Method for N-methyl-N-nitrosourea Mutagenesis on Hermaphroditic Germ Cells of Rice

doi:10.11983/CBB18243

Abstract

Abstract: N-methyl-N-nitrosourea (MNU) has been used in mutagenesis of fertilized eggs of rice (Oryza sativa). In this study, the method for MNU mutagenesis on hermaphroditic germ cells of rice was established by in vivo synchronous MNU treatment of mature germ-organs in the rice cultivar Liaoyan 6 and the screening of the progeny populations. As compared with MNU treatment of fertilized eggs in Liaoyan 6, MNU treatment of hermaphroditic germ cells under each group of conditions significantly decreased the growth and development indexes of M₁ populations and increased the incidence of mutant characters in M₁-M₂ populations. When the hermaphroditic germ cells were treated for 60 min in MNU buffer (pH 4.8) containing 1.5 mmol∙L ^-1 MNU and 10 mmol∙L ^-1 phosphate, the incidence of mutant characters was about 3 times higher than that based on the MNU treatment of fertilized eggs. A series of homozygous mutants including novel plant and grain mutants were obtained by further screening M₃ populations. The method for MNU mutagenesis on mature hermaphroditic germ-cells of rice significantly improved the efficiency of broad-spectrum mutagenesis. Its application can provide efficient technical support for large-scale development of various mutants for identifying unknown functional genes and breeding rice.

Key words: rice, hermaphroditic germ cells, N-methyl-N-nitrosourea, mutagenesis

Huaidong Tian,Jing Li,Baohua Tian,Pengfei Niu,Zhen Li,Zhongxiao Yue,Yajuan Qu,Jianfang Jiang,Guangyuan Wang,Huihui Cen,Nan Li,Feng Yan. Method for N-methyl-N-nitrosourea Mutagenesis on Hermaphroditic Germ Cells of Rice[J]. Chinese Bulletin of Botany, 2019, 54(5): 625-633.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB18243

https://www.chinbullbotany.com/EN/Y2019/V54/I5/625

Figures/Tables 8

Table 1 Design for MNU treatments of germ cells in rice

Treatments	MNU (mmol∙L^-1)	Time (min)	pH	PO₄^3– (mmol∙L^-1)
Germ cells	0.5 1.0 1.5 2.0	20 40 60 80	4.2 4.8 5.4 6.0	5 10 20 40
Control	1.0	40	4.8	10

Table 2 Effects of MNU-treatment factors on growth and development of M1 populations of germ cells of Liaoyan 6 rice cultivar

Treatments of germ cells		Setting rate (%)	Seedling rate (%)	Adult plant rate (%)	Fertility rate(%)
Variable MNU (mmol∙L^-1)	0.5 1.0 1.5 2.0	86.1 78.6 73.5 62.2	82.3 74.5 68.3 59.8	82.4 77.1 70.4 61.7	89.3 80.4 76.7 68.2
Variable time (min)	20 40 60 80	88.5 78.6 74.7 65.8	83.1 74.5 69.3 60.8	83.2 77.1 72.3 66.9	91.0 80.4 73.8 67.6
Variable pH	4.2 4.8 5.4 6.0	85.3 78.6 74.5 82.9	80.3 74.5 79.6 83.7	85.3 77.1 81.0 84.9	89.6 80.4 85.2 87.9
Variable PO₄^3- (mmol∙L^-1)	5 10 20 40	83.7 78.6 82.5 87.4	82.4 74.5 78.1 85.0	86.3 77.1 70.6 82.8	89.7 80.4 85.3 86.9
Control treatment		89.5	87.3	90.4	92.8

Table 3 Effects of MNU-treatment factors on mutant-character expression in M1-M2 populations of germ cells of Liaoyan 6 rice cultivar

Treatments of germ cells		Number of mutant characters							Incidences (%)
Treatments of germ cells		Seedling	Culm	Leaf	Panicle	Glume	Seed	Total	Incidences (%)
Variable MNU (mmol∙L^-1)	0.5 1.0 1.5 2.0	3 4 8 5	2 4 6 3	3 4 5 2	1 2 3 1	1 1 2 -	8 15 15 6	18 30 39 17	4.6 7.6 9.9 4.3
Variable time (min)	20 40 60 80	3 4 5 5	2 4 3 3	3 4 5 3	2 2 1 2	- 1 2 1	5 15 15 6	15 30 31 20	3.8 7.6 7.9 5.1
Variable pH	4.2 4.8 5.4 6.0	5 4 5 3	3 4 3 3	2 4 3 3	1 2 1 -	1 1 1 1	9 15 10 6	21 30 23 16	5.3 7.6 5.9 4.1
Variable PO₄^3- (mmol∙L^-1)	5 10 20 40	5 4 6 5	2 4 3 3	3 4 4 2	2 2 1 1	1 1 2 1	7 15 7 6	20 30 23 18	5.1 7.6 5.9 4.6
Control treatment		3	2	2	1	1	4	13	3.3

