Waxy Rice Mutants with Unique Processing Properties for Waxy Rice Breeding

Transcription

1 Breeding Science 57 : (2007) Note Waxy Rice Mutants with Unique Processing Properties for Waxy Rice Breeding Kazuyuki Kobayashi* 1,2) and Minoru Nishimura 3) 1) Niigata Agricultural Junior College, Maki-kou, Niigata , Japan 2) Graduate School of Science and Technology, Niigata University, Ikarashi, Niigata , Japan 3) Institute of Radiation Breeding, National Institute of Agrobiological Sciences, 2425 Kamimurata, Hitachiomiya, Ibaraki , 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 mochi rice) has been widely and predominantly used for annual events or festivities in Japan since ancient times and is essential to Japanese culture. Food products made from waxy rice include rice cakes (kirimochi), rice crackers (arare and okaki), steamed rice with red beans (okowameshi) and rice pastries (wagashi). Different processing characteristics of rice are required for each product. During the manufacture of the popular rice cakes and rice crackers, rice cake dough is refrigerated for ease of cutting. If the dough could be made to harden sooner, the required refrigeration period would be shortened, the costs of refrigeration would decrease, and the whole manufacturing process could become more efficient. The rate of hardening of rice cakes varies with the ripening conditions and the rice cultivars. Among the waxy rice cultivars in commercial use in Japan, Oryza sativa L. ssp. japonica Koganemochi grown in Niigata Prefecture produces rice cakes with a superior hardening rate (Saito 1987). Koganemochi had been targeted as breeding objective cultivar in waxy rice breeding in Japan until now. The rice cake processing industry currently requires new waxy rice cultivars with unique processing and grain characteristics to expand the range of use of waxy rice and to increase the consumption of waxy rice products in Japan. However, the genetic resources available in the pedigree records of paddy waxy rice cultivars in Japan are limited. There is a need to use new genetic resources to meet this demand (Akama and Arisaka 1992). We have focused on the genetic resources in artificially induced waxy mutants, in which we expect to find diverse mutations. We studied the processing properties of several artificially induced waxy mutants in order to breed cultivars with unique processing properties suitable for the development of new waxy rice cake products. In the waxy mutant Communicated by T. Nishio Received August 14, Accepted December 8, *Corresponding author ( kazuyuki-k@m2.nct9.ne.jp) lines, we identified rice cake processing properties that had not been previously observed in commercial paddy waxy rice cultivars in Japan. These mutants could become potential gene sources for the breeding of new cultivars. In the present paper, we described waxy rice mutant lines conferring unique rice cake processing characteristics. Materials and methods We used 24 artificial mutant lines stored at the Institute of Radiation Breeding located at the National Institute of Agrobiological Sciences, Ibaraki, Japan. Waxy rice cultivars Koganemochi and Mangetsumochi (japonica), and Hokurikumochi 181 (indica) were used for comparison (Table 1). Seedlings were transplanted at the Institute of Radiation Breeding and at the Niigata Crop Research Center and grown at various air temperatures during ripening by early (late April), conventional (late May) and late (late June) transplanting in 2001 to Other cultivation conditions were identical at each station until harvest. We examined the hardness of the rice cakes after refrigeration according to the method of Ishizaki (1994), using samples harvested in 2001 and The validity of this procedure for evaluating the hardness of the rice cakes was reported elsewhere (Kobayashi 2004). We determined the rate of hardening at room temperature according to the method of Kobayashi et al. (2003a), using samples from the Niigata Crop Research Center in Rice cakes were left at room temperature (23 C), and their hardness was measured at 3, 7 and 14 days after the preparation using a Tensipresser (TTP-50BX, Taketomo Electric Inc., Tokyo, Japan). Because the samples were left at room temperature during such a long period of time, we disinfected their surfaces with 90% ethanol after each measurement to prevent colonization by mould. We examined the heat gelatinization properties by using samples harvested in 2001, 2002 and The heat gelatinization properties of the brown rice flour were measured with a Rapid Visco Analyser (RVA-3D, Newport Scientific Pty. Ltd., Warriewood, NSW, Australia; hereafter referred

