不结球白菜游离小孢子培养及植株再生研究
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摘要
为进一步优化不结球白菜游离小孢子培养技术体系,本研究以20个不同基因型的不结球白菜为试验材料,研究不同基因型、4℃低温胁迫处理时间和头孢噻胯浓度对游离小孢子胚胎发生的影响,并对小孢子胚胎发育和再生过程进行观察和倍性鉴定。结果表明,当花瓣/花药(P/A)长度比值为0.85～1.10时,不结球白菜小孢子主要处于单核晚期;共有10个基因型不结球白菜出胚,其中出胚率最大的为H20,平均出胚率为7.75胚·10蕾~(-1);4℃低温胁迫处理1 d时不结球白菜出胚率最高;头孢噻胯对不结球白菜出胚率的影响与基因型密切相关;从胚状体到再生幼苗阶段,不结球白菜基因型不同,其胚胎再生频率也不同;倍性鉴定共检测出21个双单倍体和2个单倍体,自然加倍率为91.3%,不结球白菜植株形态表现出明显的多样性。本研究结果为不结球白菜游离小孢子高效培养体系的优化提供了一定的技术支撑。
In order to optimize the microspore culture technology in non-heading Chinese cabbage, the effects of genotype, low temperature stress and the addition amount of cefotaxime on the embryogenesis of isolated microspore were studied with 20 genotypes of non-heading Chinese cabbage. The development of microspore-derived embryos, the embyros regeneration and the ploidy identification were also observed, The results were as follows: When petal/anther(P/A) was 0.85～1.10, the microspores of non-heading Chinese cabbage were mainly in the late stage of uninucleate. There were 10 genotypes of non-heading Chinese cabbage showing embryogenesis, the incidence of embryo was highest for the genotype of H20, which was 7.75 embryoids·10 bud~(-1). Low tempeature stress pretreatment at 4℃ for 1 d was helpful for embryogenesis. The effect of cefotaxime on the rate of embryo formation was highly dependent on genotype. From the embryoids to seedling stage, the frequency of embryo regeneration was varied within different genotypes; 21 microspore-derived plants were doubled haploids(DH) and 2 were haploids through ploidy identification, the natural doubling rate was 91.3%. The morphology of the obtained microspore-derived plants showed obvious diversity. These researches provide a way for the optimization of microspore culture in non-heading Chinese cabbage.
In order to optimize the microspore culture technology in non-heading Chinese cabbage, the effects of genotype, low temperature stress and the addition amount of cefotaxime on the embryogenesis of isolated microspore were studied with 20 genotypes of non-heading Chinese cabbage. The development of microspore-derived embryos, the embyros regeneration and the ploidy identification were also observed, The results were as follows: When petal/anther(P/A) was 0.85～1.10, the microspores of non-heading Chinese cabbage were mainly in the late stage of uninucleate. There were 10 genotypes of non-heading Chinese cabbage showing embryogenesis, the incidence of embryo was highest for the genotype of H20, which was 7.75 embryoids·10 bud~(-1). Low tempeature stress pretreatment at 4℃ for 1 d was helpful for embryogenesis. The effect of cefotaxime on the rate of embryo formation was highly dependent on genotype. From the embryoids to seedling stage, the frequency of embryo regeneration was varied within different genotypes; 21 microspore-derived plants were doubled haploids(DH) and 2 were haploids through ploidy identification, the natural doubling rate was 91.3%. The morphology of the obtained microspore-derived plants showed obvious diversity. These researches provide a way for the optimization of microspore culture in non-heading Chinese cabbage.
