Creation of Ogura cytoplasmic male sterility (Ogura CMS) material of Brassica juncea

doi:10.3969/j.issn.1004-1524.2023.05.09

Acta Agriculturae Zhejiangensis ›› 2023, Vol. 35 ›› Issue (5): 1058-1068.DOI: 10.3969/j.issn.1004-1524.2023.05.09

• Horticultural Science • Previous Articles Next Articles

Creation of Ogura cytoplasmic male sterility (Ogura CMS) material of Brassica juncea

LI Chongjuan¹^,²(), LYU Fengxian¹^,², YANG Ding¹^,², ZHANG Liqin²^,³, LAN Mei²^,³, YANG Hongli²^,³, XU Xuezhong²^,³, HU Jingfeng²^,³, SHEN Shipin⁴, WU Yufei⁴, HE Jiangming²^,³^,^*(), DONG Xiangshu¹

1. Resource Plant Research Institute, Yunnan University, Kunming 650504, China
2. Horticulture Research Institute, Yunnan Academy of Agricultural Sciences, Kunming 650205, China
3. Yunnan Branch of National Vegetable Improvement Center, Kunming 650205, China
4. Agricultural Science and Technology Promotion Center of Zhenxiong County, Zhenxiong 657200, Yunnan, China

Received:2022-04-13 Online:2023-05-25 Published:2023-06-01
Contact: HE Jiangming

Abstract

Abstract:

In order to obtain Ogura cytoplasmic male sterility Brassica juncea, the B genome of Brassica juncea was introduced into Chinese cabbage by hybridization, and the male sterility gene of Chinese cabbage was created by backcrossing combined with embryo rescue technique. With CCR17001 Ogura cytoplasmic male sterility Chinese cabbage and leaf mustard JC17000 hybridization, and leaf mustard as recurrent parent for 5 consecutive backcross generations, the Ogura cytopathic male sterile mustard material was obtained and identified by morphology, cytology and molecular biology. The results showed that the plant morphology of this material was similar to that of its recurrent parent, with well-developed nectaries, highly degenerated stamens into petals, 100% sterility and sterility rate, stable sterility, normal seed setting ability, which could be used as a reliable material for breeding advantage of mustard.

Key words: Brassica juncea, interspecies cross, Ogura cytoplasmic male sterility, embryo rescue

CLC Number:

S637.2

LI Chongjuan, LYU Fengxian, YANG Ding, ZHANG Liqin, LAN Mei, YANG Hongli, XU Xuezhong, HU Jingfeng, SHEN Shipin, WU Yufei, HE Jiangming, DONG Xiangshu. Creation of Ogura cytoplasmic male sterility (Ogura CMS) material of Brassica juncea[J]. Acta Agriculturae Zhejiangensis, 2023, 35(5): 1058-1068.

Figures/Tables 13

Fig.1 Breeding model for the transfer of Ogura CMS in mustard

Table 1 Components of embryo rescue medium for interspecific hybrid

编号 No.	基础培养基 Minimal medium	NAA质量浓度 NAA concentration/ (mg·L^-1)	6-BA质量浓度 6-BA concentration/ (mg·L^-1)	KT质量浓度 KT concentration/ (mg·L^-1)	AC质量浓度 AC concentration/ (g·L^-1)
M1	MS	0	0	0	0
M2	MS	0.1	0.1	1	1
M3	MS	0.3	0.3	1	1
M4	MS	0.5	0.5	1	1
M5	MS	0.7	0.7	1	1
M6	MS	0.9	0.9	1	1
M7	MS	0.5	0.3	1	1
M8	MS	0.3	0.5	1	1

Table 2 Information of primers for identifying Ogura cytoplasm male sterility

引物名称 Primer name	上游引物 Forward primer(5'→3')	下游引物 Reverse primer(5'→3')
ogu-1	ATGATTACCTTTTTCGAAAAAATTGC	TTTATTTTCTCGGTCCATTTTC
ogu-2	TAGCGCTATCTTTCGGCCCT	ACCCGAAGGTCCTGGTCTCT
ogu-3	GCCTCAAACTGGATAAATCT	CAAGGATCTCGTTCATTC
ogu-4	CCATATTTGGCTAAGCTGGTTTTCT	TATCATCTCGGTCCATTGTCCAC

Fig.2 Embryogenesis rate of different culture mediums M1-M8 were different culture mediums, their components were the same as in Table 1. Data on the bars marked without the same lowercase letter indicated significant differences at P<0.05. The same as below.

Fig.3 Embryo rescue situation of interspecific hybrid of F1×JC17000 a, Ovule germination; b, Proliferation and differentiation of survival embryos; c and d, Embryo rescued seedling.