2 176 Kobayashi and Nishimura to as RVA ) by the method described by Kobayashi et al. (2003b). In order to rapidly detect a structural mutation of waxy starch, the apparent amylose content, as an indicator of the content of iodine-binding amylopectin, was measured with an AutoAnalyzer 3 (Bran + Luebbe Co., Ltd., Norderstedt, Germany), whose base sensitivity was amplified four times, compared with ordinary measurements, according to the method of Kobayashi et al. (2003b). The concentration of amylopectin in starch increased linearly at 5 days after flowering and peaked at about 20 days after flowering (Asaoka et al. 1985). Hence, we defined the cumulative daily average air temperature over the 20-day period after heading as the temperature during the ripening period (hereafter referred to as ripening temperature ). We studied the relationships between various ripening temperatures and the above-mentioned parameters to determine the effect of the ripening temperature on the rice cake processing properties. Rice cake hardening properties The relationship between the hardness of the rice cakes and heat gelatinization properties based on amylographic analysis of rice flour has been well documented. Among the heat gelatinization properties, the gelatinization temperature (temperature at the start of gelatinization of a rice flour suspension) is strongly correlated with the hardness of the rice cakes after refrigeration (Yanase et al. 1982a, 1982b, Saito 1987). Both properties are closely related to the air temperature during the ripening period: higher air temperature enhances 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. We should also elucidate the mechanisms controlling the hyper-hardness and hardening rates of the rice cakes made from O. glaberrima (wx). In contrast, in the mutant lines of O. sativa, hardness of the rice cakes after refrigeration was approximately 50% lower and the gelatinization temperature was 3 C lower than those in Koganemochi (Fig. 1). At room temperature, rice cakes that were softer at 3 days after preparation tended to harden more slowly. Even at 2 weeks after preparation, rice cakes made from the mutant lines derived from O. sativa were only about half as hard as those made from Koganemochi (Fig. 2). Therefore, rice cakes made from these O. sativa mutant lines would require a longer period of time before they could be cut. The production of rice cakes that remain soft over a long period of time is important for traditional Japanese confectioneries such as waxy rice pastries, manufactured from waxy rice flour. Rice cakes made from Mangetsumochi, a Japanese commercial cultivar, and Hokurikumochi 181, a waxy indica rice cultivar, displayed lower hardness and hardening rates than those made from Koganemochi, close to those of the mutant lines (Fig. 1 and Fig. 2). Hence, we should also elucidate the mechanisms controlling the lower hardening rates of the rice cakes made from the mutant lines of O. sativa by comparing them with those made from waxy rice cultivars. Heat gelatinization properties When we examined the heat gelatinization properties of rice flour by RVA, many of the lines derived from Norin 8 and Nihonmasari showed lower maximum viscosity values, lower minimum and final viscosity values, and consequently a lower consistency than Koganemochi. However, Fig. 1. Two important processing indicators in waxy rice mutants. Comparison of hardness of the rice cakes and gelatinization temperature of rice flour in each line grown at the ripening temperature of 500 C and calculation based on linear regression in each plant material.,, : Average value of lines derived from each cultivar, respectively.

3 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 amylose in the rice endosperm, it is considered to be one of 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 appear 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). Thus, it is possible that an increased content of iodinebinding amylopectin increased the maximum viscosity. Similarly, none of the lines derived from Nihonmasari synthesized Wx protein, while line 25 (WX23) showed a Fig. 3. Heat gelatinization properties of plant materials. The numbers at the bottom denote the lines listed in Table 1. Fig. 4. Relationship between the apparent amylose content and maximum viscosity. Horizontal lines for each symbol denote the standard deviation.

4 178 Kobayashi and Nishimura Table 1. Plant materials Code No. Cultivar/Line Original cultivar Mutagen Apparent amylose Ripening temperature ( C) 1) Gene Wx protein 2) content (%) 3) locus 2) (µg/mg) Min. Max. Range Ave. S.D. 1 Koganemochi Mangetsumochi wx Hokurikumochi wx1N1A Norin 8 EMS wx wx1N1A Norin 8 EMS wx wx4N1A Norin 8 EMS wx7N1A Norin 8 EMS wx wx5 Norin 8 EMS wx wx1N1 Norin 8 EMS wx2 Norin 8 EMS wx wx5 Norin 8 EMS KURwx4N1 Norin 8 Neutrons wx Gwx1 Norin 8 Acute γ-rays wx RGwx2wx Reimei Acute γ-rays wx REwx3wx Reimei EMS REwx5 Reimei EMS wx REwx11 Reimei EMS wx REwx3 Reimei EMS wx REwx7 Reimei EMS wx WX1 Nihonmasari Acute γ-rays wx WX5 Nihonmasari Acute γ-rays wx WX14 Nihonmasari EI WX15 Nihonmasari EI wx WX19 Nihonmasari EI WX23 Nihonmasari EI wx WX25 Nihonmasari Chronic γ-rays wx O. glaberrima (wx) O. glaberrima EMS ) 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 assumed 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 storage proteins (Yoshii and Arisaka 1994) affect the processing properties. Therefore, we consider that the variations in the components of the rice grain, such as cellular tissues, lipids and storage proteins, together with the variations in the structure of waxy starch, exerted an influence on the heat gelatinization properties of these lines. Fig. 5. Response of hardness of the rice cakes and gelatinization temperature of rice flour to the ripening temperature defined as the cumulative daily average air temperature over the 20-day period after heading. : Koganemochi, : Mangetsumochi, : Hokurikumochi 181, : WX14 (induced from Nihonmasari), : WX15 (induced from Nihonmasari), : O. glaberrima (wx).

