January 2, 2019.
Aboriginal people in Southeast Asia have been cultivating rice as staple food for more than 5,000 years since the prehistoric period. The evidence of rice cultivation was found in the excavation of Ban Chiang civilization in Udon Thani Province. Thailand has thousands of native rice varieties  and Thai people have high skill in rice planting. It is not uncommon for Thailand to be in the top three of the rice exporters of the world's rice market.
However, Thai farmers are still in the top of the career groups in debt. The main reason is that Thai farmers are planting rice for consumption (table rice). Competitors such as Vietnamese and Indian farmers can produce rice at lower costs which force the Thai government to lower the price of Thai rice for the rice competition in the world’s rice market. Consequently, the rice production in the country is not worth for investment and hence Thai farmers are poor in Thailand.
In the near future, it is an urgent task for the Thai government to let Thai farmers have opportunities in rice businesses. That is to provide Thai farmers with new high quality rice varieties having good agricultural characteristics, such as dwarfism or intermediate stature, photoperiod insensitivity, sturdy short stems, small dark green straight up leaves, high tillering capacity, and high number of panicle per bunch, broad resistances to diseases and insects, and response to nitrogen fertilizers . In addition to the good agricultural characteristics, the rice varieties should be extraordinary rice such as fragrant rice, good cooking quality rice as KDML 105, high nutrition rice (i.e. purple rice, red rice, etc.), and rice for use as the materials for high-value rice products such as rice bran oil, etc., rice for the health and beauty market, rice for use as a body herbal, rice suitable to the production of rice flour and glutinous rice flour for being used as raw materials for processed products such as rice noodles, etc., and high-yield rice for feeding animals such as pigs.
Research in development of high-quality rice varieties has been performed for many years at Chiang Mai University (CMU). The research activities have been funded by the Thailand Center of Excellence in Physics, Committee on Higher Education, Ministry of Education under the research project of "Innovative physics with low energy ion beam for quality rice breeding". Thai researchers have discovered new rice varieties that meet the above-mentioned requirements. Using new techniques, the inventors developed their own processes and tools. The following is a summary of the progress of this research project.
2. Rice breeding by low-energy ion beam technology
The conventional rice breeding technique using biological marker assistants would only provide a low possibility to obtain stable rice lines within a short period of time, because this technique would require 10 generations or approximately 10 years to study rice line stability. Applying physical methods such as gamma rays is another option for the rice breeding. The gamma-ray radiation technique would require a half breeding duration compared with the conventional biological breeding technique, as the time required to study the mutational stability is 5 generations. For instance, seeds of Thai Jasmine rice (Oryza sativa L. cv. KDML105) were radiated by gamma rays at doses of 20 and 15 krad and consequently RD6 and RD15 rice lines were produced, respectively. Both rice lines contained the 2-acetyl-1-pyrroline (2AP) content similar to that of the wild type. However, undesirable characteristics were found in the new lines. These included low crop yield, photoperiod sensitivity (annual cultivation only in the in-season period), tall variety (unsuitable for expanding the rice cultivation), small and delicate culm (easily affected by wind and rain before harvest) and susceptibility to rice blast disease and brown planthopper . Furthermore, gamma rays are generated by Co-60 source and it has several limitations. For example, since the application and maintenance of the source are harmful, a special design to store the source needs to be made. High intensity Co-60 sources are costly and have to be imported. Moreover, the source has a limited lifetime for use as the half-life of Co-60 is 5.3 years.
In 2002, our Chiang Mai University research team launched a program of developing a novel rice mutation technique of using low energy ion beam (<100 keV) to induce mutation in Thai rice, instead of using gamma ray . In 2005, the research team issued a press release of the first success in the application of low-energy heavy-ion beam for Thai jasmine rice breeding at Chiang Mai University (Figure 1a). The research team has carried out the Thai jasmine rice breeding program and successfully obtained rice mutants such as the purple BKOS6 rice mutant [5,6], short HyKOS1 and PKOS1 rice mutants, tall TKOS1 rice mutant , high-crop-yield and blast-resistant HyKOS3-1 rice mutant , and storage-durable-purple HyKOS21 rice mutant . Moreover, Chao Hom CMU-ARDA 09-1 (HyKOS3) rice mutant, Chao CMU-ARDA 09-2 (HyKOS3-1) rice mutant, Chao Hom CMU-ARDA 09-3 (HyKOS16) rice mutant, Chao Kham CMU-ARDA 09-4 (HyKOS21) rice mutant and Chao CMU-ARDA 09-5 (HyKOS22) rice mutant were registered under the Plants Act B.E. 2518, the Department of Agriculture, Ministry of Agriculture and Cooperatives, Thailand (Figure 1b) . The rice mutation breeding by low-energy-ion-beam technology has been continuously studied and developed. So, the method for the rice mutation breeding by this technology to obtain most of desirable traits in Thai rice has been eventually established in Thailand.
