叶用芥菜小孢子培养技术体系的完善及DH系创制
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摘要
以3个优良的地方品种(杂合体)为试材进行游离小孢子培养,对胚诱导、植株再生、倍性分析和单倍体加倍技术进行研究,完善叶用芥菜小孢子培养技术体系,创制优良DH系应用于育种实践中。在小孢子培养中,选取花蕾是小孢子培养成功的关键,花瓣长/花药长为3/4,花药为黄绿色可作为叶用芥菜小孢子培养选取花蕾的形态指标;供试3个品种都获得胚状体,但品种间出胚差异较大,MR-1共45个胚/皿,MR-2共198个胚/皿,MR-3共4个胚/皿;子叶形胚接种于再生培养基上成株率达98%,获得128株再生植株,利用流式细胞仪鉴定出105株双单倍体,自然加倍率为82%,在组培苗期用0.1%的秋水仙素浸泡单倍体植株茎尖1h,加倍率为75%。从DH群体中筛选出4个优良DH系,通过品种比较试验鉴选出1个耐抽薹DH系,4月上旬开始抽薹开花,叶柄宽大肥厚,产量高,适合鲜食加工,可补充当地春淡市场。
The microspores of 3 leaf mustard genotypes(MR-1,MR-2 and MR-3)were cultured to research embryos induction,plant regeneration,ploidy analysis,haploid doubling,optimize the isolated microspore culture technical system of leaf mustard and create excellent DH lines of leaf mustard for production practice.Buds selection was crucial to microspore culture.The morphological index of buds were:Petal length∶anther length was 3∶4;its color was yellow green.Embryos were obtained for the 3 cultivars.There were differences of the number of embryos generating between varieties and MR-1,MR-2 and MR-3 were 45,198 and 4 per dish, respectively.The natural doubling rate of leaf mustard chromosomes was 82% by flow cytometry and the haploid doubling rate was 75% with 0.1% colchicine soaking stem tip 1 hour in tissue culture seedling.Cotyledon-stage embryos were cultivated on regeneration culture and128 plants were obtained and the rate of plant formation reached 98%.A total of 105 double haploids were identified by flow cytometer and 4 excellent DH lines were screened from them.A double haploid line was supplied to the spring market for postponed bolting date,thick petioles and high yield and it was suitable for fresh food processing.
The microspores of 3 leaf mustard genotypes(MR-1,MR-2 and MR-3)were cultured to research embryos induction,plant regeneration,ploidy analysis,haploid doubling,optimize the isolated microspore culture technical system of leaf mustard and create excellent DH lines of leaf mustard for production practice.Buds selection was crucial to microspore culture.The morphological index of buds were:Petal length∶anther length was 3∶4;its color was yellow green.Embryos were obtained for the 3 cultivars.There were differences of the number of embryos generating between varieties and MR-1,MR-2 and MR-3 were 45,198 and 4 per dish, respectively.The natural doubling rate of leaf mustard chromosomes was 82% by flow cytometry and the haploid doubling rate was 75% with 0.1% colchicine soaking stem tip 1 hour in tissue culture seedling.Cotyledon-stage embryos were cultivated on regeneration culture and128 plants were obtained and the rate of plant formation reached 98%.A total of 105 double haploids were identified by flow cytometer and 4 excellent DH lines were screened from them.A double haploid line was supplied to the spring market for postponed bolting date,thick petioles and high yield and it was suitable for fresh food processing.
