René Smulders: New DNA techniques in the enhancement of plants | WURcast

René Smulders: New DNA techniques in the enhancement of plants | WURcast


Good evening, I would like to tell something about tinkering with DNA in plants. As this has been done since ten thousand years BC, we will make a proper time travel during the coming 20-25 minutes. we will make a proper time travel during the coming 20-25 minutes. I would like to start with the domestication of our crops and with how we have changed these over time. Thereafter we will give an overview of the tools currently used by plant breeders. One aspect of this is mutation breeding and on the basis of this I would like to look at new techniques used in breeding such as Crispr/Cas. In the end we draw our conclusions and introduce the next speaker. When we look at the wild kindred of our crops, it is not always easy to recognize them. For example, this one comes form New-Guinea, it is a wild banana. Nowadays it looks like this. When you search on the internet you will find all kind of pictures, of which I have selected a few. This is a beautiful picture of maize and its wild kindred and if you look closely to its scale, you will see that the plan on the left, the wild maize has a lot of stems and leaves, but very little seeds. So the ovary is about this size. You can see that we remodeled the whole plant, in order to generate the most yield. You will thus not be surprised that also the eggplant has changed a lot over years. It originally comes from America, but its roots have been domesticated in the Middle East. It there became a white, edible root. The orange color has been selected in the Netherlands, in the sixteenth century. When you browse a little further, you for example come across this domestication of a watermelon from South-Africa. Which originally looked like this and was quite hard. And also a peach from China, which also was very small originally. The author of these figures thought that current fruits were artificial, since they have been substantially adjusted form their natural forms. However, you could also look at is as a form of evolution, but driven by humans. This is the way Vavilov, the father of the gene banks, formulated it. And when you look at evolution, this is a very nice study of one of my colleagues, who is also sitting somewhere in this room, together with a groups of Chinese. We know that with cabbage we have a lot of different variaties: variaties with thicker stems, or with a lot of leaves in different forms. Or variaties which extended their way of flowering. The nice thing about cabbage is that there have been two ways in which it has been domesticated. The brassica oleracea here on the right in Europa, and the brassica rapa in China on the left. And when you look at the different types which are added in the figure, you can see the same types occur in both variaties. You know they are different, for example when looking at Chinese and white cabbage, you notice they are similar but that there still is a difference between them. In this study they looked at the genetical relationships of all these types. Also they investigated the underlying mutations. Here you can see that the same way of changing the way of flowering or leaves, Is caused by almost the same mutations, or mutations in related genes. What happens independently in two different parts of the world, in around one thousand years. So, in some respects these kind of domestication processes are very repeatable. However, sometimes it is not that repeatable. This for example is study in which we participated about the domestication of apples. The cultivated apple from the malus sieversii from the mountains in Central Asia, are actually quite large. But during their travel of around 1000 years towards western Europe, we identified them with other varieties, whereby this variety now more resembles to the wild variety which now appears here, than to the one it started with. Domestication thus uses present genetic variation, and makes selections from this. Within the species but also with related ones. And this is actually what plan breeders still do. But it also uses new mutations which occur spontaneous. For example in different wheat variaties mutaties occur through which seeds do not fall, but stay attached to the plant. These kind of mutations would not survive in the wild, since it is bad for the spreading of the seeds. But when this happened one the first agricultural plots, it was also immediately applied by farmers. And this is especially true for plants with sterile fruits. Since they could not reproduce in an natural way. We could only vegetative increase them by cutting them. And that is what we selectively often did, because fruits without seeds are easier to eat. From all these selections we could conclude that often the products of breeding and selecting are save, since we extensively select and test these selections. To give an example, when we breed apples we have one potential breed which derives from 10.000 to 20.000 seeds. So, we select a very small fraction of the plants and all others are not used for different reasons. Plant breeding as a science is defined since around 1900, because then the laws of Mendel were rediscovered. So from then on we knew exactly what genetics means and does. But actually in the centuries before, one already mixed different characteristics, for example in roses. European roses traditionally only flower once, only