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Acta Agronomica Sinica ›› 2021, Vol. 47 ›› Issue (5): 860-868.doi: 10.3724/SP.J.1006.2021.04153


Genetic and cytological analysis of male sterile mutant msm2015-1 in mungbean

WU Ran-Ran(), LIN Yun, CHEN Jing-Bin, XUE Chen-Chen, YUAN Xing-Xing, YAN Qiang, GAO Ying, LI Ling-Hui, ZHANG Qin-Xue, CHEN Xin*()   

  1. Institute of Industrial Crops, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, Jiangsu, China
  • Received:2020-07-11 Accepted:2020-10-14 Online:2021-05-12 Published:2020-10-30
  • Contact: CHEN Xin E-mail:rrwu@jaas.ac.cn;cx@jaas.ac.cn
  • Supported by:
    National Key Research and Development Program of China(2019YFD1001301);National Key Research and Development Program of China(2019YFD1001300);Natural Science Foundation of Jiangsu Province(BK20190257);National Natural Science Foundation of China(31871696);Jiangsu Agricultural Industry Technology System(JATS[2019]399)


In China, the low yield per unit has always been a problem in the mungbean industry. The heterosis of mungbean is an effective way to increase yield. The male sterile line is a valuable resource to realize the utilization of hybrid. In this study, 60Co-γ radiation mutagenesis technology was used to induce male sterile mutants in mungbean, and we gained one mutant named male sterile mungbean 2015-1 (msm2015-1) for the first time. There was no significant phenotype difference between msm2015-1 and Sulyu 1 during the vegetative growth stage. The floral organ of msm2015-1 was normal, but the anthers of msm2015-1 were white and could not crack and disperse powder normally in the flowering stage. In the mature stage, hardly any normal pods grew up. Genetic analysis showed the separation ratio of fertility and sterility in the fertile segregation population was in line with 3:1, indicating that the sterility trait was controlled by a single recessive nuclear gene. Cytological analysis of pollen development revealed that there were few pollen grains of msm2015-1 attached to mature stigmas. The pollen grains of msm2015-1 did not germinate in vitro. The cytoplasm of msm2015-1’s pollen could not be stained by Alexander dye. Three abortion types showed up when stained by I2-KI dye: typical abortion, spherical abortion and stained abortion. DAPI staining showed the nuclei of msm2015-1’s pollen developed abnormally. Magenta acetate staining indicated that abortion occurred in the early stage of pollen development, while the asymmetry and more anomalous fission of meiosis during the tetrad stage were the main causes of pollen abortion.

Key words: mungbean, male sterile, genetic analysis, pollen development, cytology

Fig. 1

Phenotypic analysis of the msm2015-1 mutant in mungbean A: vegetative growth stage; B: flower buds; C: opening flowers; D: anatomy of the flower bud, from left to right in order: standard petals, wing petals, keel petals, stamens and pistils; E: the little pods on 2nd day after flowering; F: nearly ripen pods. In figure A, bar = 10 cm; in figure B to F, bar = 1 cm."

Fig. 2

Morphology observation of msm2015-1’s anthers and pollen grains in mungbean A: stamen, the white arrows indicate the indehiscent anther of msm2015-1; B: the pistil of Sulyu 1, the white arrow indicates the attached pollen grains on stigma; C: the pistil of msm2015-1, the white arrow indicates rarely pollen grains attached on stigma; D: scanning electron micrograph (SEM) image of Sulyu 1’s anther; E: SEM image of msm2015-1’s anther; F: SEM image of Sulyu 1’s mature pollen grains; G: SEM image of msm2015-1’s mature pollen grains. In figure A to C, bars = 1 mm; in figure D and E, bars = 100 µm; in fugure F and G, bars = 10 µm."

Table 1

Genetic analysis of fertility separation population"

Observed number
Theoretical number
可育株Fertile plants 187 193.5 0.873
不育株Sterile plants 71 64.5

Fig. 3

Pollen germination of the msm2015-1 mutant in vivo and in vitro in mungbean A: pollen germination on stigma of Sulyu 1, the white arrows indicate the pollen grains attached on stigma and germinated pollen tubes; B: pollen germination on stigma of msm2015-1, the white arrow indicates few pollen grains attached on stigma; C: mature pollen germination of Sulyu 1 in vitro; D: mature pollen germination of msm2015-1 in vitro. Bar = 100 µm."

