索邦和西伯利亚百合的离体再生体系建立
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摘要
【目的】确定索邦(Sorbonne)和西伯利亚(Siberia)百合的离体再生体系适合的诱导培养基、增殖培养基、生根培养基以及最佳转化受体系统。【方法】选用索邦和西伯利亚百合的鳞茎球作为外植体,比较它们在添加不同激素配比的培养基中的生长状况,得出两种百合诱导愈伤组织和不定芽、继代增殖、诱导生根培养基和对卡那霉素敏感的最适条件。【结果】索邦百合最佳诱导培养基是MS+2,4-D 1.5～2.0 mg/L,西伯利亚百合最佳诱导培养基为MS+2, 4-D 1.0～1.5 mg/L;两种百合的最佳继代增殖培养基均为MS+6-BA 0.5 mg/L+NAA 0.2mg/L:两种百合的最佳生根培养基为1/2MS+NAA 0.2 mg/L、1/2MS+IBA 0.2 mg/L;筛选转化植株时,质量浓度为150 mg/L的卡那霉素足以抑制非转化细胞或细菌的生长。【结论】为东方百合品种种球工厂化生产及遗传转化再生体系充实理论依据。
【Objective】The suitable induction medium, proliferation medium, rooting medium and the optimal transformation receptor system were determined for the in vitro regeneration systems of Lilium Sorbonne and Siberia.【Method】The bulbs of Sorbonne and Siberia were used as explants to compare their growth status in the medium with different hormone ratios. And the optimal conditions of callus and adventitious bud induction, subculture proliferation, induction/rooting medium and kanamycin sensitivity were obtained.【Result】The best induction medium for Sorbonne was MS+2,4-D 1.5-2.0 mg/L, while the best induction medium for Siberia was MS+2,4-D 1.0-1.5 mg/L. The optimal subculture proliferation medium for the two Lilium species was MS+6-BA 0.5 mg/L+NAA 0.2 mg/L. The optimal rooting media for the two Lilium species were 1/2 MS+NAA 0.2 mg/L and 1/2 MS+IBA 0.2 mg/L, respectively. When screening transformed plants, kanamycin at a mass concentration of 150 mg/L was sufficient to inhibit the growth of non-transformed cells or bacteria.【Conclusion】The theoretical basis was provided for the industrialized production and genetic transformation and regeneration system of Lilium oriental varieties.
【Objective】The suitable induction medium, proliferation medium, rooting medium and the optimal transformation receptor system were determined for the in vitro regeneration systems of Lilium Sorbonne and Siberia.【Method】The bulbs of Sorbonne and Siberia were used as explants to compare their growth status in the medium with different hormone ratios. And the optimal conditions of callus and adventitious bud induction, subculture proliferation, induction/rooting medium and kanamycin sensitivity were obtained.【Result】The best induction medium for Sorbonne was MS+2,4-D 1.5-2.0 mg/L, while the best induction medium for Siberia was MS+2,4-D 1.0-1.5 mg/L. The optimal subculture proliferation medium for the two Lilium species was MS+6-BA 0.5 mg/L+NAA 0.2 mg/L. The optimal rooting media for the two Lilium species were 1/2 MS+NAA 0.2 mg/L and 1/2 MS+IBA 0.2 mg/L, respectively. When screening transformed plants, kanamycin at a mass concentration of 150 mg/L was sufficient to inhibit the growth of non-transformed cells or bacteria.【Conclusion】The theoretical basis was provided for the industrialized production and genetic transformation and regeneration system of Lilium oriental varieties.
