M4? All right. We have two independent isolets. Two independent isolets of a mutant of the class PCM. 

Maybe you can tell me what you think PCM stands for. 
(inaudible)
 
(laughter)
 Okay, I like that. Well, what's different between these plants and these plants? 
- Colors. The petals. 
- A lighter color. 
- This is a class we call the petal color mutant class. 

And you'll see that the petals, obviously, whereas the wild type are vibrant green or yellow, these are very pale. We have other isolets that are even more severely affected, almost a bone white color in the petals. 
(inaudible)
 Well, all right. So let's go there. What we want to do is understand how this phenotype came about. And the first question I think was asked over here, is it just physiological? May be these plants are a little sicker, and they are not able to produce as much pigment as the wild type. Or is it due to a stable, heritable genetic change? So what would you do to start the genetic analysis? 

- Another generation? 
- So you just self it. 
- Yes. 
- Well, in fact, we do that as a preliminary test. But does it really answer the question? Let's say that the hypothesis is that this is a physiological change. That these plants, for whatever reason, are just a little sicker, so they can't produce as much pigment. You might think that a plant that starts out a little bit sick isn't going to raise particularly healthy children, upon selfing, and so those children themselves would come out with pale colors in the next generation. So selfing is important. It's the easiest way to see whether the phenotype is heritable. 

But the true geneticist... 
(inaudible)
 We'll get there. Excellent, excellent. The first thing that we do, we're interested in understanding several fundamental features of these mutants. Is this mutant phenotype due to a dominant or a recessive allele? Relative to the wild type? That's probably a good guess. And in most cases, as I said, changing the sequence of a gene is going to disrupt function and it will be a loss of function allele. But some mutations do endow the gene products with additional activities that are evident even in the presence of the wild type allele that is in the presence of a heterozygote. 

So the first thing that we do is we cross each of our mutant lines, we back cross it to the wild type. We create that heterozygote, where we have the wild type allele juxtaposed with on the homologous chromosome the mutant allele brought in from this parent in that cross. Then you ask, what's the phenotype of the F1, the progeny from that cross. So what's your prediction? 
(inaudible)
 
- It would go back to the bright yellow. 
- Right. I think in most cases, as I say, most of the mutations that we're going to generate are going to behave as recessive. And so, the F1 from a back cross will be phenotypically wild type. 

(inaudible)
 Well, as it so happens, you have-- In fact, here are plants resulting from that back cross. I told you, they're Fast Plants! 
(laughter)
 This is like the cooking show approach to genetics. 
(inaudible)
 All right. So there's two things that you can draw from these results. First of all, what can we infer about the dominance or recessive nature of the mutant alleles? They are most likely recessive. 

Now the other question. The other question is, how many mutant loci exist or are necessary in order to generate this phenotype? Is it due to a mutation of a single locus, or is it due to mutations at multiple loci? In some cases, mutant phenotypes require the concerted loss of activity of several different loci, in which case the inherited patterns are somewhat more complex. We're interested in understanding whether these are single gene or multiple gene phenotypes. So we already know from the back cross that they're recessive alleles. Right? We also can infer then. We can infer the genotype of these F1 back crossed plants. We'll actually use the real symbol. 

No, this isn't going to work, is it? Bad combination of letters. So we'll let "+" stand for the wild type allele and "-" for the mutant locus, the PCM alleles. So we know, or have good reason to suspect, that the genotype of these F1 back cross plants are heterozygous for the mutation. Now what's going to happen when we self them? Well, we've seen this a couple of times now, when you self a heterozygote, what are the genotypic classes that result? Three genotypic classes. How many phenotypic classes are there going to be? How many different types of phenotypes will result? 
- Two. 

- Two, right. And in what proportions? 
(inaudible)
 Exactly, three-quarters. This will be wild type. This constitutes one-fourth of all the zygotes. This is one-half. And this is one-fourth. These will be phenotypically wild type. And only this homozygous mutant class will recapitulate the phenotype after selfing those back cross progeny. 

