Chin Bull Bot ›› 2019, Vol. 54 ›› Issue (5): 625-633.doi: 10.11983/CBB18243

• TECHNIQUES AND METHODS • Previous Articles     Next Articles

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:2020-03-10 Published:2019-09-01
  • Contact: Tian Huaidong E-mail:huaidongt@sxu.edu.cn

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

Table 1

Design for MNU treatments of germ cells in rice"

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

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
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

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 (%)
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

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."

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