利用黄籽羽衣甘蓝人工合成甘蓝型油菜初步研究
|
摘要
甘蓝型黄籽油菜的种子含油量、饼粕蛋白质含量通常比同一遗传来源的甘蓝型黑籽油菜的高，而且商品性好，选育甘蓝型油菜黄籽油菜已成为当前油菜育种的主要目标之一。控制黄色籽粒基因遗传资源的缺乏是甘蓝型黄籽油菜育种的主要障碍，发掘新的黄籽遗传资源势在必行。1994年，西南农业大学油菜研究室在十字花科芸薹属甘蓝种的变种羽衣甘蓝天然群体中发现了黄籽单株，经过多年人工选择，选育出几个表现较好的羽衣甘蓝黄籽品系，它们是甘蓝型黄籽油菜育种和油菜籽粒颜色遗传研究的宝贵基因资源。
本试验对羽衣甘蓝进行了农艺性状考查和品质分析。结果表明，羽衣甘蓝的农艺性状表现与甘蓝型油菜极为相似。种子含油量、胚饼蛋白质含量比对照中油821的高，综合品质表现优良。
为了了解羽衣甘蓝C染色体组黄籽基因与A染色体组黄籽基因间的相互关系，探索黄籽基因转移到甘蓝型油菜中的可行性，实验以白菜型黄籽油菜和黄籽羽衣甘蓝为材料，采用远缘杂交子房培养和原生质体融合两种不同方法进行了人工合成甘蓝型油菜研究。远缘杂交子房培养结果表明，以黄籽白菜型油菜为母本，黄籽羽衣甘蓝作父本，取授粉后10d左右的子房在MS+2．0mg／L6-BA+0．2mg／LNAA+7％Sucrose+300mg／LCH+0．8％Agar培养基上胚胎挽救效果最为理想。子房培养共收获19粒种子(结籽率为3．4粒／100子房)，其中8粒种子在培养基上萌发(萌发率为42．1％)，通过离体繁殖获得了4个无性系。白菜型油菜和羽衣甘蓝之间原生质体融合难度较大，试验中仅观察到细胞团的形成，没有获得理想的再生植株。对子房培养获得的无性系进行形态学、细胞学、生物化学等多种方法综合鉴定表明，有两个为白菜型油菜和羽衣甘蓝杂交的真杂种，它们将是开展甘蓝型黄籽油菜育种的重要材料。
Oil and protein content in seeds of yellow rapeseed (Brassica napus L.) are higher than that of black rapeseed from the same genetic background. It is much easier for yellow rapeseed to go into the market. The breeding of yellow rapeseed has become one of the main objectives in rapeseed breeding. It is hard for breeders to select effective yellow rapeseed lines due to the scarcity of yellow-seeded gene resources. It is very important to explore new genetic resources of yellow rapeseed. Yellow seed plant was excitably found in natural population of B. oleracea var. aceaphala, which is a variety of Cruciferae Brassica oleracea, in summer of 1994 by researcher of Oilcrop Lab of SWAU. After consecutive artificial selection for many years, some lines with good performance were obtained. They are valuable genetic materials for the study of the breeding of yellow rapeseed(5. napus).
The agronomic traits and qualitative traits of B. oleracea var. aceaphala were analyzed in present paper. The results demonstrated that performance of agronomic traits in B. oleracea var. aceaphala is almost the same as that in B. napus. Qualitative analysis indicated that not only seed oil and embryo protein content of B. oleracea var. aceaphala were higher than that of Zhongyou 821,but also the other qualitative traits were better.
To investigate the relationship between yellow-seeded gene(s) on genome C and genome A , to explore the feasibility of transfer of yellow-seeded gene(s) to B. napus, the studies were conducted on artificial synthesis of B. napus by using culture of ovary originated from interspecific hybridization and protoplast fusion between
yellow-seeded B. campestris and yellow-seeded D. oleracea var. aceaphala. The results indicated that the culture of ovary taken 10 days after pollination on MS+2.0mg/L6-BA-K).2mg/LNAA+7%Sucrose+300mg/LCH+0.8%Agar medium had most efficient for seed production in the cross of B. campestris X B. oleracea var. aceaphala. Altogether 19 seeds were produced in the experiment, 8 of them germinated on medium and 4 regenerated lines were obtained at last in vitro. Protoplast fusion between B. campestris and B. oleracea var. aceaphala failed to give expected results. Cell cluster was observed in this study, but no regenerated plantlets were obtained. Regeneration lines from ovary culture in vitro were identified with morphological, cytological and biochemical methods. The results showed that two lines (2-3 and 5-1) were true hybrids from B. campestris Xfi. oleracea var. aceaphala cross. The two lines will be important materials for yellow rapeseed breeding.
