- Email: [email protected]
Determination of Amylose Content and Its Relationship with RVA Profile Within Genetically Similar Cultivars of Rice (Oryza sativa L. ssp. japonica)
Agricultural Sciences in China
August 2010
2010, 9(8): 1101-1107
Determination of Amylose Content and Its Relationship with RVA Profile Within Genetically Similar Cultivars of Rice (Oryza sativa L. ssp. japonica) WANG Xin-qi1, YIN Lin-qing1, SHEN Ge-zhi1, XU Li2 and LIU Qiao-quan2 1 2
Crop Breeding and Cultivation Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai 201106, P.R.China Key Laboratory of Plant Functional Genomics, Ministry of Education/Yangzhou Agricultural College, Yangzhou 225009, P.R.China
Abstract Anther culture pure lines with antisense Wx gene were generated by Agrobacterium tumefaciens-mediated co-transformation followed by anther culture in transgenic rice (Oryza sativa L. ssp. japonica). The antisense Wx transgenic pure lines were used to analyze the differences and the relationship between RVA eigenvalues among the lines with different amylose contents. 77 pure lines of anther culture having antisense Wx gene were studied for the amylose content of its offspring cultivar, Wuyunjing 7. It was found that levels of amylose content were similar to the control in 19 lines (16.0%); 1-5% lower than control in 50 lines, of which, within 11.0-13.9% in 40 lines; and 3.1-4.0% in 8 lines. The effect of the amylose content on RVA eigenvalue was analyzed through the RVA profile graphic comparison and test of significance for RVA eigenvalues. The analysis of RVA profiles with different amylose contents indicated that there were two kinds of RVA profiles within genetically similar cultivars. The lines with 3.1-4.0% of amylose content were distinctly different from other lines and conventional glutinous rice. The comparison of the similarity curve of RVA profile of the lines with different amylose contents showed that the lines with lower amylose content had a distinguished RVA profile with the curve increasing gradually, but not exceeding the first apex. The test of significance for eigenvalues of the RVA profile in the lines with different amylose contents indicated that there were five eigenvalues remarkably different among the lines with 3.1-4.0% of amylose content compared with other two groups. Further, there were three eigenvalues remarkably different among the lines with 11.0-13.5% of amylose content compared with control group. An optimized mathematical model, which characterizes the relationships between the amylose content and RVA eigenvalue, has been established in this study. From this model, it was clearly indicated that the parameters PeT and SBV were more related to amylose content. It was concluded that the differences in amylose content not only affected the eigenvalues of RVA but also caused different RVA profiles within genetically similar cultivars. The introduction of antisense Wx gene could lead to reduce the amylose content and lay theoretical foundation for quality improvement in rice breeding programs. Key words: antisense Wx gene, amylose content, RVA profile, rice
INTRODUCTION Grain quality is one of the most important objectives of most rice breeding programs. In many rice-producing areas of the world, cooking and eating quality
always represents a major criterion in evaluating rice grain. Cooking and eating quality in rice is mainly determined by the starch composition, especially by three physicochemical characteristics of the starch, viz., amylose content (AC), gel consistency (GC), and gelatinization temperature (GT) (Juliano 1985; Mo
This paper is translated from its Chinese version in Scientia Agricultura Sinica. WANG Xin-qi, Associate Professor, E-mail: [email protected]; Correspondence SHEN Ge-zhi, Professor, Tel: +86-21-62208184, E-mail: gzshen2003@yahoo. com.cn © 2010, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S1671-2927(09)60196-6
1102
et al. 1990). High AC in the endosperm is usually associated with dry, fluffy, and separated cooked rice grains, and represents the key determinant of poor cooking and eating quality (Juliano 1985). In rice endosperm, amylose synthesis is initiated by GBSS, and encoded by the Wx gene on chromosome 6 (Okagaki and Wessler 1988). The waxy gene (Wx) has been cloned and sequenced from both the japonica and indica rice cultivars (Wang et al. 1990), its regulation and expression has been studied (Wang et al. 1995), and its structure and function has been figured out (Wang et al. 1990, 1995; Shimada et al. 1993). Moreover, scientists have synthesized different antisense Wx gene fragments and integrated into rice genome with the help of transgenic technology, and succeeded in obtaining pure transgenic lines with significantly reduced amylose content. Practically, it has proven that antisense Wx gene in rice transgenic breeding technology has become an effective way to improve the quality of starch in rice (Chen et al. 2002; Liu et al. 2003; Shen et al. 2004). In recent years, the commencement of rapid viscosity analyzer (RVA) and the related analysis software facilitate the rapid detection of stability and viscosity character of rice starch, and comparison of RVA eigenvalues in different subspecies (Shu et al. 1999; Wu et al. 2003). The RVA profile of stability, genotypic and environmental interactions in indica rice was analysed (Bao and Xia 2001; Wan et al. 2004). The relationship between the RVA eigenvalues and the quality indicators (Hu et al. 2004; Sui et al. 2005), impact of starch synthesis related genes on the RVA eigenvalues (Bao et al. 1999, 2003) were studied to assist the early selection of indica rice with good cooking and eating quality in rice quality improvement programmes by RVA profile (Wu et al. 2001). These studies further explored the awareness and use of RVA profile eigenvalues in selection of starch quality in rice improvement progammes. In present study, anther culture pure lines with antisense Wx gene were generated by Agrobacterium tumefaciens-mediated co-transformation, followed by anther culture in transgenic rice. The antisense Wx transgenic pure lines were used to analyze the differences and the relationship in RVA eigenvalue among lines with different amylose contents with the aim to select varieties with good cooking and eating quality.
WANG Xin-qi et al.
MATERIALS AND METHODS Plant materials The plant materials consisted of the transgenetic rice (Oryza sativa L. ssp. japonica) cultivar, Wuyunjing 7, and its anther culture offspring with antisense Wx gene. The Agrobacterium tumefaciens strain EHA105, and TDNA zone of the fusion plasmid pCAMBIA1300 and p13W8 carrying hygromycin resistance gene (hpt), and antisense Wx gene fragment, respectively, were kindly provided by Professor Wang Zongyang, Institute of Plant Physiology, Chinese Academy of Sciences, Shanghai, China. The genetic transformation was carried out according to Shen et al. (2004).
