Waxy Rice Mutants with Unique Processing Properties for Waxy Rice Breeding

Breeding Science 57 : 175–180 (2007)

Note

Kazuyuki Kobayashi*1,2) and Minoru Nishimura3)

1) Niigata Agricultural Junior College, 12021 Maki-kou, Niigata 953-0041, Japan 2) Graduate School of Science and Technology, Niigata University, 2-8050 Ikarashi, Niigata 950-2181, Japan 3) Institute of Radiation Breeding, National Institute of Agrobiological Sciences, 2425 Kamimurata, Hitachiomiya, Ibaraki 319-2293, Japan

Key Words: Oryza glaberrima Steud., Oryza sativa L., breeding materials, induced mutants, processing properties, rice cake hardening rate, waxy (or glutinous) rice.

Waxy rice (also called glutinous rice, sticky rice and lines, we identified rice cake processing properties that had mochi rice) has been widely and predominantly used for an- not been previously observed in commercial paddy waxy nual events or festivities in Japan since ancient times and is rice cultivars in Japan. These mutants could become poten- essential to Japanese culture. Food products made from tial gene sources for the breeding of new cultivars. In the waxy rice include rice cakes (kirimochi), rice crackers present paper, we described waxy rice mutant lines confer- (arare and okaki), steamed rice with red beans ring unique rice cake processing characteristics. (okowameshi) and rice pastries (wagashi). Different processing characteristics of rice are required for each product. Materials and methods During the manufacture of the popular rice cakes and We used 24 artificial mutant lines stored at the Institute rice crackers, rice cake dough is refrigerated for ease of cut- of Radiation Breeding located at the National Institute of ting. If the dough could be made to harden sooner, the re- Agrobiological Sciences, Ibaraki, Japan. Waxy rice cultivars quired refrigeration period would be shortened, the costs of ‘Koganemochi’ and ‘Mangetsumochi’ (japonica), and refrigeration would decrease, and the whole manufacturing ‘Hokurikumochi 181’ (indica) were used for comparison process could become more efficient. (Table 1). Seedlings were transplanted at the Institute of The rate of hardening of rice cakes varies with the rip- Radiation Breeding and at the Niigata Crop Research Center ening conditions and the rice cultivars. Among the waxy rice and grown at various air temperatures during ripening by cultivars in commercial use in Japan, Oryza sativa L. ssp. early (late April), conventional (late May) and late (late japonica ‘Koganemochi’ grown in Niigata Prefecture pro- June) transplanting in 2001 to 2003. Other cultivation condi- duces rice cakes with a superior hardening rate (Saito 1987). tions were identical at each station until harvest. ‘Koganemochi’ had been targeted as breeding objective cul- We examined the hardness of the rice cakes after refrig- tivar in waxy rice breeding in Japan until now. eration according to the method of Ishizaki (1994), using The rice cake processing industry currently requires samples harvested in 2001 and 2002. The validity of this new waxy rice cultivars with unique processing and grain procedure for evaluating the hardness of the rice cakes was characteristics to expand the range of use of waxy rice and to reported elsewhere (Kobayashi 2004). increase the consumption of waxy rice products in Japan. We determined the rate of hardening at room tempera- However, the genetic resources available in the pedigree ture according to the method of Kobayashi et al. (2003a), us- records of paddy waxy rice cultivars in Japan are limited. ing samples from the Niigata Crop Research Center in 2003. There is a need to use new genetic resources to meet this de- Rice cakes were left at room temperature (23°C), and their mand (Akama and Arisaka 1992). hardness was measured at 3, 7 and 14 days after the prepara- We have focused on the genetic resources in artificially tion using a Tensipresser (TTP-50BX, Taketomo Electric induced waxy mutants, in which we expect to find diverse Inc., Tokyo, Japan). Because the samples were left at room mutations. We studied the processing properties of several temperature during such a long period of time, we disinfect- artificially induced waxy mutants in order to breed cultivars ed their surfaces with 90% ethanol after each measurement with unique processing properties suitable for the develop- to prevent colonization by mould. ment of new waxy rice cake products. In the waxy mutant We examined the heat gelatinization properties by using samples harvested in 2001, 2002 and 2003. The heat ge- Communicated by T. Nishio latinization properties of the brown rice flour were measured Received August 14, 2006. Accepted December 8, 2006. with a Rapid Visco Analyser (RVA-3D, Newport Scientific *Corresponding author (e-mail: [email protected]) Pty. Ltd., Warriewood, NSW, Australia; hereafter referred 