引文
[1] Ferrie A M R, Caswell K L. Isolated microspore culture techniques and recent progress for haploid and doubled haploid plant production[J]. Plant Cell Tissue and Organ Culture, 2011, 104(3):301-309
[2] Germanã M A. Gametic embryogenesis and haploid technology as valuable support to plant breeding[J]. Plant Cell Reports, 2011, 30(5):839-857
[3] 侯喜林. 不结球白菜育种研究新进展[J].南京农业大学学报, 2003, 26(4):111-115
[4] Lichter R. Induction of haploid plants from isolated pollen of Brassica napus[J]. Zeitschrift Für Pflanzenphysiologie, 1982, 105(5):427-434
[5] Chuong P Ⅴ, Beversdorf W D. High-frequency embryogenesis through isolated microspore culture in Brassica napus L. and B-carinata braun[J]. Plant Science, 1985, 39(3):219-226
[6] Sato T, Nishio T, Hirai M. Plant-regeneration from isolated microspore cultures of Chinese cabbage (Brassica campestris spp.pekinensis)[J]. Plant Cell Reports, 1989, 8(8):486-488
[7] Takahata Y, Keller W A. High-frequency embryogenesis and plant-regeneration in isolated microspore culture of Brassica oleracea L.[J]. Plant Science, 1991, 74(2):235-242
[8] Xu L, Najeeb U, Naeem M S, Wan G L, Jin Z L, Khan F, Zhou W J. In vitro Mutagenesis and Genetic Improvement[M]. New York: Springer, 2012:151-173
[9] Germana M A, Lambardi M. In vitro Embryogenesis in Higher Plants[M]. New York: Springer, 2016
[10] 原玉香, 张晓伟, 耿建峰, 蒋武生, 韩永平. 利用游离小孢子培养技术育成早熟大白菜新品种‘豫新60’[J]. 园艺学报, 2004, 31(5): 704-709
[11] 宋立晓, 曾爱松, 高兵, 严继勇. 青花菜新品种苏青3号的选育[J]. 江苏农业科学, 2014, 42(12): 216-218
[12] 曾爱松, 高兵, 宋立晓, 严继勇. 秋季专用牛心甘蓝新品种锦秋55的选育[J]. 江苏农业科学, 2015, 43(11): 230-231
[13] 万光龙. 基于甘蓝型油菜小孢子培养和胚状体诱导的植株再生、转化与诱变体系的构建[D]. 杭州: 浙江大学, 2010
[14] Lu Y, Dai S, Gu A, Liu M, Wang Y, Luo S, Zhao Y, Wang S, Xuan S, Chen X, Li X, Bonnema G, Zhao J, Shen S. Microspore induced doubled haploids production from ethyl methanesulfonate (EMS) soaked flower buds is an efficient strategy for mutagenesis in Chinese cabbage[J]. Frontiers in Plant Science, 2016, 7: 1780
[15] Verónica P V, Patricia C M, Alba R S, Seguí-Simarro J M. Formation and excretion of autophagic plastids (plastolysomes) in Brassica napus embryogenic microspores[J]. Frontiers in Plant Science, 2015, 6: 94
[16] Pilarska M, Malec P, Salaj J, Bartnicki F, Konieczny R. High expression of somatic embryogenesis receptor-like kinase coincides with initiation of various developmental pathways in in vitro culture of Trifolium nigrescens[J]. Protoplasma, 2016, 253(2): 345-355
[17] 蒋武生, 耿建峰, 张晓伟, 原玉香, 韩永平, 姚秋菊. 基因型和有机附加物对小白菜花药培养胚状体诱导的影响[J]. 中国瓜菜, 2006(2): 1-4
[18] 蒋武生, 原玉香, 张晓伟, 耿建峰, 韩永平, 郅玉宝. 小白菜游离小孢子培养及其再生植株[J]. 河南农业大学学报, 2005, 39(4): 40-43
[19] 蒋武生, 张晓伟, 姚秋菊, 原玉香, 耿建峰. 活性炭对小白菜游离小孢子培养的影响[J]. 华北农学报, 2008,23(5): 93-96
