Creating novel plants using heavy-ion beams
15 February 2008 (Volume 3 Issue 2)
Seeds, cultured cells, shoots, and other parts of a wide variety of plants, including roses, chrysanthemums, rice, buckwheat, satsuma oranges, and green peppers, are brought to the Radiation Biology Team at the Nishina Center for Accelerator-based Science in the RIKEN Wako Institute. The team is working to create new varieties of plants through mutations by exposing these seeds and other plant parts to heavy-ion beams generated by accelerating atomic ions using a particle accelerator. Plant breeding with heavy-ion beams is a technology unique to Japan. The following is an interview with Tomoko Abe, Deputy Laboratory Head of the Radiation Biology Team, who talks about the fascination of heavy-ion-beam breeding and its future prospects. Also described below is ‘Nishina Zao’, a variety of cherry with a new flower color created using heavy-ion beams, which became the first plant that RIKEN registered under the Seeds and Seedlings Law.
Flowers conceived at RIKEN
Abe’s cards feature pictures of flowers such as dahlias, petunias, and verbenas. “All these are new varieties we created using heavy-ion-beam irradiation,” says Abe. ‘World’—a vivid red variety of dahlia—was bred from ‘Miharu’, a pink variety of the flower, through a joint project with the Hiroshima City Agriculture, Forestry and Fisheries Promotion Center (Fig. 1). As a result of the treatment, the flower size increased, with the number of petals increasing from 60 to 80. Pilot marketing began in the fall of 2001. ‘World’ is the first new variety of plant created by heavy-ion beam irradiation to become commercially available in the world.
Figure 1: World,’ a new variety of dahlia with a new flower color (left) and the parent variety ‘Miharu’ (right).
As a result of treatment with heavy-ion beams, the flower color became dark red, and the flower became larger in size with an increased number of petals. ‘World’ was developed through joint research with the Hiroshima City Agriculture, Forestry and Fisheries Promotion Center and was launched in the fall of 2001 for pilot marketing. ‘World’ is the first new variety of plant created by heavy-ion beam breeding in the world to become commercially available.
Abe’s favorite flower of those she has bred to date is ‘Surfinia Rose Veined’, a petunia variety (Fig. 2). Heavy-ion beam irradiation resulted in a change in the original color from purple to brilliant pink. The same treatment rendered verbenas seedless (sterile) so that an increased number of flowers can be enjoyed for a longer time. Three Temari varieties are available: ‘Bright Pink’, ‘Sakura Pink’, and ‘Momo’. Petunias and verbenas are commercially available from Suntory Flowers Limited. “I noticed that ‘Surfinia Rose Veined’ seedlings featured in a photograph in an advertisement at a home center. I was very impressed by the fact that they were actually on sale.” She adds that these plants are very popular overseas, as well as in Japan.
Technology unique to Japan
In agriculture, including the ornamental plant industry, breeding to create new varieties with improved characteristics is widespread. Available approaches to breeding include hybridization, in which parent varieties with each of the desired characteristics are crossed with each other, exploration, in which variant varieties are sought in nature, gene recombination, and mutation induction (mutagenesis).
In mutagenesis, genes are artificially damaged using chemical mutagens, X-rays, gamma rays and the like, and a variety with the desired characteristics is selected from among the resulting mutants. Mutagenesis using radiation is based on the same principle as naturally and commonly occurring mutations from cosmic rays that impinge on the Earth from outer space. The number of varieties that have been bred by this method exceeds 2,200 worldwide. Heavy-ion beams are drawing attention as a groundbreaking method for mutagenesis.
Figure 2: ‘Surfinia Rose veined’, a new variety of petunia with a new flower color, and members of the Radiation Biology Team.
As a result of heavy-ion beam irradiation, the petal color changed from purple to brilliant pink. Its seedlings are commercially available from Suntory Flowers Limited. Photo taken in front of the Nishina Building.