176 Kobayashi and Nishimura to as “RVA”) by the method described by Kobayashi et al. lower and the gelatinization temperature was 3°C lower than (2003b). those in ‘Koganemochi’ (Fig. 1). At room temperature, rice In order to rapidly detect a structural mutation of waxy cakes that were softer at 3 days after preparation tended to starch, the apparent amylose content, as an indicator of the harden more slowly. Even at 2 weeks after preparation, rice content of iodine-binding amylopectin, was measured with cakes made from the mutant lines derived from O. sativa an AutoAnalyzer 3 (Bran + Luebbe Co., Ltd., Norderstedt, were only about half as hard as those made from Germany), whose base sensitivity was amplified four times, ‘Koganemochi’ (Fig. 2). Therefore, rice cakes made from compared with ordinary measurements, according to the these O. sativa mutant lines would require a longer period of method of Kobayashi et al. (2003b). time before they could be cut. The concentration of amylopectin in starch increased The production of rice cakes that remain soft over a linearly at 5 days after flowering and peaked at about 20 days long period of time is important for traditional Japanese con- after flowering (Asaoka et al. 1985). Hence, we defined the fectioneries such as waxy rice pastries, manufactured from cumulative daily average air temperature over the 20-day waxy rice flour. Rice cakes made from ‘Mangetsumochi’, a period after heading as the temperature during the ripening Japanese commercial cultivar, and ‘Hokurikumochi 181’, a period (hereafter referred to as “ripening temperature”). We waxy indica rice cultivar, displayed lower hardness and studied the relationships between various ripening tempera- hardening rates than those made from ‘Koganemochi’, close tures and the above-mentioned parameters to determine the to those of the mutant lines (Fig. 1 and Fig. 2). Hence, we effect of the ripening temperature on the rice cake process- should also elucidate the mechanisms controlling the lower ing properties. hardening rates of the rice cakes made from the mutant lines of O. sativa by comparing them with those made from waxy Rice cake hardening properties rice cultivars. The relationship between the hardness of the rice cakes and heat gelatinization properties based on amylographic Heat gelatinization properties analysis of rice flour has been well documented. Among the When we examined the heat gelatinization properties of heat gelatinization properties, the gelatinization temperature rice flour by RVA, many of the lines derived from ‘Norin 8’ (temperature at the start of gelatinization of a rice flour sus- and ‘Nihonmasari’ showed lower maximum viscosity val- pension) is strongly correlated with the hardness of the rice ues, lower minimum and final viscosity values, and conse- cakes after refrigeration (Yanase et al. 1982a, 1982b, Saito quently a lower consistency than ‘Koganemochi’. However, 1987). Both properties are closely related to the air temperature during the ripening period: higher air temperature en- hances these properties (Saito 1987, Matsue et al. 2002). We calculated the hardness of the rice cakes after refrigeration and the gelatinization temperature in each line grown at a ripening temperature of 500°C, using a linear regression in order to compare the properties. Comparison of each property at the same ripening temperature revealed that in a waxy mutant line derived from O. glaberrima (hereafter referred to as ‘O. glaberrima (wx)’), the hardness of the rice cakes after refrigeration was two times higher and the gelatinization temperature was more than 7°C higher than those of ‘Koganemochi’, a leading cultivar in Japan (Fig. 1). Oryza glaberrima (wx) rice cakes also hardened more readily than those of ‘Koganemochi’, even at room temperature (Fig. 2). Okamoto and Nemoto (1998), Okamoto et al. (2002) and Okamoto et al. (2003) had already reported that rice cakes made from the waxy rice cultivar ‘Kantomochi 172’, produced by the crossing of the waxy rice cultivar ‘Tsukubahatamochi’ (japonica) and nonwaxy rice cultivar ‘IRAT109’ (tropical japonica), hardened more quickly than those made from many other japonica waxy rice cultivars. Fig. 1. Two important processing indicators in waxy rice mutants. We should also elucidate the mechanisms controlling the Comparison of hardness of the rice cakes and gelatinization hyper-hardness and hardening rates of the rice cakes made temperature of rice flour in each line grown at the ripening from O. glaberrima (wx). temperature of 500°C and calculation based on linear regres- In contrast, in the mutant lines of O. sativa, hardness of sion in each plant material. , , : Average value of lines the rice cakes after refrigeration was approximately 50% derived from each cultivar, respectively. Waxy rice mutants with unique processing properties 177