Figure 1 (a) Press conference to issue the first success in the application of low-energy heavy-ion beam for Thai Jasmine rice breeding on June 24, 2005 at Chiang Mai University. (b) Five Thai Jasmine rice mutants registered under the Plants Act B.E. 2518, the Department of Agriculture, Ministry of Agriculture and Cooperatives, Thailand.
In the research project “Innovative Physics Using Low Energy Ion Beam for Quality Rice Breeding”, RD6 and Sangyod Phatthalung rice were selected for mutation induction. The selected rice varieties have gained popularity because of good cooking quality. RD6 rice is fragrant glutinous rice and Sangyod Phatthalung rice is red non-glutinous rice due to high anthocyanin contents. However, both rice varieties are photoperiod sensitive (annual cultivation), tall and produce low crop yield. Moreover, the cultivation of Sangyod Phatthalung rice takes 8 months when the rice plantation starts in June .
The novel rice mutation breeding was initially carried out by dormancy breaking of the rice seeds at 49°C for 5 days and the incubated rice seeds were then dehusked. The undamaged brown rice seeds were put into a homemade copper biosample holder. The embryos of the brown rice seeds were placed on the top to face the ion beam (Figure 2a). The rice seeds were bombarded by mixed molecular and atomic nitrogen ions using the home-developed compact ion implanter in the off-season period of February 20-28, 2015 and 2016, respectively (Figure 2b). The ion-bombarded rice seeds were then planted on March 1, 2015 and 2016, respectively. Rice mutants with photoperiod insensitivity indicated by rice panicle appearing before June 10, 2015 and 2016 were selected for further mutational stability study. In M2 to M5 generations, rice mutants with desirable traits  were selected. RD6 and Sangyod Phatthalung rice mutants obtained in 2015 were named MRD6 and MSY, respectively. And, RD6 and Sangyod Phatthalung rice mutants obtained in 2016 were named OSRD6 and OSSY, respectively. Figure. 3 shows a summary of the rice mutation breeding by using this technique.
Figure 2 (a) Copper bio-sample holder with a diameter of 10 cm for loading of RD6 and Sangyod Phatthalung brown rice seeds, and (b) the compact ion implanter developed and installed in the ion beam laboratory, Chiang Mai University for ion-beam induced rice mutation breeding  without residual radioactivity.
Figure 3 Diagram of a summary of the Sangyod Phatthalung rice mutation breeding induced by applying low-energy ion-beam technology.
3. Eighty-five RD6 and Sangyod Phatthalung rice mutants
In 2015, we obtained 27 RD6 rice mutants (MRD6-1 to MRD6-27) and 7 Sangyod Phatthalung rice mutants (MSY-1-1 to MSY-4) from the first rice mutation induction. One year later, we obtained 25 RD6 rice mutants (OSRD6-1 to OSRD6-27) and 26 Sangyod Phatthalung rice mutants (OSSY-1-1 to OSSY-4). All obtained 85 rice mutants showed photoperiod insensitivity and desirable traits related to the high-crop-yield rice as described earlier .
Low-energy ion-beam technology provided a much higher mutation rate than gamma ray, because the linear energy transfer (LET) of ions is 300-6,000 times higher than that of gamma ray from Co-60. Furthermore, this technology produced desirable phenotypic variations in one rice mutant such as shorter culm length, photoperiod insensitivity and higher crop yield due to higher number of panicles per plant. Most of RD6 and Sangyod Phatthalung rice mutants produced higher crop yield than their wild types. Crop yields of RD6 and Sangyod Phatthalung rice mutants were in the range of 544 - 1,424 kg/rai (3.4 – 8.9 t/ha) and 512 - 1,536 kg/rai (3.2 – 9.6 t/ha), respectively, whereas RD6 and Sangyod Phatthalung wild types had crop yields of 528 and 688 kg/rai (3.3 and 4.3 t/ha), respectively (Figures 4 and 5).
Figure 4 Examples of (a) RD6 and (b) Sangyod Phatthalung rice mutants compared with their wild types.
Figure 5 The crop yield, plant height, and number of panicle per plant of RD6 rice mutants (MRD6 and OSRD6) and Sangyod Phatthalung rice mutants (MSY and OSSY) compared with those of RD6 and Sangyod Phatthalung (SYP) wild types. Red dashed lines are the values of the phenotypes of the wild types for comparisons.
