Screening and phenotypic analysis of EMS-induced mutants in Lagenaria siceraria

doi:10.3969/j.issn.1004-1524.20240920

Acta Agriculturae Zhejiangensis ›› 2025, Vol. 37 ›› Issue (9): 1914-1923.DOI: 10.3969/j.issn.1004-1524.20240920

• Horticultural Science • Previous Articles Next Articles

Screening and phenotypic analysis of EMS-induced mutants in Lagenaria siceraria

ZHENG Cheng¹^,²(), WANG Ying², WANG Jian², GUO Xiao¹^,², WANG Baogen², WU Xinyi², ZHU Biao¹, LI Guojing², WU Xiaohua²^,^*()

¹ College of Horticultural Science, Zhejiang A&F University, Hangzhou 311300, China
² Institute of Vegetables, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China

Received:2024-10-28 Online:2025-09-25 Published:2025-10-15
Contact: WU Xiaohua

Abstract

Abstract:

To investigate suitable ethyl methanesulfonate (EMS) mutagenesis conditions and screen for mutants in bottle gourd (Lagenaria siceraria), seeds of the local variety Hangzhou Long Gourd were treated with different volume fractions of EMS and soaking time to determine the optimal mutagenesis parameters. Subsequently, these optimal conditions were applied to construct an EMS-induced mutant library.The results showed that the optimal EMS mutagenesis condition for bottle gourd was soaking seeds in 1.2% EMS solution for 12 h, under which the seed germination rate was 49.4%. Using this mutagenesis condition, an EMS mutant library containing 667 M₁ generation plants was established. From this library, 14 mutants exhibiting various phenotypes were identified, including yellowing leaf, wrinkled leaf, albino lethal plant, albino leaf, disease spot leaf, dwarf plant, short fruit and albino peel. The mutation rate was 2.10%. Phenotypic characterization and ultrastructural observation revealed that one yellowing leaf mutant, X-67, possessed lower photosynthetic pigment content and incomplete chloroplast development, potentially explaining its juvenile leaf yellowing phenotype. This study identified the optimal EMS mutagenesis conditions for bottle gourd and obtained a collection of EMS-induced mutants, providing valuable material resources for functional genomics research in bottle gourd.

Key words: bottle gourd, ethyl methane sulfonate, mutant, yellowing leaf

CLC Number:

S642.9

ZHENG Cheng, WANG Ying, WANG Jian, GUO Xiao, WANG Baogen, WU Xinyi, ZHU Biao, LI Guojing, WU Xiaohua. Screening and phenotypic analysis of EMS-induced mutants in Lagenaria siceraria[J]. Acta Agriculturae Zhejiangensis, 2025, 37(9): 1914-1923.

Figures/Tables 9

Table 1 Germination rate of Lagenaria siceraria under different treatments %

EMS体积分数 Volume fraction of EMS/%	发芽率 Germination rate
EMS体积分数 Volume fraction of EMS/%	6 h	12 h	18 h
0	98.6±0.7 a	98.0±0.8 a	97.8±0.5 a
0.6	81.8±1.5 b	75.4±2.8 c	63.6±2.8 d
0.8	72.2±1.9 c	62.8±2.6 d	57.4±0.8 d
1.2	61.0±2.9 d	49.4±1.1 e	36.0±2.2 f
1.6	53.8±2.2 e	31.0±1.7 g	16.4±2.3 i
2.0	37.8±1.4 f	24.6±2.2 h	2.2±0.4 j

Fig.1 Leaf mutants phenotypes of EMS-mutagenized M1 populations of Lagenaria siceraria A, Wild-type; B-D, Yellowing leaf; E, Wrinkled leaves; F-H, Albino lethal plant; I, Albino leaf; J, Disease spot leaf.

Fig.2 Plant type mutants phenotypes of EMS-mutagenized M1 populations of Lagenaria siceraria A, Wild-type; B-D, Dwarf plants.

Fig.3 Fruit mutants phenotypes of EMS-mutagenized M1 populations of Lagenaria siceraria A, Wild-type; B, Short fruit; C, Albino peel.

Fig.4 Partial KASP genotyping results of EMS-mutagenized M1 populations of Lagenaria siceraria Red dots indicate genotype HEX. Blue dots indicate genotype FAM. Black dots indicate non-template control.

Table 2 Leaf phenotypes of yellowing leaf mutant and wild type of Lagenaria siceraria cm

材料	叶长	叶宽	叶柄长
Material	Leaf length	Leaf width	Petiole length
WT	24.04±1.20 a	32.46±3.20 a	16.26±3.40 a
X-67	23.96±1.10 a	32.20±3.10 a	16.22±3.40 a

Fig.5 Plant phenotype of yellowing leaf mutant X-67 and wild type of Lagenaria siceraria A, Wild-type; B, Yellowing leaf mutant X-67.