本试验对羽衣甘蓝进行了农艺性状考查和品质分析。结果表明，羽衣甘蓝的农艺性状表现与甘蓝型油菜极为相似。种子含油量、胚饼蛋白质含量比对照中油821的高，综合品质表现优良。
为了了解羽衣甘蓝C染色体组黄籽基因与A染色体组黄籽基因间的相互关系，探索黄籽基因转移到甘蓝型油菜中的可行性，实验以白菜型黄籽油菜和黄籽羽衣甘蓝为材料，采用远缘杂交子房培养和原生质体融合两种不同方法进行了人工合成甘蓝型油菜研究。远缘杂交子房培养结果表明，以黄籽白菜型油菜为母本，黄籽羽衣甘蓝作父本，取授粉后10d左右的子房在MS+2．0mg／L6-BA+0．2mg／LNAA+7％Sucrose+300mg／LCH+0．8％Agar培养基上胚胎挽救效果最为理想。子房培养共收获19粒种子(结籽率为3．4粒／100子房)，其中8粒种子在培养基上萌发(萌发率为42．1％)，通过离体繁殖获得了4个无性系。白菜型油菜和羽衣甘蓝之间原生质体融合难度较大，试验中仅观察到细胞团的形成，没有获得理想的再生植株。对子房培养获得的无性系进行形态学、细胞学、生物化学等多种方法综合鉴定表明，有两个为白菜型油菜和羽衣甘蓝杂交的真杂种，它们将是开展甘蓝型黄籽油菜育种的重要材料。
Oil and protein content in seeds of yellow rapeseed (Brassica napus L.) are higher than that of black rapeseed from the same genetic background. It is much easier for yellow rapeseed to go into the market. The breeding of yellow rapeseed has become one of the main objectives in rapeseed breeding. It is hard for breeders to select effective yellow rapeseed lines due to the scarcity of yellow-seeded gene resources. It is very important to explore new genetic resources of yellow rapeseed. Yellow seed plant was excitably found in natural population of B. oleracea var. aceaphala, which is a variety of Cruciferae Brassica oleracea, in summer of 1994 by researcher of Oilcrop Lab of SWAU. After consecutive artificial selection for many years, some lines with good performance were obtained. They are valuable genetic materials for the study of the breeding of yellow rapeseed(5. napus).
The agronomic traits and qualitative traits of B. oleracea var. aceaphala were analyzed in present paper. The results demonstrated that performance of agronomic traits in B. oleracea var. aceaphala is almost the same as that in B. napus. Qualitative analysis indicated that not only seed oil and embryo protein content of B. oleracea var. aceaphala were higher than that of Zhongyou 821,but also the other qualitative traits were better.
To investigate the relationship between yellow-seeded gene(s) on genome C and genome A , to explore the feasibility of transfer of yellow-seeded gene(s) to B. napus, the studies were conducted on artificial synthesis of B. napus by using culture of ovary originated from interspecific hybridization and protoplast fusion between
yellow-seeded B. campestris and yellow-seeded D. oleracea var. aceaphala. The results indicated that the culture of ovary taken 10 days after pollination on MS+2.0mg/L6-BA-K).2mg/LNAA+7%Sucrose+300mg/LCH+0.8%Agar medium had most efficient for seed production in the cross of B. campestris X B. oleracea var. aceaphala. Altogether 19 seeds were produced in the experiment, 8 of them germinated on medium and 4 regenerated lines were obtained at last in vitro. Protoplast fusion between B. campestris and B. oleracea var. aceaphala failed to give expected results. Cell cluster was observed in this study, but no regenerated plantlets were obtained. Regeneration lines from ovary culture in vitro were identified with morphological, cytological and biochemical methods. The results showed that two lines (2-3 and 5-1) were true hybrids from B. campestris Xfi. oleracea var. aceaphala cross. The two lines will be important materials for yellow rapeseed breeding.