Physical properties of rice mutants’ seeds and brown rice grains were found different in size and shape, as well as in color of brown rice, from those of their wild types. For instance, some brown rice grains of RD6 rice mutants were purple, while brown rice of the RD6 wild type was white. Brown rice grains of most Sangyod Phatthalung rice mutants were white and purple; however, brown rice of the Sangyod Phatthalung wild type was red (Figure 6). Chemical properties of the rice mutants’ grains were also found changed, such as the amylose content. Some RD6 rice mutants were low-amylose-non-glutinous rice (10 - 20 % amylose content) and some were high-amylose-non-glutinous rice (>25 % amylose content), whereas the RD6 wild type was glutinous rice (<10 % amylose content). Some Sangyod Phatthalung rice mutants were also changed into glutinous rice and high-amylose-non-glutinous rice, while the Sangyod Phatthalung wild type was low-amylose-non-glutinous rice. Increase and decrease in the nutritional values of the rice mutants’ brown rice, such as protein, fat and β-carotene contents in brown rice, were found (Figure 7). Moreover, the 2AP content of 11 RD6 rice mutants’ brown rice was in a range of 0.37 – 1.59 mg/kg, whereas brown rice of the RD6 wild type contained much higher 2AP at 5.11 mg/kg. Seven Sangyod Phatthalung rice mutants possessed the 2AP content in brown rice were in a range of 0.57 – 1.89 mg/kg, while this fragrance content could not be detected in brown rice of the Sangyod Phatthalung wild type (Figure 8).
Figure 6 Examples of rice seeds and brown rice grains of (a) RD6 rice mutants and (b) Sangyod Phatthalung rice mutants compared with their wild types
Figure 7 Color of brown rice and major chemical properties of RD6 rice mutants (MRD6 and OSRD6) and Sangyod Phatthalung rice mutants (MSY and OSSY), compared with the RD6 and Sangyod Phatthalung (SYP) wild types. Amylose content was analyzed at Faculty of Agro-Industry, Chiang Mai University. Protein and fat contents were analyzed at Agricultural Technology Services Center, Faculty of Agriculture, Chiang Mai University. Carotenoid content was analyzed at Faculty of Engineering and Agro-industry, Maejo University. Red dashed lines are used for the comparison in the grain properties of rice mutants with those of their wild types.
Figure 8 2AP content in brown rice of RD6 rice mutants (MRD6) and Sangyod Phatthalung rice mutants (MSY) compared with that of the RD6 wild type. The 2AP content was analyzed at Rice Chemistry Research Laboratory and Center of Excellence for Innovation in Chemistry (PERCH-CIC), Department of Chemistry, Chiang Mai University. Red dashed lines are used for the comparison of the 2AP content in brown rice mutants with that of RD6 wild type.
4. Selection and categorization of rice mutants to supply for the demand of consumers and the use of various rice industries
According to the tests of RD6 and Sangyod Phatthalung rice mutants, we selected some rice mutants with outstanding plant and grain qualities and categorized them into 7 groups for use in several rice industries.
Group 1: Six fragrant non-glutinous Sangyod Phatthalung rice mutants (Table 1). These rice mutants are potentially suitable for being used as material in rice flour and rice noodle industries such as fermented rice vermicelli (Kanom-jeen) and fine cut white rice noodle (Kuay Teaw), owing to these fragrant-high-amylose rice mutants possessing higher final viscosity in rice grain than non-fragrant Chainat 1 and Khao Gaw Diaw 35 rice, which are officially known most suitable for rice flour and fermented rice vermicelli productions (Figure 9).
Table 1 Crop yield, amylose content, 2AP content and final viscosity of the selected fragrant Sangyod Phatthalung rice mutants for rice flour industry.
Figure 9 Fermented rice vermicelli by using the selected fragrant Sangyod Phatthalung rice mutants compared with that of Chainat 1 (the 1st from left on the top row) and Khao Gaw Diaw 35 rice (the 2nd from left on the top row). All fermented rice vermicelli samples were produced from 350 gram of rice.
Group 2: 3 fragrant glutinous RD6 rice mutants (Table 2). These rice mutants are potentially suitable for consumption and dessert production.
Table 2 Crop yield, amylose content and 2AP content of the selected fragrant RD6 rice mutants for consumption.
Group 3: Six pigmented RD6 and Sangyod Phatthalung rice mutants (Table 3). These rice mutants are potentially suitable for health care consumption and for being used as materials in functional food and cosmetic productions.