[20] 耿建峰, 侯喜林, 张晓伟, 蒋武生, 原玉香, 韩永平, 姚秋菊, 成妍, 李英. 影响白菜游离小孢子培养关键因素分析[J]. 园艺学报, 2007, 34(1): 111-116
[21] 高素燕, 李英, 单晓政, 侯喜林. 预处理对不结球白菜游离小孢子胚胎发生的影响[J]. 江苏农业学报, 2008, 24(6): 878-881
[22] 刘环环. 不结球白菜和青花菜游离小孢子培养技术的研究[D].南京: 南京农业大学, 2014
[23] 张振超, 耿鑫鑫, 戴忠良, 潘跃平, 王兵, 许玲, 颜志明, 周伟军. 甘蓝类植物小孢子培养及植株再生研究[J]. 核农学报, 2013, 27(7): 929-937
[24] Bhatia R, Dey S S, Sood S, Sharma K, Sharma V K, Parkash C, Kumar R. Optimizing protocol for efficient microspore embryogenesis and doubled haploid development in different maturity groups of cauliflower (B-oleracea var. botrytis L.) in India[J]. Euphytica, 2016, 212(3):439-454
[25] Gu H, Zhao Z, Sheng X, Yu H, Wang J. Efficient doubled haploid production in microspore culture of loose-curd cauliflower (Brassica oleracea var. botrytis)[J]. Euphytica, 2014, 195(3):467-475
[26] Shumilina D V, Shmykova N A, Bondareva L L, Suprunova T P. Effect of genotype and medium culture content on microspore-derived embryo formation in Chinese cabbage (Brassica rapa ssp. chinensis) Cv. Lastochka[J]. Biology Bulletin, 2015, 42(4):302-309
[27] 姜凤英, 冯辉, 王超楠. 小白菜小孢子培养胚状体的诱导[J]. 沈阳农业大学学报, 2006, 37(5): 763-765
[28] 毛忠良, 张振超, 姚悦梅, 戴忠良, 秦文斌, 潘跃平. 羽衣甘蓝小孢子胚胎发生观察及再生植株倍性鉴定[J]. 西北植物学报, 2012, 32(10): 2016-2022
[29] 成妍, 班青宇, 王倩, 侯喜林. 不结球白菜游离小孢子培养及再生植株的倍性鉴定[J]. 南京农业大学学报, 2009, 32(2): 25-29
[30] 陈琳, 刘克德, 吴江生, 孙秀丽, 汪频, 石淑稳. 中国白菜小孢子离体培养和双单倍体植株再生[J]. 中国农学通报, 2010, 26(14): 78-81
[31] Gu H H, Zhou W J, Hagberg P.High frequency spontaneous production of doubled haploid plants in microspore cultures of Brassica rapa ssp. Chinensis[J]. Euphytica, 2003, 134 (3): 239-245
[32] Mathias R J, Mukasa C. The effect of cefotaxime on the growth and regeneration of callus from four varieties of barley (Hordeumvulgare L.)[J]. Plant Cell Reports, 1987, 6(6):454-457
[33] Danilova S A, Dolgikh Y I. The stimulatory effect of the antibiotic cefotaxime on plant regeneration in maize tissue culture[J]. Russian Journal of Plant Physiology, 2004, 51(4):559-562
[34] Bhau B S, Wakhlu A K. Effect of some antibiotics on the in vitro morphogenetic response from callus cultures of Coryphantha elephantidens[J]. Biologia Plantarum, 2001, 44(1):19-24
[35] Ahmadi B, Shariatpanahi M E, Ojaghkandi M A, Azadi P, Keshavarz-Alizadeh M. Improved microspore embryogenesis induction and plantlet regeneration using putrescine, cefotaxime and vancomycin in Brassica napus L.[J]. Plant Cell Tissue and Organ Culture, 2014, 118(3):497-505
[36] 曾爱松, 宋立晓, 闫圆圆, 李健绮, 严继勇. 抗生素在甘蓝类蔬菜小孢子胚胎发生中的双重作用[J]. 核农学报, 2017, 31(3): 455-460
[37] 袁素霞, 刘玉梅, 方智远, 杨丽梅, 庄木, 张扬勇, 孙培田. 甘蓝类蔬菜小孢子再生植株染色体倍性与气孔保卫细胞叶绿体数的相关性[J]. 中国农业科学, 2009, 42(1): 189-197
