连州菜心小孢子培养体系的建立
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摘要
游离小孢子培养技术是快速创制连州菜心双单倍体纯系的一条有效途径,为建立高效的连州菜心小孢子培养体系,应用于优势杂交育种实践,以引进的3个广东省连州市地方菜心品种迟熟连州菜心(17AY007)、连州尺寸心(17AY008)和连州尖叶迟菜心(17AY009)为试材进行小孢子培养,研究不同基因型和植物生长调节剂(芸薹素内酯,BR及噻苯隆,TDZ)对小孢子胚胎发生及植株再生的影响,并对获得的连州菜心小孢子再生植株进行倍性鉴定。结果表明:3个不同基因型连州菜心的小孢子胚胎发生率无显著差异;在NLN-13培养基中添加适宜浓度的BR或TDZ可显著提高连州菜心小孢子胚胎发生率和直接成苗率,其中,当BR浓度为0.04mg·L-1时,17AY007和17AY009的胚诱导率和直接成苗率均达到最高(2.38胚·蕾-1,72.82%;2.83胚·蕾-1,54.62%);当TDZ浓度为0.1mg·L-1和0.2mg·L-1时,17AY007的胚诱导率和直接成苗率分别达到最高(2.67胚·蕾-1,57.76%);TDZ浓度为0.3mg·L-1时,17AY009的胚诱导率和直接成苗率均达到最高(2.42胚·蕾-1,57.36%);3种连州菜心再生植株的双单倍体率均在56%以上,其中,17AY007双单倍体率最高,为64.29%。综上,创制了双单倍体植株42株,为连州菜心优势杂交育种奠定了材料基础。
Microspore culture is a method of creating doubled haploid lines of Lianzhou flowering Chinese cabbage. This study aims were to establish a highly efficient microspore culture protocol and applied it to the practice of heterosis breeding program.Three local varieties of flowering Chinese cabbage in Lianzhou City, Guangdong Province, were used as donor plants which were late-maturing Lianzhou flowering Chinese cabbage(17AY007), Lianzhou short flowering Chinese cabbage(17AY008) and Lianzhou late flowering Chinese cabbage with sharp leaves(17AY009). The effects of genotypes and plant growth regulators brassinolide(BR) and thidiazuron(TDZ) on microspore embryogenesis and plant regeneration were studied, and the ploidy of microspore regenerated plants was identified. The results showed that there was no significant difference in the frequency of microspore embryogenesis among the three genotypes of Lianzhou flowering Chinese cabbage. Adding appropriate concentration of BR or TDZ to NLN-13 medium could significantly increase the frequencies of microspore embryogenesis and direct conversion to plants.When the concentration of BR was 0.04 mg·L-1, both the frequencies of microspore embryogenesis and direct conversion to plants of 17AY007 and 17AY009 were the highest(2.38 embryos per bud,72.82%; 2.83 embryos per bud,54.62%). When the TDZ concentrations were 0.1 mg·L-1and 0.2 mg·L-1, the frequencies of microspore embryogenesis and direct conversion to plants of17AY007 reached the highest(2.67 embryos per bud,57.76%), respectively. When the TDZ concentration was 0.3 mg·L-1, the frequencies of microspore embryogenesis and direct conversion to plants of 17AY009 reached the highest(2.42 embryos per bud,57.36%). The doubled haploid rates of three varieties of regenerated plants were above 56%, and the doubled haploid rate of17AY007 was the highest(64.29%). 42 doubled haploid lines were obtained in this study, which were materials for heterosis breeding program for Lianzhou flowering Chinese cabbage.
Microspore culture is a method of creating doubled haploid lines of Lianzhou flowering Chinese cabbage. This study aims were to establish a highly efficient microspore culture protocol and applied it to the practice of heterosis breeding program.Three local varieties of flowering Chinese cabbage in Lianzhou City, Guangdong Province, were used as donor plants which were late-maturing Lianzhou flowering Chinese cabbage(17AY007), Lianzhou short flowering Chinese cabbage(17AY008) and Lianzhou late flowering Chinese cabbage with sharp leaves(17AY009). The effects of genotypes and plant growth regulators brassinolide(BR) and thidiazuron(TDZ) on microspore embryogenesis and plant regeneration were studied, and the ploidy of microspore regenerated plants was identified. The results showed that there was no significant difference in the frequency of microspore embryogenesis among the three genotypes of Lianzhou flowering Chinese cabbage. Adding appropriate concentration of BR or TDZ to NLN-13 medium could significantly increase the frequencies of microspore embryogenesis and direct conversion to plants.When the concentration of BR was 0.04 mg·L-1, both the frequencies of microspore embryogenesis and direct conversion to plants of 17AY007 and 17AY009 were the highest(2.38 embryos per bud,72.82%; 2.83 embryos per bud,54.62%). When the TDZ concentrations were 0.1 mg·L-1and 0.2 mg·L-1, the frequencies of microspore embryogenesis and direct conversion to plants of17AY007 reached the highest(2.67 embryos per bud,57.76%), respectively. When the TDZ concentration was 0.3 mg·L-1, the frequencies of microspore embryogenesis and direct conversion to plants of 17AY009 reached the highest(2.42 embryos per bud,57.36%). The doubled haploid rates of three varieties of regenerated plants were above 56%, and the doubled haploid rate of17AY007 was the highest(64.29%). 42 doubled haploid lines were obtained in this study, which were materials for heterosis breeding program for Lianzhou flowering Chinese cabbage.
引文
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[9]许玲,刘旦,何水平,等.“双高双低”油菜抗病新品种浙大619的选育与高产栽培技术研究[J].核农学报,2013,27(12):1839-1844.