引文
1．陈树忠 殷家明 唐章林等 甘蓝型油菜与羽衣甘蓝远缘杂交初步研究 西南农业大学学报，2000，22(3)：208-210
2．陈玉萍 花椰菜和甘蓝型油菜种间杂交的研究 Ⅰ油菜和花椰莱种间杂种组织培养 效率 湖北农业科学，1995，(5)：46-48
3．程振东 卫志明 甘蓝型油莱下胚轴原生质体培养的研究 生物工程学报，1994，10(1)：30-33
4．冯午 邓岳芬 李懋学等 白菜甘蓝种杂种后代细胞质遗传及其利用前景探讨园艺学报，1993，20(3)：267-273
5．葛扣麟 人工合成欧洲油菜(B.napus)的研究 复旦大学学报，1964，9(3)：392-398
6．何凤发 遗传学实验技术与方法 西南农业大学出版社，1996
7．何康 刘瑞龙 中国农业百科全书(生物卷) 农业科学出版社，1991
8．胡能书 万国权 同工酶技术及其应用 湖南科学技术出版社，1985
9．贾士荣 杨美珠 芸薹属作物的原生质体培养和细胞融合 植物生理学通讯，1988，(5)：7-12
10．李加纳 唐章林 张学昆等 不同遗传背景的甘蓝型黄籽油菜粒色遗传初步研究 中国油料作物学报，1998，20(4)：16-19
11．李明山 大白菜与萝卜属间杂种幼胚离体培养的研究 园艺学报，1992，19(4)：353-357
12．李世君 孟征 盛方镜等 大白菜无菌苗叶肉原生质体植株再生 生物工程学报，1992，8(3)：249-254
13．李向辉 植物遗传操作技术 科学出版社，1988
14．梁红 冯午 白菜与甘蓝的种间杂交及其杂种后代的研究 园艺学报，1990，17(3)：203-210
15．梁红 何丽贞 覃广泉 甘蓝与菜心种间杂种的幼胚培养及F_1代的快速繁殖 中国蔬菜，1994，(6)：25-26
16．刘后利 甘蓝型黄籽油菜的遗传研究 作物学报，1992，18(4)：241-248
17．刘后利 油菜的遗传和育种 上海科学技术出版社，1985
18．刘后利 油菜遗传和育种学 中国农业大学出版社，2000
19．罗公禄 李名扬 甘蓝型油菜原生质体培养和植株再生 西南农业大学学报，1993，15(6)：518-521
20．罗鹏 油菜遗传文献记要 四川大学出版社，1985
21．孟金陵 甘蓝型油菜与甘蓝杂交不亲和性研究：Ⅰ花粉—雌蕊相互作用及杂种早期胚胎发育 华中农业大学学报，1987，6(5)：203-208
22．戚存扣 傅寿仲 浦惠明 甘蓝型油菜低芥酸黄籽新品种宁油10号的选育 江苏农业科学，1998，(4)：28-29
23．饶璐璐 羽衣甘蓝 蔬菜，1997，(1)：11-12
24．宋同明 脉冲核磁共振仪对作物种子含油量的快速测定 作物学报，1989，15(2)：160-166
25．孙敬三 桂耀林 植物细胞工程实验技术 科学出版社，1995
26．田志宏 孟金陵 芸薹属作物原生质体融合及基因转移 中国油料，1997，19(1)：70-75
27．王汉中 刘后利 油菜皮壳中多酚酶活性的组织化学检定及其与粒色的关系 中国油料，1991，(1)：30-33
28．王怀名 杜广芩 贾翠莹等 观赏羽衣甘蓝的离体培养 华北农学报，1995，10(1)：68-69
29．王良中 刘玉贞 甘蓝型油菜的人工合成 西南农业学报，1992，5(3)：12-17
30．文雁成等 对甘蓝与大白菜种间杂交合成的甘蓝型油菜的研究 中国油料作物学报，1999，21(4)：8-11
31．吴江生，石淑稳，刘后利 黄籽突变体的遗传研究 全国作为育种学术讨论会论文集，1998，4：299-303
32．熊新生 贾士荣 傅幼英 甘蓝原生质体无性系的变异 园艺学报，1988，(15)，120-123
33．徐利元 甘蓝型油菜与蓝花子远缘杂交及双二倍体的合成 遗传学报，1996，23(2)：124-130
34．许智宏 植物生物技术 上海科学技术出版社，1998
35．许智宏 卫志明 植物原生质体培养和遗传操作 上海科学技术出版社 1997
36．杨美珠 贾士荣 埃塞俄比亚芥的原生质体培养与植株再生 植物生理学报 1989，31(2)：89-94
37．杨乃傅 用组织培养方法繁殖十植物实验 植物生理学报，1979，5(4)：369-377
38．赵军良 陈柔如 白菜型油菜原生质体培养的研究 山西大学学报，1989，12(4)：465-469
39．赵云 王茂林 油菜苔茎段髓部原生质体培养再生植株 作物学报，1998，24(3)：291-297
40．朱利泉 基础生物化学(实验原理与方法) 成都科学技术出版社1997
41. B.Rosen,C.Hallden,W.K,Heneen Diploid B.napus somatic hybrids,characterization of nuclear and organellar DNA.Theor Appi Genet,1988,76:197-203
42. Barsby,-T.L.,Chuong,-P.V.,Yarrow,-S.A.The combination of Polima CMS and cytoplasmic triazine resistance in Brassica napus.Theor-Appl-Genet,1987,73(6):809-814
43. Barsby,-T.L.,Yarrow,-S.A,Shepard,-J.F.A rapid and efficient alternative procedure
for the regeneration of plants from hypocotyl protoplasts of Brassica napus. Plant-Cell-Rep,1986,5(2) :101-103
44． Chen B. Y., Heneen W.K., Jonsson R. Resynthesis of Brassica napus L. through interspecific hybridization between B. alboglabra Bailey and B. campestris L. with special emphasis on seed colour. Plant Breeding, 1988,101: 52-59
45． Christey M.C., Makaroff C.A., Earle E.D., Atrazine-resistant cytoplasmic male-sterile-nigra broccoli obtainted by protoplast fusion between cytoplasmic male-sterile Brassica oleracea and atrazine-resistant Brassica campestris. Theor Appl Genet, 1991,83:201-208
