基于体细胞胚发生的细叶百合和兰州百合多倍体诱导及鉴定
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摘要
以细叶百合(Lilium pumilum DC.Fisch.)和兰州百合(Lilium davidii var.unicolor)的鳞片和胚性愈伤组织作为诱导材料,用不同浓度秋水仙素以浸泡和混培两种处理方式进行多倍体诱导。以形态学观测、根尖染色体计数、气孔观测对多倍体植株进行倍性鉴定,分别获得了19株细叶百合四倍体植株和6株兰州百合四倍体植株,最佳处理方式为0.1%秋水仙素浸泡胚性愈伤组织24 h。使用ISSR对经体细胞胚发生的二倍体和多倍体再生植株进行遗传分析,发现两种百合二倍体植株遗传较稳定,而多倍体植株均发生遗传变异,其中细叶百合和兰州百合遗传变异率分别为15.48%和9.75%。
Using scales and embryogenic callus as the target tissues,Lilium pumilum DC. Fisch. and Lilium davidii var. unicolor were accomplished the ployploid inducement by soaking and mixed culture with different concentrations of colchicine. Nineteen tetraploids of Lilium pumilum DC. Fisch. and 6 tetraploids of Lilium davidii var. unicolor have been identified,through morphological observation,root tip chromosome count and stomatal observation. The optimum condition is immersing embryogenic callus with 0.1% colchicine for 24 hours to achieve the highest efficiency of chromosome doubling. Dipold plants of both lilies are genetically stable while polyploid plants experience the genetic variation by using ISSR to conduct the genetic analysis of plants via somatic embryogenesis. The variation rates of polyploid in Lilium pumilum DC. Fisch. and Lilium davidii var. unicolor are 15.48% and 9.75%,respectively.
Using scales and embryogenic callus as the target tissues,Lilium pumilum DC. Fisch. and Lilium davidii var. unicolor were accomplished the ployploid inducement by soaking and mixed culture with different concentrations of colchicine. Nineteen tetraploids of Lilium pumilum DC. Fisch. and 6 tetraploids of Lilium davidii var. unicolor have been identified,through morphological observation,root tip chromosome count and stomatal observation. The optimum condition is immersing embryogenic callus with 0.1% colchicine for 24 hours to achieve the highest efficiency of chromosome doubling. Dipold plants of both lilies are genetically stable while polyploid plants experience the genetic variation by using ISSR to conduct the genetic analysis of plants via somatic embryogenesis. The variation rates of polyploid in Lilium pumilum DC. Fisch. and Lilium davidii var. unicolor are 15.48% and 9.75%,respectively.
引文
Alsi?a I,Duma M,Dubova L,?enberga A,Da?is S.2016 Comparison of different chlorophylls determination methods for leafy vegetables.Agronomy Research,14(2):309-316.
Bekheet S A,Gabr A M M,Reda A A,El-Bahr M K.2015.Micropropagation and assessment of genetic stability of in vitro raised jojoba(Simmondsia chinensis Link.)plants using SCo T and ISSR markers.Plant Tiss Cult Biotech,25(2):165-179.
Cabral G B,Carneiro V T C,Dusi D,Martinelli A P.2016.Somatic embryogenesis and plant regeneration of Brachiaria brizantha.Meth Mol Bio,1359:395-402.
Eeckhaut T G R,Werbrouck S P O,Leus L W H,Debergh C P.2004.Chemically induced polyploidization in Spathiphyllum wallisii Regel through somatic embryogenesis.Plant Cell Tiss Org Cul,78(3):241-246.
Endemann M,Hristoforoglu K,Stauber T,Wilhelm E.2001.Assessment of age-related polyploidy in Quercus robur L.somatic embryos and regenerated plants using DNA flow cytometry.Bio Plantarum,44(3):339-345.
Ge Zhen-yang,Jiang Li-min,Yu Ying-jie,Li Peng,Yi Huai-feng,Li Li-cheng.2013.Growth modelling and visualization simulation of lily bulblet.Trans Chin Soci Agr Mach,44(3):209-214.(in Chinese)戈振扬,姜利民,于英杰,李鹏,易怀峰,李立成.2013.百合鳞茎的生长建模与可视化模拟.农业机械学报,44(3):209-214.