Table 3 Embryogenesis rate at different sampling time

授粉后时间 Time after pollination/d		取胚数 Number of ovules	出胚数 Number of embryogenesis	出胚率 Embryogenesis rate/%
第1次回交	11	169	17	10.06±0.05 c
The first backcross	12	263	40	15.21±0.11 b
	13	121	32	26.44±0.03 a
	14	101	16	15.84±0.04 b
	15	166	14	8.36±0.06 d
	16	109	4	4.73±1.55 e
第2次回交	11	71	8	11.07±0.16 d
The second backcross	12	91	16	17.58±0.07 b
	13	150	49	32.66±0.16 a
	14	204	33	16.71±0.02 c
	15	139	15	10.89±0.08 d
	16	87	5	5.75±0.11 e

Fig.4 Plant morphology of parents, hybrid and backcross progeny a, Recurrent parent mustard JC17000; b, Ogura CMS Chinese cabbage CCR17001; c, Interspecific hybridition F1; d, BC5.

Fig.5 Comparison of flower morphology between BC5 and recurrent parents a, The flower of JC17000; b, The flower of CCR17001; c, The flower of BC5; d, Dissection of JC17000 flower; e, Dissection of CCR17001 flower; f, Dissection of BC5 flower. Scale bar=1 cm.

Table 4 Comparison of floral organ structure characteristics between cytoplasmic male sterility of Brassica juncea and its parents cm

材料 Material	花蕾长 Bud length	花蕾直径 Bud diameter	花瓣长 Corolla length	花瓣宽 Corolla diameter	花柱长 Nectar length	雄蕊长 Stamens length
JC17000	0.66 ab	0.34 ab	1.17 a	0.56 b	0.55 b	0.77 ab
CCR17001	0.67 a	0.38 a	1.24 a	0.79 a	0.54 b	0.70 b
BC₅	0.58 b	0.27 b	0.70 b	0.26 c	0.76 a	—

Table 5 Comparison of fruitfulness between parents, cross and backcross generations

材料 Material	授粉花数 Number of flowers	结籽数 Number of seeds	平均单花结籽数 Average number of seeds per fiower
JC17000	243	2 638	10.85
F1	109	394	3.61
BC₁	321	-	-
BC₂	298	-	-
BC₃	466	2 319	4.98
BC₄	267	1 623	7.08
BC₅	352	2 923	10.30

Fig.6 Chromosome observation of cross and backcross generations a, Mitotic cells of ogura CMS Chinese cabbage CCR17001, 2n=20; b, Mitotic cells of the recurrent parent mustard JC17000, 2n=36; c, Mitotic cells of interspecific hybrid progeny F1, 2n=28; d, Mitotic cells of BC5, 2n=36. Scale bar=10 μm.

Fig.7 Flow cytometry of parent, cross and backcross progeny a, Ogura CMS Chinese cabbage CCR17001; b, Recurrent parent mustard JC17000; c, Interspecific hybrid progeny F1; d, BC5.

Fig.8 Ogura cytoplasmic male sterility molecular identification BC5 and its parents M, DL2000 marker; 1, Ogura CMS Chinese cabbage CCR17001; 2, Recurrent parent mustard JC17000; 3, BC5; 4, Fertile Chinese cabbage CCR21001.