Molecular detection of experimental material Transgenic plants and its lines of anther culture offspring were analyzed by PCR amplification using the antisense Wx gene primers. The size of PCR product for antisense Wx gene and hygromycin resistance gene was 450 and 598 bp, respectively. Total DNA extraction and PCR conditions used was according to Edwards et al. (1991).
Determination of amylose content and its RVA profile The amylose content of transgenic line and its anther culture offspring was determined according to Anonymous (1988). The determination of RVA was carried out with a rapid visco analyzer (Newport Scientific, Australia), and analysed by TCW software. The test of significance and model optimization was carried out with the Data Processing System (DPSTM ver. 9.50 standard edition).
RESULTS Plant materials and determination of amylose content The transgenic plants and its anther culture pure lines were identified by PCR amplification with antisense Wx
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
Determination of Amylose Content and Its Relationship with RVA Profile Within Genetically Similar Cultivars of Rice
gene primers. 77 pure lines of anther culture having antisense Wx gene were studied for the amylose content of its offspring cultivar, Wuyunjing 7. It was found that levels of amylose content was similar to the control in 19 lines (16.0%); 1-5% lower than control in 50 lines, of which, 11.0-13.9% in 40 lines; and 3.1-4.0% in 8 lines (Table 1). The determination of amylose content in the 38 lines from 5 different independent transgenic plants followed by anther culture showed that the differences in amylose content of anther culture lines and 3 independent transgenic plants (35-15, 35-17 and 9-44) were not significantly different. Further, the amylose content of anther culture lines from 2 independent transgenic plants (11-43, 26-5) were significantly lower than that of the control, and the differences were larger among lines.
Of the 12 lines derived from 26-5, 7 lines had wax-like grain appearance (AC < 5%), and 5 lines (AC=12.914.3%) showed a clear distinction (Table 2) .
Graphical comparison of RVA from anther lines with different amylose contents The Fig.1 shows the RVA profile of the amylose content from anther culture progeny lines from the same independent transgenic plant (26-5), and the conventional glutinous in rice. The curve A of amylose content was more than 12.0% with transparent lines grain appearance (japonica); the curve B of amylose content was below 5% with waxy lines grain appearance (glutinous); and the curve C was conventional glutinous with amylose content below 2.0%. Even though, the lines of A and B were from the the same source of an independent transgenic plant (26-5), there were differences in their amylose content and grain appearance. Apart from the existence of differences on PeT, there were differences in peak values. Despite the waxy appearance (glutinous) in both of B and C groups, the curves were also different. The peak in the C group was early and lower.
Table 1 The amylose content (AC) in antisense Wx gene anther culture lines1) AC (%)
Reduction in over control (AC, %)
Number of lines
0.0-1.0 1.1-1.9 2.1-2.4 4.1-5.0 12.0-12.9
19 10 38 2 8
15.0-16.0 14.0-15.0 12.0-13.9 11.0-11.9 3.1-4.0 1)
1103
The amylose content (AC) in control was 16.0%.
Table 2 The amylose content (AC) of lines derived from anther culture of different transgenic plants Lines of T1 plant 35-15 35-17 9-44 11-43 26-5 Wuyunjing 7
Amylose content of plant derived from anther culture (%) 1
2
3
4
5
6
13.5 15.9 12.5 11.8 12.9 16.0 *
13.5 15.9 13.5 11.5 13.5
13.7 15.5 13.3 12.9 3.1
14.1 15.3 13.3 12.3 3.3
14.0 15.5 13.8 14.0 4.0
13.7 12.5 14.3 3.7
7
8
9
10
11
12
14.3 12.9
14.6 4.0
14.4 13.6
14.3
4.0
3.7
*
, negative control.
Fig. 1 The RVA profiles of anther lines derived from same transgenic plant (26-5).
As the number of lines in anther culture descendant of 26-5 with amylose content more than 11.0% were limited, it couldn’t fully reflect the RVA profile for appearance of grain lines. Therefore, the RVA curves were compared for the amylose contents more than 11.0% lines derived from different independent transgenic plants. The results showed that inter-lines differences were in the level of peak and valley, but the trend of peak time and curve trend were similar to each other (Fig.2). In order to analyze the impact of amylose content on the curve trend, the lines were divided with amylose
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
1104
WANG Xin-qi et al.
content close to the control (15.0-16.0%), and in the range of 11.5-13.5% (reduction over control 2-3% as compared to their RVA curve, respectively) as a group. The results showed that inter-lines differences were not only in the peak and valley but also in the curve trend of their RVA profiles. The group of 11.5-13.5% amylose content had a RVA profile with the curve increasing gradually, but didn’t exceed the first apex. On the contrary, the group with amylose content 15.0-16.0% exceeded its curve to the first apex (Figs.3 and 4).
Fig. 4 The RVA profile of amylose content in range of 15.0-16.0%.
Comparison of RVA eigenvalues with different amylose contents in anther lines Multiple comparisons of RVA eigenvalues by method of LSR for the three groups having different amylose contents (15.0-16.0%, 11.5-13.5% and below 5.0%) is shown in Table 3. It was found that the lines with amylose content below 5% group, except BDV and PeT of eigenvalues were closer to other two groups (compared with the group of 15.0-16.0% with no significant differences, and the group of 11.5-13.5% with significant differences). The remaining six eigenvalues were significantly lower than other two groups among eight eigenvalues of RVA profile, and the differences were extremely significant. In the group with amylose content of 15.0-16.0%, except BDV and PeT, other eigenvalues were the maximum in other two groups. The group with amylose content less than 5% had the lowest eigenvalues than other two groups. The group with amylose content of 11.5-13.5% was the intermediate group to the other two groups (except its the BDV and PaT were higher than the group of 15.0-16.0%). The test of significance with HPV, CPV, and SBV showed that the differences were extremely significant and CSV was significant in the three groups. The BDV
Fig. 2 The RVA profiles of anther lines derived from different transgenic plants.
Fig. 3 The RVA profiles of amylose content in range of 11.5-13.5%.