引文
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[11]郑洁,王有国,崔光芬,王祥宁,蒋亚莲,马璐琳,段青,杜文文,贾文杰.东方百合可育品种及其不育突变体花药形成的细胞学观察[J].西南农业学报,2015,28(3):1246-1250.doi:10.16213/j.cnki.scjas.2015.03.061.ZHENG J,WANG Y G,CUI G F,WANG X N,JIANG T L,MAL L,DUAN Q,DU W W,JIA W J.Cytological observation of anther formation of Lilium‘Oriental Hybrids’normal line and male sterile mutant line[J].Southwest China Journal of Agricultural Sciences,2015,28(3):1246-1250.doi:10.16213/j.cnki.scjas.2015.03.061
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[13]龙彬,廖晓珊,郑思乡,龚建华,邓雅文.株洲红东方百合种球繁育技术[J].湖南农业科学,2018(7):5-7.doi:10.16498/j.cnki.hnnykx.2018.07.002.LONG B,LIAO X S,ZHENG S X,GONG J H,DENG Y W.Breeding technology of Oriental Lily of Zhuzhou Red[J].Hunan Agricultural Sciences,2018(7):5-7.doi:10.16498/j.cnki.hnnykx.2018.07.002.
[14]农艳丰,杨美纯,宁云芬,周珊.东方百合“甜梦”花器官组织培养再生植株研究[J].农业研究与应用,2016(1):1-6.NOGN Y F,YANG M C,NIGN Y F,ZHOU S.Study on tissue culture and regeneration of floral organ of Oriental Lilium“Sweet dreams”[J].Agricultural Research and Application,2016(1):1-6.
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[16]闫海霞,蒋月喜,邓杰玲,黄昌艳,王晓国,何荆洲,卜朝阳.3种东方百合离体培养[J].南方农业学报,2016,47(6):889-894.doi:10.3969/j.issn.2095-1191.2016.06.889.YAN H X,JIANG Y X,DENG J L,HUANG C Y,WANG X G,HEJ Z,PU Z Y.In vitro culture for 3 varieties of Oriental Lily[J].Journal of Southern Agriculture,2016,47(6):889-894.doi:10.3969/j.issn.2095-1191.2016.06.889.
[17]辛轶.东方百合‘贝尼尼’体细胞胚的诱导及蓝色基因遗传转化的研究[D].杨凌:西北农林科技大学,2012.XIN T.Studies on somatic embryogenesis and genetic transformation with blue gene of oriental Lily'BERNINI'[D].Yangling:Northwest A&F University,2012.
[18]崔祺.花青素合成调节基因Bi/Cl转化东方百合‘索邦’的研究[D].北京:北京林业大学,2014.CUI Q.Transformation of Lilium Oriental Hybrid‘Sorbonne'with anthocyanin regulatory gene Bi/Cl[D].Beijing:Beijing Forestry University,2014.
[19]马惠,郭扶兴,周俊彦,罗新谈.百合叶片愈伤组织的诱导和植株再生[J].植物生理学报,1992(4):284.MA H,GUO F X,ZHOU J Y,LUO X T.Callus induction and plant regeneration in young leaves of Lilium concolor[J].Plant Physiology Communications,1992(4):284.
[20]余桂红,马鸿翔,周淼平,黄益洪,陆维忠.百合农杆菌介导的遗传转化受体系统的建立[J].江西农业学报,2004(2):15-19.doi:10.19386/j.cnki.jxnyxb.2004.02.004.YU G H,MA H X,ZHOU M P,HUANG Y H,LU W Z.Establishment of acceptor system of agrobacterium-mediated transformation of Lily[J].Acta Agriculturae Jiangxi,2004(2):15-19.doi:10.19386/j.cnki.jxnyxb.2004.02.004.
[21]唐东芹,钱虹妹,黄丹枫,唐克轩.百合基因转化直接分化受体系统的建立[J].江苏农业科学,2003(3):48-51.doi:10.15889/j.issn.1002-1302.2003.03.019.TANG D Q,QIAN H M,HUANG D F,TANG K X.Establishment of direct differentiation receptor system for Lily gene transformation[J].Jiangsu Agricultural Sciences,2003(3):48-51.doi:10.15889/j.issn.1002-1302.2003.03.019.
[2]周春华,尤超,陈凝华.百合组织培养研究进展[J].北方园艺,2013(14):193-195.ZHOU C H,YOU C,CHEN N H.Research advances on tissue culture of Lilium brownie[J].Northern Horticulture.2013(14):193-195.