References 28

[1]	陈学群, 陈材林, 周源, 等. 芥菜的种内进化及进化机制探讨[J]. 西南农业学报, 1994, 7(1): 6-12.
	CHEN X Q, CHEN C L, ZHOU Y, et al. Intraspecific evolution and possible evolution mechanism in mustard, Bramssica juncea[J]. Southwest China Journal of Agricultural Sciences, 1994, 7(1): 6-12. (in Chinese with English abstract)
[2]	范永红, 沈进娟, 董代文. 芥菜类蔬菜产业发展现状及研究前景思考[J]. 农学学报, 2016, 6(2): 65-71.
	FAN Y H, SHEN J J, DONG D W. Development status and research prospect of mustard vegetables industry[J]. Journal of Agriculture, 2016, 6(2): 65-71. (in Chinese with English abstract)
[3]	邓英, 唐兵, 付文苑, 等. 叶用芥菜小孢子培养技术体系的完善及DH系创制[J]. 中国农业大学学报, 2018, 23(9): 60-67.
	DENG Y, TANG B, FU W Y, et al. Opertimization of the isolated microspore culture system of leaf mustard and double haploid line producing[J]. Journal of China Agricultural University, 2018, 23(9): 60-67. (in Chinese with English abstract)
[4]	OGURA H. Studies on the new male-sterility in Japanese radish, with special reference to the utilization of this sterility towerds the practical raising of hybrid seeds[J]. Memoirs of the Faculty of Agriculture Kagoshima University, 1967, 6(3):1446-1459.
[5]	兰梅, 胡靖锋, 刘家佳, 等. 利用分子标记鉴定白菜细胞质雄性不育类型的研究[J]. 中国农学通报, 2018, 34(6): 137-142. DOI
	LAN M, HU J F, LIU J J, et al. Identifying cytoplasmic male sterile types of Chinese cabbage(Brassica campestris L.) by molecular markers[J]. Chinese Agricultural Science Bulletin, 2018, 34(6): 137-142. (in Chinese with English abstract)
[6]	万正杰, 李海渤, 姚培杰, 等. 芥菜类蔬菜杂种优势利用的研究进展与展望[J]. 华中农业大学学报, 2018, 37(1): 115-120.
	WAN Z J, LI H B, YAO P J, et al. Progress and prospect of heterosis utilization in mustard(Brassica juncea L.) vegetables[J]. Journal of Huazhong Agricultural University, 2018, 37(1): 115-120. (in Chinese with English abstract)
[7]	NIKORNPUN M, SENAPA N. 胞质雄性不育性在叶用芥菜上的利用[J]. 中国蔬菜, 2004(1): 10-11.
	NIKORNPUN M, SENAPA N. The application of cytoplasmic male sterility on leaf mustard[J]. China Vegetables, 2004(1): 10-11. (in Chinese with English abstract)
[8]	金海霞, 冯辉, 徐书法. 通过大白菜胞质不育系与芥菜远缘杂交选育新的芥菜胞质不育系[J]. 园艺学报, 2006, 33(4): 737-740.
	JIN H X, FENG H, XU S F. Breeding of new CMS lines of leaf mustard by means of interspecific cross with CMS line of Chinese cabbage[J]. Acta Horticulturae Sinica, 2006, 33(4): 737-740. (in Chinese with English abstract)
[9]	章时蕃, 张淑江, 李菲, 等. 萝卜胞质结球芥和长柄芥雄性不育系的选育[J]. 中国蔬菜, 2014(2): 20-23.
	ZHANG S F, ZHANG S J, LI F, et al. Selective breeding of heading mustard and long petiole mustard ogu CMS lines[J]. China Vegetables, 2014(2): 20-23. (in Chinese with English abstract)
[10]	邹瑞昌, 万正杰, 徐跃进, 等. 叶用芥菜细胞质雄性不育系0912A的胞质效应和杂种优势分析[J]. 植物科学学报, 2012, 30(3): 261-268.
	ZOU R C, WAN Z J, XU Y J, et al. Cytoplasmic effects and heterosis in cytoplasmic male sterile line of leafy mustard 0912A[J]. Plant Science Journal, 2012, 30(3): 261-268. (in Chinese with English abstract) DOI URL
[11]	李自娟, 黄文, 应芳卿, 等. 番茄花粉保存与生活力测定[J]. 长江蔬菜, 2014(12): 53-54.
	LI Z J, HUANG W, YING F Q, et al. Preservation methods and determination of tomato pollen vitality[J]. Journal of Changjiang Vegetables, 2014(12): 53-54. (in Chinese with English abstract)
[12]	杨光圣, 傅廷栋, BROWN F F. 甘蓝型油菜细胞质雄性不育的遗传分类研究[J]. 中国农业科学, 1998, 31(1): 28-32.
	YANG G S, FU T D, BROWN F F. The genetic classification of cytoplasmic male sterility systems in Brassica napus L[J]. Scientia Agricultura Sinica, 1998, 31(1): 28-32. (in Chinese with English abstract)
[13]	GALBRAITH D W, HARKINS K R, MADDOX J M, et al. Rapid flow cytometric analysis of the cell cycle in intact plant tissues[J]. Science, 1983, 220(4601): 1049-1051. DOI PMID
[14]	刘娇娇. 结球甘蓝细胞质雄性不育系“QM”和“PM”的鉴定[D]. 哈尔滨: 东北农业大学, 2015.
