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

doi:10.3969/j.issn.1004-1524.2022.08.08

Acta Agriculturae Zhejiangensis ›› 2022, Vol. 34 ›› Issue (8): 1638-1647.DOI: 10.3969/j.issn.1004-1524.2022.08.08

• Horticultural Science • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S634.5

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.

Figures/Tables 14

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

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

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

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

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

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.

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

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.

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.

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.

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.

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.

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.

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.

References 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)

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

RichHTML

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 14

References 21

Related Articles 3

Recommended Articles

Metrics

Comments

[1]	CHEN Jipeng, LIU Xiaolin, LI Shengqiang, LIU Xianjun, HU Yueqing, CHEN Tao. Obtaining and genetic analysis of hybrid progeny between Brassica napus cv. Xiangyou 15 and Brassica campestris ssp. pekinensis [J]. Acta Agriculturae Zhejiangensis, 2021, 33(7): 1170-1176.
[2]	WANG Shuzhen, ZHOU Liping, QIU Jieren, TONG Jianxin, YU Hong, CHAI Weiguo, LAI Wenguo. Comparative study on traits of progeny and parents of strawberry interspecific cross [J]. , 2020, 32(10): 1780-1787.
[3]	ZHANG Fu-xian;ZHANG Xin-hui;CHEN Run-xing;XU Jian-xiang;XIANG Xiao-min. Compatibility of interspecific hybridization between Cucurbita pepo L. and Cucurbita maxima D． [J]. , 2010, 22(1): 0-29.