Table 3 The comparison of eigenvalues for RVA profile in different groups of amylose contents Type 15.0-16.0% 11.5-13.5% < 5%
PKV
HPV
BDV
CPV
SBV
PeT
PaT
CSV
230.9 ± 13.5 A 227.4 ± 10.9 A 147.8 ± 7.2 B
150.2 ± 8.7 A 136.7 ± 14.4 B 65.3 ± 4.4 C
80.7 ± 5.6 a 90.7 ± 7.1 b 82.5 ± 5.1 a
257.2 ± 12.6 A 219.3 ± 12.9 B 89.5 ± 5.2 C
26.3 ± 2.4 A -8.1 ± 5.2 B -58.3 ± 5.4 C
6.3 ± 0.05 A 6.2 ± 0.12 A 4.4 ± 0.10 B
75.2 ± 5.8 ab 80.9 ± 7.2 a 72.2 ± 0.3 b
107.0 ± 4.4 aA 82.6 ± 14.8 bA 24.2 ± 0.7 cB
Letters a, b, and c denote the level of significance at 5%, and letters A, B, and C denote the level of significance at 1%.
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
Determination of Amylose Content and Its Relationship with RVA Profile Within Genetically Similar Cultivars of Rice
and CSV were at the level of significance among of 15.0-16.0% group and 11.5-13.5% group. However, there were no significant differences among PKV, PeT, and PaT. The BDV and PaT were at a significant level in the group of 11.5-13.5% and amylose content less than 5%.
Relationship between amylose content and eigenvalues of RVA profile In order to compare the inter-relationship between amylose contents and RVA eigenvalues, the amylose contents were treated as the dependent variable (Y), and RVA eigenvalues as the independent variables (X1- X8 were termed for PKV, HPV, BDV, CPV, SBV, PeT, PaT, and CSV, respectively). The established multiple regression equation was Y = -6.647 - 0.285X1 + 0.68X2 + 0.873X3 - 0.4X4 + 0.623X5 + 3.515X6 - 0.046X7 - 0.155X8. The F value was 57.298 > F0.01 (8, 25) = 3.32 and R vaule was 0.974. The level of F-test was extremely significant in regression equation. According to the absolute value of regression equation coefficient, the eigenvalues of RVA profile and amylose content were as follows: peak time (PeT) > breakdown (BDV) > hot paste viscosity (HPV) > setback (SBV) > cold paste viscosity (CPV) > peak viscosity (PKV) > consistence (CSV) > pasting temperature (PaT). The optimized equation was established to mask the effect of eigenvalues interaction to the amylose content. The equation was Y = -9.562 + 0.053X5 + 3.743X6. The level of F-test value was 210.164 > F 0.01 (2, 31) = 5.34 and R value was 0.965. F value was extremely significant as well. An optimized equation showed that PeT and SBV of RVA eigenvalues were closely related to amylose content, and were positively correlated.
DISCUSSION There are a lot of evaluation indicators in rice quality, and amylose content is one of the important physical/ chemical indicator. With development of molecular biology and biotechnology advances, genetic transformation of antisense Wx gene has been proven to be effective in reducing amylose content in rice (Wu et al. 2001; Chen et al. 2002; Shen et al. 2004). The im-
1105
provement in transformation methods and the application of anther culture technique has been able to quickly access no-resistance tags with lower amylose content and stability of the lines, which has opened up a new way in transgenic breeding (Wu et al. 2001; Shen et al. 2004). The random insertion of antisense Wx gene in rice was done to get the lines with different amylose contents, and were screened from the generations of independent transformants. In present study, the transformation of antisense Wx gene in japonica and indica rice resulted in the lines with reduced amylose content in varing degrees. The lines with even AC < 5.0% were obtained. But, lines with 6.0-10.0% of amylose content were not found in japonica, and lines with 10.014.0% of amylose content were not found in indica (data not shown). Although, the reasons explained were the different positions inserted and the low frequency of occurrence, it more likely the other related genes in the process of starch synthesis (Bao et al. 2000). Glutinous and non-glutinous distinction in rice from the grain morphology is based on the wax-like and transparent appearance. The classification on the basis of the physical/chemical properties indicates that the rice having amylose content less than 2% is treated as glutinous rice (Bao 2007). In some of the lines, grains are wax-like but it is difficult to differentiate it as glutinous on the basis of grain morphology. However, the amylose content in the range of 3.1-4.0% is difficult to classify as glutinous rice on the basis of criteria. Although, its RVA graphics with amylose content in the range of 3.1-4.0% level, similar trend to conventional glutinous rice could be observed, but there may be significant differences in their eigenvalues, which are having very high to very low amylose content (3-9%) (Bao 2007). The determination of amylose is still based on the use of traditional method, with limited samples’ determination and the problem of accuracy. The birth of rapid visco analyzer has provided the rapid and accurate method for determination of rice starch quality. The related information of rice quality indicators that had obtained from RVA eigenvalues analysis could be helpful in breeding of rice for quality improvement. Previous studies of the analysis of RVA profile in rice were focused on the comparison of RVA eigenvalues for sub-species and cultivars, and found that amylose content was closely related to the viscosity of rice (Su
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
1106
et al. 1999; Wu et al. 2001, 2002; Bao et al. 2003, 2004). The present study was based on the insertion of antisense Wx gene to obtain different lines with different amylose contents in the same varietiy, and analysis of the relationship between amylose content and RVA profile. Although, there was defect with fewer samples, the samples were genetically similar except different amylose contents, and there was no interference from the other genetic differences for the determination of RVA eigenvalues. The CPV and lines of RVA curve with different amylose contents showed that there was a close relationship between amylose content and CPV. The lines with low amylose content had CPV value lower than that of PKV, and the CPV value was lower than the first peak performance in RVA curve. In addition, the value of CPV directly affected the values of SBV and CSV. The comparison of the RVA eigenvalues in the group of varieties with different amylose contents revealed that the group with low amylose content varieties in Japan (15.7%) has CPV values lower than the group of varieties in Jiangsu species of China (about 17.6%) (Shen et al. 2006). Therefore, the CPV may have an important role in the evaluation of the starch quality.
CONCLUSION The curve and eigenvalues of the RVA profile in the lines with different phenotypic appearance derived from the same independent transformant were significantly different. The lines with wax-like phenotypic appearance were different from the conventional glutinous variety of rice. The transparent phenotypic appearance of the grain was same in anther culture lines, and the RVA profile had a similar curve, but there were significant differences in RVA eigenvalues among the lines with different amylose contents. The optimized model showed that the high amylose content was clearly related to PeT and SBV of RVA eigenvalues.
Acknowledgements The study was supported by the grants from the Ministry of Science and Technology, China (ZJY-A-02-02), the Shanghai Science Committee Program Fund, China (013912019), and Natural Science Fundation of Jiangsu
WANG Xin-qi et al.
