菜心和芥蓝种间杂交创制异源四倍体蔬菜种质

doi:10.3969/j.issn.1004-1524.2022.08.08

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (8): 1638-1647.DOI: 10.3969/j.issn.1004-1524.2022.08.08

菜心和芥蓝种间杂交创制异源四倍体蔬菜种质

吕凤仙¹^,²(), 和江明¹^,³, 李崇娟¹^,², 杨鼎¹^,², 胡靖锋¹^,³^,^*(), 杨红丽¹^,³, 兰梅¹^,³, 徐学忠¹^,³, 张丽琴¹^,³

1.云南省农业科学院园艺作物研究所,云南昆明 650205
2.云南大学农学院,云南昆明 650504
3.国家蔬菜改良中心云南分中心,云南昆明 650205

收稿日期:2021-10-29 出版日期:2022-08-25 发布日期:2022-08-26
通讯作者: 胡靖锋
作者简介:*胡靖锋,E-mail: 276635044@qq.com
吕凤仙(1997—),女,云南宣威人,硕士研究生,研究方向为十字花科蔬菜育种。E-mail: 2547682199@qq.com
基金资助:
云南省“万人计划”产业领军人才(YNWR-CYJS-2018-040);国家大宗蔬菜产业技术体系昆明综合试验站项目(CARS-23-G-33);云南省科技厅重大科技专项(2019ZG001)

Creation of allotetraploid vegetable germplasm by interspecific hybridization of Brassica rapa L. ssp chinensis var. utilis Tsen et Lee and Brassica oleracea var. Alboglabra

LYU Fengxian¹^,²(), HE Jiangming¹^,³, LI Chongjuan¹^,², YANG Ding¹^,², HU Jingfeng¹^,³^,^*(), YANG Hongli¹^,³, LAN Mei¹^,³, XU Xuezhong¹^,³, ZHANG Liqin¹^,³

1. Horticultural Crops Research Institute, Yunnan Academy of Agricultural Sciences, Kunming 650205, China
2. School of Agriculture, Yunnan University, Kunming 650504, China
3. Yunnan Branch of National Vegetable Improvement Center, Kunming 650205, China

Received:2021-10-29 Online:2022-08-25 Published:2022-08-26
Contact: HU Jingfeng

摘要/Abstract

摘要：

通过菜心和芥蓝的种间杂交可丰富遗传背景,创新种质资源,但由于受精合子发育障碍,需对种间杂种进行胚挽救,才能获得后代。以菜心作为母本,芥蓝作为父本,进行种间杂交,于杂交后取种间杂种胚进行人工培养,设置5种不同胚挽救培养基,6个不同取材时间,4种不同生根培养基,3种不同染色体加倍方式,筛选适合的种间杂种胚挽救方式、增殖扩繁方式、染色体加倍方式,提高种间杂种获得率。结果显示,ERM₄(MS+0.5 mg·L^-1 6-BA+0.5 mg·L^-1 NAA+1.0 mg·L^-1 KT+1 g·L^-1 AC)出胚率为10.47%,为最佳胚挽救培养基;授粉后12 d为最佳取材时间,出胚率为26.32%;RM₄(1/2MS+0.5 mg·L^-1 IBA)生根率最高、生根时间最短,为最佳生根培养基;秋水仙碱涂抹生长点5次,染色体加倍率可达62.26%,为最佳染色体加倍方式。通过形态学鉴定、染色体分析、倍性鉴定等方法,对F₁代杂种植株进行鉴定,结果显示,所得杂种为真杂种,可育株为异源四倍体。异源四倍体蔬菜种质的成功创制为后续育种研究奠定了基础。

关键词: 菜心, 芥蓝, 种间杂种, 胚挽救, 异源四倍体, 薹用蔬菜

Abstract:

Hybridization between Brassica rapa L. ssp chinensis var. utilis Tsen. et Lee and Brassica oleracea var. Alboglabra can enrich genetic background and innovate germplasm resources. However, due to the dysplasia of fertilization zygote, embryo rescue of the interspecific hybrid is necessary to obtain the offspring. The interspecific hybridization was performed using Brassica rapa L. ssp chinensis var. utilis Tsen. et Lee as female parents and Brassica oleracea var. Alboglabra as male parents. Hybrid embryos were harvested for artificial culture after crosses. In this experiment, five kinds of mediums, six different sampling time, four kinds of rooting mediums and three kinds of chromosome-doubling manner were set. Suitable interspecific hybrid embryo rescue methods, proliferation and expansion methods, and chromosome doubling methods were selected to improve the hybrid acquisition rate. The results showed that embryogenesis rate of ERM₄ (MS+0.5 mg·L^-1 6-BA+0.5 mg·L^-1 NAA+1.0 mg·L^-1 KT+1 g·L^-1 AC) was 10.47%, used as the optimal embryo rescue medium. 12 d after pollination was the best sampling time since the embryogenesis rate was 26.32%. RM₄ (1/2MS+0.5 mg·L^-1 IBA) had the highest rooting rate and the shortest rooting time, which was the best rooting medium. The method that applied at growth points 5 times with colchicine, was the best chromosome-doubling manner, in which chromosome doubling rate reached to 62.26%. And then the F₁ generation hybrids plants were identified through morphological identification, chromosome analysis and ploidy identification. The results showed that the hybrids obtained in the experiment were true hybrids, and the fertile plants were allotetraploidy. The successful creation of allotetraploid vegetable germplasm laid a foundation for subsequent breeding research.

Key words: Brassica rapa L. ssp chinensis var. utilis Tsen et Lee, Brassica oleracea var. Alboglabra, interspecific hybrid, embryo rescue, allotetraploid, vegetables made of fresh and tender rachis

中图分类号:

S634.5

吕凤仙, 和江明, 李崇娟, 杨鼎, 胡靖锋, 杨红丽, 兰梅, 徐学忠, 张丽琴. 菜心和芥蓝种间杂交创制异源四倍体蔬菜种质[J]. 浙江农业学报, 2022, 34(8): 1638-1647.

LYU Fengxian, HE Jiangming, LI Chongjuan, YANG Ding, HU Jingfeng, YANG Hongli, LAN Mei, XU Xuezhong, ZHANG Liqin. Creation of allotetraploid vegetable germplasm by interspecific hybridization of Brassica rapa L. ssp chinensis var. utilis Tsen et Lee and Brassica oleracea var. Alboglabra[J]. Acta Agriculturae Zhejiangensis, 2022, 34(8): 1638-1647.

图/表 14

表1 菜心和芥蓝种间杂种胚挽救培养基成分

Table 1 Components of embryo rescue culture medium for interspecific hybrid of Brassica rapa L. ssp chinensis var. utilis Tsen et Lee×Brassica oleracea var. Alboglabra

编号No.	基本培养基Basic culture medium	6-BA/(mg·L^-1)	NAA/(mg·L^-1)	KT/( mg·L^-1)	AC/(g·L^-1)
ERM₁	MS	0	0	0	1
ERM₂	MS	0.3	0	0	1
ERM₃	MS	0.5	0	0	1
ERM₄	MS	0.5	0.5	1.0	1
ERM₅	MS	5	0.5	1.0	1

表2 菜心和芥蓝种间杂种生根培养基成分

Table 2 Components of rooting medium for interspecific hybrid of Brassica rapa L.ssp chinensis var. utilis Tsen. et Lee×Brassica oleracea var. Alboglabramg·L-1

编号 No.	基本培养基 Basic culture medium	NAA	IBA	IAA
RM₁	MS	0.5	0	0
RM₂	MS	0	0.5	0
RM₃	MS	0	0	0.5
RM₄	1/2MS	0	0.5	0