5 Waxy rice mutants with unique processing properties 179 Rice cake hardness in response to ripening temperature In almost all the lines, the hardness of the rice cakes and the gelatinization temperature increased with the ripening temperature, as described in previous reports (Saito 1987, Matsue et al. 2002). However, the hardness of the rice cakes made from lines 22 (WX14) and 23 (WX15), derived from Nihonmasari, did not increase, even when the ripening temperature increased by nearly 80 C as well as the gelatinization temperatures (Fig. 5). Suzuki et al. (2002) reported the existence of a rice mutant in which amylose synthesis was insensitive to the environmental temperature. This phenomenon was caused by a constant level of Wx gene expression at normal and cool temperatures under the control of a single recessive mutation. Therefore, it is possible that the enzymatic reactions involved in the biosynthesis of waxy starch in relation to the hardness of the rice cakes did not depend on the ripening temperature in lines 22 and 23. However, the hardness of the rice cakes made from lines 22 and 23 was slightly different from that in Mangetsumochi (Fig. 1, Fig. 2 and Fig. 5). Hence, we should analyze in detail the mechanisms underlying the response of the hardness of the rice cakes to the ripening temperature by comparing them with those of other cultivars. New genetic resources for waxy rice breeding research Oryza glaberrima (wx) is an artificial mutant line developed at the National Institute of Genetics, Shizuoka, Japan (Sano 1976). There are no reports on the characteristics of natural waxy mutants of O. glaberrima. We expect that in the interspecific hybrid populations produced using O. glaberrima (wx), the genetic polymorphism of the rice cake hardening properties could be higher than that in the other populations obtained by intraspecific hybridization of O. sativa. Therefore, we consider that O. glaberrima (wx) could become an important experimental material for genetic studies on the hyper-hardening of the rice cakes. To introduce the hyper-hardening rate of the rice cakes from O. glaberrima (wx) into Koganemochi, the development of a continuous backcrossing method using O. glaberrima (wx) as the non-recurrent parent and Koganemochi as the recurrent parent would be ideal. Because the two cultivars display an extremely remote relation, it will be necessary to overcome hybrid inviability. NERICA (New Rice for Africa) lines have been successfully bred through interspecific hybridization between O. glaberrima and O. sativa using an embryo rescue technique (Jones 1999). The application of this technique may enable to introduce the hyper-hardening property of the rice cakes of O. glaberrima (wx) into O. sativa waxy rice cultivars in Japan. If these properties could be introduced into Koganemochi, the cost of refrigeration would be substantially reduced. We expect that O. glaberrima (wx) will also be a valuable breeding material for future improvement of the rice cake hardening properties of waxy rice cultivars in Japan. In recent years, Koganemochi grown in Niigata Prefecture has often shown an undesirably high degree of rice cake hardening, because of the high ripening temperatures, which adversely affected palatability and product quality (Egawa, K. unpublished). Thus, slight changes in the hardening properties of the rice cakes with the ripening temperature are important to address this problem. Detailed studies on these properties, along with the identification of mutant genes and analysis of their structure and function should be actively promoted. To introduce these mutant genes into new waxy rice cultivars, further crossing or backcrossing will be essential for breaking the linkage of adverse agronomic characters. It is thus important that analyses of mutant genes be conducted by using the results obtained in the Rice Genome Project to perform DNA marker selection for the breeding of new waxy rice cultivars. Acknowledgments We are grateful to Dr. T. Fukuyama, Faculty of Agriculture, Field Center for Sustainable Agriculture and Forestry, Niigata University, for the suggestions, encouragement and critical review of the manuscript. This work was supported by a grant for Nuclear Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Literature Cited Akama, Y. and M. Arisaka (1992) Waxy rice. In Rice breeding in Japan. 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