Guo L,Xu W,Zhang Y,Wei Z.2017.Inducing triploids and tetraploids with high temperatures in Populus sect.Tacamahaca.Plant Cell Rep,36(2):1-14.
Hassan H A,Wang H P,Shen D,Qiu Y,Li X X.2015.Sprout differentiation and mutation induction of garlic(Allium sativum L.)callus exposed to gamma radiation.Plant Growth Regul,75:465-471.
He M,Gao W J,Gao Y H,Liu Y Z,Yang X,Jiao H B,Zhou Y W.2016a.Polyploidy induced by colchicine in Dendranthema indicum var.aromaticum,a scented chrysanthemum.Eur J Hortic Sci,81(4):219-226.
He Y H,Sun Y,Zheng R,Ye A I,Cao Z,Bao M Z.2016b.Induction of tetraploid male sterile Tagetes erecta by colchicine treatment and its application for interspecific hybridization.Horticul Plant J,2(5):284-292.
Jin L,Zhang Y,Yan L,Guo Y L,Niu L X.2012.Phenolic compounds and antioxidant activity of bulb extracts of six Lilium species native to China.Molecules,17:9361-9378.
Khateeb W A,Bahar E,Lahham J,Schroeder D,Hussein E.2013.Regeneration and assessment of genetic fidelity of the endangered tree Moringa peregrina(Forsk.)Fiori using Inter Simple Sequence Repeat(ISSR).Physiol Mol Biol Plants,19(1):157-164.
Leila A S P,Moacir P,Sebastiao D O E S,Herminio S R,Helida M M,Janay A S S.2014.Inducing and identifying artificially-induced polyploidy in bananas.Afr J Bio,13(37):3748-3758.
Leroy X J,Leon K,Charles G,Branchard M.2000.Cauliflower somatic embryogenesis and analysis of regenerant stability by ISSRs.Plant Cell Rep,19:1102-1107.
Levin D A.1983.Polyploidy and novelty in flowering plants.Am Nat,122(1):1-25.
Li Dan,Luo Yi-ran,Han Guo-wei,Zhang Xue,Wu Ying-ying,He Cheng-zhong.2017.Studies on polyploidy induction of wild Lilium nepalense D.Don.J Yunnan Agri Uni(Natural Science),32(4):678-684.(in Chinese)李旦,罗一然,韩国伟,张雪,吴英英,何承忠.2017.野生紫斑百合多倍体诱导研究.云南农业大学学报(自然科学),32(4):678-684.
Li Xiao-shuang,Wang Qing,Meng Yu-cheng,Xiang Zhong-huai,He Ning-jia.2017.Polyploidy induction and identification of wild germplasm resource Yun 7 of Morus wittiorum Hand-Mazz.Sci Sericulture,43(3):369-373.(in Chinese)李晓双,王青,孟钰程,向仲怀,何宁佳.2017.野生长穗桑种质资源云7的多倍体诱导和鉴定.蚕业科学,43(3):369-373.
Li Yan-na,Wei Yi-ming,Hu Gui-bing,Chen Hou-bin,Xu Chun-xiang.2010.Plant regeneration via somatic embryogenesis after cryopreservation of embryogenic cell suspensions of banana(Musa spp.AAA)by vitrification and the genetic stability of regenerated plant.Acta Horticulturae Sinica,37(6):899-905.(in Chinese)李艳娜,尉义明,胡桂兵,陈厚彬,徐春香.2010.香蕉胚性细胞悬浮系玻璃化法超低温保存及再生植株遗传稳定性分析.园艺学报,37(6):899-905.
Loureiro J,Pinto G,Lopes T,Dolezel J,Santos C.2005.Assessment of ploidy stability of the somatic embryogenesis process in Quercus suber L.using flow cytometry.Planta,221:815-822.