Fig. 2. Changes in rice cake hardness at 23°C in each line over a two-week period after processing. : Koganemochi, : Mangetsumochi, : Hokurikumochi 181, : Mutant line induced from O. glaberrima. : Mutant lines induced from Norin 8, : Mutant lines induced from Reimei, : Mutant lines induced from Nihonmasari. many of the lines derived from ‘Reimei’ (white boxes in Fig. 3) showed higher maximum viscosity values than ‘Koganemochi’. Their values for minimum viscosity, final viscosity and breakdown were also higher than those of other mutant lines. Lines 15 (84REwx3wx) and 16 (84REwx5) showed clearly a higher consistency (Fig. 3). Although Wx protein is responsible for the synthesis of Fig. 3. Heat gelatinization properties of plant materials. The numbers amylose in the rice endosperm, it is considered to be one of at the bottom denote the lines listed in Table 1. the main proteins that bind with starch (Sano 1984). Then, examination of the relationship between the heat gelatinization properties (Fig. 3) and the presence or concentration of Wx protein (Table 1) revealed that Wx protein did not ap- pear to be directly involved in the heat gelatinization properties of our plant materials. Figure 4 shows the relationship between the maximum viscosity values and the apparent amylose contents of our plant materials. The mutant lines (15–19) derived from ‘Reimei’, with the exception of line 14, showed higher apparent amylose contents than the other cultivars (Table 1), and higher maximum viscosity values than ‘Koganemochi’ (Fig.4). Thus, although, ordinary waxy rice harbors an amylose-free rice endosperm, it is possible that the apparent the amylose content is related to a higher maximum viscosity value. Among the five lines (15–19) induced from ‘Reimei’, line 19 (85REwx7) did not synthesize Wx protein (Table 1). Fig. 4. Relationship between the apparent amylose content and maxi- Thus, it is possible that an increased content of iodine- mum viscosity. Horizontal lines for each symbol denote the binding amylopectin increased the maximum viscosity. standard deviation. Similarly, none of the lines derived from ‘Nihonmasari’ synthesized Wx protein, while line 25 (WX23) showed a 178 Kobayashi and Nishimura

Table 1. Plant materials Apparent amylose ° 1) 2) Code Ripening temperature ( C) Gene Wx protein 3) Cultivar/Line Original cultivar Mutagen content (%) No. locus2) (µg/mg) Min. Max. Range Ave. S.D. 1 Koganemochi 482 527 45 0.24 0.09 2 Mangetsumochi 452 523 71 wx 0.00 0.24 0.13 3 Hokurikumochi 181 393 540 147 0.32 0.17 4 73wx1N1A Norin 8 EMS 411 523 112 wx 0.00 0.20 0.08 5 74wx1N1A Norin 8 EMS 411 503 92 wx 1.05 0.16 0.04 6 74wx4N1A Norin 8 EMS 393 523 130 0.21 0.10 7 74wx7N1A Norin 8 EMS 393 510 117 wx 0.00 0.27 0.23 8 75wx5 Norin 8 EMS 411 510 99 wx 0.99 0.58 0.30 9 76wx1N1 Norin 8 EMS 411 513 102 0.57 0.36 10 76wx2 Norin 8 EMS 411 507 96 wx 0.71 0.25 0.13 11 76wx5 Norin 8 EMS 451 535 84 0.50 0.94 0.47 12 KURwx4N1 Norin 8 Neutrons 411 504 93 wx 0.00 0.20 0.07 13 82Gwx1 Norin 8 Acute γ-rays 411 523 112 wx 0.00 0.50 0.34 14 82RGwx2wx Reimei Acute γ-rays 464 545 81 wx 0.00 0.41 0.38 15 84REwx3wx Reimei EMS 464 540 77 0.74 0.86 0.46 16 84REwx5 Reimei EMS 464 541 77 wx 0.98 0.96 0.50 17 84REwx11 Reimei EMS 464 541 77 wx 0.62 0.50 0.28 18 85REwx3 Reimei EMS 464 545 81 wx 0.04 1.48 0.82 19 85REwx7 Reimei EMS 456 540 85 wx 0.00 1.47 0.74 20 WX1 Nihonmasari Acute γ-rays 451 532 82 wx 0.00 0.27 0.20 21 WX5 Nihonmasari Acute γ-rays 451 539 88 wx 0.00 0.23 0.16 22 WX14 Nihonmasari EI 451 534 83 0.00 0.23 0.12 23 WX15 Nihonmasari EI 451 534 83 wx 0.00 0.21 0.12 24 WX19 Nihonmasari EI 451 532 81 0.00 0.29 0.14 25 WX23 Nihonmasari EI 449 534 86 wx 0.00 2.27 0.43 26 WX25 Nihonmasari Chronic γ-rays 451 540 88 wx 0.00 0.24 0.13 27 O. glaberrima (wx) O. glaberrima EMS 376 497 122 0.29 0.07 1) Cumulative daily average air temperature during the ripening period, for 20 days after heading depending on the transplanting time. 2) Gene locus and Wx protein content of plant materials had already been estimated in 1992 at the Institute of Radiation Breeding. 3) Apparent amylose content was measured with AutoAnalyzer 3. higher apparent amylose content and a higher maximum viscosity value than the other lines derived from the same cultivar (Table 1, Fig. 3 and Fig. 4). We therefore consider that the line contained an increased amount of iodine-binding amylopectin. It is possible that the apparent amylose content (or amylose-like starch structure) was associated with the heat gelatinization properties of these lines. In addition, we as- sumed that the larger variation in the apparent amylose content of these lines (Fig. 4) was affected by various ripening temperatures depending in the transplanting time. The heat gelatinization properties of brown rice flour are strongly affected by the amount of cellular tissue present (Arisaka 1994). Moreover, lipids (Kuge 1992) and seed stor- Fig. 5. Response of hardness of the rice cakes and gelatinization tem- age proteins (Yoshii and Arisaka 1994) affect the processing perature of rice flour to the ripening temperature defined as the properties. Therefore, we consider that the variations in the cumulative daily average air temperature over the 20-day peri- components of the rice grain, such as cellular tissues, lipids od after heading. : Koganemochi, : Mangetsumochi, : and storage proteins, together with the variations in the Hokurikumochi 181, : WX14 (induced from Nihonmasari), structure of waxy starch, exerted an influence on the heat ge- : WX15 (induced from Nihonmasari), : O. glaberrima latinization properties of these lines. (wx). Waxy rice mutants with unique processing properties 179