46． Chuong,-P.V., Pauls,-K.P., Beversdorf,-W.D. A simple culture method for Brassica hypocotyl protoplasts. Plant-Cell-Rep, 1985,4 (1) : 4-6
47． Chuong,-P.V., Pauls,-K.P., Beversdorf,-W.D. Protoplast culture and plant regeneration from Brassica carinata Braun. Plant-Cell-Rep, 1987, 6 (1) : 67-69
48． E.Sundberg, M.Landgren, K.Glimelius Fertility and chromosome stability in B. napus resynthesised by protoplast fusion Theor Appl Genet, 1987,75:96-104
49． Fan,-Z., Stefansson,-B. R., Sernyk,-J.L. Maintainers and restorers for three male-sterility-inducing cytoplasms in rape (Brassica napus L.). Can-J-Plant-Sci-Rev-Can-Phytotechnie, 1986, 66 (2) :229-234
50． Glimelius,-K., Djupsjobacka,-M., Fellner-Feidegg,-H. Selection and enrichment of plant protoplast heterokaryons of Brassicaceae by flow sorting. Plant-Sci, 1986, 45 (2) :133-141
51． Gowers S., Christey M.C. Intercrossing B. napus and B. oleracea to introgress characters from kale to rape. Proceedings of the 10th International Rapeseed Congres.1999
52． Horak J. Regeneration of diploid and polyploid plant from the stem pith explants of diploid marrow stem Kale(5． oleracea) Ann bot(London),1975,39:571
53． J. Fahlison, L.Rahlen and K.Glimelius, Analysis of plants regenerated from protoplast fusion between B. napus and Eruca sativa, Theor Appl Genet. 1988, 76: 507-512
54． Jana Muller , Karin Sonntag Electrical fusion of protoplasts in Brassicaceae. Proceedings of the 10th International Rapeseed Congres,1999
55． K.Glimelius High growth rate and regeneration capacity of hypocotyl protoplasts in some Brassicaceae Physiol. Plant, 1984,61:38-44
56． Lillo,-C., Shahin,-E.A. Rapid regeneration of plants from hypocotyl protoplasts and root segments of cabbage. Hortscience, 1986, 21 (2) : 315-317．
57． Lustinec J. and Horak J. Induced regeneration of plant in tissue cultures of Brassica Oleracea. Experientia, 1970,26:919
58． M. Landgren, K. Glimelius A high frequency of intergenomic mitochondrial recombination and an overall biased segregation of B. campestris or recombined B. compestris mitochondria were found in somatic hybrids made within Brassicaceae, Theor Appl Genet, 1994,87:854-862
59． Mattheij W M.,Puite K. Tetraploid potato hybrids through protoplast fusion and analysis of their performance in the field. Theor Appl Genet, 1992,83:807-818．
60． Menczel L. Wolfe K. High frequency of fusion induced in freely suspende protoplast
mixture by polyethylene glycol and dimethylsulfoxide at high pH. Plant Cell Rep,1987,6(2) :98-101
61． Menczel.-L., Morgan.-A., Brown,-S. Fusion-mediated combination of Ogura-type cytoplasmic male sterility with Brassica napus plastids using X-irradiated CMS protoplasts. Plant-Cell-Rep, 1989,8(1) : 116-118
62． Niu Yingze, Wang Liangzhong, Liu Yuzhen Creation of new germplasm in B. napus using landraces of B. chinensis and B. oleracea var. alboglbra from S.W. china, Proceedings of the 10th International Rapeseed Congres.1999