Province, China (BK2007510).
References Bao J S. 2007. Progress in studies on inheritance and improvement of rice starch quality. Molecular Plant Breeding, 6s, 1-20. Bao J S, Corke H, He P, Zhu L H. 2003. Analysis of quantitative trait loci for starch properties of rice based on an RIL population. Acta Botanica Sinica, 8, 986-994. Bao J S, He P, Li S G, Xia Y W, Chen Y, Zhu L H. 2000. Comparative mapping quantitative trait loci controlling the cooking and eating quality of rice (Oryza sativa L.). Scientia Agricultura Sinica, 5, 8-14. (in Chinese) Bao J S, He P, Xia Y W, Chen Y, Zhu L H. 1999. The RVA profile characteristic of paddy rice mainly controlled in Wx gene. Chinese Science Bulletin, 18, 1973-1976. Bao J S, Shen S Q, Xie J K. 2004. A study on the improvement of eating quality of early indica rice with the assistance of RVA. Molecular Plant Breeding, 1, 49-53. Bao J S, Xia Y W. 2001. Genetic effects and genotype X environment interactions for the starch RVA profiles in indica rice. Scientia Agricultura Sinica, 2, 123-127. (in Chinese) Chen X H, Liu Q Q, Wang Z Y, Wang X W, Cai X L, Zhang J L, Gu M H. 2002. Introduction of antisense waxy gene into the main parent lines of indica hybrid rice. Chinese Science Bulletin, 9, 684-688. Edwards K, Johnstone C, Thompson C. 1991. A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Research, 6, 1349. Hu P S, Zhai H Q, Tang S Q, Wan J M. 2004. Rapid evaluation rice cooking and palatability quality by RVA profile. Acta Agronomica Sinica, 6, 519-524. (in Chinese) Juliano B O. 1985. Criteria and test for rice grain quality. In: Juliano B O, ed, Rice Chemistry and Technology. American Association of Cereal Chemists, Inc., St. Paul, Minn, USA. pp. 443-513. Liu Q Q, Wang Z Y, Chen X H, Cai X L, Tang S Z, Yu H X, Zhang J L, Hong M M, Gu M H. 2003. Stable inheritance of the antisense Wx gene in transgenic rice with reduced amylose level and improved quality. Transgenic Resarch, 1, 71-82. Ministry of Agiculture of P.R.China. 1988. Method for Rice Quality Measurement, Standard of Ministry of Agriculture, P.R.China NY147-88. Chinese Standard Press, Beijing. pp. 4-6. (in Chinese) Mo H D, Gu M H, Jiang C J. 1990. Progress on Genetic Study About Quality Characters of Cereal Crops. Jiangsu Science and Technique Press, Nanjing. (in Chinese) Okagaki R J, Wessler S R. 1988. Comparison of non-mutant and mutant waxy genes in rice and maize. Genetics, 120, 11371143. Shen G Z, Wang X Q, Yin L Q, Cai X L, Wang Z Y. 2004.
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
Determination of Amylose Content and Its Relationship with RVA Profile Within Genetically Similar Cultivars of Rice
1107
Rapidly obtaining the markerless transgenic rice with reduced amylose content by co-transformation and anther culture.
H Q, Wan J M. 2004. Stability analysis for the RVA profile properties of rice starch. Acta Agronomica Sinica, 12, 1185-
Journal of Plant Physiology and Molecular Biology, 6, 637643.
1191. (in Chinese) Wang Z Y, Zheng F Q, Shen G Z, Gao J P, Snustand D P, Li M
Shen G Z, Yin L Q, Wang X Q, Cai X L, Wang Z Y. 2003. Transgenic rice with markerless antisense rice waxy gene
G, Zhang J L, Hong M M. 1995. The amylose content in rice endosperm is related to the post-transcriptional regulation
obtained by agrobacterium-mediated co-transformation. Acta Photophysiologica Sinica, 6, 569-575. (in Chinese)
of the waxy gene. The Plant Journal, 4, 613-622. Wang Z Y, Wu Z L, Xing Y Y, Zheng F G, Guo X L, Zhang W G,
Shen X P, Shen M X, Gong L P, Ji H J, YaoY M, Wang J P, Gao F, Gu Q Q. 2006. Analysis of RVA profile diversity in local
Hong M M. 1990. Nucleotide sequence of rice waxy gene. Nucleic Acids Research, 19, 58-98. (in Chinese)
late japonica rice at Taihu lake area. Acta Agronomica Sinica, 12, 319-322. (in Chinese)
Wu D X, Duan Z Y, Shu Q Y, Xia Y W. 2003. Transfer of starch viscosity characteristics between subspecies (variety) and
Shimada H, Tada Y, Kawasaki T, Fujimura T A. 1993. Antisense regulation of the rice waxy gene expression using a PCR-
variety in rice. Chinese Journal of Rice Science, 4, 319-322. (in Chinese)
amplified fragment of the rice genome reduces the amylose content in grain starch. Theoretical and Applied Genetics,
Wu D X, Shu Q Y, X ia Y W. 2001. Assisted-selection for early indica rice with good eating quality by RVA profile. Acta
86, 665-672. Shu Q Y, Wu D X, Xia Y W, Gao M W, McClung A. 1999.