[38] 王玉书, 王欢, 范震宇, 冯辉. 观赏羽衣甘蓝小孢子培养及再生植株倍性变异[J]. 核农学报, 2015, 29(6): 1037-1043
[2] Germanã M A. Gametic embryogenesis and haploid technology as valuable support to plant breeding[J]. Plant Cell Reports, 2011, 30(5):839-857
[3] 侯喜林. 不结球白菜育种研究新进展[J].南京农业大学学报, 2003, 26(4):111-115
[4] Lichter R. Induction of haploid plants from isolated pollen of Brassica napus[J]. Zeitschrift Für Pflanzenphysiologie, 1982, 105(5):427-434
[5] Chuong P Ⅴ, Beversdorf W D. High-frequency embryogenesis through isolated microspore culture in Brassica napus L. and B-carinata braun[J]. Plant Science, 1985, 39(3):219-226
[6] Sato T, Nishio T, Hirai M. Plant-regeneration from isolated microspore cultures of Chinese cabbage (Brassica campestris spp.pekinensis)[J]. Plant Cell Reports, 1989, 8(8):486-488
[7] Takahata Y, Keller W A. High-frequency embryogenesis and plant-regeneration in isolated microspore culture of Brassica oleracea L.[J]. Plant Science, 1991, 74(2):235-242
[8] Xu L, Najeeb U, Naeem M S, Wan G L, Jin Z L, Khan F, Zhou W J. In vitro Mutagenesis and Genetic Improvement[M]. New York: Springer, 2012:151-173
[9] Germana M A, Lambardi M. In vitro Embryogenesis in Higher Plants[M]. New York: Springer, 2016
[10] 原玉香, 张晓伟, 耿建峰, 蒋武生, 韩永平. 利用游离小孢子培养技术育成早熟大白菜新品种‘豫新60’[J]. 园艺学报, 2004, 31(5): 704-709
[11] 宋立晓, 曾爱松, 高兵, 严继勇. 青花菜新品种苏青3号的选育[J]. 江苏农业科学, 2014, 42(12): 216-218
[12] 曾爱松, 高兵, 宋立晓, 严继勇. 秋季专用牛心甘蓝新品种锦秋55的选育[J]. 江苏农业科学, 2015, 43(11): 230-231
[13] 万光龙. 基于甘蓝型油菜小孢子培养和胚状体诱导的植株再生、转化与诱变体系的构建[D]. 杭州: 浙江大学, 2010
[14] Lu Y, Dai S, Gu A, Liu M, Wang Y, Luo S, Zhao Y, Wang S, Xuan S, Chen X, Li X, Bonnema G, Zhao J, Shen S. Microspore induced doubled haploids production from ethyl methanesulfonate (EMS) soaked flower buds is an efficient strategy for mutagenesis in Chinese cabbage[J]. Frontiers in Plant Science, 2016, 7: 1780
[15] Verónica P V, Patricia C M, Alba R S, Seguí-Simarro J M. Formation and excretion of autophagic plastids (plastolysomes) in Brassica napus embryogenic microspores[J]. Frontiers in Plant Science, 2015, 6: 94
[16] Pilarska M, Malec P, Salaj J, Bartnicki F, Konieczny R. High expression of somatic embryogenesis receptor-like kinase coincides with initiation of various developmental pathways in in vitro culture of Trifolium nigrescens[J]. Protoplasma, 2016, 253(2): 345-355
[17] 蒋武生, 耿建峰, 张晓伟, 原玉香, 韩永平, 姚秋菊. 基因型和有机附加物对小白菜花药培养胚状体诱导的影响[J]. 中国瓜菜, 2006(2): 1-4
[18] 蒋武生, 原玉香, 张晓伟, 耿建峰, 韩永平, 郅玉宝. 小白菜游离小孢子培养及其再生植株[J]. 河南农业大学学报, 2005, 39(4): 40-43
[19] 蒋武生, 张晓伟, 姚秋菊, 原玉香, 耿建峰. 活性炭对小白菜游离小孢子培养的影响[J]. 华北农学报, 2008,23(5): 93-96
[20] 耿建峰, 侯喜林, 张晓伟, 蒋武生, 原玉香, 韩永平, 姚秋菊, 成妍, 李英. 影响白菜游离小孢子培养关键因素分析[J]. 园艺学报, 2007, 34(1): 111-116
