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

• 技术方法 • 上一篇    下一篇

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. 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 E-mail:huaidongt@sxu.edu.cn
  • 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

Tian Huaidong1,3,*(),Li Jing1,Tian Baohua1,Niu Pengfei1,Li Zhen1,Yue Zhongxiao1,Qu Yajuan1,Jiang Jianfang1,Wang Guangyuan2,Cen Huihui1,Li Nan1,Yan Feng1   

  1. 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: Tian Huaidong E-mail:huaidongt@sxu.edu.cn

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

表1

水稻生殖细胞MNU实验设计"

Treatments MNU
(mmol∙L-1)
Time
(min)
pH PO43– (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"

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
PO43-
(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"

Treatments of
germ cells
Number of mutant characters Incidences (%)
Seedling Culm Leaf Panicle Glume Seed Total
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
PO43-
(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。"

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。"

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。"

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。"

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。"

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