[3]宛淑艳,柳美红.铁炮百合快速繁殖技术研究[J].广东农业科学,2013,40(23):31-34.doi:10.3969/j.issn.1004-874X.2013.23.009.WAN S Y,LIU M H.Study on the rapid propagation of Lilium formolongi[J].Guangdong Agricultural Sciences,2013,40(23):31-34.doi:10.3969/j.issn.1004-874X.2013.23.009.
[4]常琳,杜方,王丽婷,李兴桃,张鲜艳,樊俊苗.金石和伯爵百合鳞茎诱导及膨大研究[J].山西农业科学,2018,46(8):1279-1281,1370.CHANG L,DU F,WANG L T,LI X T,ZHANG X Y,FAN J M.Study on bulblet induction and enlargement of Golden Stone and Album Lily[J].Journal of Shanxi Agricultural Sciences,2018,46(8):1279-1281,1370.
[5]李彪,莫鸿伟,卜顺法,汤桂钧,黄雪龙.百合优质种球的无性繁殖技术研究[J].上海农业科技,2018(5):93-94.LI B,MO X W,BU S F,TANG G J,HUANG X L.Study on asexual propagation technology of Lily high quality germ balls[J].Shanghai Agricultural Science and Technology,2018(5):93-94.
[6]王丽婷.百合组培苗鳞茎诱导及膨大研究[D].太原:山西农业大学,2017.WAGN L T.Study on bulb induction and expansion of Lily by tissue culture[D].Taiyuan:Shanxi Agricultural University,2017.
[7]仇硕,唐凤鸾,夏科,李秀娟,赵志国,蒋庆鸿,赵健.龙牙百合增殖培养及鳞茎生长发育研究[J].广东农业科学,2017,44(4):61-66.doi:10.16768/j.issn.1004-874X.2017.04.010.CHOU S,TANG F L,XIA K,LI X J,ZHAO Z G,JIANG Q H,ZHAOJ.Propagation culture and bulbs development of Lilium brownii var.viridulum[J].Guangdong Agricultural Sciences,2017,44(4):61-66.doi:10.16768/j.issn.1004-874X.2017.04.010.
[8]王雅楠,薛莉,雷家军.新疆百合鳞片离体培养研究[J].东北农业大学学报,2017,48(4):30-35.doi:10.19720/j.cnki.issn.1005-9369.2017.04.005WANG Y L,XUE L,LEI J J.Study on culture in vitro of scales in Lilium martagon var.pilosiusculum[J].Journal of North-east Agricultural University,2017,48(4):30-35.doi:10.19720/j.cnki.issn.1005-9369.2017.04.005.
[9]吴昀.基于离体模式体系的百合小鳞茎发生发育机制研究[D].杭州:浙江大学,2016.WU Y.The mechanisms of Lily bulblet initiation and development based on the in vitro model system[D].Hangzhou:Zhejiang University,2016.
[10]于雪,张铭芳,封紫,袁晓娜,贾桂霞.6个东方百合品种的染色体核型分析[J].广东农业科学,2012,39(10):137-140,144.doi:10.3969/j.issn.1004-874X.2012.10.041.YU X,ZHANG M F,FENG Z,YUAN X N,JIA G X.Karyotype analysis on 6 cultivars of Oriental Lilies[J].Guangdong Agricultural Sciences,2012,39(10):137-140,144.doi:10.3969/j.issn.1004-874X.2012.10.041.
[11]郑洁,王有国,崔光芬,王祥宁,蒋亚莲,马璐琳,段青,杜文文,贾文杰.东方百合可育品种及其不育突变体花药形成的细胞学观察[J].西南农业学报,2015,28(3):1246-1250.doi:10.16213/j.cnki.scjas.2015.03.061.ZHENG J,WANG Y G,CUI G F,WANG X N,JIANG T L,MAL L,DUAN Q,DU W W,JIA W J.Cytological observation of anther formation of Lilium‘Oriental Hybrids’normal line and male sterile mutant line[J].Southwest China Journal of Agricultural Sciences,2015,28(3):1246-1250.doi:10.16213/j.cnki.scjas.2015.03.061
[12]朱庆松,徐函兵,刘秀青.东方百合索邦高效再生体系的建立[J].现代园艺,2018(15):7-10.doi:10.14051/j.cnki.xdyy.2018.15.003.ZHU Q S,XU H B,LIU X Q.Establishment of Sorbonne regeneration system of Oriental Lily[J].Xiandai Horticulture,2018(15):7-10.doi:10.14051/j.cnki.xdyy.2018.15.003.