Fig.6 Chlorophyll content in leaves of yellowing leaf mutant X-67 and wild type of Lagenaria siceraria WT, Wild type; X-67, Yellowing leaf mutant. ** indicates significant difference compared with WT at p<0.01 level; * indicates significant difference compared with WT at p<0.05 level.

Fig.7 Ultrastructure of chloroplasts in leaves of yellowing leaf mutant X-67 and wild type of Lagenaria siceraria A, Chloroplast of wild type; B, Chloroplast of leaf yellowing mutant.

References 34

[8]	宋燕妮, 杜黎黎, 王学征, 等. 甜瓜突变体库的构建及M₂群体表型变异的研究[J]. 植物遗传资源学报, 2015, 16(6): 1338-1344.
	SONG Y N, DU L L, WANG X Z, et al. Construction of mutant library and research on phenotypic variation of M₂ population in melon[J]. Journal of Plant Genetic Resources, 2015, 16(6): 1338-1344. (in Chinese with English abstract)
[9]	薛红霞, 蒋举卫, 李晓丽, 等. 黄瓜突变体库的构建及表型变异的初步研究[J]. 核农学报, 2019, 33(3): 432-439.
	XUE H X, JIANG J W, LI X L, et al. Construction and preliminary screening of EMS induced cucumber mutants[J]. Journal of Nuclear Agricultural Sciences, 2019, 33(3): 432-439. (in Chinese with English abstract)
[10]	弭宝彬, 谢玲玲, 王鑫, 等. 冬瓜EMS诱变条件优化及突变体筛选[J]. 中国农学通报, 2018, 34(21): 35-41.
	MI B B, XIE L L, WANG X, et al. EMS mutagenesis optimization and mutants screening of wax gourd[J]. Chinese Agricultural Science Bulletin, 2018, 34(21): 35-41. (in Chinese with English abstract)
[1]	ERICKSON D L, SMITH B D, CLARKE A C, et al. An Asian origin for a 10, 000-year-old domesticated plant in the Americas[J]. Proceedings of the National Academy of Sciences of the United States of America, 2005, 102(51): 18315-18320.
[2]	BEEVY S S, KURIACHAN P. Chromosome numbers of south Indian Cucurbitaceae and a note on the cytological evolution in the family[J]. Journal of Genetics and Genomics, 1996, 31: 65-71.
[11]	尹丽娟, 王春霞, 李陈浩, 等. 西瓜EMS诱导的突变体筛选及表型分析[J]. 园艺学报, 2023, 50(11): 2401-2416.
	YIN L J, WANG C X, LI C H, et al. Screening and phenotypic analysis of EMS induced mutants in watermelon[J]. Acta Horticulturae Sinica, 2023, 50(11): 2401-2416. (in Chinese with English abstract)
[3]	崔清志, 刘晓虹, 陈惠明. EMS诱变技术研究进展[J]. 湖南农业科学, 2013(5): 7-9.
	CUI Q Z, LIU X H, CHEN H M. Research progress of EMS mutation technology[J]. Hunan Agricultural Sciences, 2013(5): 7-9. (in Chinese)
[12]	康保珊, 李俊香, 刘丽锋, 等. 甲基磺酸乙酯诱变瓠瓜种子的条件筛选[J]. 中国瓜菜, 2017, 30(2): 7-10.
	KANG B S, LI J X, LIU L F, et al. Screening optimal condition of mutagenesis with EMS for bottle gourd(Lagenaria siceraria) seed[J]. China Cucurbits and Vegetables, 2017, 30(2): 7-10. (in Chinese with English abstract)
[4]	刘翔. EMS诱变技术在植物育种中的研究进展[J]. 激光生物学报, 2014, 23(3): 197.
	LIU X. Progresses on EMS mutagenesis in plant breeding[J]. Acta Laser Biology Sinica, 2014, 23(3): 197. (in Chinese with English abstract)
[5]	叶俊, 吴建国, 杜婧, 等. 水稻“9311”突变体筛选和突变体库构建[J]. 作物学报, 2006, 32(10): 1525-1529.
	YE J, WU J G, DU J, et al. The screening of mutants and construction of mutant population for cultivar “9311” in rice (Oryza sativa L.)[J]. Acta Agronomica Sinica, 2006, 32(10): 1525-1529. (in Chinese with English abstract)
[6]	曹亚萍, 武银玉, 范绍强, 等. EMS诱变技术在小麦上的应用[J]. 激光生物学报, 2019, 28(5): 394.
	CAO Y P, WU Y Y, FAN S Q, et al. Application of induction technology with EMS on wheat[J]. Acta Laser Biology Sinica, 2019, 28(5): 394. (in Chinese with English abstract)