“This technology is unique to Japan,” says Abe. Heavy-ion beams are produced by accelerating atomic ions (atoms deprived of electrons) to half the speed of light (the speed of light is 300,000 km s–1) using particle accelerators. Large heavy-ion accelerators emerged in the 1970s, and are now used not only in nuclear physics, but also in the applied sciences. For example, RIKEN has been engaged in research into cancer treatment using heavy-ion beams in cooperation with the National Institute of Radiological Sciences. Heavy-ion irradiation kills cancer cells by damaging their genes. With this as a pointer, heavy-ion-beam breeding emerged.
When an accelerator for cancer treatment was to be built at the National Institute of Radiological Sciences, and testing at RIKEN was about to be completed, Abe and her colleagues were enjoying viewing the cherry blossoms. Among those present was Yasushige Yano, now the Director of the Nishina Center for Accelerator-based Science. He said, “Abe-san, the beam line will soon be out of use—don’t you have any plans to use it for biological experiments?”
“How about plants?” Abe replied. “If genes are artificially damaged to a degree that does not cause cell death, a wide variety of mutations will occur and new plants will be produced.”
“That’s interesting, isn’t it?” said Yano. This conversation was the starting point of heavy-ion beam breeding.
Although basic research on inducing mutations using heavy-ion beams was already underway, no one in Japan or the rest of the world had attempted to apply this technology to full-scale breeding. “This could not be achieved anywhere else besides RIKEN,” Abe adds. “Most accelerators are used only for experiments in nuclear physics. However, Dr. Yoshio Nishina, who led the project to construct Japan’s first cyclotron at RIKEN, is said to have urged, ‘Make use of the accelerator not only in nuclear physics, but also in various other fields.’ His advice is still valid now.” Fields other than nuclear physics account for 20–30% of the total annual operating time of the radioactive-isotope-beam factory (RIBF), and experiments by the Radiation Biology Team account for about 3% of the total operating time.
High mutation rates and diverse mutations after only a few seconds of irradiation
“Low exposure levels, high mutation rates, and a wide variation of mutations—these are the advantages of heavy-ion beams,” says Abe. “Because heavy-ion beams have a lot of energy per particle, several tens to one thousand times more than those of X-ray and gamma-ray beams, a gene can be significantly damaged even with just a single particle. In addition, because only one site on the DNA double-strand is broken by the passage of the beam, a mutant lacking just a single gene is produced with high probability.”
Such mutants are ideal for breeding. An attempt to create a new variety with increased disease resistance by crossing a pair of different varieties often results in the desired increased disease resistance but with decreased palatability. In such cases, it is necessary to restore the original palatability by repeating many cycles of hybridization with a highly palatable variety. This takes five to ten years. If X-rays or gamma rays are used instead, numerous particles are required to obtain the desired effect because the damage caused by each particle is low; as a result, it is more likely that more than one gene will be damaged. This poses the problem of the likely alteration of more than one characteristic as with hybridization. “The use of heavy-ion beams often alters only a single characteristic,” says Abe. A new variety can be obtained by selecting a mutant with a modification to the target characteristic while retaining the existing valuable characteristics. The time span for breeding can be shortened significantly to two or three years.
The Radiation Biology Team uses heavy-ion beams generated by accelerating ions in the RIKEN ring cyclotron at RIBF. At present, heavy-ion-beam breeding is conducted at three sites in Japan: RIKEN, the Takasaki Advanced Radiation Research Institute of the Japan Atomic Energy Agency, and the Wakasa Wan Energy Research Center. The RIKEN accelerator boasts the highest performance, enabling use of the ions of carbon, nitrogen, neon, argon, iron, and other elements, thereby offering a greater variation in the mutations. Another advantage is the high-energy output from the accelerator and the longer range of the heavy ions in plant cells. “If the heavy ions stop in the cell, the cell is damaged to excess,” says Abe. “So I want to allow the ions to pass through the cell and obtain a uniform dose. The RIKEN accelerators make this possible.”
A wide variety of plant parts can be targets for exposure, including dry seeds, water-soaked seeds, cultured cells, and shoots called scions (Fig. 3). These materials are grown after irradiation and mutants with the desired characteristics are selected. Heavy-ion beams do not allow ‘targeted shooting’ of a particular gene. However, because of the short duration and low level of exposure to the irradiation, the survival rate is high, and mutations occur with a high probability exceeding 10%. Additionally, because a large number of samples can be treated at one time, the ‘many shots for one hit’ rule applies.