Mallick P,Chattaraj S,Sikdar S R.2017.Molecular characterizations of somatic hybrids developed between Pleurotus florida and Lentinus squarrosulus through inter-simple sequence repeat markers and sequencing of ribosomal RNA-ITS gene.Biotech,7(5):298.
Marzougui N,Elfalleh W,Boubaya A,Guasmi F,Ferchichi A,Lachieheb B.2010.Effect of polyploidy on ISSR molecular profile and the contents of vitamins and proteins in Trigonella foenum-graecum L.Acta Bot Gallica,157(1):89-100.
Mcgregor C E,Lambert C A,Greyling M M,Louw J H,Warnich L.2000.A comparative assessment of DNA fingerprinting techniques(RAPD,ISSR,AFLP and SSR)in tetraploid potato(Solanum tuberosum L.)germplasm.Euphytica,113(2):135-144.
Nakel T,Tekleyohans D G,Mao Y,Fuchert G,Vo D,GroβR.2017.Triparental plants provide direct evidence for polyspermy induced polyploidy.Nat Commun,8(1):10-33.
Naz R,Anis M,Alatar A A.2016.ISSR marker-based detection of genomic stability in Cassia occidentalis L.plantlets derived from somatic embryogenesis.Eng Life Sci,16:17-24.
Noori S A S,Norouzi M,Karimzadeh G,Shirkool K,Niazian M.2017.Effect of colchicine-induced polyploidy on morphological characteristics and essential oil composition of ajowan(Trachyspermum ammi L.).Plant Cell Tiss Org,130(7):1-9.
Pitschmann A,Purevsuren S,Obmann A,Natsagdorj D,Gunbing S,Narantuya Ch,Kletter S.2013.Traditional mongolian medicine:history and status quo.Phytochem Rev,12(4):943-959.
Rai M K,Phulwaria M H,Gupta A K,Shekhawat N S,Jaiswal U.2012.Genetic homogeneity of guava plants derived from somatic embryogenesis using SSR and ISSR markers.Plant Cell Tiss Org Cult,111:259-264.
Sattler M C,Carvalho C R,Clarindo W R.2016.The polyploidy and its key role in plant breeding.Planta,243(2):281-296.
Soltis P S,Soltis D E.2016.Ancient WGD events as drivers of key innovations in angiosperms.Curr Opin Plant Bio,30:159-165.
Wang J,Huang L,Bao M Z,Liu G F.2009.Production of interspecific hybrids between Lilium longiflorum and Lilium lophophorum var.linearifolium via ovule culture at early stage.Euphytica,167(1):45-55.
Widoretno W.2016.In vitro induction and characterization of tetraploid Patchouli(Pogostemon cablin,Benth.)plant.Plant Cell Tiss Org Cult,125(2):261-267.
Wu Ze,Zhong Xiong-hui,Cao Xing,Gong Ben-he,Cheng Yun-he,Chen Quan-quan,Yi Ming-fang.2014.Morpho-histological study of somatic embryo-like structures and somatic embryos in lily.Acta Horticulturae Sinica,41(8):1716-1722.(in Chinese)吴泽,钟雄辉,曹兴,宫本贺,程运河,陈全全,义鸣放.2014.百合类体细胞胚和体细胞胚的形态学与组织学研究.园艺学报,41(8),1716-1722.
Yang Ying-jie,Ge Bei-bo,Wei Qian,Gao Jun-ping,Hong Bo.2013.Colchicines-induced polyploid plants and identification in Lilium pumilum DC.J Chin Agr Uni,18(1):128-133.(in Chinese)杨英杰,葛蓓孛,魏倩,高俊平,洪波.2013.秋水仙素诱导细叶百合多倍体研究.中国农业大学学报,18(1):128-133.
Zhang J,Gai M Z,Li X Y,Li T L,Sun H M.2016.Somatic embryogenesis and direct as well as indirect organogenesis in Lilium pumilum DC.Fisch.an endangered ornamental and medicinal plant.Biosci Biotech Bioch,80(10):1898-1906.