in May or June. Chinese roses however flower during the whole year and since the 18th century, roses from China have been shipped to holland and have been crossed. Because people wanted to have these flowering characteristics in the European roses as well. And as a result, most roses you can nowadays buy in Holland, will also flower through the whole year. So plant breeders look for plants with a stable and high yield, and with a tolerance for different kinds of stress and resistance to diseases and pests. In addition with an increased quality. Which can for example mean bigger or sweeter fruits or less seeds. And all other preferences such as shelf-life, In order to make sure that in the case the producers is far from the consumer, the products will not decay before the product has reached the consumer. This actually has been very successful. This is an example of the yield of maize in America. You can see half way it represents the beginning of the 20th century And after that you see yields increase significantly due to different technological developments. This is not only due to breeding, but also other aspect such as irrigation and manure play a role. But the transitions have clearly to do with new technologies in breeding. And when looking at the tools used, one can distinguish a lot of different methods which have gradually been introduced. Methods which make it easier to sort variations and to combine and then to preserve and replicate them. When looking into the modification of DNA, sometimes you want to create new variations. Or you might want to change some specific characteristics. One of the ways one could do this is mutation breeding, which has been introduced around the 40’s. The idea behind this is that mutations always can occur, most of them are recovered by plants themselves, but this is not always the case. And we can select these kind of mutations. In if we increase the frequency we come across the mutations we want more often. These kind of changes are the basis of all genetic variations within plants, but also within human. It is roughly estimated that between 2 persons here in this room, around one million aspects on their DNA are different. And this could increase by 40 – 80 random differences per generation. So this is the basis of selection, and when this is not enough you could increase it. In mutation breeding, ionizing radiation or chemical mutation is already being used. Chrysanthemums are a good example, because it works well in them. If you irradiate a pink chrysanthemum, you can detect all sorts of different colors from the offspring. What chrysanthemum breeders often do as standard, is to breed the shape and other characteristics of a chrysanthemum into a pink variant. And then breed the whole range of colors in that certain variant, so that you will have a whole series of variants that look the same, except for the color of the flower. Mutation breeding is also offered by an agency of the FAO in Vienna. You can go there for mutilating your tropical variety, in the hope of finding a disease-resistant plant. It is also used by companies, like the example above of the herbicide-resistant canola, also known as rapeseed, which is herbicide resistant because of a form of natural mutation from mutation breeding. It is widely accepted and this is an example. It is a very famous example, because this is the first seedless grapefruit, which made the whole grapefruit cultivation popular in America and which is also very popular in organic farming. A small side step: mutations always occur, so instead of following the mutation frequency in a few plants, you could also look at the occurrence of mutations in very large areas. This, for example, happened with apples. So if you have 100,000 hectares of apples and you have growers who look carefully at those apples, they sometimes find an apple that looks a bit different. They can then register it themselves as a new breed. This has been done extensively with Elstar apples: these are 3 out of 10-15 Elstar mutants that have been compared a few years ago, on our trial fields in Randwijk. The expert can spot the differences, but they are very small. It was a very successful process, but it basically comes down to the fact that the original Elstar is barely for sale in the stores. Almost all Elstars which are for sale are mutants. And often they are a bit more smooth and shiny Since the original ones are a bit more lustreless So if you buy a more dull Elstar, it might be an original one. But in other cases you’ll buy a mutant. I though this would be a nice picture to show you, taking away one base from the DNA could disrupt the gene And this is one of the reasons why mutation breeding is so effective. The other reason is that, taking away one of the characteristics, because normally disable genes, could bring something positive for us. For example when you think about seedlessness or one part of the plant that doesn’t grow well anymore so it could invest in its fruits. And in addition, I will give an example of this later, but sometimes genes are used by pathogens to enter, and if you disable these you will create a form of disease resistance. In conclusion, there are several positive characteristics in plants which could be created by us by simply disabling genes. The only drawback in this, is that is works randomly. So you have to mutate a lot of plants in order to create the mutation you actually need. But this means that in these