	LIU J J. Identification of QM and PM cabbage cytoplasmic male sterile lines[D]. Harbin:Northeast Agricultural University, 2015. (in Chinese with English abstract)
[15]	刘志新, 徐玉颖, 姚培杰, 等. 芥菜胞质雄性不育系结荚性状的筛选和细胞学鉴定[J]. 华中农业大学学报, 2021, 40(3): 133-140.
	LIU Z X, XU Y Y, YAO P J, et al. Screening and cytological identification of pod-setting traits in cytoplasmic male sterile lines of mustard(Brassica juncea)[J]. Journal of Huazhong Agricultural University, 2021, 40(3): 133-140. (in Chinese with English abstract)
[16]	张明方, 陈竹君, 汪炳良, 等. 榨菜胞质雄性不育系选育初探[J]. 北京农业大学学报, 1993(S4): 129-133.
	ZHANG M F, CHEN Z J, WANG B L, et al. A preliminary study on breeding of cytoplasmic male sterile line for Tuber mustard[J]. Journal of China Agricultural University, 1993(S4): 129-133. (in Chinese with English abstract)
[17]	曾志红, 王永清, 陈学群, 等. 芥菜胞质雄性不育系花器形态结构与育性表现[J]. 西南农业学报, 1998, 11(2): 40-44.
	ZENG Z H, WANG Y Q, CHEN X Q, et al. Morphology and fertility of flower of cytoplasmic male sterile lines in vegetable mustard[J]. Southwest China Journal of Agricultural Sciences, 1998, 11(2): 40-44. (in Chinese with English abstract)
[18]	范永红, 周光凡, 陈材林. 茎瘤芥胞质雄性不育系的选育及其主要性状调查[J]. 中国蔬菜, 2001(5): 4-7.
	FAN Y H, ZHOU G F, CHEN C L. Breeding of cytoplasm male sterility(CMS) lines of Tuber mustard and investigati on of their main characters[J]. China Vegetables, 2001(5): 4-7. (in Chinese with English abstract)
[19]	周庆红, 周灿, 范淑英. 远缘杂交在芸薹属作物育种中的应用研究进展[J]. 北方园艺, 2015(2):165-170.
	ZHOU QH, ZHOU C, FAN S Y. Research advance in application of distant hybridization on byeeding of Brassica crops[J]. Northern Horticulture, 2015(2):165-170. (in Chinese with English abstract)
[20]	王丹, 王文和, 史滟澦. 芥菜型油菜与白菜正反杂交的胚胎学研究[J]. 植物学通报, 2006, 41(2): 158-163.
	WANG D, WANG W H, SHI Y Y. Embryologic research into reciprocal crosses between Brassica juncea and B. pekinesis[J]. Chinese Bulletin of Botany, 2006, 41(2): 158-163. (in Chinese with English abstract)
[21]	刘后利. 油菜遗传育种学[M]. 北京: 中国农业大学出版社, 2000.
[22]	李祥升, 常立春, 陈海旭, 等. 白菜基因组导入芥菜的远缘杂交及后代分离偏向性[J]. 新疆农业科学, 2021, 58(7): 1306-1315. DOI
	LI X S, CHANG L C, CHEN H X, et al. Segregation distortion of progeny of distant hybridization of Brassica juncea introduced into Chinese cabbage genome[J]. Xinjiang Agricultural Sciences, 2021, 58(7): 1306-1315. (in Chinese with English abstract)
[23]	柳霖坡. 结球甘蓝三倍体的创建及其细胞和胚胎发育研究[D]. 保定: 河北农业大学, 2004.
	LIU L P. A study on the obtainning of triploid and its cell, embryo development in head cabbage[D]. Baoding: Hebei Agricultural University, 2004. (in Chinese with English abstract)
[24]	扈新民, 李锡香, 梁燕, 等. 甘蓝与萝卜属间杂交及其胚挽救技术优化[J]. 中国蔬菜, 2009(10): 7-12.
	HU X M, LI X X, LIANG Y, et al. Optimization of technology for embryo rescue in distant hybridization between cabbage and radish[J]. China Vegetables, 2009(10): 7-12. (in Chinese with English abstract)
[25]	HARBERD D J. A simple effective embryo culture technique for brassica[J]. Euphytica, 1969, 18(3): 425-429. DOI URL
[26]	吕凤仙, 李崇娟, 杨鼎, 等. 菜薹和芥蓝种间杂交胚挽救技术研究[J]. 福建农业学报, 2021, 36(11): 1288-1294.
	LU F X, LI C J, YANG D, et al. Embryo rescue of interspecific Brassica campestris×B. albograbra hybrids[J]. Fujian Journal of Agricultural Sciences, 2021, 36(11): 1288-1294. (in Chinese with English abstract)
[27]	陈竹君, 张明方, 汪炳良, 等. 榨菜胞质雄性不育及其农艺性状的研究[J]. 园艺学报, 1995, 22(1): 40-46.
	CHEN Z J, ZHANG M F, WANG B L, et al. A study on fertility and agronomic characters of CMS lines for Tuber mustard[J]. Acta Horticulturae Sinica, 1995, 22(1): 40-46. (in Chinese with English abstract)
[28]	邹瑞昌. 叶用芥菜细胞质雄性不育系0912A的评价及杂种优势利用[D]. 武汉: 华中农业大学, 2012.
	ZOU R C. Evaluation and exploitation of heterosis in cytoplasmic male sterile line of leaf Mustard 0912A[D]. Wuhan: Huazhong Agricultural University, 2012. (in Chinese with English abstract)