Agronomica Sinica, 2, 165-172. (in Chinese) Wu D X, Shu Q Y, Xia Y W. 2001. Rapid identification of starch
Preliminary analysis of the difference of RVA profile character among of subspecies Oryza sativa L. Acta Agronomic Sinica,
viscosity property of early indica rice varieties with different apparent amylose content by RVA profile. Chinese Journal
3, 279-283. (in Chinese) Sui J M, Li X, Yan S, Yan C J, Zhang R, Tang S Z, Lu J F, Chen
of Rice Science, 1, 57-59. (in Chinese) Wu D X, Shu Q Y, Xia Y W. 2002. Endosperm appearance
Z X, Gu M H. 2005. Studies on the rice RVA profile characteristics and its correlation with the quality. Scientia
marker and physical/chemical indicator of RVA profile combined assisted selection for the improvement of eating
Agricultura Sinica, 4, 657-663. (in Chinese) Wan X Y, Cheng L M, Wang H L, Xiao Y H, Bi J C, Liu X, Zhai
quality of early indica rice. Chinese Journal of Rice Science, 1, 80-82. (in Chinese) (Managing editor ZHANG Yi-min)
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
August 2010
2010, 9(8): 1101-1107
Determination of Amylose Content and Its Relationship with RVA Profile Within Genetically Similar Cultivars of Rice (Oryza sativa L. ssp. japonica) WANG Xin-qi1, YIN Lin-qing1, SHEN Ge-zhi1, XU Li2 and LIU Qiao-quan2 1 2
Crop Breeding and Cultivation Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai 201106, P.R.China Key Laboratory of Plant Functional Genomics, Ministry of Education/Yangzhou Agricultural College, Yangzhou 225009, P.R.China
Abstract Anther culture pure lines with antisense Wx gene were generated by Agrobacterium tumefaciens-mediated co-transformation followed by anther culture in transgenic rice (Oryza sativa L. ssp. japonica). The antisense Wx transgenic pure lines were used to analyze the differences and the relationship between RVA eigenvalues among the lines with different amylose contents. 77 pure lines of anther culture having antisense Wx gene were studied for the amylose content of its offspring cultivar, Wuyunjing 7. It was found that levels of amylose content were similar to the control in 19 lines (16.0%); 1-5% lower than control in 50 lines, of which, within 11.0-13.9% in 40 lines; and 3.1-4.0% in 8 lines. The effect of the amylose content on RVA eigenvalue was analyzed through the RVA profile graphic comparison and test of significance for RVA eigenvalues. The analysis of RVA profiles with different amylose contents indicated that there were two kinds of RVA profiles within genetically similar cultivars. The lines with 3.1-4.0% of amylose content were distinctly different from other lines and conventional glutinous rice. The comparison of the similarity curve of RVA profile of the lines with different amylose contents showed that the lines with lower amylose content had a distinguished RVA profile with the curve increasing gradually, but not exceeding the first apex. The test of significance for eigenvalues of the RVA profile in the lines with different amylose contents indicated that there were five eigenvalues remarkably different among the lines with 3.1-4.0% of amylose content compared with other two groups. Further, there were three eigenvalues remarkably different among the lines with 11.0-13.5% of amylose content compared with control group. An optimized mathematical model, which characterizes the relationships between the amylose content and RVA eigenvalue, has been established in this study. From this model, it was clearly indicated that the parameters PeT and SBV were more related to amylose content. It was concluded that the differences in amylose content not only affected the eigenvalues of RVA but also caused different RVA profiles within genetically similar cultivars. The introduction of antisense Wx gene could lead to reduce the amylose content and lay theoretical foundation for quality improvement in rice breeding programs. Key words: antisense Wx gene, amylose content, RVA profile, rice
INTRODUCTION Grain quality is one of the most important objectives of most rice breeding programs. In many rice-producing areas of the world, cooking and eating quality
always represents a major criterion in evaluating rice grain. Cooking and eating quality in rice is mainly determined by the starch composition, especially by three physicochemical characteristics of the starch, viz., amylose content (AC), gel consistency (GC), and gelatinization temperature (GT) (Juliano 1985; Mo
This paper is translated from its Chinese version in Scientia Agricultura Sinica. WANG Xin-qi, Associate Professor, E-mail: [email protected]; Correspondence SHEN Ge-zhi, Professor, Tel: +86-21-62208184, E-mail: gzshen2003@yahoo. com.cn © 2010, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S1671-2927(09)60196-6
1102
et al. 1990). High AC in the endosperm is usually associated with dry, fluffy, and separated cooked rice grains, and represents the key determinant of poor cooking and eating quality (Juliano 1985). In rice endosperm, amylose synthesis is initiated by GBSS, and encoded by the Wx gene on chromosome 6 (Okagaki and Wessler 1988). The waxy gene (Wx) has been cloned and sequenced from both the japonica and indica rice cultivars (Wang et al. 1990), its regulation and expression has been studied (Wang et al. 1995), and its structure and function has been figured out (Wang et al. 1990, 1995; Shimada et al. 1993). Moreover, scientists have synthesized different antisense Wx gene fragments and integrated into rice genome with the help of transgenic technology, and succeeded in obtaining pure transgenic lines with significantly reduced amylose content. Practically, it has proven that antisense Wx gene in rice transgenic breeding technology has become an effective way to improve the quality of starch in rice (Chen et al. 2002; Liu et al. 2003; Shen et al. 2004). In recent years, the commencement of rapid viscosity analyzer (RVA) and the related analysis software facilitate the rapid detection of stability and viscosity character of rice starch, and comparison of RVA eigenvalues in different subspecies (Shu et al. 1999; Wu et al. 2003). The RVA profile of stability, genotypic and environmental interactions in indica rice was analysed (Bao and Xia 2001; Wan et al. 2004). The relationship between the RVA eigenvalues and the quality indicators (Hu et al. 2004; Sui et al. 2005), impact of starch synthesis related genes on the RVA eigenvalues (Bao et al. 1999, 2003) were studied to assist the early selection of indica rice with good cooking and eating quality in rice quality improvement programmes by RVA profile (Wu et al. 2001). These studies further explored the awareness and use of RVA profile eigenvalues in selection of starch quality in rice improvement progammes. In present study, anther culture pure lines with antisense Wx gene were generated by Agrobacterium tumefaciens-mediated co-transformation, followed by anther culture in transgenic rice. The antisense Wx transgenic pure lines were used to analyze the differences and the relationship in RVA eigenvalue among lines with different amylose contents with the aim to select varieties with good cooking and eating quality.
WANG Xin-qi et al.
MATERIALS AND METHODS Plant materials The plant materials consisted of the transgenetic rice (Oryza sativa L. ssp. japonica) cultivar, Wuyunjing 7, and its anther culture offspring with antisense Wx gene. The Agrobacterium tumefaciens strain EHA105, and TDNA zone of the fusion plasmid pCAMBIA1300 and p13W8 carrying hygromycin resistance gene (hpt), and antisense Wx gene fragment, respectively, were kindly provided by Professor Wang Zongyang, Institute of Plant Physiology, Chinese Academy of Sciences, Shanghai, China. The genetic transformation was carried out according to Shen et al. (2004).
Molecular detection of experimental material Transgenic plants and its lines of anther culture offspring were analyzed by PCR amplification using the antisense Wx gene primers. The size of PCR product for antisense Wx gene and hygromycin resistance gene was 450 and 598 bp, respectively. Total DNA extraction and PCR conditions used was according to Edwards et al. (1991).