[21] 高素燕, 李英, 单晓政, 侯喜林. 预处理对不结球白菜游离小孢子胚胎发生的影响[J]. 江苏农业学报, 2008, 24(6): 878-881
[22] 刘环环. 不结球白菜和青花菜游离小孢子培养技术的研究[D].南京: 南京农业大学, 2014
[23] 张振超, 耿鑫鑫, 戴忠良, 潘跃平, 王兵, 许玲, 颜志明, 周伟军. 甘蓝类植物小孢子培养及植株再生研究[J]. 核农学报, 2013, 27(7): 929-937
[24] Bhatia R, Dey S S, Sood S, Sharma K, Sharma V K, Parkash C, Kumar R. Optimizing protocol for efficient microspore embryogenesis and doubled haploid development in different maturity groups of cauliflower (B-oleracea var. botrytis L.) in India[J]. Euphytica, 2016, 212(3):439-454
[25] Gu H, Zhao Z, Sheng X, Yu H, Wang J. Efficient doubled haploid production in microspore culture of loose-curd cauliflower (Brassica oleracea var. botrytis)[J]. Euphytica, 2014, 195(3):467-475
[26] Shumilina D V, Shmykova N A, Bondareva L L, Suprunova T P. Effect of genotype and medium culture content on microspore-derived embryo formation in Chinese cabbage (Brassica rapa ssp. chinensis) Cv. Lastochka[J]. Biology Bulletin, 2015, 42(4):302-309
[27] 姜凤英, 冯辉, 王超楠. 小白菜小孢子培养胚状体的诱导[J]. 沈阳农业大学学报, 2006, 37(5): 763-765
[28] 毛忠良, 张振超, 姚悦梅, 戴忠良, 秦文斌, 潘跃平. 羽衣甘蓝小孢子胚胎发生观察及再生植株倍性鉴定[J]. 西北植物学报, 2012, 32(10): 2016-2022
[29] 成妍, 班青宇, 王倩, 侯喜林. 不结球白菜游离小孢子培养及再生植株的倍性鉴定[J]. 南京农业大学学报, 2009, 32(2): 25-29
[30] 陈琳, 刘克德, 吴江生, 孙秀丽, 汪频, 石淑稳. 中国白菜小孢子离体培养和双单倍体植株再生[J]. 中国农学通报, 2010, 26(14): 78-81
[31] Gu H H, Zhou W J, Hagberg P.High frequency spontaneous production of doubled haploid plants in microspore cultures of Brassica rapa ssp. Chinensis[J]. Euphytica, 2003, 134 (3): 239-245
[32] Mathias R J, Mukasa C. The effect of cefotaxime on the growth and regeneration of callus from four varieties of barley (Hordeumvulgare L.)[J]. Plant Cell Reports, 1987, 6(6):454-457
[33] Danilova S A, Dolgikh Y I. The stimulatory effect of the antibiotic cefotaxime on plant regeneration in maize tissue culture[J]. Russian Journal of Plant Physiology, 2004, 51(4):559-562
[34] Bhau B S, Wakhlu A K. Effect of some antibiotics on the in vitro morphogenetic response from callus cultures of Coryphantha elephantidens[J]. Biologia Plantarum, 2001, 44(1):19-24
[35] Ahmadi B, Shariatpanahi M E, Ojaghkandi M A, Azadi P, Keshavarz-Alizadeh M. Improved microspore embryogenesis induction and plantlet regeneration using putrescine, cefotaxime and vancomycin in Brassica napus L.[J]. Plant Cell Tissue and Organ Culture, 2014, 118(3):497-505
[36] 曾爱松, 宋立晓, 闫圆圆, 李健绮, 严继勇. 抗生素在甘蓝类蔬菜小孢子胚胎发生中的双重作用[J]. 核农学报, 2017, 31(3): 455-460
[37] 袁素霞, 刘玉梅, 方智远, 杨丽梅, 庄木, 张扬勇, 孙培田. 甘蓝类蔬菜小孢子再生植株染色体倍性与气孔保卫细胞叶绿体数的相关性[J]. 中国农业科学, 2009, 42(1): 189-197
[38] 王玉书, 王欢, 范震宇, 冯辉. 观赏羽衣甘蓝小孢子培养及再生植株倍性变异[J]. 核农学报, 2015, 29(6): 1037-1043
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