表3 菜心×芥蓝杂种胚珠在不同培养基上出胚率

Table 3 Embryogenesis rate of hybrid ovules of Brassica rapa L. ssp chinensis var. utilis Tsen. et Lee×Brassica oleracea var. Alboglabra on different culture mediums

编号 No.	胚珠培养数 Number of ovules	成胚数 Number of embryogenesis	出胚率 Embryogenesis rate/%
ERM₁	305	0	0 d
ERM₂	375	6	1.60±0.78 c
ERM₃	338	7	2.07±0.54 c
ERM₄	382	40	10.47±0.27 a
ERM₅	423	32	7.57±0.37 b

表4 菜心×芥蓝不同取材时间出胚率

Table 4 Embryogenesis rate of hybrid ovules of Brassicarapa L. ssp chinensis var. utilis Tsen. et Lee×Brassica oleracea var. Alboglabra at different sampling time

授粉后时间 Time after pollination/d	胚珠培养数 Number of ovules	出胚数 Number of embryogenesis	出胚率 Embryogenesis rate/%
10	90	4	4.44±1.93 b
11	88	9	10.23±1.97 b
12	95	25	26.32±2.77 a
13	382	40	10.47±0.92 b
14	150	10	6.67±3.34 b
15	60	2	3.33±2.89 c

表5 菜心×芥蓝种间杂种不同培养基生根情况

Table 5 Rooting conditions of Brassica rapa L. ssp chinensis var. utilis Tsen. et Lee×Brassica oleracea var. Alboglabra interspecific hybrids on different culture mediums

编号 No.	培养幼苗数 Number of cultured seedlings	生根幼苗数 Number of rooting seedlings	生根率 Rooting rate/%	生根时间 Rooting time/d
RM₁	28	4	14.29±3.07 c	11.33±0.58 b
RM₂	25	9	36.00±2.57 a	8.67±0.58 c
RM₃	27	7	25.93±2.75 b	15.00±1.00 a
RM₄	43	16	37.21±1.47 a	8.33±0.58 c

图1 菜心×芥蓝种间杂种幼苗生根过程生长情况 A,RM1上发生盐害现象的幼苗;B,RM2上发生盐害现象的幼苗;C,RM3上发生盐害现象的幼苗;D,RM4上正常生长幼苗。标尺=1 cm。

Fig.1 Growth during rooting of Brassica rapa L. ssp chinensis var. utilis Tsen. et Lee×Brassica oleracea var. Alboglabra interspecific hybrid seedlings A, Seedlings with salt damage on RM1; B, Seedlings with salt damage on RM2; C, Seedlings with salt damage on RM3; D, Normal growth of seedlings on RM4; Bar scale=1 cm.

表6 菜心×芥蓝种间杂种染色体加倍方法处理效果

Table 6 Effect of chromosome doubling method on Brassica rapa L. ssp chinensis var. utilis Tsen. et Lee×Brassica oleracea var. Alboglabra interspecific hybrids

处理 Treatment	处理株数 Number of plant	生根率 Rooting rate/%	成活率 Survival rate/%	可育株数 Number of fertile plant	加倍率 Rate of chromosome doubling/%	嵌合体株数 Number of chimera
A	52	13.46	5.77	1	33.33	1
B	104	22.12	5.77	0	0	0
C	53	100	100	33	62.26	33

图2 菜心×芥蓝种间杂种染色体加倍过程死亡现象 1和2,A处理;3和4,B处理。标尺=1 cm。

Fig.2 Death of seeldings during the chromosome doubling process of Brassica rapa L. ssp chinensis var. utilis Tsen. et Lee×Brassica oleracea var. Alboglabra interspecific hybrids 1 and 2, Treatment A; 3 and 4, Treatment B. Bar scale=1 cm.

图3 菜心×芥蓝种间杂种经染色体加倍处理后幼苗炼苗后死亡现象 A,A处理;B,B处理。标尺=1 cm。

Fig.3 Death of seeldings with chromosome doubling treatment after the seedling hardening of Brassica rapa L. ssp chinensis var. utilis Tsen. et Lee× Brassica oleracea var. Alboglabra interspecific hybrids A, Treatment A; B, Treatment B. Bar scale=1 cm.