引文
[1]邓英,宋明,吴康云,郭惊涛,孟平红.不同叶用芥菜品种营养成分分析[J].中国蔬菜,2010,1(2):42-45Deng Y,Song M,Wu K Y,Guo J T,Meng P H.Analysis of nutririve components in different leaf mustard[Brassica juncea L Czern et coss]varieties[J].China Vegetables,2010,1(2):42-45(in Chinese)
[2]栗根义,高睦枪,赵秀山.大白菜游离小孢子培养[J].园艺学报,1993,20(2):167-170Li G Y,Gao M Q,Zhao X S.Isolated microspore culture of Chinese cabbage(Brassica campestris ssp pekinensis)[J].Acta Horticulturae Sinica,1993,20(2):167-170(in Chinese)
[3]栗根义,高睦枪,杨建平,徐小利,史宣杰,蒋武生,张晓伟,耿建峰,龙云铭.利用游离小孢子培养技术育成豫白菜7号(豫园1号)[J].中国蔬菜,1998,1(4):16-19Li G Y,Gao M Q,Yang J P,Xu X L,Shi X J,Jiang W S,Zhang X W,Geng J F,Long Y M.A New Chinese cabbage F1 Hybrid Yubaicai No7 developed by isolated microspore culture[J].China Vegetables,1998,1(4):16-19(in Chinese)
[4]耿建峰,原玉香,张晓伟,蒋武生,韩永平,高睦枪,栗根义.利用游离小孢子培养育成早熟大白菜新品种“豫新5号”[J].园艺学报,2003,30(2):249Geng J F,Yuan Yu X,Zhang X W,Jiang W S,Han Y P,Gao M Q,Li G Y.A new Chinese Cabbage-Yuxin 5[J].Acta Horticulturae Sinica,2003,30(2):249(in Chinese)
[5]姚秋菊,蒋武生,原玉香,耿建峰,张晓伟,王志勇.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):929Yao Q J,Jiang W S,Yuan Y X,Geng J F,Zhang X W,Wang Z Y.A new early:Maturing Chinese Cabbage cultivar by isolated microspore Culture‘Yuxin 58’[J].Acta Horticulturae Sinica,2008,35(6):929(in Chinese)
[6]张凤兰,张德双,余阳俊,徐家炳,赵岫云,于拴仓,卢桂香.早熟青麻叶大白菜新品种京翠60号的选育[J].中国蔬菜,2011,1(2):92-93Zhang F L,Zhang D S,Yu Y J,Xu J B,Zhao X Y,Yu S C,Lu G X.A new chinese cabbage F1hybrid-‘Jingcui No.60’[J].China Vegetables,2011,1(2):92-93(in Chinese)
[7]曾爱松,宋立晓,高兵,严继勇.秋季专用平头甘蓝新品种锦秋60的选育[J].长江蔬菜,2015,4(4):10-12Zeng A S,Song L X,Gao B,Yan J Y.Breeding of Jinqiu 60,a new cabbage F1hybrid with flattened-shaped head in autumn[J].Journal of Chang Jiang Vegetables,2015,4(4):10-12(in Chinese)
[8]姚秋菊,张晓伟,王志勇,魏小春,原玉香,蒋武生,赵艳艳,张强.甘蓝新品种‘豫甘5号’的选育[J].中国瓜菜,2015,28(6):36-38Yao Q J,Zhang X W,Wang Z Y,Wei X C,Yuan Y X,Jiang W S,Zhao Y Y,Zhang Q.A new cabbage cultivar‘Yugan No5[J].China Cucurbits and Vegetables,2015,28(6):36-38(in Chinese)
[9]胡永霞,李晓峰,轩淑欣,申书兴,王彦华.利用小孢子培养创建大白菜:结球甘蓝易位系[J].园艺学报,2014,41(7),1361-1368Hu Y X,Li X F,Xuan S X,Shen S X,Wang Y H.Obtaining of one Chinese cabbage-cabbage translocation lines by microspore culture[J].Acta Horticulturae Sinica,2014,41(7):1361-1368(in Chinese)
[10]Prem D,Gupta K,Abha A.Effect of exogenous and endogenous factors on microspore embryogenesis in indian mustard(Brassica Juncea(L)czern and coss)[J].Vitro Cellular&Development Biology Plant,2005,41(3):266-273