Figure 1 Mutant characters for culm form in M1-M3 populations obtained by the MNU treatments of germ cells of Liaoyan 6 rice cultivar (A) The wild type culm; (B) Multiple-tiller culm; (C) Less-tiller culm; (D), (E) Dwarf culm. Bars=10 cm. MNU see Table 1.

Figure 2 Mutant characters for leaf form and leaf color in M1-M3 populations obtained by the MNU treatments of germ cells of Liaoyan 6 rice cultivar (A) The wild type leaf; (B) Wide leaf; (C) Narrow and rolled leaf; (D) Vertical leaf; (E) Deep-green leaf; (F) Light-green leaf; (G) Yellow margin; (H) Spotted leaf. Bars=5 cm. MNU see Table 1.

Figure 3 Mutant characters for panicle form and panicle color in M1-M3 populations obtained by the MNU treatments of germ cells of Liaoyan 6 rice cultivar (A) The wild type panicle; (B) Short panicle; (C) Sterile panicle; (D) Reddish panicle. Bars=5 cm. MNU see Table 1.

Figure 4 Mutant characters for glume form and glume color in M1-M3 populations obtained by the MNU treatments of germ cells of Liaoyan 6 rice cultivar (A) The wild type glume; (B) Awning glume; (C) Purple-black glume; (D) Reddish-brown glume. Bars=5 mm. MNU see Table 1.

Figure 5 Mutant characters of seed in M1-M3 populations obtained by the MNU treatments of germ cells of Liaoyan 6 rice cultivar (A) The wild type seed; (B) Slender seed; (C) Wrinkled endosperm; (D) Abdomen-sunken endosperm; (E) Shrunken endosperm; (F) Waxy endosperm; (G) Dull endosperm; (H) Floury endosperm; (I) Core-white endosperm; (J) Periphery-white endosperm; (K) Reddish-brown endosperm; (L) Green endosperm. Bars=5 mm. MNU see Table 1.

