水稻两性生殖细胞的N-甲基-N-亚硝基脲诱变方法

doi:10.11983/CBB18243

植物学报 ›› 2019, Vol. 54 ›› Issue (5): 625-633.DOI: 10.11983/CBB18243

水稻两性生殖细胞的N-甲基-N-亚硝基脲诱变方法

田怀东^1,^3,^*(),李菁¹,田保华¹,牛鹏飞¹,李珍¹,岳忠孝¹,屈雅娟¹,姜建芳¹,王广元²,岑慧慧¹,李南¹,闫枫¹

^1. 山西大学生命科学学院植物种质与作物遗传资源实验室, 太原 030006
^2. 山西省农业科学院作物科学研究所, 太原 030031
^3. 山西景康农业技术推广有限公司, 晋中 030600

收稿日期:2018-11-13 接受日期:2019-06-18 出版日期:2019-09-01 发布日期:2020-03-10
通讯作者: 田怀东
基金资助:
山西省科技创新计划(2014101025)

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

Huaidong Tian^1,^3,^*(),Jing Li¹,Baohua Tian¹,Pengfei Niu¹,Zhen Li¹,Zhongxiao Yue¹,Yajuan Qu¹,Jianfang Jiang¹,Guangyuan Wang²,Huihui Cen¹,Nan Li¹,Feng Yan¹

^1. Laboratory of Plant Germplasm and Genetic Resources of Crop, College of Life Science, Shanxi University, Taiyuan 030006, China
^2. Institute of Crop Sciences, Shanxi Academy of Agricultural Sciences, Taiyuan 030031, China
^3. Shanxi Jingkang Agriculture Technology Promotion Co. Ltd, Jinzhong 030600, China

Received:2018-11-13 Accepted:2019-06-18 Online:2019-09-01 Published:2020-03-10
Contact: Huaidong Tian

摘要/Abstract

摘要： N-甲基-N-亚硝基脲(MNU)被用于水稻(Oryza sativa)受精卵的诱变。通过水稻辽盐6号成熟生殖器官的MNU体内同步处理及后代群体筛查, 确立了水稻两性生殖细胞的MNU诱变方法。与辽盐6号受精卵的MNU处理相比, 各组条件下两性生殖细胞的MNU处理明显使M₁群体生长发育的指标降低及M₁-M₂群体中突变性状的发生率升高。两性生殖细胞在含有1.5 mmol∙L ^-1 MNU和10 mmol∙L ^-1 PO₄ ^3-的缓冲液(pH4.8)中处理60分钟, 突变性状发生率是基于受精卵MNU处理的3倍。进一步筛查M₃群体, 获得了包含新型植株和籽粒突变体的纯合突变体系列。研究结果表明, 水稻两性生殖细胞的MNU诱变可显著提高广谱诱变效率。该技术的应用可为水稻的未知功能基因鉴定和育种所需的各种突变体规模化开发提供高效的技术支撑。

关键词: 水稻, 两性生殖细胞, N-甲基-N-亚硝基脲, 诱变

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

田怀东,李菁,田保华,牛鹏飞,李珍,岳忠孝,屈雅娟,姜建芳,王广元,岑慧慧,李南,闫枫. 水稻两性生殖细胞的N-甲基-N-亚硝基脲诱变方法. 植物学报, 2019, 54(5): 625-633.

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. Chinese Bulletin of Botany, 2019, 54(5): 625-633.

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图/表 8

表1 水稻生殖细胞MNU实验设计

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

表2 MNU处理因素对辽盐6号水稻生殖细胞M1群体生长发育的影响

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

表3 辽盐6号水稻生殖细胞的MNU处理因素对M1-M2群体中突变性状表达的影响

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

图1 辽盐6号水稻生殖细胞经MNU处理所得的M1-M3群体中的秆形突变性状 (A) 野生型秆; (B) 多分蘖秆; (C) 少分蘖秆; (D), (E) 矮化秆。Bars=10 cm。MNU同表1。

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.

图2 辽盐6号水稻生殖细胞经MNU处理所得M1-M3群体中的叶形与叶色突变性状 (A) 野生型叶; (B) 宽叶; (C) 细卷叶; (D) 竖叶; (E) 深绿叶; (F) 浅绿叶; (G) 黄叶缘; (H) 斑点叶。Bars=5 cm。MNU同表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.

图3 辽盐6号水稻生殖细胞经MNU处理所得M1-M3群体中的穗形与穗色突变性状 (A) 野生型穗; (B) 短穗; (C) 不育穗; (D) 泛红穗。Bars=5 cm。MNU同表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.

图4 辽盐6号水稻生殖细胞经MNU处理所得M1-M3群体中的颖形与颖色突变性状 (A) 野生型颖; (B) 有芒颖; (C) 紫黑颖; (D) 红棕颖。Bars=5 mm。MNU同表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.

图5 辽盐6号水稻生殖细胞经MNU处理所得M1-M3群体中的种子突变性状 (A) 野生型种子; (B) 细长种子; (C) 皱纹胚乳; (D) 腹部凹陷胚乳; (E) 皱缩胚乳; (F) 蜡质胚乳; (G) 暗浊色胚乳; (H) 粉质胚乳; (I) 心白胚乳; (J) 周白胚乳; (K) 红褐色胚乳; (L) 绿色胚乳。Bars=5 mm。MNU同表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.

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水稻两性生殖细胞的N-甲基-N-亚硝基脲诱变方法

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

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