The situation with a two locus model is more complex. Let me say that it's the genotypic distribution is 9-3-3-1, that might ring a bell for some of you. But the phenotypic distribution would be 15 to 1 wild type to mutant. Right? We make a couple of hidden assumptions in there, but that will suffice. So then the question is what sort of segregation ratios do we observe in the F2? And voila! What I would like you to do is to score the segregation. Count the number of mutant individuals and the total number of plants. 
(inaudible)

 
- We have to count each plant? 
(indistinct talking)
 
- Here's one of the light ones. 
(laughter)
 
- Why don't we just look for light ones, because there's still so many dark ones. 
- Oh, there's a light one. 
(laughter)

 
- Four lights. 
- Did you count this one? 
- Five lights. Yes, I did. 
(indistinct talking)
 
- You think? 
- No, six lights. I didn't count that one either. 

Seven lights. 
- You have a good eye. 
(indistinct talking)
 
- What about this one? 
(indistinct talking)
 
- Let's start from this side. 
- Okay. 
- Any from this group here? 

- Come on, we're expecting fourth graders to do this. 
(laughter)
 
- Are we smarter than fourth graders? 
- Actually, no, let me back off that a little bit. You're at a bit of a disadvantage. These petal color phenotypes are much more distinguishable from the wild type at an earlier stage. As the flowers mature, as the plants mature you begin to see some essence of leaves on pollinated flowers. So these are beginning to dry up and will drop off after while. They look pretty pale to me. 

That's just natural. So the phenotype is a bit difficult to distinguish at this later stage. So it's a little unfair. It's just the way the timing worked out for this generation. 
(inaudible)
 Well, I don't know. Do you think that-- What information would you need in order to know whether that's a valid approach? 
(inaudible)
 Right, and you would have to have a wild type population in order to see that, right? What's the variation in height among the wild type? 

What's the variation in the mutant class? Do those overlap? Does that overlap preclude you from actually using height as a proxy for petal color? I think that it does. I don't think that the difference is enough. All we're looking for-- We want to keep things as simple as possible. We don't want to impose upon any burden for, you know, technology really, on the fourth grade, or the sixth grade, or the tenth grade classroom. So all of our mutants need to have that to be visually distinguishable. So, no. You could, if you were interested in quantitating the phenotype. 

You could also do some extractions to see what pigments you could expect from these petals, as compared to the amount of pigment that you can extract from wild type. 
(indistinct talking)
 Right, and it's all up to the teacher. They can take these tools and use them in whatever level of sophistication that is appropriate for their grade levels. So those are great ideas. In fact, you know, my students, my undergraduates are, each of them working on a class of mutants. These PCM classes are Melissa's. And so at some point, we're going to map them, we're going to quantitate them, we're going to compare them. Let me ask one other question. Now, you guys haven't had a chance to see the mutants in this other population. 

But you can glance over and see they're pretty much the same as this. And yet, these were derived from independently generated mutagenized families. So the question arises, is the mutant phenotype-- Is the mutant locus in that line the same as the mutant locus in this line? 
- You know. 
(laughter)
 
- Okay, let me ask you this. How would you answer the question? 
- It would seem to be the same. 
- I think somebody had-- 

- It would seem to be the same. 
- Do you cross pollinate them? 
- Okay, what's the prediction then? 
- Well, let's see, if they're the same, they should end up with the same distribution of the offspring. About one-quarter of them should be pale. 
- All right, let's draw this out. So we have PCM one. We know now, because of the one-fourth segregation ratio that the mutant phenotype in this line is due to a single recessive locus. And that mutant individuals, phenotypically mutant individuals, have this genotype. Agreed? 

They're mutant. It would be minus over minus. Likewise, did you guys observe about a quarter? So, those are also homozygous for the recessive mutant locus. We crossed them together. Now what are the prediction if these are due to mutations in the same locus? What's the phenotype of the resulting progeny going to be? Is it going to be wild type or mutant? 
- Both. 
- Both? 