plants there will also be mutations that you actually do not need. But through backcrossing and selection it will still be usable. So there are new techniques, which were created since the year 2000 We had around 15 years of experience with mutation breeding We have GMO’s which get a lot of attention and which are very expensive to make. Ever since there have been developed some new techniques which can change DNA in a fairly precise way, and can also accelerate the cycle of breeding. This means you can obtain your product in a more effective way, this is especially important for cross fertilization and crops for which breeding works very slow. I will give an example of this later. Sometimes they are difficult to show in the final product, and sometimes they are only used in the breeding process and will not be present in the final product. A point I want to mention finally is about the question whether a crop is genetically modified or not. It could be the case that not a new gene has been added, but that the techniques which are used are, in Europe, regarded as genetic modification. This is a very relevant question, since launching a genetically modified crop in Europe is so expensive, that for some crops it is not worth it. This means that the potential for this technique could be very high, but it will barely be used when it is too expensive to start up. I will not discuss all techniques, but I would like to focus on the examples of two methods. The first one is referred to as gen editing, which can be done by using Crispr/Cas And the other is called ‘early flowering’, I will explain later what is meant by this. This will yield crops with only small changes, this could for example be only four base pairs. Or even no change due to the technique, since it was only used during the process and will not be present in the final product. My first example is a bacteria that creates these kind of stains on rice. I will call this ‘the case of the hijacked gene’. You should imagine, the bacteria will land on the leave as a spore, and will not have enough reserve food to grow and to colonize the crop. It needs the plant in order to establish an infection. So it will activate the gene within the plant, in this case the gene of glucose transporter, which pumps sugar out of the cells, so the bacteria could live from this. Researchers, from the ERI a research center of the philipines, firstly disabled this gene, which created a crop that was resistent but it barely grew, since the plant itself also needs these genes. Because the transport of glucose is an important characteristic, especially for the growth of the plant. And then they thought, where does this protein actually affect the promoter? And they saw this was another place than where the plant itself would affect it. What we see here, on the small upper part, is a small piece of gene sequence, which is located just before the gene. The gene is located further on the right and the promoter elements used by the plant further on the left. This is the part where the protein of the pathogen engages to enable the gene. Afterwards they used gen editing to cut the DNA. This means the plants has to recover, and this normally works out well, but sometimes is does not. So from a large lineage, you could find mutants on exactly this place where the recuperation went wrong. The sequences below, are sequences from several plants. In most there has been a small part of the DNA removed, or there has been added a small part, this is the case when reparation does not work out completely. On the bottom on the right you see that some mutations ensure that the plants have become resistant to the bacterium. So here the protein can not affect the bacterium anymore, so it is not able to grow. If you look more closely, you can see that taking away 4 base pairs from this promoter, which does not really have a function but is only used by the bacterium, could already induce disease resistance. This is relatively a easy technical thing, and very targeting. This plant is identical to rice without this mutation, as long as there is no bacteria involved. And in the case that there is a bacteria, then it will not become sick but it will stay identical to itself without bacteria. This editing of genes is possible for very specific location in the genome. It could also be done for all alleles at once, and also for crops such as potatoes. In addition, it is also possible for small and underdeveloped crops. What is interesting is the fact that it is also possible for characteristics, which are actually too complicated for mutation breeding. And one of the studies, for example about disabling allergens, which often are multiple genes in the genome that code for a protein to which allergens react. One of the studies we do here is to try to achieve this in the case for wheat. In order to disable all the epitopes on which people with Celiac disease react, and at the same time keeping the quality of the wheat constant. This would create wheat that is suitable for people who actually have an intolerance for wheat. This is normally not possible through random mutation breeding, but it is possible through Crispr/Cas, since with this you can reach specific epitomes. My second example, yields plants which do not contain a new feature, but can be produced faster. This was created due to scabies in apples. Scabies is the disease for which crops are most often treated in Holland, often every week in order