Fig. 4

Pollen viability of the msm2015-1 mutant analyzed by staining analysis in mungbean A: Alexander staining of Sulyu 1’s mature pollen; B: Alexander staining of msm2015-1’s mature pollen; C: statistics of pollen abortion rate of Alexander staining; D: I2-KI staining of Sulyu 1’s mature pollen; E: I2-KI staining of msm2015-1’s mature pollen; F: statistics of pollen abortion rate of I2-KI staining; G: Alexander staining of Sulyu 1’s anthers of mature flower bud; H: Alexander staining of msm2015-1’s anthers of mature flower bud. Bar = 100 µm."

Fig. 5

Detection of pollen nuclear development in the msm2015-1 mutant A: DAPI staining of Sulyu 1’s mature pollen, the white arrow indicates the generative nucleus, and the red arrow indicates the vegetative nucleus; B: DAPI staining of msm2015-1’s mature pollen. Bar = 20 µm."

Fig. 6

Developmental early stage analysis of msm2015-1’s pollen in mungbean A: the early stages of pollen development, the white arrows indicate the uneven-size microspores caused by the asymmetry and inhomogeneity meiosis during the tetrad stage; B: the anomalous more fission of meiosis happened during the tetrad stage, the numbers of msm2015-1’s microspores are indicated by the Arabic numbers. PMC: pollen mother cell; Msp: microspore. Bar = 20 µm."