- I would predict-- I may be wrong. 
(laughter)
 I would predict a quarter of them would be mutant. 
- Let me change something here, and see if it maybe clarifies things. So what we're hypothesizing is that we're crossing, in effect, PCM1 mutant homozygous by PCM1 homozygous mutant. What's the genotype going to be? Homozygous recessive for PCM1. What's the phenotype going to be? What color are the petals going to be? 
(inaudible)

 Right, they'll be mutant. If the mutation in that line is at the same locus as the mutation in this line, the progeny from an intercross will be phenotypically mutant. On the other hand, if these are in fact, due to mutations at different loci, then what's the gene type at locus PCM1 in the progeny? With respect to the PCM1 locus, what is the genotype of the gametes, all of the gametes produced by this parent? 
- You lost us! 
(laughter)
 This parent can only supply mutant PCM1 loci to the gamete. Right. Then you're combining it, so what's the genotype of the gametes produced from this parent at the PCM1 locus? Wild type. 

Right? So now you're going to have a PCM1 over plus heterozygote. And likewise, this parent can only provide the mutant PCM2 allele. This fellow is wild type at the PCM2 locus. It's defective at PCM1. So it's going to be wild type. You're going to have to be heterozygous at both loci. So this parent can provide the gene product that's missing in this one. This parent can provide the gene product that's lost in this parent. The resulting progeny are heterozygous, and therefore phenotypically wild type. 

So we have two completely different predictions, depending on whether we think that these are due to mutations at the same or different loci that we can determine by a simple cross. Right? And I can't tell you the answer. 
(laughter)
 We actually have plants that are about a week away from telling us, unambiguously, whether these mutant phenotypes are due to mutations at the same or different loci. We have to repeat this. We have in our collection, how many PCM mutants do we have, Melissa? We have nine independent isolets of PCM mutants. We have to winnow our collection down and say, do we really need nine different, you know, mutants at PCM1? Well, it's really only of interest to us if we can find one that's additional loci that yield the same phenotype. 

And think about that. Somehow or another, you have to get to yellow. Starting from some molecule, some building block in the metabolic pathways that operates in these plants to produce that vibrant yellow pigmentation. And so you might imagine that there are a number of enzymatic steps that must occur in order to build the complex-- and molecule that yields the yellow pigment. Disruption of any of the genes that encode the enzymes that catalyze these steps will limit or prevent the amount of yellow pigmentation produced in the plant? So PCM1 might affect the enzymes or the gene that encodes the enzyme responsible for this step. PCM2 may encode the enzyme responsible for this step. So this allows students to start to think about things like biochemical pathways, metabolic pathways. The difference in petal colors allows the students and the teachers to discuss things about why are petals colored in the first place. Right? 

We talk about insect and pollinator interactions. So these tools not only serve as a guide to understanding genetics, but they serve as a jumping off point for all sorts of things from the biochemical to the ecological. So we're going to be, you know, we're doing these same sorts of analyses on all of these mutants that we've generated. We have probably 12 high mutants in our collection. We have a large number of pale greens. We have five petal mutant lines, and we have nine PCMs, etc., and we have other phenotypes. We're going to sort them out. And hopefully, by about next summer, we're going to have a collection of mutants that we say here's the criteria. Any fourth grader can tell the phenotype. 

The phenotype doesn't limit the fertility of the plant. You can't do genetics, really, if you can't get seeds. So that's another criteria that we have to satisfy. Once we had that down, and we say we have this collection of mutants which yield the same phenotype. We have this collection of mutants that allows independent segregation of two phenotypes. And so you can watch the two loci segregate into the F2. Then we're going to start going out into classrooms. We're going to bring teachers to UW-Madison over the summer. And we're going to train them. And we're going to deploy them, and hopefully get them into classrooms. 

