Development of new breeds with mutation technology using heavy ion beams
独立行政法人理化学研究所 仁科加速器研究センター 生物照射チーム チームリーダー 阿部知子
In nature, mutation may be triggered by cosmic rays, UV rays or chemical substances which accidentally damage genes. Plant breeding using heavy ion beams aims to induce mutation artificially. This technique is drawing attention not only for being useful in developing new plants with useful characteristics such as resistance to salt or the ability to collect heavy metals, but also as an important research tool to elucidate the functions and meanings of genes.
Inducing mutation with heavy ion beams for plant breeding
These days people want greater diversity among plants, and breeding the right kind of plant is getting more and more important in agriculture and horticulture. For example, popular flowers for home gardens are usually those with wide variation of colors, or petals, such as single or double petals. What kind of plant breeding approaches are there to meet these market demands and how exactly do the heavy ion beams developed by RIKEN come into this?
Abe: The most common approach to changing the color or shape of a flower is hybridization. You first create a cross-breed group with varied characteristics by cross pollinating the original plant and a related plant with the desirable characteristic that you want to bring into the original plant. From this mixed bunch, you select the individuals with the desirable characteristic. Blue carnations and roses, which had never existed in nature before, are the result of this kind of genetic recombination introducing a blue color gene from another plant into the carnation or rose. There’s another approach, called mutation induction (mutagenesis), where mutation is artificially induced, then desirable plants are selected from the results and a new variety is created. Chemical substances and radiation such as x-ray and gamma ray, have been used as mutation inducers (mutagens) so far. At RIKEN, we are studying mutagenesis using heavy ion beams generated by the RIKEN Ring Cyclotron (a piece of equipment in which particles are accelerated along a circular track) as the mutagen.
What is the heavy ion beam that you use to induce mutation?
E5 Beam line
Abe: Heavy ion refers to an ionized atom, created by removing electrons, which is heavier than helium. We produce heavy ion beams by accelerating heavy ions up to 50% of the speed of light (approx. 300,000 km/sec) in the Ring Cyclotron. There are only a few facilities in the world where such heavy ion beams are used for thremmatology and RIKEN’s is the most suitable for plant breeding.
What results are you achieving?
Abe: A new variety of flowering cherry named Nishina Zao was created in this way in October 2007. First we irradiated a cutting of a green flower variety called Gyo-i-ko with a heavy ion beam, then we grafted this irradiated cutting onto a green leaf variety, which eventually produced yellowish flowers. Over the next few years, we created more and more grafted plants and they all produced yellowish flowers. In this way, a new variety was created through mutation induced by heavy ion beams. The name Nishina Zao comes from two persons.One is Dr. Yoshio Nishina, who gave his name to our institute, the Nishina Center for Accelerator-based Science, and is referred to as the father of modern physics in Japan. The other name, Zao, is of the name of the region in Yamagata prefecture where Dr Nishina worked with the plant breeder. This new variety adds value to flowering cherry trees, combining beautifully with the other pink varieties. We have so far developed 17 new varieties of garden plants in the genus of dahlia, petunia, verbena and Torenia, all of which have been commercialized and are on the market.
Are there no harmful effects to the eco-system in introducing mutant varieties?
Abe:Mutation occurs naturally in nature though the occurrence rate is low. Since the rate is low it takes a long time for a new variety to develop, but mutation is a part of evolution. Mutation occurs in every living thing, including weeds, insects and microbes. The mutants we use for breeding are a very small part of this. Also, the mechanism of mutation is the same whether it occurs naturally or is artificially induced. The same result might appear anyway if we waited for a long enough time; what we are doing is to shorten the time by making mutation more effective through artificial means. So I would say there is no harmful impact on the eco-system.
What is the most significant advantage of heavy ion beam breeding?
Abe:With traditional mutagens, the treatment was such that in order to destroy a target gene, genes other than the target were damaged as well which resulted in weakening the growth or killing half of the samples. In these cases, the selected mutants with the desirable characteristics are repeatedly hybridized in order to regain the other characteristics destroyed by the treatment. With the heavy ion beam, it is possible to induce mutations without weakening the plant. That is because the heavy ion beam, which passes through the plant at high speed, affects only a minute area of a few nanometers. It cuts the DNA strand and the plant tries to repair the damage but cannot, and becomes a mutant with a part of its DNA strand missing. Mutants selected like this are thought to have a high probability of a smaller number of damaged genes other than the targets. In this way the mutants selected by using heavy beam breeding can become, almost immediately, new varieties. This shortens the developing time for a new plant variety from ten years to three years. Another advantage is that, with the development of genome science, we can now compare the DNA sequences between normal strains and mutants, leading to the elucidation of gene functions. By integrating this knowledge, we can accumulate knowhow on creating varieties that contribute to solving environmental and food problems.
Examples of plant varieties created by this approach
What kind of results are you getting in terms of solving environmental problems?
Abe:Let me give you several examples.
【Plants which accumulate rare metals】
Some ferns and moss are known to accumulate heavy metals such as cadmium and lead. We are conducting research into improving the quality of water and soils contaminated by heavy metals, by enhancing this characteristic of moss to make its heavy metal collecting mechanism more efficient, in collaboration with the RIKEN Plant Science Center, Tokyo University of Science, and the Dowa Holdings Co., Ltd. Research in the recovery of precious mineral resources using plants is a growing field, with the breeding of mutants to recover certain heavy metals, such as rare metals, high on the agenda.