图4 菜心×芥蓝种间杂种经染色体加倍处理后花粉染色观察结果 A,可育株花粉; B,不育株花粉;标尺=25 μm。

Fig.4 Observation of pollen staining of interspecific hybrids of Brassica rapa L. ssp chinensis var. utilis Tsen. et Lee×Brassica oleracea var. Alboglabra after chromosome doubling treatment A, Pollen of fertile plant; B, Pollen of sterile plant. Bar scale=25 μm.

图5 菜心×芥蓝种间杂种植株形态 A和D,菜心;B和E,种间杂种;C和F,芥蓝;A、B、C的标尺=5 cm;D、E、F标尺=0.5 cm。

Fig.5 Plant morphology of Brassica rapa L. ssp chinensis var. utilis Tsen. et Lee×Brassica oleracea var. Alboglabra interspecific hybrids A and D, Brassica rapa L. ssp chinensis var. utilis Tsen. et Lee; B and E, Einterspecific hybrid; C and F, Brassica oleracea var. Alboglabra; Bar scales of figure A, B and C were 5 cm; Bar scales of figure D, E and F were 0.5 cm.

图6 菜心×芥蓝种间杂种花朵和角果形态 A、D、E、G、I为可育株;B、C、F、H、J为不育株;标尺=1 cm。

Fig.6 Flower and pod forms of Brassica rapa L. ssp chinensis var. utilis Tsen. et Lee×Brassica oleracea var. Alboglabra interspecific hybrids A, D, E, G and I were fertile plants; B, C, F, H and J were sterile plant. Bar scale=1 cm.

图7 菜心×芥蓝种间杂种染色体数量 A,不育株柱头染色体;B,可育株柱头染色体;C,可育株花药染色体。

Fig.7 Chromosome number of Brassica rapa L. ssp chinensis var. utilis Tsen. et Lee×Brassica oleracea var. Alboglabra interspecific hybrids A, Stigma chromosome of sterile plant; B, Stigma chromosome of fertile plant; C, Anther chromosomes of fertile plants.

图8 菜心×芥蓝种间杂种倍性鉴定直方图 A,菜心;B,芥蓝C,甘蓝型油菜;D,不育株;E和F,可育株。

Fig.8 Histogram of ploidy identification of Brassica rapa L. ssp chinensis var. utilis Tsen. et Lee×Brassica oleracea var. Alboglabra interspecific hybrids A, Brassica rapa L. ssp chinensis var. utilis Tsen. et Lee; B, Brassica oleracea var. Alboglabra; C, Brassica napus L.; D, Sterile plant; E and F, Fertile plant.