to prevent it. So scabies resistant crops are needed, in regular as well as in ecological production. There are several scabies resistant crops. This resistance is extracted from the wild apple type here on the left. These apples are as large as cherries. Crossing took 5 generations, We crossed several times, and each time the apple gets a little bigger. Until you reach a normally sized consumption apple. On the right you can see the santana. In case this resistance is stopped or one wants to add a second resistance this would take another 5 generations to do. And all this time one should use pesticides. How could one solve this? One of the reasons why this will take so lon is that when you cross apples, you take the seeds from its result, and you will sow this the next year, then it will take 5 or 6 years before it will flower for the first time. And only after that you can start with a second crossing. This is typical for apples and pears, because for example related species such as cherries or peaches could do this way faster. The same applies to roses, you could have a small rose which already has its flower. So actually you would like to have similar apples, with two leaves and an flower In this case you could do the crossing. Of course, this is not a commercially viable apple. But it is able to skip one generation, so you could cross already the next year and you do not have to wait 5 or 6 years In case you need 5 generations, you could save a lot of time by doing this. As a results the following is developed; in different ways you can make a plants start flowering by overexpression of a gene. In this way you create a GMO plant, by flowering through overexpression. The idea is as follows: you cross the wild plant with a GMO plant. Then half of the progeny will inherit the GM construct and will start flowering and you will use this half to do a second crossing, and you will keep doing this for 4 or 5 times until you will reach the apple you initially wanted. But in the last crossing, or one crossing before, you will select the plants which do not inherit the GM construct, these are still conventional plants. You have to wait 5 or 6 years for one time, but then you can start a normal breeding again. If you add this up, you could cross 5 generations but still be needing 15 years in order to combine these characteristics. The end product of this plant contains a resistancy gene from a crossable type of plant. This is identical to what you could produce in 50 years, but you can do it within 15 years by using this GM construct, which you use temporarily before you cross it out again. So these new techniques for more precise breeding make more sustainable, effective and quick breeding possible. But actually they do not fit in to the list of GM or nog GM, since in the end they do not have a gene from another type, because you only used this temporarily or you only made a small change to it. So they are actually somewhere in between conventional breeding GM and mutation breeding So they are effective and quick and by using them we could do a lot more than we used to. Also for example hypoallergenic crops. This wouldn’t have happened if it was as expensive as the introduction of GM plants. On these slides a summarize this. Actually the technique you use does not say a lot about the plant which is created by it. Neither about its risks. Actually you should use a process related system, instead of a product related system. And if these plants are identical to the plant you could get using conventional breeding, then you would not get something that would fit the regulation system of genetically modified plants. About these kinds of topics we have debates and dialogues with people who are concerned. In this slide I summarize the objections we often come across during these conversations. Part is about possible new dangers of the techniques, partly because people are worried that by focussing on one technique other techniques and thus solutions are neglected. For example by forgetting cultivation by using ecological pesticides. Another concern is about power. Techniques could be patented so only certain firms could use them. And in these debates we also look at the costs of not using these techniques, in terms of health and environment since you have to keep using pesticides, and also economical costs. So, to conclude my story: the new techniques have the potential to speed up plant breeding. And therefor breeding could add to the challenge we face of creating more food with less inputs and also with less land. Breeding is not the only solution to this, but it does play an important role. By going back to the beginning, my conclusions are: People have changed the DNA of crops already since the beginning of agriculture. In some cases genomes have been doubled and caused huge changes. In addition, one could make more precise changes. We think these changes are safe because it includes the process of screening and selecting. So it is not dependent on the used techniques in order to create the crops. And to make my story connected to the one of the following lecturer: if we are so concerned about the safety of crops that we prohibit gene editing, this could result in for example a gen edited baby, who has been cured from a disease, that can not eat gen edited food since it is assumed to be unsafe. And with this I would like to end my presentation, thank you.

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