Zhang X,Cao Q,Jia G.2017 A protocol for fertility restoration of F1 hybrid derived from Lilium×formolongi‘Raizan 3’×Oriental hybrid‘Sorbonne’.Plant Cell Tiss Org,129(3):375-386.
Zhang Xi-qing,Wang Lian-juan,Cao Qin-zheng,Jia Gui-xia.2017.Polyploidy induction and identification in Lilium concolor var.pulchellum.JBeijing Forestry Uni,39(7):96-102.(in Chinese)张锡庆,汪莲娟,曹钦政,贾桂霞.2017.有斑百合多倍体诱导及鉴定.北京林业大学学报,39(7):96-102.
Zhong Cheng,Tian Xin,Liu Lunpei,Li Xingyuan,Yang Cen.2015.Polyploid induced from wild Lilium sulphureum with colchicine in Guizhou.Guizhou Agri Sci,43(8):9-11.(in Chinese)钟程,田鑫,刘伦沛,李性苑,杨芩.2015.秋水仙素诱导贵州野生淡黄花百合的多倍体.贵州农业科学,43(8):9-11.
Zhou S,Ramanna M S,Visser R G,Tuyl J M.2008.Genome composition of triploid lily cultivars derived from sexual polyploidization of Longiflorum×Asiatic hybrids(Lilium).Euphytica,160(2):207-215.
Zimmerman J L.1993.Somatic embryogenesis:a model for early development in higher plants.Plant Cell,5(10):1411-1423.
Bekheet S A,Gabr A M M,Reda A A,El-Bahr M K.2015.Micropropagation and assessment of genetic stability of in vitro raised jojoba(Simmondsia chinensis Link.)plants using SCo T and ISSR markers.Plant Tiss Cult Biotech,25(2):165-179.
Cabral G B,Carneiro V T C,Dusi D,Martinelli A P.2016.Somatic embryogenesis and plant regeneration of Brachiaria brizantha.Meth Mol Bio,1359:395-402.
Eeckhaut T G R,Werbrouck S P O,Leus L W H,Debergh C P.2004.Chemically induced polyploidization in Spathiphyllum wallisii Regel through somatic embryogenesis.Plant Cell Tiss Org Cul,78(3):241-246.
Endemann M,Hristoforoglu K,Stauber T,Wilhelm E.2001.Assessment of age-related polyploidy in Quercus robur L.somatic embryos and regenerated plants using DNA flow cytometry.Bio Plantarum,44(3):339-345.
Ge Zhen-yang,Jiang Li-min,Yu Ying-jie,Li Peng,Yi Huai-feng,Li Li-cheng.2013.Growth modelling and visualization simulation of lily bulblet.Trans Chin Soci Agr Mach,44(3):209-214.(in Chinese)戈振扬,姜利民,于英杰,李鹏,易怀峰,李立成.2013.百合鳞茎的生长建模与可视化模拟.农业机械学报,44(3):209-214.
Guo L,Xu W,Zhang Y,Wei Z.2017.Inducing triploids and tetraploids with high temperatures in Populus sect.Tacamahaca.Plant Cell Rep,36(2):1-14.
Hassan H A,Wang H P,Shen D,Qiu Y,Li X X.2015.Sprout differentiation and mutation induction of garlic(Allium sativum L.)callus exposed to gamma radiation.Plant Growth Regul,75:465-471.
He M,Gao W J,Gao Y H,Liu Y Z,Yang X,Jiao H B,Zhou Y W.2016a.Polyploidy induced by colchicine in Dendranthema indicum var.aromaticum,a scented chrysanthemum.Eur J Hortic Sci,81(4):219-226.
He Y H,Sun Y,Zheng R,Ye A I,Cao Z,Bao M Z.2016b.Induction of tetraploid male sterile Tagetes erecta by colchicine treatment and its application for interspecific hybridization.Horticul Plant J,2(5):284-292.
Jin L,Zhang Y,Yan L,Guo Y L,Niu L X.2012.Phenolic compounds and antioxidant activity of bulb extracts of six Lilium species native to China.Molecules,17:9361-9378.