[11]Agarwal P K,Agarwal P,Custers J B M,Liu C M,Bhojwani S S.PCIB an antiauxin enhances microspore embryogenesis in microspore culture of Brassica juncea[J].Plant Cell Tissue&Organ Culture,2006,86(2):201-210
[12]Prem D,Gupta K,Agnihotri A.Harnessing mutant donor plants for microspore culture in Indian mustard[Brassica juncea(L)Czern and Coss][J].Euphytica,2012,184(2):207-222
[13]吴康云,陶莲,李正丽,邓英,孟平红,赵大芹.叶用芥菜游离小孢子的培养及其植株再生[J].贵州农业科学,2011,39(8):9-10Wu K Y,Tao L,Li Z L,Deng Y,Meng P H,Zhao D Q.Microspore culture and plantlet regeneration in leaf mustard Brassica Juncea[J].Guizhou Agricultural Sciences,2011,39(8):9-10(in Chinese)
[14]吴慧敏,何勇,邵泱峰,朱祝军,符庆功.叶用芥菜小孢子离体培养条件[J].浙江农林大学学报,2014,31(2):315-321Wu H M,He Y,Shao Y F,Zhu Z J,Fu Q G.In vitro microspore culture of leaf mustard[J].Journal of Zhejiang A&F University,2014,31(2):315-321(in Chinese)
[15]顾祥昆,李菲,张淑江,章时蕃,张慧,孙日飞.芥菜游离小孢子培养技术研究[J].中国蔬菜,2013,(12):23-30Gu X K,Li F,Zhang S J,Zhang S F,Zhang H,Sun R F.Studies on free microspore culturing technology in Brassica juncea(L)Czern et Coss[J].China Vegetables,2013(12):23-30(in chinese)
[16]刘冬,郭平仲,刘凡,曹鸣庆.芥菜(Brassica juncea L)小孢子胚发生和植株再生[J].首都师范大学学报,1997,18(1):76-80Liu D,Guo P Z,Liu F,Cao M Q.Embryogenesis and plant regeneration via isolated mierospore culture in Brassica juncea L[J].Journal of Capital Normal University,1997,18(1):76-80(in Chinese)
[17]Ali M M,Khaleque M A,Custers J B M,Kurram M M H.Microspore culture and the performance of microspore derived doubled haploid in Brassica juncea(L)[J].Bangladesh Journal of Agricultural Research,2008,33(4):571-578
[18]陈玉萍,田志宏,陈爱武,孟金陵.包心芥菜游离小孢子培养的初步研究[J].华中农业大学学报,1998,17(1):93-95Chen Y P,Tian Z H,Chen A W,Meng J L.Study on microspore culuture of Brassica Juncea var Capitata[J].Jounal of Huazhong Agicultural University,1998,17(1):93-95(in Chinese)
[19]虞慧芳,钟新民,顾宏辉,李必元,张晓辉.航天搭载雪里蕻小孢子培养获得再生植株[J].浙江农业学报,2007,19(5):356-359Yu H F,Zhong X M,Gu H H,Li B Y,Zhang X H.Obtaining microspore cultured plants in potherb mustard,Brassica juncea var multiceps,by space-breeding satellite[J].ACTA Agriculture Zhejiangensis,2007,19(5):356-359(in Chinese)
[20]王长龙,杨培新,郑奕雄.结球芥菜游离小孢子培养的胚状体发生[J].江西农业学报,2011,23(11):31-33Wang C L,Yang P X,Zheng Y X.Embryogenesis in culture of isolated mierospores of Brassica juncea var Capitat[J].Aeta Agricuhurae Jiangxi,2011,23(11):31-33(in Chinese)
[21]曾柳,胡承伟,张学昆,程勇,陆光远,邹锡玲.秋水仙碱对油菜小孢子产胚率和再生植株二倍体率的影响.中国油料作物学报[J].2016,38(2):254-259Zeng L,Hu C W,Zhang X K,Cheng Y,Lu G Y,Zou X L.Colchicine effects on microspore embryo yield and diploid regeneration in rapeseed[J].Chinese Journal of Oil Crop Sciences,2016,38(2):254-259(in Chinese)