[1] Rabbi S M F, Rahman M M, Mondal M M A, Bhowal S K, Haque M A. Effect of chitosan application on plant characters, yield attributes and yield of mungbean. Res J Agric Environ Manage, 2016,5:95-100.
[2] Lambrides C J, Godwin I D. Mungbean. Genome Map Mol Breed Plants, 2007,3:69-90.
[3] Kang Y J, Kim S K, Kim M Y, Lestari P, Kim K H, Ha B K, Jun T H, Hwang W J, Lee T, Lee J, Shim S, Yoon M Y, Jang Y E, Han K S, Taeprayoon P, Yoon N, Somta P, Tanya P, Kim K S, Gwag J G, Moon J K, Lee Y H, Park B S, Bombarely A, Doyle J J, Jackson S A, Schafleitner R, Srinives P, Varshney R K, Lee S H. Genome sequence of mungbean and insights into evolution within Vigna species. Nat Commun, 2014,5:5543.
[4] Singh N, Mallick J, Sagolsem D, Mandal N, Bhattacharyya S. Mapping of molecular markers linked with MYMIV and yield attributing traits in mungbean. Indian J Genet Plant Breed, 2018,78:118-126.
[5] 邢宝龙, 殷丽丽. 绿豆育种及分子遗传学研究进展. 现代农业科技, 2017, (10):39-40.
Xing B L, Yin L L. Research progress of breeding and molecular genetics on Vigna radiata L. Modern Agric Sci Technol, 2017, (10):39-40 (in Chinese with English abstract).
[6] Tester M, Langridge P. Breeding technologies to increase crop production in a changing world. Science, 2010,327:818-822.
[7] Kim Y J, Zhang D B. Molecular control of male fertility for crop hybrid breeding. Trends Plant Sci, 2018,23:53-65.
[8] Longin C F H, Muhleisen J, Maurer H P, Zhang H L, Gowda M, Reif J C. Hybrid breeding in autogamous cereals. Theor Appl Genet, 2012,125:1087-1096.
[9] Duvick D N. Biotechnology in the 1930s: the development of hybrid maize. Nat Rev Genet, 2001,2:69-74.
[10] Li G, Dong Y, Zhao Y, Tian X, Liu W. Genome-wide prediction in a hybrid maize population adapted to northwest China. Crop J, 2020,8:830-842.
[11] Li H L, Zhou Y, Xin W L, Wei Y Q, Zhang J L, Guo L L. Wheat breeding in northern China: achievements and technical advances. Crop J, 2019,7:718-729.
[12] Muhleisen J, Maurer H P, Stiewe G, Bury P, Reif J C. Hybrid breeding in barley. Crop Sci, 2013,53:819-824.
[13] Luo X, Tan Y Q, Ma C Z, Tu J X, Shen J X, Yi B, Fu T D. High-throughput identification of SNPs reveals extensive heterosis with intra-group hybridization and genetic characteristics in a large rapeseed ( Brassica napus L.) panel. Euphytica, 2019,215:2-10.
[14] Tang G L, Quan Y W, Mi H F, Zhai L X, Li J J, Li W L. Breeding of high yield, high quality and disease-resistant hybrid cotton varieties Hanza 160 and Hanza 1692. Agric Biotechnol, 2018,7:37-39.
[15] Chen X, Sorajjapinun W, Reiwthongchum S, Srinives P. Identification of parental mungbean lines for production of hybrid varieties. CMU J, 2003,2:97-106.
[16] Chen L T, Liu Y G. Male sterility and fertility restoration in crops. Annu Rev Plant Biol, 2014,65:579-606.
[17] Bohra A, Jha U C, Adhimoolam P, Bisht D, Singh N P. Cytoplasmic male sterility (CMS) in hybrid breeding in field crops. Plant Cell Rep, 2016,35:967-993.
[18] Van Ginkel M, Ortiz R. Cross the best with the best, and select the best: HELP in breeding selfing crops. Crop Sci, 2018,58:17-30.
[19] Li J J, Nadeem M, Sun G L, Wang X B, Qiu L J. Male sterility in soybean: Occurrence, molecular basis and utilization. Plant Breed, 2019,138:659-676.
[20] Wu Y, Fox T W, Trimnell M R, Wang L, Xu R J, Cigan A M, Huffman G A, Garnaat C W, Hershey H, Albertsen M C. Development of a novel recessive genetic male sterility system for hybrid seed production in maize and other cross-pollinating crops. Plant Biotechnol J, 2016,14:1046-1054.
[21] Wan X Y, Wu S W, Li Z W, Dong Z Y, An X L, Ma B, Tian Y H, Li J P. Maize genic male-sterility genes and their applications in hybrid breeding: progress and perspectives. Mol Plant, 2019,12:321-342.
[22] Kim Y J, Zhang D, Jung K H. Molecular basis of pollen germination in cereals. Trends Plant Sci, 2019,24:1126-1136.
[23] Zhang D, Luo X, Zhu L. Cytological analysis and genetic control of rice anther development. J Genet Genomics, 2011,38:379-390.
[24] Shi J X, Cui M H, Yang L, Kim Y J, Zhang D B. Genetic and biochemical mechanisms of pollen wall development. Trends Plant Sci, 2015,20:741-753.
[25] 陈海元. 水稻种间杂种不育位点S40的精细定位及其败育机理的研究. 南京农业大学博士学位论文, 江苏南京, 2017.
Chen H Y. Fine Mapping and Study of Sterility Mechanism of the Interspecific Hybrid Sterility Locus S40 in Rice. PhD Dissertation of Nanjing Agricultural University, Nanjing, Jiangsu, China, 2017 (in Chinese with English abstract).