Table 3 Crop yield, amylose content and color of brown rice of the selected pigmented RD6 and Sangyod Phatthalung rice mutants for health care consumption and for being used as materials in functional food and cosmetic productions.
Group 4: 4 high-fat glutinous RD6 rice mutants (Table 4). These rice mutants are potentially suitable for being used as materials in the rice bran oil production and consumption.
Table 4 Crop yield and fat content of selected high-fat RD6 rice mutants for rice bran oil production.
Group 5: 4 high-β-carotene Sangyod Phatthalung rice mutants (>10 µg/g β -carotene) (Table 5). These rice mutants could be used as a food source for the people with vitamin A deficiency or vitamin A intake lower than 4,000 IU.
Table 5 Crop yield and β-carotene content of the selected high-β-carotene Sangyod Phatthalung rice mutants.
Group 6: 9 glutinous RD6 and Sangyod Phatthalung rice mutants with high-yield potential (> 1,200 kg/rai) (Table 6). These rice mutants could be used to increase the production of glutinous rice of Thailand and could increase the capability of the glutinous Thai rice export in the world market.
Table 6 Crop yield and amylose contents of the selected high-yield-potential glutinous RD6 and Sangyod Phatthalung rice mutants for increase in capability of the glutinous Thai rice export.
Group 7: 2 non-glutinous Sangyod Phatthalung rice mutants with high-yield potential (> 1,200 kg/rai) (Table 7). These rice mutants could be used to increase the production of non-glutinous rice of Thailand and could increase the capability of the Thai non-glutinous rice export in the world market by exporting as parboiled rice. Furthermore, these rice mutants could be used to feed livestock.
Table 7 Crop yield and amylose content of the selected high-yield-potential non-glutinous RD6 and Sangyod Phatthalung rice mutants for increase in capability of glutinous Thai rice export and use to feed livestock.
These selected rice mutants tended to produce high crop yields (750-1,500 kg/rai) and their grain qualities were in agreement with the demands of consumers and rice industries. The cultivation of these rice mutants could be worth for rice farmers and the use in rice industries. Thus, these rice mutants could well respond to the needs of Thailand in terms of rice for development in life quality of Thai rice farmers and the increase in export opportunities of Thai rice and rice products as well.
5. Alleviation of Thai farmers’ poverty by using new rice varieties that served for national strategy Thailand 4.0: Ratchaburi Model
To solve the problem of the poverty of Thai farmers and the problem of agricultural labor lacking, the Farmers Council of Ratchaburi Province is strengthening farmer groups in Ratchaburi by organizing the farmers to large cooperative agricultural groups. Ratchaburi Province has a rice cultivation area in total of about 596,657 rais, among which 326,896 rais are for in-season rice planting and 267,203 rais for off-season rice planting. The yield of rice production is about 425,717 tons per year. There are about 1,874 households participating in rice farming. The cooperation of the farmers leads to agricultural progress and development since it is an effective and sustainable way to solve the problems of farmers. The cooperation promotes their bargaining power in both production and marketing. And it also brings collaboration of the farmer groups. Development in a large-scale rice production of the groups as well as good management in rice production makes the groups easy to find funding sources and external supports.
Ratchaburi rice farmers face not worth for rice production investment because of low-yield rice varieties planting. They also suffer from major rice diseases and insect invasion during rice planting. The rice varieties they use in the rice production are not resistant to major rice disease (Blast) and insects including brown planthopper and white backed planthopper. The farmers collect grains in plots and use them as seeds every year but they are not pure bred. As a result, the rice crop yields decrease. The price of rice is low because the rice grains are not pure, resulting in low rice quality. In addition, rice varieties grown by the farmers in Ratchaburi province are cultivated for consumption only. The marketing channels for rice drainage are limited and cannot be processed into higher value rice products. Today, Ratchaburi is also the largest pig farm of the country. There are about 3 million pigs, and the main food of the pigs is rice. Therefore, pig farms have a high demand for rice. For example, large farms with 50,000 pigs require about 32 tons of rice per day. Therefore, the Farmers Council of Ratchaburi tries to solve the problem of shortage of rice varieties. The farmer groups have been paying close attention to the rice development project of CMU and also visiting the CMU rice development facilities for more than four years, as shown in Figures 10-12, and subsequently they have requested cooperation for rice transfer to the farmers in Ratchaburi province. The rice will be used as a motivator of the collaborative farming (large scale farming) policy of the Farmers Council of Ratchaburi to develop a potential of Ratchaburi farmers in both integrated rice production and marketing and reduce the dependence on governmental subsidies. The Farmers Council of Ratchaburi can select rice varieties that are suitable to the local rice planting areas. If this working mode is successful, it will be a prototype mode for rice farmer groups in other provinces as well as available for other areas.