At some point, it becomes almost a marketing exercise, because we have to go into the classroom and say why we think these are useful. Why should you, Mrs. Jones, use these in the classroom. Mrs. Jones is very, very busy. Mrs. Jones is very, very stressed. But Mrs. Jones also has to cope with state standards and exams. These will help them achieve those standardized test results. 

So their students will actually understand genetics. They'll understand plant growth and development. They'll understand, to some extent, biochemistry. They'll understand better ecological relationships between plants and their pollinators. So these are the tools that we're developing. 
- It will be easier because of Paul Williams' Fast Plants already in the classroom. 
- I think so. So just in conclusion, I thought that you guys, if you're interested, these are some wild type B3 seeds which you're welcome to take home. Put them in some soil, give them some water, put them in a window box. I think I might have enough. 

I have one left. Oh, okay, good. Good. Excellent. So that's happening over the next couple of years. And hopefully, they will be in your children or your grandchildren's classroom very soon. So with that, I'll be happy to take any questions. Yeah? 
(inaudible)

- People are afraid of organically modified materials. Genetic modification. Do you think there will be any reluctance from teachers to be handling something like that? 
- Well, in fact, that is a concern. That limits the sorts of things that we might imagine doing. But the fact is that these are simple base pair changes. There's no foreign DNA in these plant species, or in these mutant lines. There are only mutations. And mutations occur in every generation. No one can argue that these are loosing some material out into the environment, or you know an antibiotic resistant marker, so these don't contain that, so that should not be an issue. 

(inaudible)
 Well, they will. They will. There's some really bizarre issues. We have a class of mutants, they're dwarf mutants. And as their name implies, they're very short. Typically, dwarf mutants result from an inability to synthesize or an inability to perceive the presence of a plant hormone acid, or GA. GA simulates elongation and cell expansion. And so absent GA, let's say you're a mutant dwarf that can't synthesize GA, you're disrupted in this complex 20-step biochemical pathway required to synthesize the active GA so you're dwarfed. Well, if your phenotype, the dwarf phenotype is the result of your inability to synthesize GA, you can actually in many cases rescue the phenotype, as we say. 

You can cure the phenotype by applying the acid to the apex of the plant. That's distinct from the class of dwarf mutants, which are defective in their ability to perceive the presence of GA, in which case, adding GA back doesn't do any good. So again-- Let me finish this. So grapes for instance, are very, very rich in GA, either from endogenous supplies, or from grower applied treatments. No one objects to having grapes in the classroom. But GA is on the USDA sanctioned list of materials that should not be used in the classroom. So it's kind of a CYA sort of operation. So those concerns are ones that we'll have to address. But fortunately, I think we have the right action. We have no transgenic materials in these plant lines. 

They're just genetic variants, in which any wild type population there will exist plenty. Any other questions? 
- Have you considered giving the information and the teaching strategies to student teachers, so they could take them out? 
- Well, we're actually interested. As I mentioned, this program is funded by the Howard Hughes Medical Institute. Their interest is in improving undergraduate education, attracting students to research. Many undergraduates, you know, at the UW, will go through their four or five years, or more, without ever stepping foot in an actual research lab. We serve the purposes of the Hughes programs in two ways. First, the bar to entry is very low. Melissa, you know, you're very talented and stuff, but-- 

(laughter)
 I'm sorry! Someone of an intellectual stature much lower than Melissa can still come into the lab and do these experiments. A lot of it is just gardening, right? And the ability to look at plants and see, one of these things is not like the other. And then follow the phenotype through the successive generations, and the sort of analysis that we're talking about. And then we also have, we haven't began to exploit it much, but we're going to begin this summer, molecular aspects. So we can map the locations of these mutations using polymerase chain reaction or PCR based markers. So students, you know, when we deploy these in the classroom, we can have students actually do some mapping. We can use PCR to amplify polymorphic fragments. 