[7]	刘根忠, 王云飞, 孟祥飞, 等. EMS诱变的‘里格尔87-5 '番茄M₂群体突变体表型及其抗坏血酸研究[J]. 园艺学报, 2017, 44(1): 120-130.
	LIU G Z, WANG Y F, MENG X F, et al. Phenotype mutation and ascorbate characterization in M₂ population derived from EMS treatment in ‘Ligeer 87-5' tomato[J]. Acta Horticulturae Sinica, 2017, 44(1): 120-130. (in Chinese with English abstract)
[13]	陈婷婷, 符卫蒙, 余景, 等. 彩色稻叶片光合特征及其与抗氧化酶活性、花青素含量的关系[J]. 中国农业科学, 2022, 55(3): 467-478.
	CHEN T T, FU W M, YU J, et al. The photosynthesis characteristics of colored rice leaves and its relation with antioxidant capacity and anthocyanin content[J]. Scientia Agricultura Sinica, 2022, 55(3): 467-478. (in Chinese with English abstract)
[14]	徐明远, 何鹏, 赖伟, 等. 植物叶色变异分子机制研究进展[J]. 分子植物育种, 2021, 19(10): 3448-3455.
	XU M Y, HE P, LAI W, et al. Advances in molecular mechanism of plant leaf color variation[J]. Molecular Plant Breeding, 2021, 19(10): 3448-3455. (in Chinese with English abstract)
[15]	马道承, 王凌晖, 梁机. 形态标记在植物中的应用研究进展[J]. 江苏农业科学, 2022, 50(8): 55-62.
	MA D C, WANG L H, LIANG J. Research progress on application of morphological markers in plants[J]. Jiangsu Agricultural Sciences, 2022, 50(8): 55-62. (in Chinese with English abstract)
[16]	沈镝, 李锡香. 瓠瓜种质资源描述规范和数据标准[M]. 北京: 中国农业出版社, 2008: 10-22.
[17]	WANG Y, WU X H, LI Y W, et al. Identification and validation of a core single-nucleotide polymorphism marker set for genetic diversity assessment, fingerprinting identification, and core collection development in bottle gourd[J]. Frontiers in Plant Science, 2021, 12: 747940.
[18]	汪颖. 水稻白条纹基因WSL4的图位克隆与功能分析[D]. 北京: 中国农业科学院, 2017.
	WANG Y. Map-based cloning and functional analysis of a white stripe leaf gene WSL4 in rice (Oryza sativa L.)[D]. Beijing: Chinese Academy of Agricultural Sciences, 2017. (in Chinese with English abstract)
[19]	崔霞, 梁燕, 李翠, 等. 化学诱变及其在蔬菜育种中的应用[J]. 西北农林科技大学学报(自然科学版), 2013, 41(3): 205-212.
	CUI X, LIANG Y, LI C, et al. Chemical mutagenesis and its application in vegetable breeding[J]. Journal of Northwest A&F University(Natural Science Edition), 2013, 41(3): 205-212. (in Chinese with English abstract)
[20]	尚娜. EMS诱变番茄突变体库的构建及表型分析[D]. 哈尔滨: 东北农业大学, 2017.
	SHANG N. Construction and phenotypic analysis of tomato mutant library induced by EMS[D]. Harbin: Northeast Agricultural University, 2017. (in Chinese with English abstract)
[21]	闵子扬, 韩小霞, 李勇奇, 等. 中国南瓜突变体库构建及表型变异的初步分析[J]. 核农学报, 2021, 35(4): 761-768.
	MIN Z Y, HAN X X, LI Y Q, et al. Construction and preliminary screening of EMS induced mutant library of pumpkin (Cucurbita moschata D.)[J]. Journal of Nuclear Agricultural Sciences, 2021, 35(4): 761-768. (in Chinese with English abstract)
[22]	谢玲玲, 周火强, 弭宝彬, 等. EMS诱变技术研究概况及应用进展[J]. 湖南农业科学, 2020(6): 92-95.
	XIE L L, ZHOU H Q, MI B B, et al. Research progress and application of EMS mutagenesis technology[J]. Hunan Agricultural Sciences, 2020(6): 92-95. (in Chinese with English abstract)
[23]	赵越. EMS诱导小麦突变及优良突变鉴选[D]. 杨凌: 西北农林科技大学, 2020.
	ZHAO Y. The mutantion of wheat induced by EMS and identification and screening of excellent mutants[D]. Yangling: Northwest A&F University, 2020. (in Chinese with English abstract)