Rice cake hardness in response to ripening temperature In recent years, ‘Koganemochi’ grown in Niigata Pre- In almost all the lines, the hardness of the rice cakes and fecture has often shown an undesirably high degree of rice the gelatinization temperature increased with the ripening cake hardening, because of the high ripening temperatures, temperature, as described in previous reports (Saito 1987, which adversely affected palatability and product quality Matsue et al. 2002). However, the hardness of the rice cakes (Egawa, K. unpublished). Thus, slight changes in the hard- made from lines 22 (WX14) and 23 (WX15), derived from ening properties of the rice cakes with the ripening tempera- ‘Nihonmasari’, did not increase, even when the ripening ture are important to address this problem. Detailed studies temperature increased by nearly 80°C as well as the gelatini- on these properties, along with the identification of mutant zation temperatures (Fig. 5). genes and analysis of their structure and function should be Suzuki et al. (2002) reported the existence of a rice mu- actively promoted. tant in which amylose synthesis was insensitive to the envi- To introduce these mutant genes into new waxy rice ronmental temperature. This phenomenon was caused by a cultivars, further crossing or backcrossing will be essential constant level of Wx gene expression at normal and cool for breaking the linkage of adverse agronomic characters. It temperatures under the control of a single recessive muta- is thus important that analyses of mutant genes be conducted tion. Therefore, it is possible that the enzymatic reactions in- by using the results obtained in the Rice Genome Project to volved in the biosynthesis of waxy starch in relation to the perform DNA marker selection for the breeding of new hardness of the rice cakes did not depend on the ripening waxy rice cultivars. temperature in lines 22 and 23. However, the hardness of the rice cakes made from lines 22 and 23 was slightly different Acknowledgments from that in Mangetsumochi (Fig. 1, Fig. 2 and Fig. 5). Hence, we should analyze in detail the mechanisms underly- We are grateful to Dr. T. Fukuyama, Faculty of Agri- ing the response of the hardness of the rice cakes to the rip- culture, Field Center for Sustainable Agriculture and Forest- ening temperature by comparing them with those of other ry, Niigata University, for the suggestions, encouragement cultivars. and critical review of the manuscript. This work was sup- ported by a grant for Nuclear Research from the Ministry of New genetic resources for waxy rice breeding research Education, Culture, Sports, Science and Technology of Oryza glaberrima (wx) is an artificial mutant line de- Japan. veloped at the National Institute of Genetics, Shizuoka, Japan (Sano 1976). There are no reports on the character- Literature Cited istics of natural waxy mutants of O. glaberrima. We expect that in the interspecific hybrid populations produced using Akama, Y. and M. Arisaka (1992) Waxy rice. In “Rice breeding in O. glaberrima (wx), the genetic polymorphism of the rice Japan. The challenge posed by super-rice” Kushibuchi, K. (ed.), cake hardening properties could be higher than that in the Association for Advancement of Agricultural Science, Tokyo. other populations obtained by intraspecific hybridization of p. 202–208 (in Japanese). O. sativa. Therefore, we consider that O. glaberrima (wx) Arisaka, M. 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