63． Nobumichi Inomata Production of interspecific hybrids in B. campestris and B. oleracea by culture in vitro of excised ovaries I .Development of excised ovaries in the crosses of various cultivars. Japan. J.Genet,1978,53:161-173
64． Nobumichi Inomata .Production of interspecific hybrids in B. campestris and B. oleracea by culture in vitro of excised ovaries II .Development of excised ovaries on various culture media. Japan. J. Breed,1979, 29(2) :115-120
65． Olsson,G. Study on interspecific hybridition of Brassicaeae. Plant breeding Abst, 1977
66． Pelletier,-G, Primard.-C., Vedel,-F. Intergeneric cytoplasmic hybridization in cruciferae by protoplast fusion [Brassica napus, Raphanus sativus, Brassica campestris]. M-G-G-Mol-Gen-Genet, 1983,191 (2) : 244-250
67． Rashid A., Rakow G. and Downey R. K.: Development of yellow B. napus through interspecifid cross, Plant Breeding, 1994,112,127-134
68． Richard H., Ozminkowski Jr. And Pablo Jourdan Comparing the resynthesis of B. napus L. By interspecific somatic and sexual hybridization II Hybrid morphology and identifying organelle genomes.Journal-of-the-American-Society-for-Horticultural-Science. 1994, 119:4,816-823
69． Richard H.,Ozminkowski Jr., Pablo Jourdan Comparing the resynthesis of B. napus L. By interspecific somatic and sexual hybridization I Producing and identifying hybrids. Journal-of-the-American-Society-for-Horticultural-Science, 1994,119: 4, 808-815
70． Gowers,M. C.Christey Intercrossing B. napus and B. oleracea to introgress characters from kale to rape. Proceedings of the 10th International Rapeseed Congres,1999
71． Senda M., Takeda J., Abe S. Etl. Induction of cell fusion of plant protoplasts by electrical stimulation. Plant Cell Physiol,1979,20:1441-1443
72． Spangenberg,-G., Koop,-H.U., Lichter,-R. Microculture of single protoplasts of Brassica napus. Physiol-Plant, 1986, 66 (1) : 1-8
73． Sunberg E., Glimelius K. Effects of parental ploidy level and genetic divergence on chromosome elimination and chloroplast segregation in sometic hybrids with Brassicaeae. Theor Appl Genet, 1991,83:81-88
74． Thomzik J E., Hain R. Transfer and segregation of triazine tolerant chloroplasts in Brassica napus. Theor Appl Genet, 1988,76:165-171
75． U. Nagahara Genome analysis in Brassica with special reference to the experimental formation of B. napus and peculiar mode of ferlitization. Jap. Jour. Bot, 1935,7:389-452
76． Xu,-Z.H., Davey,-M.R., Cocking,-E.C. Plant regeneration from root protoplasts of Brassica. Plant-Sci-Let,24 (1) : 117-121
77． Yamahaja H., Kuginuki Y. Efficient production of somatic hybrids between Raphanus sativus and Brassica oleracea. Jpn J Breed, 1992,42:329-339
78． Yarrow,-S.A., Wu,-S.C., Barsby,-T.L. The introduction of CMS mitochondria to triazine tolerant Brassica napus L., var. "Regent", by micromanipulation of individual heterokaryons. Plant-Cell-Rep, 1986, 5 (6) :415-418
79． Zee SY, Hui LH In vitro plant regeration from hyplcoty and cotyledons of Chinese kale. Z Pflanzenphysiol, 1977,82:440
80． Zimmermann U., Scheurich P. High frequency fusion of planta protoplasts by electric fields. Planta, 1981,151:26-32