Khateeb W A,Bahar E,Lahham J,Schroeder D,Hussein E.2013.Regeneration and assessment of genetic fidelity of the endangered tree Moringa peregrina(Forsk.)Fiori using Inter Simple Sequence Repeat(ISSR).Physiol Mol Biol Plants,19(1):157-164.
Leila A S P,Moacir P,Sebastiao D O E S,Herminio S R,Helida M M,Janay A S S.2014.Inducing and identifying artificially-induced polyploidy in bananas.Afr J Bio,13(37):3748-3758.
Leroy X J,Leon K,Charles G,Branchard M.2000.Cauliflower somatic embryogenesis and analysis of regenerant stability by ISSRs.Plant Cell Rep,19:1102-1107.
Levin D A.1983.Polyploidy and novelty in flowering plants.Am Nat,122(1):1-25.
Li Dan,Luo Yi-ran,Han Guo-wei,Zhang Xue,Wu Ying-ying,He Cheng-zhong.2017.Studies on polyploidy induction of wild Lilium nepalense D.Don.J Yunnan Agri Uni(Natural Science),32(4):678-684.(in Chinese)李旦,罗一然,韩国伟,张雪,吴英英,何承忠.2017.野生紫斑百合多倍体诱导研究.云南农业大学学报(自然科学),32(4):678-684.
Li Xiao-shuang,Wang Qing,Meng Yu-cheng,Xiang Zhong-huai,He Ning-jia.2017.Polyploidy induction and identification of wild germplasm resource Yun 7 of Morus wittiorum Hand-Mazz.Sci Sericulture,43(3):369-373.(in Chinese)李晓双,王青,孟钰程,向仲怀,何宁佳.2017.野生长穗桑种质资源云7的多倍体诱导和鉴定.蚕业科学,43(3):369-373.
Li Yan-na,Wei Yi-ming,Hu Gui-bing,Chen Hou-bin,Xu Chun-xiang.2010.Plant regeneration via somatic embryogenesis after cryopreservation of embryogenic cell suspensions of banana(Musa spp.AAA)by vitrification and the genetic stability of regenerated plant.Acta Horticulturae Sinica,37(6):899-905.(in Chinese)李艳娜,尉义明,胡桂兵,陈厚彬,徐春香.2010.香蕉胚性细胞悬浮系玻璃化法超低温保存及再生植株遗传稳定性分析.园艺学报,37(6):899-905.
Loureiro J,Pinto G,Lopes T,Dolezel J,Santos C.2005.Assessment of ploidy stability of the somatic embryogenesis process in Quercus suber L.using flow cytometry.Planta,221:815-822.
Mallick P,Chattaraj S,Sikdar S R.2017.Molecular characterizations of somatic hybrids developed between Pleurotus florida and Lentinus squarrosulus through inter-simple sequence repeat markers and sequencing of ribosomal RNA-ITS gene.Biotech,7(5):298.
Marzougui N,Elfalleh W,Boubaya A,Guasmi F,Ferchichi A,Lachieheb B.2010.Effect of polyploidy on ISSR molecular profile and the contents of vitamins and proteins in Trigonella foenum-graecum L.Acta Bot Gallica,157(1):89-100.
Mcgregor C E,Lambert C A,Greyling M M,Louw J H,Warnich L.2000.A comparative assessment of DNA fingerprinting techniques(RAPD,ISSR,AFLP and SSR)in tetraploid potato(Solanum tuberosum L.)germplasm.Euphytica,113(2):135-144.
Nakel T,Tekleyohans D G,Mao Y,Fuchert G,Vo D,GroβR.2017.Triparental plants provide direct evidence for polyspermy induced polyploidy.Nat Commun,8(1):10-33.
Naz R,Anis M,Alatar A A.2016.ISSR marker-based detection of genomic stability in Cassia occidentalis L.plantlets derived from somatic embryogenesis.Eng Life Sci,16:17-24.
Noori S A S,Norouzi M,Karimzadeh G,Shirkool K,Niazian M.2017.Effect of colchicine-induced polyploidy on morphological characteristics and essential oil composition of ajowan(Trachyspermum ammi L.).Plant Cell Tiss Org,130(7):1-9.