[2]栗根义,高睦枪,赵秀山.大白菜游离小孢子培养[J].园艺学报,1993,20(2):167-170Li G Y,Gao M Q,Zhao X S.Isolated microspore culture of Chinese cabbage(Brassica campestris ssp pekinensis)[J].Acta Horticulturae Sinica,1993,20(2):167-170(in Chinese)
[3]栗根义,高睦枪,杨建平,徐小利,史宣杰,蒋武生,张晓伟,耿建峰,龙云铭.利用游离小孢子培养技术育成豫白菜7号(豫园1号)[J].中国蔬菜,1998,1(4):16-19Li G Y,Gao M Q,Yang J P,Xu X L,Shi X J,Jiang W S,Zhang X W,Geng J F,Long Y M.A New Chinese cabbage F1 Hybrid Yubaicai No7 developed by isolated microspore culture[J].China Vegetables,1998,1(4):16-19(in Chinese)
[4]耿建峰,原玉香,张晓伟,蒋武生,韩永平,高睦枪,栗根义.利用游离小孢子培养育成早熟大白菜新品种“豫新5号”[J].园艺学报,2003,30(2):249Geng J F,Yuan Yu X,Zhang X W,Jiang W S,Han Y P,Gao M Q,Li G Y.A new Chinese Cabbage-Yuxin 5[J].Acta Horticulturae Sinica,2003,30(2):249(in Chinese)
[5]姚秋菊,蒋武生,原玉香,耿建峰,张晓伟,王志勇.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):929Yao Q J,Jiang W S,Yuan Y X,Geng J F,Zhang X W,Wang Z Y.A new early:Maturing Chinese Cabbage cultivar by isolated microspore Culture‘Yuxin 58’[J].Acta Horticulturae Sinica,2008,35(6):929(in Chinese)
[6]张凤兰,张德双,余阳俊,徐家炳,赵岫云,于拴仓,卢桂香.早熟青麻叶大白菜新品种京翠60号的选育[J].中国蔬菜,2011,1(2):92-93Zhang F L,Zhang D S,Yu Y J,Xu J B,Zhao X Y,Yu S C,Lu G X.A new chinese cabbage F1hybrid-‘Jingcui No.60’[J].China Vegetables,2011,1(2):92-93(in Chinese)
[7]曾爱松,宋立晓,高兵,严继勇.秋季专用平头甘蓝新品种锦秋60的选育[J].长江蔬菜,2015,4(4):10-12Zeng A S,Song L X,Gao B,Yan J Y.Breeding of Jinqiu 60,a new cabbage F1hybrid with flattened-shaped head in autumn[J].Journal of Chang Jiang Vegetables,2015,4(4):10-12(in Chinese)
[8]姚秋菊,张晓伟,王志勇,魏小春,原玉香,蒋武生,赵艳艳,张强.甘蓝新品种‘豫甘5号’的选育[J].中国瓜菜,2015,28(6):36-38Yao Q J,Zhang X W,Wang Z Y,Wei X C,Yuan Y X,Jiang W S,Zhao Y Y,Zhang Q.A new cabbage cultivar‘Yugan No5[J].China Cucurbits and Vegetables,2015,28(6):36-38(in Chinese)
[9]胡永霞,李晓峰,轩淑欣,申书兴,王彦华.利用小孢子培养创建大白菜:结球甘蓝易位系[J].园艺学报,2014,41(7),1361-1368Hu Y X,Li X F,Xuan S X,Shen S X,Wang Y H.Obtaining of one Chinese cabbage-cabbage translocation lines by microspore culture[J].Acta Horticulturae Sinica,2014,41(7):1361-1368(in Chinese)
[10]Prem D,Gupta K,Abha A.Effect of exogenous and endogenous factors on microspore embryogenesis in indian mustard(Brassica Juncea(L)czern and coss)[J].Vitro Cellular&Development Biology Plant,2005,41(3):266-273
[11]Agarwal P K,Agarwal P,Custers J B M,Liu C M,Bhojwani S S.PCIB an antiauxin enhances microspore embryogenesis in microspore culture of Brassica juncea[J].Plant Cell Tissue&Organ Culture,2006,86(2):201-210