2．陈玉萍 花椰菜和甘蓝型油菜种间杂交的研究 Ⅰ油菜和花椰莱种间杂种组织培养 效率 湖北农业科学，1995，(5)：46-48
3．程振东 卫志明 甘蓝型油莱下胚轴原生质体培养的研究 生物工程学报，1994，10(1)：30-33
4．冯午 邓岳芬 李懋学等 白菜甘蓝种杂种后代细胞质遗传及其利用前景探讨园艺学报，1993，20(3)：267-273
5．葛扣麟 人工合成欧洲油菜(B.napus)的研究 复旦大学学报，1964，9(3)：392-398
6．何凤发 遗传学实验技术与方法 西南农业大学出版社，1996
7．何康 刘瑞龙 中国农业百科全书(生物卷) 农业科学出版社，1991
8．胡能书 万国权 同工酶技术及其应用 湖南科学技术出版社，1985
9．贾士荣 杨美珠 芸薹属作物的原生质体培养和细胞融合 植物生理学通讯，1988，(5)：7-12
10．李加纳 唐章林 张学昆等 不同遗传背景的甘蓝型黄籽油菜粒色遗传初步研究 中国油料作物学报，1998，20(4)：16-19
11．李明山 大白菜与萝卜属间杂种幼胚离体培养的研究 园艺学报，1992，19(4)：353-357
12．李世君 孟征 盛方镜等 大白菜无菌苗叶肉原生质体植株再生 生物工程学报，1992，8(3)：249-254
13．李向辉 植物遗传操作技术 科学出版社，1988
14．梁红 冯午 白菜与甘蓝的种间杂交及其杂种后代的研究 园艺学报，1990，17(3)：203-210
15．梁红 何丽贞 覃广泉 甘蓝与菜心种间杂种的幼胚培养及F_1代的快速繁殖 中国蔬菜，1994，(6)：25-26
16．刘后利 甘蓝型黄籽油菜的遗传研究 作物学报，1992，18(4)：241-248
17．刘后利 油菜的遗传和育种 上海科学技术出版社，1985
18．刘后利 油菜遗传和育种学 中国农业大学出版社，2000
19．罗公禄 李名扬 甘蓝型油菜原生质体培养和植株再生 西南农业大学学报，1993，15(6)：518-521
20．罗鹏 油菜遗传文献记要 四川大学出版社，1985
21．孟金陵 甘蓝型油菜与甘蓝杂交不亲和性研究：Ⅰ花粉—雌蕊相互作用及杂种早期胚胎发育 华中农业大学学报，1987，6(5)：203-208
22．戚存扣 傅寿仲 浦惠明 甘蓝型油菜低芥酸黄籽新品种宁油10号的选育 江苏农业科学，1998，(4)：28-29
23．饶璐璐 羽衣甘蓝 蔬菜，1997，(1)：11-12
24．宋同明 脉冲核磁共振仪对作物种子含油量的快速测定 作物学报，1989，15(2)：160-166
25．孙敬三 桂耀林 植物细胞工程实验技术 科学出版社，1995
26．田志宏 孟金陵 芸薹属作物原生质体融合及基因转移 中国油料，1997，19(1)：70-75
27．王汉中 刘后利 油菜皮壳中多酚酶活性的组织化学检定及其与粒色的关系 中国油料，1991，(1)：30-33
28．王怀名 杜广芩 贾翠莹等 观赏羽衣甘蓝的离体培养 华北农学报，1995，10(1)：68-69
29．王良中 刘玉贞 甘蓝型油菜的人工合成 西南农业学报，1992，5(3)：12-17
30．文雁成等 对甘蓝与大白菜种间杂交合成的甘蓝型油菜的研究 中国油料作物学报，1999，21(4)：8-11
31．吴江生，石淑稳，刘后利 黄籽突变体的遗传研究 全国作为育种学术讨论会论文集，1998，4：299-303
32．熊新生 贾士荣 傅幼英 甘蓝原生质体无性系的变异 园艺学报，1988，(15)，120-123
33．徐利元 甘蓝型油菜与蓝花子远缘杂交及双二倍体的合成 遗传学报，1996，23(2)：124-130
34．许智宏 植物生物技术 上海科学技术出版社，1998
35．许智宏 卫志明 植物原生质体培养和遗传操作 上海科学技术出版社 1997
36．杨美珠 贾士荣 埃塞俄比亚芥的原生质体培养与植株再生 植物生理学报 1989，31(2)：89-94
37．杨乃傅 用组织培养方法繁殖十植物实验 植物生理学报，1979，5(4)：369-377
38．赵军良 陈柔如 白菜型油菜原生质体培养的研究 山西大学学报，1989，12(4)：465-469
39．赵云 王茂林 油菜苔茎段髓部原生质体培养再生植株 作物学报，1998，24(3)：291-297
40．朱利泉 基础生物化学(实验原理与方法) 成都科学技术出版社1997
41. B.Rosen,C.Hallden,W.K,Heneen Diploid B.napus somatic hybrids,characterization of nuclear and organellar DNA.Theor Appi Genet,1988,76:197-203
42. Barsby,-T.L.,Chuong,-P.V.,Yarrow,-S.A.The combination of Polima CMS and cytoplasmic triazine resistance in Brassica napus.Theor-Appl-Genet,1987,73(6):809-814
43. Barsby,-T.L.,Yarrow,-S.A,Shepard,-J.F.A rapid and efficient alternative procedure
for the regeneration of plants from hypocotyl protoplasts of Brassica napus. Plant-Cell-Rep,1986,5(2) :101-103
44． Chen B. Y., Heneen W.K., Jonsson R. Resynthesis of Brassica napus L. through interspecific hybridization between B. alboglabra Bailey and B. campestris L. with special emphasis on seed colour. Plant Breeding, 1988,101: 52-59
45． Christey M.C., Makaroff C.A., Earle E.D., Atrazine-resistant cytoplasmic male-sterile-nigra broccoli obtainted by protoplast fusion between cytoplasmic male-sterile Brassica oleracea and atrazine-resistant Brassica campestris. Theor Appl Genet, 1991,83:201-208
46． Chuong,-P.V., Pauls,-K.P., Beversdorf,-W.D. A simple culture method for Brassica hypocotyl protoplasts. Plant-Cell-Rep, 1985,4 (1) : 4-6