Creation of Ogura cytoplasmic male sterility (Ogura CMS) material of Brassica juncea

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Figures/Tables 13

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编号 No.	基础培养基 Minimal medium	NAA质量浓度 NAA concentration/ (mg·L^-1)	6-BA质量浓度 6-BA concentration/ (mg·L^-1)	KT质量浓度 KT concentration/ (mg·L^-1)	AC质量浓度 AC concentration/ (g·L^-1)
M1	MS	0	0	0	0
M2	MS	0.1	0.1	1	1
M3	MS	0.3	0.3	1	1
M4	MS	0.5	0.5	1	1
M5	MS	0.7	0.7	1	1
M6	MS	0.9	0.9	1	1
M7	MS	0.5	0.3	1	1
M8	MS	0.3	0.5	1	1

编号 No.	基础培养基 Minimal medium	NAA质量浓度 NAA concentration/ (mg·L^-1)	6-BA质量浓度 6-BA concentration/ (mg·L^-1)	KT质量浓度 KT concentration/ (mg·L^-1)	AC质量浓度 AC concentration/ (g·L^-1)
M1	MS	0	0	0	0
M2	MS	0.1	0.1	1	1
M3	MS	0.3	0.3	1	1
M4	MS	0.5	0.5	1	1
M5	MS	0.7	0.7	1	1
M6	MS	0.9	0.9	1	1
M7	MS	0.5	0.3	1	1
M8	MS	0.3	0.5	1	1

编号 No.	基础培养基 Minimal medium	NAA质量浓度 NAA concentration/ (mg·L^-1)	6-BA质量浓度 6-BA concentration/ (mg·L^-1)	KT质量浓度 KT concentration/ (mg·L^-1)	AC质量浓度 AC concentration/ (g·L^-1)
M1	MS	0	0	0	0
M2	MS	0.1	0.1	1	1
M3	MS	0.3	0.3	1	1
M4	MS	0.5	0.5	1	1
M5	MS	0.7	0.7	1	1
M6	MS	0.9	0.9	1	1
M7	MS	0.5	0.3	1	1
M8	MS	0.3	0.5	1	1

编号 No.	基础培养基 Minimal medium	NAA质量浓度 NAA concentration/ (mg·L^-1)	6-BA质量浓度 6-BA concentration/ (mg·L^-1)	KT质量浓度 KT concentration/ (mg·L^-1)	AC质量浓度 AC concentration/ (g·L^-1)
M1	MS	0	0	0	0
M2	MS	0.1	0.1	1	1
M3	MS	0.3	0.3	1	1
M4	MS	0.5	0.5	1	1
M5	MS	0.7	0.7	1	1
M6	MS	0.9	0.9	1	1
M7	MS	0.5	0.3	1	1
M8	MS	0.3	0.5	1	1

编号 No.	基础培养基 Minimal medium	NAA质量浓度 NAA concentration/ (mg·L^-1)	6-BA质量浓度 6-BA concentration/ (mg·L^-1)	KT质量浓度 KT concentration/ (mg·L^-1)	AC质量浓度 AC concentration/ (g·L^-1)
M1	MS	0	0	0	0
M2	MS	0.1	0.1	1	1
M3	MS	0.3	0.3	1	1
M4	MS	0.5	0.5	1	1
M5	MS	0.7	0.7	1	1
M6	MS	0.9	0.9	1	1
M7	MS	0.5	0.3	1	1
M8	MS	0.3	0.5	1	1

编号 No.	基础培养基 Minimal medium	NAA质量浓度 NAA concentration/ (mg·L^-1)	6-BA质量浓度 6-BA concentration/ (mg·L^-1)	KT质量浓度 KT concentration/ (mg·L^-1)	AC质量浓度 AC concentration/ (g·L^-1)
M1	MS	0	0	0	0
M2	MS	0.1	0.1	1	1
M3	MS	0.3	0.3	1	1
M4	MS	0.5	0.5	1	1
M5	MS	0.7	0.7	1	1
M6	MS	0.9	0.9	1	1
M7	MS	0.5	0.3	1	1
M8	MS	0.3	0.5	1	1