[10]姚秋菊,张晓伟,王志勇,等.甘蓝新品种‘豫甘5号’的选育[J].中国瓜菜,2015,28(6):36-38.
[11]赵昌斌,邓武明,田露申,等.早熟高产优质杂交油菜新品种德恒油900的选育[J].贵州农业科学,2016,44(4):1-3.
[12]冯辉,杨硕,王超楠,等.青梗菜优异DH系的创制与利用[J].中国农业科学,2009,42(9):3195-3202.
[13]朱允华,刘明月,吴朝林.影响菜心游离小孢子培养的因素[J].长江蔬菜,2003(9):46-47.
[14]曾小玲,方淑桂,朱朝辉,等.不同基因型菜心游离小孢子培养和植株再生[J].热带作物学报,2014,35(12):2397-2402.
[15]姜凤英,冯辉.植物生长调节剂对羽衣甘蓝小孢子胚发生的影响[J].园艺学报,2006,33(3):642-644.
[16]章云,冯辉.大白菜×小白菜双单倍体永久作图群体的创建[J].沈阳农业大学学报,2012,43(4):425-429.
[17]付颖,杨硕,包美丽,等.小菘菜游离小孢子培养技术研究[J].中国蔬菜,2011(8):51-54.
[18]张琳,侯喜林,郝慧楠.高菜游离小孢子培养与植株再生[J].南京农业大学学报,2011,34(1):143-146.
[19]杨业正.TDZ与植物组织细胞培养[J].贵州农业科学,1991(6):43-46.
[20] BHATIA R,DEYS S,SOOD S,et al.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,2016,212:439-454.
[21] ZENGA S,YANY Y,YANJ Y,et al.Microspore embryogenesis and plant regeneration in Brussels sprouts(Brassica oleracea L.var. gemmifera)[J].Scientia Horticulturae,2015,191:31-37.
[22] WANGT T,LI H X,ZHANG J H,et al.Initiation and development of microspore embryogenesis in recalcitrant purple flowering stalk(Brassica campestris ssp. chinensis var. purpurea Hort.)genotypes[J].Scientia Horticulturae,2009,121:419-424.
[2] BELMONTE M,ELHITI M,WALDNER B,et al.Depletion of cellular brassinolide decreases embryo production and disrupts the architecture of the apicalmeristems in Brassica napus microspore-derived embryos[J].Journal of Experimental Botany,2010,61(10):2779-2794.
[3]王武,李秀兰,张献龙,等.油菜素内醋对陆地棉体细胞胚胎发生和根器官发生的影响[J].棉花学报,1994(6):83-88.
[4]高海娜.甘蓝子叶型胚状体诱导及其成苗因素的研究[D].杨凌:西北农林科技大学,2012.
[5]蒋武生,张晓伟,耿建峰,等.极早熟大白菜新品种豫新50的选育[J].中国瓜菜,2005(4):27-29.
[6]姚秋菊,蒋武生,原玉香,等.游离小孢子培养育成早熟大白菜新品种‘豫新58’[J].园艺学报,2008,35(6):929.
[7]徐学忠,胡靖锋,杨红丽,等.抗根肿病大白菜新品种CCR11242的选育[J].山东农业科学,2015,47(5):19-22.
[8]吴江生,石淑稳,周永明,等.甘蓝型双低油菜品种华双3号的选育和研究[J].华中农业大学学报,1999,18(1):1-4.
[9]许玲,刘旦,何水平,等.“双高双低”油菜抗病新品种浙大619的选育与高产栽培技术研究[J].核农学报,2013,27(12):1839-1844.
[10]姚秋菊,张晓伟,王志勇,等.甘蓝新品种‘豫甘5号’的选育[J].中国瓜菜,2015,28(6):36-38.
[11]赵昌斌,邓武明,田露申,等.早熟高产优质杂交油菜新品种德恒油900的选育[J].贵州农业科学,2016,44(4):1-3.
[12]冯辉,杨硕,王超楠,等.青梗菜优异DH系的创制与利用[J].中国农业科学,2009,42(9):3195-3202.
[13]朱允华,刘明月,吴朝林.影响菜心游离小孢子培养的因素[J].长江蔬菜,2003(9):46-47.
[14]曾小玲,方淑桂,朱朝辉,等.不同基因型菜心游离小孢子培养和植株再生[J].热带作物学报,2014,35(12):2397-2402.
[15]姜凤英,冯辉.植物生长调节剂对羽衣甘蓝小孢子胚发生的影响[J].园艺学报,2006,33(3):642-644.
[16]章云,冯辉.大白菜×小白菜双单倍体永久作图群体的创建[J].沈阳农业大学学报,2012,43(4):425-429.
[17]付颖,杨硕,包美丽,等.小菘菜游离小孢子培养技术研究[J].中国蔬菜,2011(8):51-54.
[18]张琳,侯喜林,郝慧楠.高菜游离小孢子培养与植株再生[J].南京农业大学学报,2011,34(1):143-146.
[19]杨业正.TDZ与植物组织细胞培养[J].贵州农业科学,1991(6):43-46.
[20] BHATIA R,DEYS S,SOOD S,et al.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,2016,212:439-454.
[21] ZENGA S,YANY Y,YANJ Y,et al.Microspore embryogenesis and plant regeneration in Brussels sprouts(Brassica oleracea L.var. gemmifera)[J].Scientia Horticulturae,2015,191:31-37.
[22] WANGT T,LI H X,ZHANG J H,et al.Initiation and development of microspore embryogenesis in recalcitrant purple flowering stalk(Brassica campestris ssp. chinensis var. purpurea Hort.)genotypes[J].Scientia Horticulturae,2009,121:419-424.
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