[12]Prem D,Gupta K,Agnihotri A.Harnessing mutant donor plants for microspore culture in Indian mustard[Brassica juncea(L)Czern and Coss][J].Euphytica,2012,184(2):207-222
[13]吴康云,陶莲,李正丽,邓英,孟平红,赵大芹.叶用芥菜游离小孢子的培养及其植株再生[J].贵州农业科学,2011,39(8):9-10Wu K Y,Tao L,Li Z L,Deng Y,Meng P H,Zhao D Q.Microspore culture and plantlet regeneration in leaf mustard Brassica Juncea[J].Guizhou Agricultural Sciences,2011,39(8):9-10(in Chinese)
[14]吴慧敏,何勇,邵泱峰,朱祝军,符庆功.叶用芥菜小孢子离体培养条件[J].浙江农林大学学报,2014,31(2):315-321Wu H M,He Y,Shao Y F,Zhu Z J,Fu Q G.In vitro microspore culture of leaf mustard[J].Journal of Zhejiang A&F University,2014,31(2):315-321(in Chinese)
[15]顾祥昆,李菲,张淑江,章时蕃,张慧,孙日飞.芥菜游离小孢子培养技术研究[J].中国蔬菜,2013,(12):23-30Gu X K,Li F,Zhang S J,Zhang S F,Zhang H,Sun R F.Studies on free microspore culturing technology in Brassica juncea(L)Czern et Coss[J].China Vegetables,2013(12):23-30(in chinese)
[16]刘冬,郭平仲,刘凡,曹鸣庆.芥菜(Brassica juncea L)小孢子胚发生和植株再生[J].首都师范大学学报,1997,18(1):76-80Liu D,Guo P Z,Liu F,Cao M Q.Embryogenesis and plant regeneration via isolated mierospore culture in Brassica juncea L[J].Journal of Capital Normal University,1997,18(1):76-80(in Chinese)
[17]Ali M M,Khaleque M A,Custers J B M,Kurram M M H.Microspore culture and the performance of microspore derived doubled haploid in Brassica juncea(L)[J].Bangladesh Journal of Agricultural Research,2008,33(4):571-578
[18]陈玉萍,田志宏,陈爱武,孟金陵.包心芥菜游离小孢子培养的初步研究[J].华中农业大学学报,1998,17(1):93-95Chen Y P,Tian Z H,Chen A W,Meng J L.Study on microspore culuture of Brassica Juncea var Capitata[J].Jounal of Huazhong Agicultural University,1998,17(1):93-95(in Chinese)
[19]虞慧芳,钟新民,顾宏辉,李必元,张晓辉.航天搭载雪里蕻小孢子培养获得再生植株[J].浙江农业学报,2007,19(5):356-359Yu H F,Zhong X M,Gu H H,Li B Y,Zhang X H.Obtaining microspore cultured plants in potherb mustard,Brassica juncea var multiceps,by space-breeding satellite[J].ACTA Agriculture Zhejiangensis,2007,19(5):356-359(in Chinese)
[20]王长龙,杨培新,郑奕雄.结球芥菜游离小孢子培养的胚状体发生[J].江西农业学报,2011,23(11):31-33Wang C L,Yang P X,Zheng Y X.Embryogenesis in culture of isolated mierospores of Brassica juncea var Capitat[J].Aeta Agricuhurae Jiangxi,2011,23(11):31-33(in Chinese)
[21]曾柳,胡承伟,张学昆,程勇,陆光远,邹锡玲.秋水仙碱对油菜小孢子产胚率和再生植株二倍体率的影响.中国油料作物学报[J].2016,38(2):254-259Zeng L,Hu C W,Zhang X K,Cheng Y,Lu G Y,Zou X L.Colchicine effects on microspore embryo yield and diploid regeneration in rapeseed[J].Chinese Journal of Oil Crop Sciences,2016,38(2):254-259(in Chinese)