47． Chuong,-P.V., Pauls,-K.P., Beversdorf,-W.D. Protoplast culture and plant regeneration from Brassica carinata Braun. Plant-Cell-Rep, 1987, 6 (1) : 67-69
48． E.Sundberg, M.Landgren, K.Glimelius Fertility and chromosome stability in B. napus resynthesised by protoplast fusion Theor Appl Genet, 1987,75:96-104
49． Fan,-Z., Stefansson,-B. R., Sernyk,-J.L. Maintainers and restorers for three male-sterility-inducing cytoplasms in rape (Brassica napus L.). Can-J-Plant-Sci-Rev-Can-Phytotechnie, 1986, 66 (2) :229-234
50． Glimelius,-K., Djupsjobacka,-M., Fellner-Feidegg,-H. Selection and enrichment of plant protoplast heterokaryons of Brassicaceae by flow sorting. Plant-Sci, 1986, 45 (2) :133-141
51． Gowers S., Christey M.C. Intercrossing B. napus and B. oleracea to introgress characters from kale to rape. Proceedings of the 10th International Rapeseed Congres.1999
52． Horak J. Regeneration of diploid and polyploid plant from the stem pith explants of diploid marrow stem Kale(5． oleracea) Ann bot(London),1975,39:571
53． J. Fahlison, L.Rahlen and K.Glimelius, Analysis of plants regenerated from protoplast fusion between B. napus and Eruca sativa, Theor Appl Genet. 1988, 76: 507-512
54． Jana Muller , Karin Sonntag Electrical fusion of protoplasts in Brassicaceae. Proceedings of the 10th International Rapeseed Congres,1999
55． K.Glimelius High growth rate and regeneration capacity of hypocotyl protoplasts in some Brassicaceae Physiol. Plant, 1984,61:38-44
56． Lillo,-C., Shahin,-E.A. Rapid regeneration of plants from hypocotyl protoplasts and root segments of cabbage. Hortscience, 1986, 21 (2) : 315-317．
57． Lustinec J. and Horak J. Induced regeneration of plant in tissue cultures of Brassica Oleracea. Experientia, 1970,26:919
58． M. Landgren, K. Glimelius A high frequency of intergenomic mitochondrial recombination and an overall biased segregation of B. campestris or recombined B. compestris mitochondria were found in somatic hybrids made within Brassicaceae, Theor Appl Genet, 1994,87:854-862
59． Mattheij W M.,Puite K. Tetraploid potato hybrids through protoplast fusion and analysis of their performance in the field. Theor Appl Genet, 1992,83:807-818．
60． Menczel L. Wolfe K. High frequency of fusion induced in freely suspende protoplast
mixture by polyethylene glycol and dimethylsulfoxide at high pH. Plant Cell Rep,1987,6(2) :98-101
61． Menczel.-L., Morgan.-A., Brown,-S. Fusion-mediated combination of Ogura-type cytoplasmic male sterility with Brassica napus plastids using X-irradiated CMS protoplasts. Plant-Cell-Rep, 1989,8(1) : 116-118
62． Niu Yingze, Wang Liangzhong, Liu Yuzhen Creation of new germplasm in B. napus using landraces of B. chinensis and B. oleracea var. alboglbra from S.W. china, Proceedings of the 10th International Rapeseed Congres.1999
63． Nobumichi Inomata Production of interspecific hybrids in B. campestris and B. oleracea by culture in vitro of excised ovaries I .Development of excised ovaries in the crosses of various cultivars. Japan. J.Genet,1978,53:161-173