References 36

1	淳雁, 李学勇 (2017). 水稻穗型的遗传调控研究进展. 植物学报 52, 19-29.
2	郭丹, 施勇烽, 王惠梅, 张晓波, 宋莉欣, 徐霞, 贺彦, 郭梁, 吴建利 (2016). 一个水稻显性斑点叶突变体的鉴定和基因精细定位. 作物学报 42, 966-975.
3	刘艺, 朱小品, 刘喜, 田云录, 刘世家, 王云龙, 张文伟, 江玲, 王益华, 万建民 (2018). 水稻胚乳粉质突变体flo9的表型分析和基因定位. 南京农业大学学报 41, 616-624.
4	刘子文, 胡曼曼, 王致远, 张瑜竣, 谷晗, 游佳, 王益华, 江玲, 刘玲珑 (2018). 水稻心白突变体whc的理化性质和基因定位. 南京农业大学学报 41, 231-239.
5	涂政军, 邹国兴, 黄李超, 陈龙, 代丽萍, 高易宏, 冷语佳, 朱丽, 张光恒, 胡江, 任德勇, 高振宇, 董国军, 陈光, 郭龙彪, 钱前, 曾大力 (2017). 水稻淡绿叶基因PGL11的鉴定与精细定位. 中国水稻科学 31, 489-499.
6	王影, 李相敢, 邱丽娟 (2018). CRISPR/Cas9基因组定点编辑中脱靶现象的研究进展. 植物学报 53, 528-541.
7	肖景华, 吴昌银, 袁猛, 王妮丽, 范优荣, 杨猛, 欧阳亦聃, 阮一骏, 张启发 (2015). 中国水稻功能基因组研究进展与展望. 科学通报 60, 1711-1722.
8	谢佳, 张孝波, 陶怡然, 熊毓贞, 周倩, 孙莹, 杨正林, 钟秉强, 桑贤春 (2018). 水稻短穗小粒突变体sps1的鉴定与基因精细定位. 中国农业科学 51, 1617-1626.
9	张敏娟, 李帅军, 陈琼琼, 景秀清, 陈坤明, 石春海, 李文强 (2018). 水稻矮化少蘖突变体dlt3的基因定位和蛋白质组学分析. 中国水稻科学 32, 529-537.
10	周亭亭, 饶玉春, 任德勇 (2018). 水稻卷叶细胞学与分子机制研究进展. 植物学报 53, 848-855.
11	Cooke MS, Evans MD, Dizdaroglu M, Lunec J (2003). Oxidative DNA damage: mechanisms, mutation, and disease. FASEB J 17, 1195-1214.
12	Engelbergs J, Thomale J, Rajewsky MF (2000). Role of DNA repair in carcinogen-induced ras mutation. Mutat Res 450, 139-153.
13	Fukuda M, Kawagoe Y, Murakami T, Washida H, Sugino A, Nagamine A, Okita TW, Ogawa W, Kumamaru T (2016). The dual roles of the Golgi transport 1 (GOT1B): RNA localization to the cortical endoplasmic reticulum and the export of proglutelin and α-globulin from the cortical ER to the golgi. Plant Cell Physiol 57, 2380-2391.
14	Fukuda M, Satoh-Cruz M, Wen LY, Crofts AJ, Sugino A, Washida H, Okita TW, Ogawa M, Kawagoe Y, Maeshima M, Kumamaru T (2011). The small GTPase Rab5a is essential for intracellular transport of proglutelin from the golgi apparatus to the protein storage vacuole and endosomal membrane organization in developing rice endosperm. Plant Physiol 157, 632-644.
15	Fukuda M, Wen LY, Satoh-Cruz M, Kawagoe Y, Nagamura Y, Okita TW, Washida H, Sugino A, Ishino S, Ishino Y, Ogawa M, Sunada M, Ueda T, Kumamaru T (2013). A guanine nucleotide exchange factor for Rab5 proteins is essential for intracellular transport of the proglutelin from the golgi apparatus to the protein storage vacuole in rice endosperm. Plant Physiol 162, 663-674.
16	Goff SA, Ricke D, Lan TH, Presting G, Wang RL, Dunn M, Glazebrook J, Sessions A, Oeller P, Varma H, Hadley D, Hutchison D, Martin C, Katagiri F, Lange BM, Moughamer T, Xia Y, Budworth P, Zhong JP, Miguel T, Paszkowski U, Zhang SP, Colbert M, Sun WL, Chen LL, Cooper B, Park S, Wood TC, Mao L, Quail P, Wing R, Dean R, Yu Y, Zharkikh A, Shen R, Sahasrabudhe S, Thomas A, Cannings R, Gutin A, Pruss D, Reid J, Tavtigian S, Mitchell J, Eldredge G, Scholl T, Miller RM, Bhatnagar S, Adey N, Rubano T, Tusneem N, Robinson R, Feldhaus J, Macalma T, Oliphant A, Briggs S (2002). A draft sequence of the rice genome ( Oryza sativa L. ssp. japonica). Science 296, 92-100.
17	Itoh JI, Nonomura KI, Ikeda K, Yamaki S, Inukai Y, Yamagishi H, Kitano H, Nagato Y (2005). Rice plant development: from zygote to spikelet. Plant Cell Physiol 46, 23-47.
18	Jin S, Zong Y, Gao Q, Zhu ZX, Wang YP, Qin P, Liang CZ, Wang DW, Qiu JL, Zhang F, Gao CX (2019). Cytosine, but not adenine, base editors induce genome-wide off- target mutations in rice. Science 364, 292-295.
19	Kumamaru T, Uemura Y, Inoue Y, Takemoto Y, Siddiqui SU, Ogawa M, Hara-Nishimura I, Satoh H (2010). Vacuolar processing enzyme plays an essential role in the crystalline structure of glutelin in rice seed. Plant Cell Physiol 51, 38-46.
20	Mao YF, Yang XX, Zhou YT, Zhang ZJ, Botella JR, Zhu JK (2018). Manipulating plant RNA-silencing pathways to improve the gene editing efficiency of CRISPR/Cas9 systems. Genome Biol 19, 149.