Figure 3: Samples to be irradiated with heavy-ion beams.
These are a wide variety of plant parts, including dry seeds, water-soaked seeds, scions, and cultured cells waiting to be irradiated with heavy-ion beams. The dry seeds are advantageous in that large numbers can be irradiated at one time, but their mutation rate is lower than that for other plant materials during cell division.
‘Nishina Zao’ – a new variety of cherry with a new flower color
If a new variety is created from a cultivated plant, it must be registered with the Ministry of Agriculture, Forestry and Fisheries under the Seeds and Seedlings Law. Although many new varieties have been created by the Radiation Biology Team, all have been registered by cooperating companies or agricultural stations. Last October, however, RIKEN registered a variety under the Seed and Seedlings Law for the first time in its long history.
“It is a cherry with yellowish flowers, which we created by treating the bright yellow green parent breed with heavy-ion beams. In response to our request, President Noyori named it ‘Nishina Zao’ (Fig. 4)” ‘Nishina’ is the surname of the scientist who is the father of the RIKEN accelerators, and ‘Zao’ is the name of a mountain in Yamagata Prefecture, where the co-researcher in this project lives. “I will be given a young seedling of the new variety, and I am now thinking about where to plant it. The yellow color is very beautiful when the flowers are in pre-bloom,” Adds Abe. She feels that ‘Nishina Zao’ will become a popular variety.
The Radiation Biology Team distributes morning glory seeds irradiated by heavy-ion beams to people who visit the Wako Institute on the open house day every April. “Horticultural books published in the Edo period feature illustrations of distinctive morning glories, some of which are no longer available,” explains Abe. “Visitors are given the seeds so that they can help us look for ‘henka asagao’ [variant morning glories].”
Figure 4: Nishina Zao,’ a variety of cherry with a new flower color.
Pale-yellow ‘Nishina Zao’ was created by treating greenish ‘Gyoiko’ with heavy-ion beams. It was named by Ryoji Noyori, the president of RIKEN, and became the first plant registered by RIKEN under the Seeds and Seedlings Law. In both photos, ‘Nishina Zao’ is on the right, and ‘Gyoiko’ is on the left.
The visitors are requested to sow the seeds, grow the resulting seedlings until they flower, collect their seeds, and sow them the following year. Because the mutations induced by heavy-ion beams are usually recessive, variant morning glories with distinctive colors, shapes and other characteristics can emerge in the second filial generation and beyond. “We have organized a ‘Morning Glory Club’, which has a web site at http://www.rarf.riken.go.jp/asagao/.” Abe says that here, people can submit their photographs of the morning glory varieties, including those characterized by lobate petals. If a photographed morning glory seems to be a variant, we ask the submitter to send the seeds to us, and we study them extensively for genetic changes and features. For example, there are no yellow morning glories in the world. I would be happy to see yellow morning glories in bloom.” One of these visitors could possibly discover the only yellow morning glory in the world.
First salt-resistant rice in the world
Flowers do not represent the only subject for heavy-ion beam breeding. Abe recently succeeded in producing a salt-resistant rice plant by irradiating the ‘Nipponbare’ variety with heavy-ion beams (Fig. 5). If an ordinary cultivar is grown in a paddy field affected by salt damage, its plants wither. Even after planting seeds, the rice grains obtained may become cracked or white-cored during milling. However, the new variety grows normally and produces grains that are qualitatively equivalent to those from rice plants grown in ordinary paddy fields. “I ate the rice, and it was as delicious as the rice in the control group. The same may be achievable with other cereal crops,” continues Abe. The area of land not suitable for agricultural purposes is increasing due to salt damage, and this technology is expected to contribute to solving the food issue.
Figure 5: Candidate seedlings for salt-resistant rice.