Determination of amylose content and its RVA profile The amylose content of transgenic line and its anther culture offspring was determined according to Anonymous (1988). The determination of RVA was carried out with a rapid visco analyzer (Newport Scientific, Australia), and analysed by TCW software. The test of significance and model optimization was carried out with the Data Processing System (DPSTM ver. 9.50 standard edition).
RESULTS Plant materials and determination of amylose content The transgenic plants and its anther culture pure lines were identified by PCR amplification with antisense Wx
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
Determination of Amylose Content and Its Relationship with RVA Profile Within Genetically Similar Cultivars of Rice
gene primers. 77 pure lines of anther culture having antisense Wx gene were studied for the amylose content of its offspring cultivar, Wuyunjing 7. It was found that levels of amylose content was similar to the control in 19 lines (16.0%); 1-5% lower than control in 50 lines, of which, 11.0-13.9% in 40 lines; and 3.1-4.0% in 8 lines (Table 1). The determination of amylose content in the 38 lines from 5 different independent transgenic plants followed by anther culture showed that the differences in amylose content of anther culture lines and 3 independent transgenic plants (35-15, 35-17 and 9-44) were not significantly different. Further, the amylose content of anther culture lines from 2 independent transgenic plants (11-43, 26-5) were significantly lower than that of the control, and the differences were larger among lines.
Of the 12 lines derived from 26-5, 7 lines had wax-like grain appearance (AC < 5%), and 5 lines (AC=12.914.3%) showed a clear distinction (Table 2) .
Graphical comparison of RVA from anther lines with different amylose contents The Fig.1 shows the RVA profile of the amylose content from anther culture progeny lines from the same independent transgenic plant (26-5), and the conventional glutinous in rice. The curve A of amylose content was more than 12.0% with transparent lines grain appearance (japonica); the curve B of amylose content was below 5% with waxy lines grain appearance (glutinous); and the curve C was conventional glutinous with amylose content below 2.0%. Even though, the lines of A and B were from the the same source of an independent transgenic plant (26-5), there were differences in their amylose content and grain appearance. Apart from the existence of differences on PeT, there were differences in peak values. Despite the waxy appearance (glutinous) in both of B and C groups, the curves were also different. The peak in the C group was early and lower.
Table 1 The amylose content (AC) in antisense Wx gene anther culture lines1) AC (%)
Reduction in over control (AC, %)
Number of lines
0.0-1.0 1.1-1.9 2.1-2.4 4.1-5.0 12.0-12.9
19 10 38 2 8
15.0-16.0 14.0-15.0 12.0-13.9 11.0-11.9 3.1-4.0 1)
1103
The amylose content (AC) in control was 16.0%.
Table 2 The amylose content (AC) of lines derived from anther culture of different transgenic plants Lines of T1 plant 35-15 35-17 9-44 11-43 26-5 Wuyunjing 7
Amylose content of plant derived from anther culture (%) 1
2
3
4
5
6
13.5 15.9 12.5 11.8 12.9 16.0 *
13.5 15.9 13.5 11.5 13.5
13.7 15.5 13.3 12.9 3.1
14.1 15.3 13.3 12.3 3.3
14.0 15.5 13.8 14.0 4.0
13.7 12.5 14.3 3.7
7
8
9
10
11
12
14.3 12.9
14.6 4.0
14.4 13.6
14.3
4.0
3.7
*
, negative control.
Fig. 1 The RVA profiles of anther lines derived from same transgenic plant (26-5).
As the number of lines in anther culture descendant of 26-5 with amylose content more than 11.0% were limited, it couldn’t fully reflect the RVA profile for appearance of grain lines. Therefore, the RVA curves were compared for the amylose contents more than 11.0% lines derived from different independent transgenic plants. The results showed that inter-lines differences were in the level of peak and valley, but the trend of peak time and curve trend were similar to each other (Fig.2). In order to analyze the impact of amylose content on the curve trend, the lines were divided with amylose
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
1104
WANG Xin-qi et al.
content close to the control (15.0-16.0%), and in the range of 11.5-13.5% (reduction over control 2-3% as compared to their RVA curve, respectively) as a group. The results showed that inter-lines differences were not only in the peak and valley but also in the curve trend of their RVA profiles. The group of 11.5-13.5% amylose content had a RVA profile with the curve increasing gradually, but didn’t exceed the first apex. On the contrary, the group with amylose content 15.0-16.0% exceeded its curve to the first apex (Figs.3 and 4).
Fig. 4 The RVA profile of amylose content in range of 15.0-16.0%.
Comparison of RVA eigenvalues with different amylose contents in anther lines Multiple comparisons of RVA eigenvalues by method of LSR for the three groups having different amylose contents (15.0-16.0%, 11.5-13.5% and below 5.0%) is shown in Table 3. It was found that the lines with amylose content below 5% group, except BDV and PeT of eigenvalues were closer to other two groups (compared with the group of 15.0-16.0% with no significant differences, and the group of 11.5-13.5% with significant differences). The remaining six eigenvalues were significantly lower than other two groups among eight eigenvalues of RVA profile, and the differences were extremely significant. In the group with amylose content of 15.0-16.0%, except BDV and PeT, other eigenvalues were the maximum in other two groups. The group with amylose content less than 5% had the lowest eigenvalues than other two groups. The group with amylose content of 11.5-13.5% was the intermediate group to the other two groups (except its the BDV and PaT were higher than the group of 15.0-16.0%). The test of significance with HPV, CPV, and SBV showed that the differences were extremely significant and CSV was significant in the three groups. The BDV
Fig. 2 The RVA profiles of anther lines derived from different transgenic plants.
Fig. 3 The RVA profiles of amylose content in range of 11.5-13.5%.