Figure 10 Training and study activities of "Research and Development of Agricultural Innovations" were held on June 1, 2017. The participants included senior managers of the Farmers Council of Ratchaburi province, rice researchers, rice entrepreneurs, and farmers of Ratchaburi.
Figure 11 Technology transfer activities of "Selection of rice mutants obtained from the mutation induction in Thai rice by low-energy ion beam" for members of the Farmers Council of Ratchaburi Province, rice mill entrepreneurs, and seed producers on December 16, 2017.
Figure 12 Technology transfer activities of "Commercial Use of Rice Varieties" for members of the Farmers Council of Ratchaburi Province and the entrepreneurs of Ratchaburi Province on May 19, 2018.
Representatives of the Farmers Council of Ratchaburi Province with rice experts came to the Chiang Mai Rice Research field to select rice for testing in the planting areas in Ratchaburi Province as shown in Figure 13. Rice varieties namely MSY-1-2, MSY-1-3 and MSY-4 were selected for use in starch industry. And one rice variety, OSSY-23, as shown in Figure 14, in rice group number 7 was selected for animal (pig) feed and parboiled rice. Finally, the Farmers Council of Ratchaburi Province and Chiang Mai University made an agreement (MOU) on August 22, 2018, to distribute the seeds to farmers in Ratchaburi Province, where one thousand farmers who want to use the rice varieties were the witnesses, as shown in Figure 15.
Today, all selected rice varieties are currently under cultivation for seed extension by the Huai Pai Agricultural Enterprise for production. As shown in Figure 16, under the operation of the Farmers Council of Ratchaburi Province, the seeds should be sufficient to meet the needs of farmers in Ratchaburi Province in year 2019.
Figure 13 On this Memorial Day, June 27, 2018, Mr. Methadsit Lakanatinawong, Head of Farmers Council of Ratchaburi Province Office (1st from right), rice experts and entrepreneurs in Ratchaburi, together with the Chiang Mai rice researchers and administrative staffs of Thailand Center of Excellence in Physics, selected suitable rice varieties for Ratchaburi farmers at the rice experiment plot, San Sai, Chiang Mai.
Figure 14 (a) Gross appearances of mutants MSY-1-2, MSY-1-3 and MSY-4 (red arrows) and (b) OSSY-23 (right pot), compared with Pathumthani 1 (left pot), the wild type, from the same rice field cultured in the off-season period in 2018. It was found that the rice mutant produced higher yields than Pathumthani 1 because of the higher tillering capacity and a higher number of panicle per bunch than that of Pathumthani 1.
Figure 15 The signing ceremony of the agreement of the rice seed (of mutants MSY-1-2, MSY-1-3, MSY-4 and OSSY-23) transfer to farmers in Ratchaburi Province on August 22, 2018 (upper left photo). Associate Professor Dr. Sumpan Singharajvapan (2nd from left in the low-right photo), Vice President of Chiang Mai University with Mr. Sutin Chadadam (2nd from right in the low-right photo), Chairman of Farmers Council of Ratchaburi Province, and Prof. Dr. Thirapat Vilaithong (1st from left in the low-right photo), Director of Thailand Center of Excellence in Physics, Mr. Arnut Wisetrajana (the middle in the low-right photo), Inspector of the Ministry of Agriculture and Cooperatives for Region 4 & 5, and more than one thousand Ratchaburi farmers (upper-right photo) witnessed at the Gymnasium, Muang county, Ratchaburi.
Figure 16 Rice seedlings field in Huai Phai district, Muang county, Ratchaburi Province. (a) In the seedling stage. (b) Mr. Chaiwit Buangam, Chairman of Huai Pai Agricultural Enterprise Group, in the field. (c) Rice researchers of Chiang Mai University, Dr. Jiranat Techarang (left) and Dr. Boonrak Phanchaisri (right) were providing advices on seed production to Mr. Chaiwit Buangam.
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Report provided by :
Dr.Jiranat Techarang1), Dr. Boonrak Phanchaisri2) and Assoc. Prof. Dr. L. D. Yu 3)
1) Postdoctoral researcher, Thailand Center of Excellence in Physics. Email: firstname.lastname@example.org
2) Researcher, Science and Technology Research Center, Chiang Mai University. Email: email@example.com
3) Senior Researcher, Thailand Center of Excellence in Physics. Email: firstname.lastname@example.org