21	Miura S, Crofts N, Saito Y, Hosaka Y, Oitome NF, Watanabe T, Kumamaru T, Fujita N (2018). Starch synthase IIa-deficient mutant rice line produces endosperm starch with lower gelatinization temperature than japonica rice cultivars. Front Plant Sci 9, 645.
22	Neale S (1976). Mutagenicity of nitrosamides and nitrosamidines in micro-organisms and plants. Mutat Res 32, 229-266.
23	Nishi A, Nakamura Y, Tanaka N, Satoh H (2001). Biochemical and genetic analysis of the effects of amylose- extender mutation in rice endosperm. Plant Physiol 127, 459-472.
24	Qu LQ, Wei XL, Satoh H, Kumamaru T, Ogawa M, Takaiwa F (2002). Inheritance of alleles for glutelin α-2 subunit genes in rice and identification of their corresponding cDNA clone. Theor Appl Genet 105, 1099-1108.
25	Qu LQ, Wei XL, Satoh H, Kumamaru T, Ogawa M, Takaiwa F (2003). Biochemical and molecular characterization of a rice glutelin allele for the GluA-1 gene. Theor Appl Genet 107, 20-25.
26	Satoh H, Matsusaka H, Kumamaru T (2010). Use of N-methyl-N-nitrosourea treatment of fertilized egg cells for saturation mutagenesis of rice. Breed Sci 60, 475-485.
27	Satoh H, Shibahara K, Tokunaga T, Nishi A, Tasaki M, Hwang SK, Okita TW, Kaneko N, Fujita N, Yoshida M, Hosaka Y, Sato A, Utsumi Y, Ohdan T, Nakamura Y (2008). Mutation of the plastidial α-glucan phosphorylase gene in rice affects the synthesis and structure of starch in the endosperm. Plant Cell 20, 1833-1849.
28	Shan QW, Wang YP, Li J, Zhang Y, Chen KL, Liang Z, Zhang K, Liu JX, Xi JJ, Qiu JL, Gao CX (2013). Targeted genome modification of crop plants using a CRISPR-Cas system. Nat Biotechnol 31, 686-688.
29	Suzuki T, Eiguchi M, Kumamaru T, Satoh H, Matsusaka H, Moriguchi K, Nagato Y, Kurata N (2008). MNUinduced mutant pools and high performance TILLING enable finding of any gene mutation in rice. Mol Genet Genomics 279, 213-223.
30	Swain CG, Scott CB (1953). Quantitative correlation of relative rates. Comparison of hydroxide ion with other nucleophilic reagents toward alkyl halides, esters, epoxides and acyl halides. J Am Chem Soc 75, 141-147.
31	Takemoto Y, Coughlan SJ, Okita TW, Satoh H, Ogawa M, Kumamaru T (2002). The rice mutant esp2 greatly accumulates the glutelin precursor and deletes the protein disulfide isomerase. Plant Physiol 128, 1212-1222.
32	Tian HD, Satoh H, Takemoto Y (2004). Inheritance of novel 57H mutations in rice and their effect on compartmentation of endosperm storage proteins. Int J Plant Sci 165, 537-544.
33	Wang NL, Long T, Yao W, Xiong LZ, Zhang QF, Wu CY (2013). Mutant resources for the functional analysis of the rice genome. Mol Plant 6, 596-604.
34	Wu JL, Wu CJ, Lei CL, Baraoidan M, Bordeos A, Madamba MRS, Ramos-Pamplona M, Mauleon R, Portugal A, Ulat VJ, Bruskiewich R, Wang GL, Leach J, Khush G, Leung H (2005). Chemical- and irradiation-induced mutants of indica rice IR64 for forward and reverse genetics. Plant Mol Biol 59, 85-97.
36	Yu J, Hu SN, Wang J, Wong GKS, Li SG, Liu B, Deng YJ, Dai L, Zhou Y, Zhang XQ, Cao ML, Liu J, Sun JD, Tang JB, Chen YJ, Huang XB, Lin W, Ye C, Tong W, Cong LJ, Geng JN, Han YJ, Li L, Li W, Hu GQ, Huang XG, Li WJ, Li J, Liu ZW, Li L, Liu JP, Qi QH, Liu JS, Li L, Li T, Wang XG, Lu H, Wu TT, Zhu M, Ni PX, Han H, Dong W, Ren XY, Feng XL, Cui P, Li XR, Wang H, Xu X, Zhai WX, Xu Z, Zhang JS, He SJ, Zhang JG, Xu JC, Zhang KL, Zheng XW, Dong JH, Zeng WY, Tao L, Ye J, Tan J, Ren XD, Chen XW, He J, Liu DF, Tian W, Tian CG, Xia HA, Bao QY, Li G, Gao H, Cao T, Wang J, Zhao WM, Li P, Chen W, Wang XD, Zhang Y, Hu JF, Wang J, Liu S, Yang J, Zhang GY, Xiong YQ, Li ZJ, Mao L, Zhou CS, Zhu Z, Chen RS, Hao BL, Zheng WM, Chen SY, Guo W, Li GJ, Liu SQ, Tao M, Wang J, Zhu LH, Yuan LP, Yang HM (2002). A draft sequence of the rice genome (Oryza sativa L. ssp. indica). Science 296, 79-92.

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