Table 3 The comparison of eigenvalues for RVA profile in different groups of amylose contents Type 15.0-16.0% 11.5-13.5% < 5%
PKV
HPV
BDV
CPV
SBV
PeT
PaT
CSV
230.9 ± 13.5 A 227.4 ± 10.9 A 147.8 ± 7.2 B
150.2 ± 8.7 A 136.7 ± 14.4 B 65.3 ± 4.4 C
80.7 ± 5.6 a 90.7 ± 7.1 b 82.5 ± 5.1 a
257.2 ± 12.6 A 219.3 ± 12.9 B 89.5 ± 5.2 C
26.3 ± 2.4 A -8.1 ± 5.2 B -58.3 ± 5.4 C
6.3 ± 0.05 A 6.2 ± 0.12 A 4.4 ± 0.10 B
75.2 ± 5.8 ab 80.9 ± 7.2 a 72.2 ± 0.3 b
107.0 ± 4.4 aA 82.6 ± 14.8 bA 24.2 ± 0.7 cB
Letters a, b, and c denote the level of significance at 5%, and letters A, B, and C denote the level of significance at 1%.
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
Determination of Amylose Content and Its Relationship with RVA Profile Within Genetically Similar Cultivars of Rice
and CSV were at the level of significance among of 15.0-16.0% group and 11.5-13.5% group. However, there were no significant differences among PKV, PeT, and PaT. The BDV and PaT were at a significant level in the group of 11.5-13.5% and amylose content less than 5%.
Relationship between amylose content and eigenvalues of RVA profile In order to compare the inter-relationship between amylose contents and RVA eigenvalues, the amylose contents were treated as the dependent variable (Y), and RVA eigenvalues as the independent variables (X1- X8 were termed for PKV, HPV, BDV, CPV, SBV, PeT, PaT, and CSV, respectively). The established multiple regression equation was Y = -6.647 - 0.285X1 + 0.68X2 + 0.873X3 - 0.4X4 + 0.623X5 + 3.515X6 - 0.046X7 - 0.155X8. The F value was 57.298 > F0.01 (8, 25) = 3.32 and R vaule was 0.974. The level of F-test was extremely significant in regression equation. According to the absolute value of regression equation coefficient, the eigenvalues of RVA profile and amylose content were as follows: peak time (PeT) > breakdown (BDV) > hot paste viscosity (HPV) > setback (SBV) > cold paste viscosity (CPV) > peak viscosity (PKV) > consistence (CSV) > pasting temperature (PaT). The optimized equation was established to mask the effect of eigenvalues interaction to the amylose content. The equation was Y = -9.562 + 0.053X5 + 3.743X6. The level of F-test value was 210.164 > F 0.01 (2, 31) = 5.34 and R value was 0.965. F value was extremely significant as well. An optimized equation showed that PeT and SBV of RVA eigenvalues were closely related to amylose content, and were positively correlated.
DISCUSSION There are a lot of evaluation indicators in rice quality, and amylose content is one of the important physical/ chemical indicator. With development of molecular biology and biotechnology advances, genetic transformation of antisense Wx gene has been proven to be effective in reducing amylose content in rice (Wu et al. 2001; Chen et al. 2002; Shen et al. 2004). The im-
1105
provement in transformation methods and the application of anther culture technique has been able to quickly access no-resistance tags with lower amylose content and stability of the lines, which has opened up a new way in transgenic breeding (Wu et al. 2001; Shen et al. 2004). The random insertion of antisense Wx gene in rice was done to get the lines with different amylose contents, and were screened from the generations of independent transformants. In present study, the transformation of antisense Wx gene in japonica and indica rice resulted in the lines with reduced amylose content in varing degrees. The lines with even AC < 5.0% were obtained. But, lines with 6.0-10.0% of amylose content were not found in japonica, and lines with 10.014.0% of amylose content were not found in indica (data not shown). Although, the reasons explained were the different positions inserted and the low frequency of occurrence, it more likely the other related genes in the process of starch synthesis (Bao et al. 2000). Glutinous and non-glutinous distinction in rice from the grain morphology is based on the wax-like and transparent appearance. The classification on the basis of the physical/chemical properties indicates that the rice having amylose content less than 2% is treated as glutinous rice (Bao 2007). In some of the lines, grains are wax-like but it is difficult to differentiate it as glutinous on the basis of grain morphology. However, the amylose content in the range of 3.1-4.0% is difficult to classify as glutinous rice on the basis of criteria. Although, its RVA graphics with amylose content in the range of 3.1-4.0% level, similar trend to conventional glutinous rice could be observed, but there may be significant differences in their eigenvalues, which are having very high to very low amylose content (3-9%) (Bao 2007). The determination of amylose is still based on the use of traditional method, with limited samples’ determination and the problem of accuracy. The birth of rapid visco analyzer has provided the rapid and accurate method for determination of rice starch quality. The related information of rice quality indicators that had obtained from RVA eigenvalues analysis could be helpful in breeding of rice for quality improvement. Previous studies of the analysis of RVA profile in rice were focused on the comparison of RVA eigenvalues for sub-species and cultivars, and found that amylose content was closely related to the viscosity of rice (Su
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
1106
et al. 1999; Wu et al. 2001, 2002; Bao et al. 2003, 2004). The present study was based on the insertion of antisense Wx gene to obtain different lines with different amylose contents in the same varietiy, and analysis of the relationship between amylose content and RVA profile. Although, there was defect with fewer samples, the samples were genetically similar except different amylose contents, and there was no interference from the other genetic differences for the determination of RVA eigenvalues. The CPV and lines of RVA curve with different amylose contents showed that there was a close relationship between amylose content and CPV. The lines with low amylose content had CPV value lower than that of PKV, and the CPV value was lower than the first peak performance in RVA curve. In addition, the value of CPV directly affected the values of SBV and CSV. The comparison of the RVA eigenvalues in the group of varieties with different amylose contents revealed that the group with low amylose content varieties in Japan (15.7%) has CPV values lower than the group of varieties in Jiangsu species of China (about 17.6%) (Shen et al. 2006). Therefore, the CPV may have an important role in the evaluation of the starch quality.
CONCLUSION The curve and eigenvalues of the RVA profile in the lines with different phenotypic appearance derived from the same independent transformant were significantly different. The lines with wax-like phenotypic appearance were different from the conventional glutinous variety of rice. The transparent phenotypic appearance of the grain was same in anther culture lines, and the RVA profile had a similar curve, but there were significant differences in RVA eigenvalues among the lines with different amylose contents. The optimized model showed that the high amylose content was clearly related to PeT and SBV of RVA eigenvalues.
Acknowledgements The study was supported by the grants from the Ministry of Science and Technology, China (ZJY-A-02-02), the Shanghai Science Committee Program Fund, China (013912019), and Natural Science Fundation of Jiangsu
WANG Xin-qi et al.
Province, China (BK2007510).