[26] Li H M, Chen H, Yang Z N, Gong J M. Cdi gene is required for pollen germination and tube growth in Arabidopsis. FEBS Lett, 2012,586:1027-1031.
[27] 蒋祥新. 绿豆大小孢子发生与雌雄配子体发育. 湖北农学院学报, 1989, (1):52-59.
Jiang X X. Mega- and microsporogenesis and development of male and female gametophytes in mungbean. J Hubei Agric Coll, 1989, (1):52-59 (in Chinese with English abstract).
[28] 孙寰, 张井勇, 王玉民, 赵丽梅. 木豆、苜蓿和大豆3种豆科作物杂种优势利用概述. 中国农业科学, 2009,42:1528-1539.
Sun H, Zhang J Y, Wang Y M, Zhao L M. A review of utilization of heterosis in three legume crops of pigeonpea, alfalfa and soybean. Sci Agric Sin, 2009,42:1528-1539 (in Chinese with English abstract).
[29] Saxena K B, Chauhan Y S, Laxman S, Kumar R V, Johansen C. Research and development of hybrid pigeonpea. Res Bull, 1996,19.
[30] Saxena K B, Kumar R V, Tikle A N, Saxena M K, Gupta P. ICPH 2671: the world’s first commercial food legume hybrid. Plant Breed, 2013,132:479-485.
[31] 赵丽梅, 孙寰, 王曙明, 王跃强, 黄梅, 李建平. 大豆杂交种杂交豆1号选育报告. 中国油料作物学报, 2004,26:16-18.
Zhao L M, Sun H, Wang S M, Wang Y Q, Huang M, Li J P. Breeding report of hybrid soybean ‘Hybrid Bean No. 1’. Chin J Oil Crop Sci, 2004,26:16-18 (in Chinese with English abstract).
[32] 赵丽梅, 彭宝, 张伟龙, 张连发, 张井勇, 李建平, 李茂海, 孙寰. 杂交大豆制种技术体系的建立. 大豆科学, 2010,29(4):165-169.
Zhao L M, Peng B, Zhang W L, Zhang L F, Zhang J Y, Li J P, Li M H, Sun H. Establishment of technology system for hybrid soybean seed production. Soybean Sci, 2010,29(4):165-169 (in Chinese with English abstract).
[33] Sangiri C, Kaga A, Tomooka N, Vaughan D, Srinives P. Genetic diversity of the mungbean ( Vigna radiata, Leguminosae) genepool on the basis of microsatellite analysis. Aust J Bot, 2007,55:837-847.
[34] Chen J, Somta P, Chen X, Cui X, Yuan X, Srinives P. Gene mapping of a mutant mungbean ( Vigna radiata L.) using new molecular markers suggests a gene encoding a YUC4-like protein regulates the chasmogamous flower trait. Front Plant Sci, 2016,7:830.
[35] Guo J X, Liu Y G. Molecular control of male reproductive development and pollen fertility in rice. J Integr Plant Biol, 2012,54:967-978.
[36] Xiao Y, You S, Kong W, Tang Q, Bai W, Cai Y, Zheng H, Wang C, Jiang L, Wang C. A GARP transcription factoranther dehiscence defected 1 (OsADD1) regulates rice anther dehiscence. Plant Mol Biol, 101:403-414.
[37] Song S, Chen Y, Liu L, Benjamin S Y H, Mao C, Gan Y, Yu H. OsFTIP7 determines auxin-mediated anther dehiscence in rice. Nat Plants, 2018,4:495-504.
[38] Zhu J, Lou Y, Xu X F, Yang Z N. A genetic pathway for tapetum development and function in Arabidopsis. J Integr Plant Biol, 2011,53:892-900.
[39] Li D D, Xue J S, Zhu J, Yang Z N. Gene regulatory network for tapetum development in Arabidopsis thaliana. Front Plant Sci, 2017,8:1559.
[40] Ranjan R, Khurana R, Malik N, Badoni S, Parida S K, Kapoor S, Tyagi A K. bHLH142 regulates various metabolic pathway-related genes to affect pollen development and anther dehiscence in rice. Sci Rep, 2017,7:43397.
[41] Yu J, Meng Z, Liang W, Kudla J, Tucker M R, Luo Z, Chen M, Xu D, Zhao G, Wang J. A rice Ca2+ binding protein is required for tapetum function and pollen formation . Plant Physiol, 2016,172:1772-1786.
[42] Li H, Yuan Z, Vizcay-Barrena G, Yang C, Liang W, Zong J, Wilson Z A, Zhang D. PERSISTENT TAPETAL CELL1 encodes a PHD-finger protein that is required for tapetal cell death and pollen development in rice. Plant Physiol, 2011,156:615-630.
[43] Yuan W, Li X, Chang Y, Wen R, Chen G, Zhang Q, Wu C. Mutation of the rice gene PAIR3 results in lack of bivalent formation in meiosis. Plant J, 2009,59:303-315.
[44] Yan X, Zeng X, Wang S, Li K, Yuan R, Gao H, Luo J, Liu F, Wu Y, Li Y, Zhu L, Wu G. Aberrant meiotic prophase I leads to genic male sterility in the novel TE5A mutant of Brassica napus. Sci Rep, 2016,6:33955.
[45] Zhao D Z. The EXCESS MICROSPOROCYTES1 gene encodes a putative leucine-rich repeat receptor protein kinase that controls somatic and reproductive cell fates in the Arabidopsis anther. Genes Dev, 2002,16:2021-2031.
[46] Yi J, Kim S R, Lee D Y, Moon S, Lee Y S, Jung K H, Hwang I, An G. The rice gene DEFECTIVE TAPETUM AND MEIOCYTES 1 (DTM1) is required for early tapetum development and meiosis. Plant J, 2012,70:256-270.
[47] Chang Z, Xu C, Huang X, Yan W, Qiu S, Yuan S, Ni H, Chen S, Xie G, Chen Z, Wu J, Tang X. The plant-specific ABERRANT GAMETOGENESIS 1 gene is essential for meiosis in rice. J Exp Bot, 2020,71:204-218.
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