If cultured in a salt-damaged paddy field (containing sodium ions at a quarter of the level found in seawater, and more than 20 times higher than that found in agricultural water), ordinary cultivars of rice wither, whereas the salt-resistant rice varieties produced by heavy-ion-beam irradiation grow well. In particular, Strain 6-99 rice grows higher than ordinary cultivars and produces heavier grains (1,000-grain weight, which is 102% the weight of Nipponbare grains), although the number of grains is smaller (90% that of Nipponbare). Overall, the salt resistance of the new strain is 1.5 times higher than that of ordinary cultivars.
Although it will take a long time to market them commercially, other crops with improved characteristics have been created at by the Radiation Biology Team and collaborators, including a variety of buckwheat with increased lodging resistance because of its lower plant height, a variety of satsuma orange that allows easy harvesting because of the absence of thorns on the branches, and a variety of green pepper that bears yellow fruits without a bitter taste. “The green pepper is wonderful,” says Abe. She explains that as a general rule, phenotypes of recessive mutations appear in the second filial generation and beyond. This is a well-known rule of Mendelism. However, despite this fact, the green pepper exhibited a recessive mutation profile in the first filial generation. The reason remains unknown. “I am determined to carry out further investigations.” Much remains unknown about the mechanism behind the induction of mutants by heavy-ion beams. One task for the Radiation Biology Team is to determine this mechanism, and establish a more effective method of mutagenesis.
Contributing to solving food problems
Now more than 120 parties are engaged in cooperative research with the Radiation Biology Team, including agricultural experiment stations, universities, and private companies. In 2001, the Cyclotron Mutagenesis Research Group was organized within the Japanese Society of Breeding. The group holds a research congress every two years to promote personal exchange among its members by means of case reports on basic research and practical applications, and to release its ‘User Breeders’ Society Report’. (The coming session is scheduled as a RIKEN symposium on January 24 and 25, 2008.) International cooperation in mutation breeding using ion-irradiated plant materials is also expanding, including joint research programs for pearl millet and sorghum with South Africa, orchids with Thailand, and wheat and barley with Australia. However, only Japan is active in actual heavy-ion beam irradiation to find new breeds. China has just emerged as the second country to develop an irradiation technique to induce mutations in plants using their heavy-ion accelarator facility. “I hope that heavy-ion beam breeding will continue to spread worldwide.”
Abe adds, “I also hope that heavy-ion beams will find applications in research in the life sciences.” If a mutant is created by heavy-ion beam irradiation, and each damaged gene is identified, its function can be determined.
Abe’s next goal is to improve the mutation rate. “Using the superconducting ring cyclotron completed in December 2006, the range of the ions can be increased, and ions of heavier elements can be accelerated. I expect that by doing so, heavy-ion beams will become a more effective tool.” There is also a major need to develop a technology for deleting a whole single gene. The technology may be achieved by precision control of heavy-ion beams.
This year the Radiation Biology Team received the Science and Technology Award (Development Division) in the Commendations from the Minister of Education, Culture, Sports, Science and Technology for ‘Development of highly efficient mutagenesis-based breeding technology using high-intensity heavy-ion accelerators’. The team has received many other awards, which indicates that heavy-ion beam breeding is drawing attention and bringing great expectations. Amid these circumstances, Abe says reflectively, “I graduated from the faculty of agriculture at my university and was not familiar with accelerators at all. But then I realized that accelerators are wonderful tools that enable us to do what has not been done using a biological approach. I want to create ‘Japan-made’ new varieties, to contribute to advancing genome science, and to resolve food and environmental problems.”
About the Researcher
Tomoko Abe
Tomoko Abe obtained her PhD in 1989 from Tohoku University. She was awarded a fellowship for Japanese Junior Scientists by the Japan Society for the Promotion of Science, and worked at the Institute of Genetic Ecology in the same university. In 1990, she joined the RIKEN Plant Functions Laboratory as a postdoctoral fellow. In 1991, she became a research scientist and was promoted to senior scientist in 1998. She became a member at the Cyclotron Center in 2003. In 2006, she moved to the Radiation Biology Team at the Nishina Center. Her research focuses on the biological effects of heavy-ion beams and the development of technology for mutation breeding.