References Bao J S. 2007. Progress in studies on inheritance and improvement of rice starch quality. Molecular Plant Breeding, 6s, 1-20. Bao J S, Corke H, He P, Zhu L H. 2003. Analysis of quantitative trait loci for starch properties of rice based on an RIL population. Acta Botanica Sinica, 8, 986-994. Bao J S, He P, Li S G, Xia Y W, Chen Y, Zhu L H. 2000. Comparative mapping quantitative trait loci controlling the cooking and eating quality of rice (Oryza sativa L.). Scientia Agricultura Sinica, 5, 8-14. (in Chinese) Bao J S, He P, Xia Y W, Chen Y, Zhu L H. 1999. The RVA profile characteristic of paddy rice mainly controlled in Wx gene. Chinese Science Bulletin, 18, 1973-1976. Bao J S, Shen S Q, Xie J K. 2004. A study on the improvement of eating quality of early indica rice with the assistance of RVA. Molecular Plant Breeding, 1, 49-53. Bao J S, Xia Y W. 2001. Genetic effects and genotype X environment interactions for the starch RVA profiles in indica rice. Scientia Agricultura Sinica, 2, 123-127. (in Chinese) Chen X H, Liu Q Q, Wang Z Y, Wang X W, Cai X L, Zhang J L, Gu M H. 2002. Introduction of antisense waxy gene into the main parent lines of indica hybrid rice. Chinese Science Bulletin, 9, 684-688. Edwards K, Johnstone C, Thompson C. 1991. A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Research, 6, 1349. Hu P S, Zhai H Q, Tang S Q, Wan J M. 2004. Rapid evaluation rice cooking and palatability quality by RVA profile. Acta Agronomica Sinica, 6, 519-524. (in Chinese) Juliano B O. 1985. Criteria and test for rice grain quality. In: Juliano B O, ed, Rice Chemistry and Technology. American Association of Cereal Chemists, Inc., St. Paul, Minn, USA. pp. 443-513. Liu Q Q, Wang Z Y, Chen X H, Cai X L, Tang S Z, Yu H X, Zhang J L, Hong M M, Gu M H. 2003. Stable inheritance of the antisense Wx gene in transgenic rice with reduced amylose level and improved quality. Transgenic Resarch, 1, 71-82. Ministry of Agiculture of P.R.China. 1988. Method for Rice Quality Measurement, Standard of Ministry of Agriculture, P.R.China NY147-88. Chinese Standard Press, Beijing. pp. 4-6. (in Chinese) Mo H D, Gu M H, Jiang C J. 1990. Progress on Genetic Study About Quality Characters of Cereal Crops. Jiangsu Science and Technique Press, Nanjing. (in Chinese) Okagaki R J, Wessler S R. 1988. Comparison of non-mutant and mutant waxy genes in rice and maize. Genetics, 120, 11371143. Shen G Z, Wang X Q, Yin L Q, Cai X L, Wang Z Y. 2004.
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
Determination of Amylose Content and Its Relationship with RVA Profile Within Genetically Similar Cultivars of Rice
1107
Rapidly obtaining the markerless transgenic rice with reduced amylose content by co-transformation and anther culture.
H Q, Wan J M. 2004. Stability analysis for the RVA profile properties of rice starch. Acta Agronomica Sinica, 12, 1185-
Journal of Plant Physiology and Molecular Biology, 6, 637643.
1191. (in Chinese) Wang Z Y, Zheng F Q, Shen G Z, Gao J P, Snustand D P, Li M
Shen G Z, Yin L Q, Wang X Q, Cai X L, Wang Z Y. 2003. Transgenic rice with markerless antisense rice waxy gene
G, Zhang J L, Hong M M. 1995. The amylose content in rice endosperm is related to the post-transcriptional regulation
obtained by agrobacterium-mediated co-transformation. Acta Photophysiologica Sinica, 6, 569-575. (in Chinese)
of the waxy gene. The Plant Journal, 4, 613-622. Wang Z Y, Wu Z L, Xing Y Y, Zheng F G, Guo X L, Zhang W G,
Shen X P, Shen M X, Gong L P, Ji H J, YaoY M, Wang J P, Gao F, Gu Q Q. 2006. Analysis of RVA profile diversity in local
Hong M M. 1990. Nucleotide sequence of rice waxy gene. Nucleic Acids Research, 19, 58-98. (in Chinese)
late japonica rice at Taihu lake area. Acta Agronomica Sinica, 12, 319-322. (in Chinese)
Wu D X, Duan Z Y, Shu Q Y, Xia Y W. 2003. Transfer of starch viscosity characteristics between subspecies (variety) and
Shimada H, Tada Y, Kawasaki T, Fujimura T A. 1993. Antisense regulation of the rice waxy gene expression using a PCR-
variety in rice. Chinese Journal of Rice Science, 4, 319-322. (in Chinese)
amplified fragment of the rice genome reduces the amylose content in grain starch. Theoretical and Applied Genetics,
Wu D X, Shu Q Y, X ia Y W. 2001. Assisted-selection for early indica rice with good eating quality by RVA profile. Acta
86, 665-672. Shu Q Y, Wu D X, Xia Y W, Gao M W, McClung A. 1999.
Agronomica Sinica, 2, 165-172. (in Chinese) Wu D X, Shu Q Y, Xia Y W. 2001. Rapid identification of starch
Preliminary analysis of the difference of RVA profile character among of subspecies Oryza sativa L. Acta Agronomic Sinica,
viscosity property of early indica rice varieties with different apparent amylose content by RVA profile. Chinese Journal
3, 279-283. (in Chinese) Sui J M, Li X, Yan S, Yan C J, Zhang R, Tang S Z, Lu J F, Chen
of Rice Science, 1, 57-59. (in Chinese) Wu D X, Shu Q Y, Xia Y W. 2002. Endosperm appearance
Z X, Gu M H. 2005. Studies on the rice RVA profile characteristics and its correlation with the quality. Scientia
marker and physical/chemical indicator of RVA profile combined assisted selection for the improvement of eating
Agricultura Sinica, 4, 657-663. (in Chinese) Wan X Y, Cheng L M, Wang H L, Xiao Y H, Bi J C, Liu X, Zhai
quality of early indica rice. Chinese Journal of Rice Science, 1, 80-82. (in Chinese) (Managing editor ZHANG Yi-min)
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.