【Rice that can flourish in a salt damaged paddy】
Salt damage, in which salt accumulates in soil causing damage to the environment and agriculture, is becoming a serious issue in Asia and the Americas. We are conducting a model experiment with the RIKEN Omics Science Center and Tohoku University, using Nipponbare, a strain of rice with a known DNA sequence. Efforts to introduce the salt resistant characteristic of wild types of rice into cultivated varieties by hybridization have been going on for more than twenty years worldwide, but none have been agriculturally successful. Using our No. 173 strain (a strain from an irradiated seed), irradiated by carbon ions at RIKEN, we have now created two strains which can grow well in salt damaged paddy fields. The occurrence ratio is as high as 1.2%. We are working to find out the optimum conditions for irradiation. Many plants are brought into RIKEN and I hope we can be of help in improving their qualities so that they can, for example, withstand unfavorable conditions.
【Promoting urban greening】
We are developing compact new plants, suitable for growing on the side of a building or a rooftop, which require minimum trimming and weeding. In cooperation with Florsaika and Tsunoda Nursery , we have developed a frost hardy variety of lampranthus, and in cooperation with Miyazaki University, we have developed a hardy variety of imperata cylindrical ornamental grass. This technology is certain to help improve the urban landscape and mitigate the heat-island effect.
【Working for stable food supply】
Farmers want to grow buckwheat or millet as an interval crop between rice crops or to make use of idle land, but there have been problems. For example, millet is taller than rice so the same harvesting machinery cannot be used, and buckwheat being so tall, it is easily knocked down by wind. We destroyed the gene that makes the plant tall, and developed a shorter millet, in cooperation with the Iwate Agricultural Research Center, and a shorter buckwheat, in cooperation with the Nagano Vegetable and Ornamental Crops Experimental Station. These efforts have produced new candidate varieties.
Research starts with a question: ‘What triggers flowering?’
We hear you studied agriculture at university. What sort of things were you interested in?
Abe:A lot of the food we eat is the fruit of a plant, so I thought if we could control the process of flowering we should be able to eat the fruit at any time and anywhere. This is why I was interested in how a plant makes flowers. In graduate school, I researched the sexuality of the asparagus. In those days, the sex of an asparagus could only be determined when it flowered. Farmers want male plants which produce about 30% more spears than female plants, but you have to wait for two to three years until the asparagus flowers before you know which is which. I searched for a protein which might be helpful in determining the sex of a young asparagus plant, but instead accidentally discovered a chemical agent which quickened the plant to flower within one month after germination. This discovery eventually led me to RIKEN.
The research on plant sexuality is still continuing with the University of Tokyo. It has advanced significantly through the use of heavy ion beam technology. White campion (Silene latifolia) has male (with XY chromosome) and female (with XX chromosome) plants and has long been a model subject for plant sexuality studies. By damaging the Y chromosome, the mutant seedlings which received irradiation can be distinguished by the shape of the flower. Now it is possible to determine the sex of a young plant with just a minute trace of DNA. In the process of this study, we inadvertently discovered a mutant male that produced hermaphroditic, not male, flowers. This characteristic proved to be hereditary with the seeds germinating into hermaphrodites. We are hopeful that through these kinds of studies we will be able to clarify the functions of a plant’s Y chromosome.
Heavy ion accelerator facilities are usually used in physics, for research on the origin of elements or nuclear structures. Isn’t it rather unusual for RIKEN to be using such facilities for biological studies?
Abe: Indeed it is. The largest accelerators in the world are constructed for the study of nuclear physics and most are used only for that. There are only six facilities in the world where heavy ion beams are used for plant breeding. We are able to use RIKEN’s RI beam factory thanks to the wisdom of Yoshio Nishina, who is considered the father of modern physics in Japan and the founder of accelerator-based studies at RIKEN. Nishina said that the accelerator should be used widely across all the disciplines, and that the cyclotron facility should not be dominated by nuclear physics, but that at least 20 to 30% of its use should be given to other disciplines, such as atomic physics, nuclear chemistry, biology and medicine. This philosophy is a deeply ingrained tradition at RIKEN. The biology irradiation beam line which we use is shared by the National Institute of Radiological Sciences, which was a part of the facility when RIKEN’s Ring Cyclotron was built, for the purpose of cancer treatment research using heavy ion beams. We are benefiting from this.
What kind of research are you going to tackle now?
Abe:The first is to improve the rate of mutation, raising it from the present 1% to 3% by, for example, changing the irradiation conditions. We have achieved two digit mutation rates for some plants. By analyzing the mutating mechanism in model plants we may be able to discover marker genes that will react to irradiation for effective mutation. This will make it possible for us to meet market needs by shortening the lead time for producing improved varieties of plants. We have already started to assess the impact of heavier ion irradiation on DNA damage. There are plants which are not affected at all or only mutate after receiving the beam at ten times the power required for other plants. By analyzing these characteristics, we may be able to create plants that can grow in outer space or are capable of restoring damaged DNA.
Does this improvement in the mutation rate mean that mutation will no longer be an arbitrary, spontaneous event but that soon we will be able to target mutations to create designer plants?
Abe:Well, not very soon. We are not at that stage yet. Targeting would mean irradiating a single target gene with the heavy ion beam, but heavy ion beams can only be focused on an area measured in micrometers (μm). We don’t have the technology yet to mark a specific gene on a chromosome in a live cell. I am hoping that with future developments in physics and biology this will eventually become possible. In any case, Still, the destruction of individual genes is happening, albeit accidentally, and heavy ion beam irradiation technology is now recognized worldwide as a very powerful tool in the development of genome science. The number of users of the technology who come from basic science backgrounds is increasing.
In concrete terms what do you expect to come out of these studies?
Abe:We may know the full DNA sequence, but that doesn’t mean we understand the roles of all the genes. In this circumstance, it is a big advantage in advancing genome science to have a technology capable of making mutants, because we can guess the function of a missing gene by comparing the genes of a normal plant with those of mutant. I will be working to meet users needs for better gene and chromosome destruction technologies by accumulating experimental track records and mutant analysis data.