[24]	陈开, 陈宇峰, 封昱均, 等. 水稻EMS诱变根系突变体的筛选及其农艺性状分析[J]. 西南农业学报, 2022, 35(6): 1233-1242.
	CHEN K, CHEN Y F, FENG Y J, et al. Screening of root mutants induced by EMS and analysis of agronomic traits in rice[J]. Southwest China Journal of Agricultural Sciences, 2022, 35(6): 1233-1242. (in Chinese with English abstract)
[25]	叶卫军, 杨勇, 张丽亚, 等. 绿豆EMS诱变突变体库的构建及表型分析[J]. 中国农学通报, 2020, 36(17): 36-41.
	YE W J, YANG Y, ZHANG L Y, et al. Construction of EMS mutagenesis mung bean mutant library and phenotypic analysis[J]. Chinese Agricultural Science Bulletin, 2020, 36(17): 36-41. (in Chinese with English abstract)
[26]	崔慧琳, 李志远, 方智远, 等. 结球甘蓝自交系YL-1的高效遗传转化体系的建立及应用[J]. 园艺学报, 2019, 46(2): 345-355.
	CUI H L, LI Z Y, FANG Z Y, et al. Establishment and application of YL-1 high-efficiency genetic transformation system in Cabbage(Brassica oleracea L. var. capitata)[J]. Acta Horticulturae Sinica, 2019, 46(2): 345-355. (in Chinese with English abstract)
[27]	张天雨, 周春雷, 刘喜, 等. 一个水稻温敏黄化突变体的表型分析和基因定位[J]. 作物学报, 2017, 43(10): 1426-1433.
	ZHANG T Y, ZHOU C L, LIU X, et al. Phenotypes and gene mapping of a thermo-sensitive yellow leaf mutant of rice[J]. Acta Agronomica Sinica, 2017, 43(10): 1426-1433. (in Chinese with English abstract)
[28]	杨小苗, 吴新亮, 刘玉凤, 等. 一个番茄EMS叶色黄化突变体的叶绿素含量及光合作用[J]. 应用生态学报, 2018, 29(6): 1983-1989.
	YANG X M, WU X L, LIU Y F, et al. Analysis of chlorophyll and photosynthesis of a tomato chlorophyll-deficient mutant induced by EMS[J]. Chinese Journal of Applied Ecology, 2018, 29(6): 1983-1989. (in Chinese with English abstract)
[29]	李洋洋, 薛冰, 周倩, 等. 黄瓜叶色黄化突变基因yl-2的鉴定[J]. 中国蔬菜, 2020(7): 30-37.
	LI Y Y, XUE B, ZHOU Q, et al. Identification of the mutant gene yl-2 conferring the yellow leaf trait in cucumber[J]. China Vegetables, 2020(7): 30-37. (in Chinese with English abstract)
[30]	戴楠, 袁光孝, 刘小敏, 等. 拟南芥黄化突变体k60的基因作图定位[J]. 北京林业大学学报, 2017, 39(7): 40-45.
	DAI N, YUAN G X, LIU X M, et al. Genetic mapping of a chlorosis mutant k60 in Arabidopsis thaliana[J]. Journal of Beijing Forestry University, 2017, 39(7): 40-45. (in Chinese with English abstract)
[31]	孙立亭, 林添资, 王云龙, 等. 水稻白条纹突变体st13的表型分析及基因定位[J]. 中国水稻科学, 2017, 31(4): 355-363.
	SUN L T, LIN T Z, WANG Y L, et al. Phenotypic analysis and gene mapping of a white stripe mutant st13 in rice[J]. Chinese Journal of Rice Science, 2017, 31(4): 355-363. (in Chinese with English abstract)
[32]	王飞, 段世名, 李彤, 等. 玉米叶色突变体遗传分析及基因定位[J]. 植物遗传资源学报, 2018, 19(6): 1205-1209.
	WANG F, DUAN S M, LI T, et al. Fine mapping and candidate gene analysis of leaf color mutant in maize[J]. Journal of Plant Genetic Resources, 2018, 19(6): 1205-1209. (in Chinese with English abstract)
[33]	蒋宏宝. 小麦叶绿素缺失突变体B23的鉴定及基因定位[D]. 杨凌: 西北农林科技大学, 2018.
	JIANG H B. Identification and gene mapping of wheat chlorophyll deletion mutant B23[D]. Yangling: Northwest A & F University, 2018. (in Chinese with English abstract)
[34]	郭丽杰. 番茄杂色叶基因vg的遗传定位分析[D]. 武汉: 华中农业大学, 2017.
	GUO L J. Genetic mapping of a variegated leaf (vg) nutant in tomato[D]. Wuhan: Huazhong Agricultural University, 2017. (in Chinese with English abstract)

Screening and phenotypic analysis of EMS-induced mutants in Lagenaria siceraria

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