参考文献 21

[1]	张华, 刘自珠. 菜薹(菜心)的市场需求与育种现状[J]. 中国蔬菜, 2010(3): 10-12.
	ZHANG H, LIU Z Z. Market demand and breeding status of Brassica rapa L. ssp chinensis var. utilis Tsen. et Lee[J]. China Vegetables, 2010(3): 10-12. (in Chinese)
[2]	秦耀国, 杨翠芹, 曹必好, 等. 芥蓝遗传育种与生物技术研究进展[J]. 中国农学通报, 2009, 25(18): 296-299.
	QIN Y G, YANG C Q, CAO B H, et al. Advances in research on genetic breeding and biotechnology of Chinese kale[J]. Chinese Agricultural Science Bulletin, 2009, 25(18): 296-299. (in Chinese with English abstract)
[3]	吴文林, 陈国菊, 郑华杰, 等. 芥蓝主要经济性状的配合力及遗传力分析[J]. 中国蔬菜, 2012(10): 31-35.
	WU W L, CHEN G J, ZHENG H J, et al. Analysis of combining ability and heritability of economic traits in Chinese kale[J]. China Vegetables, 2012(10): 31-35. (in Chinese with English abstract)
[4]	李旭峰. 芸薹属与萝卜属间远缘杂交研究进展[J]. 中国油料, 1995, 17(1): 69-73.
	LI X F. Advances in distant hybridization between Brassica and Raphanus[J]. Chinese Journal of Oil Crop Sciences, 1995, 17(1): 69-73. (in Chinese)
[5]	梁红, 覃广泉, 何丽贞. 菜心与甘蓝种间F₁代的杂种优势观察(初报)[J]. 中国蔬菜, 1994(1): 1-3.
	LIANG H, QIN G Q, HE L Z. Observation on heterosis of F₁ generation between Brassica rapa L. ssp chinensis var. utilis Tsen. et Lee and Brassica oleracea L.[J]. China Vegetables, 1994(1): 1-3. (in Chinese)
[6]	乔海云. 芸薹种与甘蓝种杂交获得新种质的研究[D]. 北京: 中国农业科学院, 2012.
	QIAO H Y. Stuty on the interspecific hybirds between Brassica rapa and Brassica oleracea[D]. Beijing: Chinese Academy of Agricultural Sciences, 2012. (in Chinese with English abstract)
[7]	满红. 菜心-芥蓝种间杂种的获得及鉴定研究[D]. 保定: 河北农业大学, 2007.
	MAN H. Obtaining and identification of interspecific hybrids from false Pakchoi×Kailan[D]. Baoding: Hebei Agricultural University, 2007. (in Chinese with English abstract)
[8]	魏云晓, 李菲, 张淑江, 等. 菜薹(菜心)-芥蓝杂交亲和性分析及后代性状表现[J]. 中国蔬菜, 2017(11): 21-27.
	WEI Y X, LI F, ZHANG S J, et al. Analysis on interspecific hybridization compatibility and progeny characteristics of flowering Chinese cabbage and Chinese kale[J]. China Vegetables, 2017(11): 21-27. (in Chinese with English abstract)
[9]	石淑稳, 吴江生, 周永明, 等. 甘蓝型油菜小孢子单倍体二倍化技术的研究[J]. 中国油料作物学报, 2002, 24(1): 1-5.
	SHI S W, WU J S, ZHOU Y M, et al. Diploidization techniques of haploids from in vitro culture microspores of rapeseed (Brassica napus L.)[J]. Chinese Journal of Oil Crop Scieves, 2002, 24(1): 1-5. (in Chinese with English abstract)
[10]	杨汉, 康雷, 李鹏飞, 等. 甘蓝型油菜-菘蓝二体附加系的创制和细胞学分析[J]. 中国油料作物学报, 2016, 38(3): 281-286.
	YANG H, KANG L, LI P F, et al. Development and cytology of disomic alien addition between Brassica napus and Isatis indigotiga[J]. Chinese Journal of Oil Crop Sciences, 2016, 38(3): 281-286. (in Chinese with English abstract)
[11]	YANG L Y, WANG Z X, HUA J. Measuring cell ploidy level in Arabidopsis thaliana by flow cytometry[J]. Methods in Molecular Biology (Clifton, N J), 2019, 1991: 101-106.
[12]	邱德勃, HARBERD D J. 萝卜和甘蓝属间杂交胚和胚乳发育过程的亚显微结构观察[J]. 植物学报, 1986, 28(5):483-491.