Pitschmann A,Purevsuren S,Obmann A,Natsagdorj D,Gunbing S,Narantuya Ch,Kletter S.2013.Traditional mongolian medicine:history and status quo.Phytochem Rev,12(4):943-959.
Rai M K,Phulwaria M H,Gupta A K,Shekhawat N S,Jaiswal U.2012.Genetic homogeneity of guava plants derived from somatic embryogenesis using SSR and ISSR markers.Plant Cell Tiss Org Cult,111:259-264.
Sattler M C,Carvalho C R,Clarindo W R.2016.The polyploidy and its key role in plant breeding.Planta,243(2):281-296.
Soltis P S,Soltis D E.2016.Ancient WGD events as drivers of key innovations in angiosperms.Curr Opin Plant Bio,30:159-165.
Wang J,Huang L,Bao M Z,Liu G F.2009.Production of interspecific hybrids between Lilium longiflorum and Lilium lophophorum var.linearifolium via ovule culture at early stage.Euphytica,167(1):45-55.
Widoretno W.2016.In vitro induction and characterization of tetraploid Patchouli(Pogostemon cablin,Benth.)plant.Plant Cell Tiss Org Cult,125(2):261-267.
Wu Ze,Zhong Xiong-hui,Cao Xing,Gong Ben-he,Cheng Yun-he,Chen Quan-quan,Yi Ming-fang.2014.Morpho-histological study of somatic embryo-like structures and somatic embryos in lily.Acta Horticulturae Sinica,41(8):1716-1722.(in Chinese)吴泽,钟雄辉,曹兴,宫本贺,程运河,陈全全,义鸣放.2014.百合类体细胞胚和体细胞胚的形态学与组织学研究.园艺学报,41(8),1716-1722.
Yang Ying-jie,Ge Bei-bo,Wei Qian,Gao Jun-ping,Hong Bo.2013.Colchicines-induced polyploid plants and identification in Lilium pumilum DC.J Chin Agr Uni,18(1):128-133.(in Chinese)杨英杰,葛蓓孛,魏倩,高俊平,洪波.2013.秋水仙素诱导细叶百合多倍体研究.中国农业大学学报,18(1):128-133.
Zhang J,Gai M Z,Li X Y,Li T L,Sun H M.2016.Somatic embryogenesis and direct as well as indirect organogenesis in Lilium pumilum DC.Fisch.an endangered ornamental and medicinal plant.Biosci Biotech Bioch,80(10):1898-1906.
Zhang X,Cao Q,Jia G.2017 A protocol for fertility restoration of F1 hybrid derived from Lilium×formolongi‘Raizan 3’×Oriental hybrid‘Sorbonne’.Plant Cell Tiss Org,129(3):375-386.
Zhang Xi-qing,Wang Lian-juan,Cao Qin-zheng,Jia Gui-xia.2017.Polyploidy induction and identification in Lilium concolor var.pulchellum.JBeijing Forestry Uni,39(7):96-102.(in Chinese)张锡庆,汪莲娟,曹钦政,贾桂霞.2017.有斑百合多倍体诱导及鉴定.北京林业大学学报,39(7):96-102.
Zhong Cheng,Tian Xin,Liu Lunpei,Li Xingyuan,Yang Cen.2015.Polyploid induced from wild Lilium sulphureum with colchicine in Guizhou.Guizhou Agri Sci,43(8):9-11.(in Chinese)钟程,田鑫,刘伦沛,李性苑,杨芩.2015.秋水仙素诱导贵州野生淡黄花百合的多倍体.贵州农业科学,43(8):9-11.
Zhou S,Ramanna M S,Visser R G,Tuyl J M.2008.Genome composition of triploid lily cultivars derived from sexual polyploidization of Longiflorum×Asiatic hybrids(Lilium).Euphytica,160(2):207-215.
Zimmerman J L.1993.Somatic embryogenesis:a model for early development in higher plants.Plant Cell,5(10):1411-1423.