[13]龙彬,廖晓珊,郑思乡,龚建华,邓雅文.株洲红东方百合种球繁育技术[J].湖南农业科学,2018(7):5-7.doi:10.16498/j.cnki.hnnykx.2018.07.002.LONG B,LIAO X S,ZHENG S X,GONG J H,DENG Y W.Breeding technology of Oriental Lily of Zhuzhou Red[J].Hunan Agricultural Sciences,2018(7):5-7.doi:10.16498/j.cnki.hnnykx.2018.07.002.
[14]农艳丰,杨美纯,宁云芬,周珊.东方百合“甜梦”花器官组织培养再生植株研究[J].农业研究与应用,2016(1):1-6.NOGN Y F,YANG M C,NIGN Y F,ZHOU S.Study on tissue culture and regeneration of floral organ of Oriental Lilium“Sweet dreams”[J].Agricultural Research and Application,2016(1):1-6.
[15]梁芳,蒋素华,崔波,李霞.两种东方系百合组织培养快繁技术[J].河南科学,2017,35(2):209-213.LIANG F,JIANG S H,CUI B,LI X.Tissue culture and rapid propagation of two Lilium Orential Hybird[J].Henan Science,2017,35(2):209-213.
[16]闫海霞,蒋月喜,邓杰玲,黄昌艳,王晓国,何荆洲,卜朝阳.3种东方百合离体培养[J].南方农业学报,2016,47(6):889-894.doi:10.3969/j.issn.2095-1191.2016.06.889.YAN H X,JIANG Y X,DENG J L,HUANG C Y,WANG X G,HEJ Z,PU Z Y.In vitro culture for 3 varieties of Oriental Lily[J].Journal of Southern Agriculture,2016,47(6):889-894.doi:10.3969/j.issn.2095-1191.2016.06.889.
[17]辛轶.东方百合‘贝尼尼’体细胞胚的诱导及蓝色基因遗传转化的研究[D].杨凌:西北农林科技大学,2012.XIN T.Studies on somatic embryogenesis and genetic transformation with blue gene of oriental Lily'BERNINI'[D].Yangling:Northwest A&F University,2012.
[18]崔祺.花青素合成调节基因Bi/Cl转化东方百合‘索邦’的研究[D].北京:北京林业大学,2014.CUI Q.Transformation of Lilium Oriental Hybrid‘Sorbonne'with anthocyanin regulatory gene Bi/Cl[D].Beijing:Beijing Forestry University,2014.
[19]马惠,郭扶兴,周俊彦,罗新谈.百合叶片愈伤组织的诱导和植株再生[J].植物生理学报,1992(4):284.MA H,GUO F X,ZHOU J Y,LUO X T.Callus induction and plant regeneration in young leaves of Lilium concolor[J].Plant Physiology Communications,1992(4):284.
[20]余桂红,马鸿翔,周淼平,黄益洪,陆维忠.百合农杆菌介导的遗传转化受体系统的建立[J].江西农业学报,2004(2):15-19.doi:10.19386/j.cnki.jxnyxb.2004.02.004.YU G H,MA H X,ZHOU M P,HUANG Y H,LU W Z.Establishment of acceptor system of agrobacterium-mediated transformation of Lily[J].Acta Agriculturae Jiangxi,2004(2):15-19.doi:10.19386/j.cnki.jxnyxb.2004.02.004.
[21]唐东芹,钱虹妹,黄丹枫,唐克轩.百合基因转化直接分化受体系统的建立[J].江苏农业科学,2003(3):48-51.doi:10.15889/j.issn.1002-1302.2003.03.019.TANG D Q,QIAN H M,HUANG D F,TANG K X.Establishment of direct differentiation receptor system for Lily gene transformation[J].Jiangsu Agricultural Sciences,2003(3):48-51.doi:10.15889/j.issn.1002-1302.2003.03.019.