64． Nobumichi Inomata .Production of interspecific hybrids in B. campestris and B. oleracea by culture in vitro of excised ovaries II .Development of excised ovaries on various culture media. Japan. J. Breed,1979, 29(2) :115-120
65． Olsson,G. Study on interspecific hybridition of Brassicaeae. Plant breeding Abst, 1977
66． Pelletier,-G, Primard.-C., Vedel,-F. Intergeneric cytoplasmic hybridization in cruciferae by protoplast fusion [Brassica napus, Raphanus sativus, Brassica campestris]. M-G-G-Mol-Gen-Genet, 1983,191 (2) : 244-250
67． Rashid A., Rakow G. and Downey R. K.: Development of yellow B. napus through interspecifid cross, Plant Breeding, 1994,112,127-134
68． Richard H., Ozminkowski Jr. And Pablo Jourdan Comparing the resynthesis of B. napus L. By interspecific somatic and sexual hybridization II Hybrid morphology and identifying organelle genomes.Journal-of-the-American-Society-for-Horticultural-Science. 1994, 119:4,816-823
69． Richard H.,Ozminkowski Jr., Pablo Jourdan Comparing the resynthesis of B. napus L. By interspecific somatic and sexual hybridization I Producing and identifying hybrids. Journal-of-the-American-Society-for-Horticultural-Science, 1994,119: 4, 808-815
70． Gowers,M. C.Christey Intercrossing B. napus and B. oleracea to introgress characters from kale to rape. Proceedings of the 10th International Rapeseed Congres,1999
71． Senda M., Takeda J., Abe S. Etl. Induction of cell fusion of plant protoplasts by electrical stimulation. Plant Cell Physiol,1979,20:1441-1443
72． Spangenberg,-G., Koop,-H.U., Lichter,-R. Microculture of single protoplasts of Brassica napus. Physiol-Plant, 1986, 66 (1) : 1-8
73． Sunberg E., Glimelius K. Effects of parental ploidy level and genetic divergence on chromosome elimination and chloroplast segregation in sometic hybrids with Brassicaeae. Theor Appl Genet, 1991,83:81-88
74． Thomzik J E., Hain R. Transfer and segregation of triazine tolerant chloroplasts in Brassica napus. Theor Appl Genet, 1988,76:165-171
75． U. Nagahara Genome analysis in Brassica with special reference to the experimental formation of B. napus and peculiar mode of ferlitization. Jap. Jour. Bot, 1935,7:389-452
76． Xu,-Z.H., Davey,-M.R., Cocking,-E.C. Plant regeneration from root protoplasts of Brassica. Plant-Sci-Let,24 (1) : 117-121
77． Yamahaja H., Kuginuki Y. Efficient production of somatic hybrids between Raphanus sativus and Brassica oleracea. Jpn J Breed, 1992,42:329-339
78． Yarrow,-S.A., Wu,-S.C., Barsby,-T.L. The introduction of CMS mitochondria to triazine tolerant Brassica napus L., var. "Regent", by micromanipulation of individual heterokaryons. Plant-Cell-Rep, 1986, 5 (6) :415-418
79． Zee SY, Hui LH In vitro plant regeration from hyplcoty and cotyledons of Chinese kale. Z Pflanzenphysiol, 1977,82:440
80． Zimmermann U., Scheurich P. High frequency fusion of planta protoplasts by electric fields. Planta, 1981,151:26-32