They can run a gel. They can score segregation of it. Presently, as I said I think I have ten or 11 undergraduates working with us on this program. Most of them are biochemistry or genetics majors. But we're very interested in getting education majors in. Because, in fact, many of the teachers who go through the UW, that will ultimately end up being science teachers never have to do science. Right? So they learn from the book, and then they teach from the book. We think that it will be much more useful if they came in and they were able to do this and say wow that's really cool. Not only would they be enthusiastic, we think, about employing these in the classroom, they will be much better positioned and able to do so. 

So we're interested in having a wide variety of students. But unfortunately that's not something that's stressed in the education program presently. We've put out ads. Right now, you know, this program, it's really exciting, and it's just great to be working with the undergraduates and seeing them as they adopt-- We've been through this mutant generation identification phase, and now we're into the genetic characterization of each of the lines. And as I said, Melissa has adopted the PCM screen. The characterization of those mutants. She's also conducting and presently conducting a screen for non-phototropic mutants. Right? Ordinarily, given a dim orthogonal, that is at the side ways oriented light, plants will bend towards that light. That's pretty complex behavior. 

Right? We're looking for mutants, which disrupt that ability, so that even when supplied only with this side light they just grow relying only on their gravi-tropic sense to tell up from down. I mean, what a cool mutant class that is. So it's great to see them. And you know she's screening families over and over, and we have a couple of candidates and we'll see if is comes out or not. So we've taken the low hanging fruit so far, with these unconditional mutant phenotypes. But we're looking for situational mutant phenotypes. Meanwhile the characterization of these mutants, of the high mutants, of the pale green mutants is ongoing in the lab, and all the students are doing these. So we have the capacity to very readily introduce and take into the program undergraduates from a variety of backgrounds. Often, students that have no science experience in labs at all. 

But in the course of it, they learn what working in lab is like. They learn how to keep a lab notebook. They learn how to think scientifically. Many of them, we hope, will then go on to say, either continue this work, or having been in a lab and say well those people aren't so strange, will then go on to another lab and consider a career in science. So we have that ability, you know, we support the Hughes program in that direction, or in that way. But of course, we also are providing the tools that we think when deployed in classrooms will help improve education both in K-12 settings, as well as in undergraduate labs. So we get kind of a double bounce on this one. Yeah? 
- What are the seeds going to produce? 
- The wild type B3 plants. 

- But I mean is there actually going to be a vegetable? 
- Oh, no, no. This is it. Actually-- Hey, hey! Don't be afraid to do this. They're actually good. You didn't think so. 
- I get enough fiber. 
(laughter)

- Yeah, exactly. You had a question? 
(inaudible)
 
- Who handles all the logistics of all of plants and the materials? 
- That's a good question. And frankly, one that we haven't thought much about yet. We're just elated to be at the point where we are. You know, when we began the mutagenesis last summer, it wasn't at all clear that we would be able to generate a large collection of mutants in this line. Many plants are polyploid. 

That is, they carry-- They're not true diploids. They carry multiple copies of a given locus. And so to generate a mutant phenotype that's is evident in the homozygous condition, is very difficult with a polyploid plant, because you have to get to a uniform loss of function condition. We don't know the level of polyploid in Brassica rapa. We were just happy to find out that we could generate mutants. And now we have them, and it's clear that we're going to be able to develop these curricular modules and do those sorts of things. Now in the case of Wisconsin Fast Plants, Paul Williams and others at Science House have outsourced the propagation of the plants to a small private company that's responsible for growing them, for sorting the seeds, to make sure they maintain a consistent quality. You know, you don't want to send out a lot of seeds which have a 50% germination rate. You need close to 100%. If you're a teacher, you've got to be able to depend on these things. 

And then further, they have licensed the Wisconsin Fast Plant brand through WARF to Carolina Biological Supply, which is a go-to place for many school teachers. They have a small, private company which does the propagation and quality control, and Carolina Biological as a distributor. It's possible that we could adopt a similar model. It depends on what the demands are. We hope it's a lot. We hope that we get to that position. But until we're faced with the need to outsource it, we're just doing everything in house. Any other questions? Well, great. Thanks again for spending a beautiful afternoon down here. 

Thank you. at UW-Madison.