	QIU D B, HARBERD D J. The fine structural observation on the development of embryo and endosperm in the intergeneric crosses of radish ♀(Raphanus sativus L.)×cabbage (Brassica oleracea L.)[J]. Journal of Integrative Plant Biology, 1986, 28(5): 483-491.
[13]	戴林建. 芸薹属植物与芸芥属间杂交的研究[D]. 长沙: 湖南农业大学, 2003.
	DAI L J. Studies on distant crossing between rocketsalad (E. saiva Mill) and plants of Brassica[D]. Changsha: Hunan Agricultural University, 2003. (in Chinese with English abstract)
[14]	吴俊, 李旭锋, 李琳, 等. 油菜诸葛菜属植物杂交亲和性研究[J]. 西南农业大学学报, 1999, 21(5): 412-416.
	WU J, LI X F, LI L, et al. Studies on pollen-pistil interaction between Brassica napus and Orychophragmus species[J]. Journal of Southwest Agricultural University, 1999, 21(5): 412-416. (in Chinese with English abstract)
[15]	刘爱华, 王建波, 朱英国. 芸薹属多倍体植物基因组进化的RAPD分析[J]. 植物分类学报, 2003, 41(6): 520-530.
	LIU A H, WANG J B, ZHU Y G. RAPD analysis on the genome evolution of polyploids in Brassica[J]. Acta Phytotaxonomica Sinica, 2003, 41(6): 520-530. (in Chinese with English abstract)
[16]	李明山, 索玉英, 周长久. 大白菜与萝卜属间杂种幼胚离体培养的研究[J]. 园艺学报, 1992, 19(4): 353-357.
	LI M S, SUO Y Y, ZHOU C J. Study on culture in vitro of hybrid embryos between Chinese cabbage and radish[J]. Acta Horticulturae Sinica, 1992, 19(4): 353-357. (in Chinese with English abstract)
[17]	HARBERD D J. A simple effective embryo culture technique for Brassica[J]. Euphytica, 1969, 18(3): 425-429. DOI URL
[18]	谭亚飞. 白菜型油菜黄籽沙逊与芥蓝种间杂种的胚挽救及后代鉴定[D]. 郑州: 郑州大学, 2016.
	TAN Y F. The embryo rescue of Brassica rapa yellow sarson and kale and progeny identification[D]. Zhengzhou: Zhengzhou University, 2016. (in Chinese with English abstract)
[19]	吴大忠. 不同杉木优良无性系组培诱导特性的研究[J]. 福建林业科技, 1999, 26(1): 26-29.
	WU D Z. Studies on the tissue culture and induction characteristics of different excellent clones of Cunninghamia lanceolata[J]. Journal of Fujian Forestry Science and Technology, 1999, 26(1): 26-29. (in Chinese with English abstract)
[20]	江莹芬, 田恩堂, 陈伦林, 等. 埃塞俄比亚芥和白菜型油菜远缘杂种F₁的鉴定[J]. 中国油料作物学报, 2007, 29(2): 209-212.
	JIANG Y F, TIAN E T, CHEN L L, et al. Identification of interspecific hybrids between Brassica carinata and B. rapa[J]. Chinese Journal of Oil Crop Sciences, 2007, 29(2): 209-212. (in Chinese with English abstract)
[21]	卢长明, 肖玲, 张斌, 等. 人工合成甘蓝型油菜的繁育特性与试管繁殖[J]. 中国油料作物学报, 2003, 25(4): 5-10.
	LU C M, XIAO L, ZHANG B, et al. Generative characteristics and in vitro multiplication of resynthesized Brassica napus[J]. Chinese Journal of Oil Crop Scieves, 2003, 25(4): 5-10. (in Chinese with English abstract)

菜心和芥蓝种间杂交创制异源四倍体蔬菜种质

Creation of allotetraploid vegetable germplasm by interspecific hybridization of Brassica rapa L. ssp chinensis var. utilis Tsen et Lee and Brassica oleracea var. Alboglabra

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[4]	赵蓉蓉;蒋明;贺蔡明;朱雅琴;周敏. 芥蓝Brassica albograbra*花青素合成酶基因BaANS的克隆与序列分析[J]. , 2010, 22(2): 0-166.
[5]	蒋明;苗立祥;胡齐赞;贺蔡明;陈珍. 芥蓝查尔酮合成酶基因BACHS的克隆与序列分析[J]. , 2009, 21(4): 0-326.