芒果‘金煌’愈伤增殖、早期体细胞胚发生及细胞学观察
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摘要
本研究以‘金煌’芒果(Mangifera indica L.)幼果的珠心组织为外植体进行愈伤组织诱导,探索了植物生长调节物质对愈伤组织增殖、早期体细胞胚发生的影响,并对培养物进行形态学与组织学的鉴定。结果表明,植物生长调节物质对愈伤组织的增殖等性状有显著的影响,其中含有1.0 mg/L 2,4-D+1.0 mg/L KT的改良B5基本培养基(B5大量元素+MS微量元素/有机物/铁盐)+4%蔗糖+10%椰子水+500 mg/L谷氨酰胺+0.7%琼脂+0.2%活性炭显著提高愈伤组织的颗粒性和胚性愈伤组织的比例,并且有利于愈伤组织体积增大和长势提高。非胚性愈伤组织呈微黄色致密状,胚性愈伤组织主要呈现黄色或白色颗粒状。胚性愈伤组织在含有5.0 mg/L ABA+500 mg/L PEG的体细胞胚发生培养基上体细胞胚产量最高,平均0.2 g胚性团上产生体细胞胚数量达40.67个,显著高于ABA或PEG单独处理。
In this study, the crucial effects of plant growth regulators on callus proliferation and somatic embryo genesis in Mangifera indica 'Jinhuang' were investigated using the callus induced by nucellar tissue of immature fruit. Morphology as well as histology identification of cultures were also carried out. The results showed that plant growth regulators had significant effects on the proliferation of callus, and the modified B5 minimal medium containing 1.0 mg/L 2,4-D + 1.0 mg/L KT(B5 macroelements + MS traces element/organic/iron salt) + 4% sucrose + 10%coconut water + 500 mg/L glutamine + 0.7% agar + 0.2% activated carbon significantly increased the ratio of callus granules and embryogenic callus, and was conducive to callus volume increase and growth. The non-embryogenic callus was yellowish and dense, while the embryogenic callus was mainly yellow or white granular. The treatment of 5.0 mg/L ABA+500 mg/L PEG significantly increased the productivity of early somatic embryos, and an average of 40.67 somatic embryos were produced from approximately 0.2 g embryogenic callus, which was sig-inificantly higher than the only ABA or PEG treatments.
In this study, the crucial effects of plant growth regulators on callus proliferation and somatic embryo genesis in Mangifera indica 'Jinhuang' were investigated using the callus induced by nucellar tissue of immature fruit. Morphology as well as histology identification of cultures were also carried out. The results showed that plant growth regulators had significant effects on the proliferation of callus, and the modified B5 minimal medium containing 1.0 mg/L 2,4-D + 1.0 mg/L KT(B5 macroelements + MS traces element/organic/iron salt) + 4% sucrose + 10%coconut water + 500 mg/L glutamine + 0.7% agar + 0.2% activated carbon significantly increased the ratio of callus granules and embryogenic callus, and was conducive to callus volume increase and growth. The non-embryogenic callus was yellowish and dense, while the embryogenic callus was mainly yellow or white granular. The treatment of 5.0 mg/L ABA+500 mg/L PEG significantly increased the productivity of early somatic embryos, and an average of 40.67 somatic embryos were produced from approximately 0.2 g embryogenic callus, which was sig-inificantly higher than the only ABA or PEG treatments.
引文
Ara H.,Jaiswal U.,and Jaiswal V.S.,2004,An improved method of proliferation of proembryogenic calli of Mangifera indica L.var.Amrapali for scale-up of somatic embryo production,Indian J.Biotechnol.,3(2):229-234
Aronen T.,Pehkonen T.,and Ryynanen L.,2009,Enhancement of somatic embryogenesis from immature zygotic embryos of Pinus sylvestris,Scand.J.For.Res.,24(5):372-383
Cheng Y.,Wan D.Y.,Zhang B.L.,Wang Y.J.,and Wen Y.Q.,2017,Induction,preservation,propagation and cytological observation of proembryonic masses of grapevine,Guoshu Xuebao(Journal of Fruit Science),34(8):968-987(程远,万东艳,张宝林,王跃进,文颖强,2017,葡萄原胚团的诱导、保存、扩繁及其细胞学观察,果树学报,34(8):968-987)
Dewald S.G.,Litz R.E.,and Moore G.A.,1989,Optimizing somatic embryo production in mango,J.Am.Soc.Hortic.Sci.,114(4):712-716
He Y.H.,Fang S.Q.,Ma J.,Hu Z.Y.,Lu M.,and Peng B.,2010,Histocytology observation on the somatic embryogenesis in Ananas comosus callus,Yuanyi Xuebao(Acta Horticulturae Sinica),37(5):689-696(何业华,方少秋,马均,胡中沂,卢敏,彭兵,2010,菠萝愈伤组织中体细胞胚起源过程的组织细胞学观察,园艺学报,37(5):689-696)
Hu R.Y.,Sun Y.H.,Wu B.,Duan H.,Zheng H.Q.,Hu D.H.,Lin H.Z.,Tong Z.K.,Xu J.L.,and Li Y.,2017,Somatic embryogenesis of immature Cunninghamia lanceolata(Lamb.)hook zygotic embryos,Sci.Rep.,7(1):56
Huang J.H.,Xie J.H.,Wang S.B.,Liang G.L.,and Ma W.H.,2009,Studies on somatic embryogenesis and plant regeneration of Mangifera persiciformis,Guoshu Xuebao(Journal of Fruit Science),26(2):176-179(黄镜浩,谢江辉,王松标,梁国鲁,马蔚红,2009,扁桃杧(Mangifera persiciformis)体细胞胚发生及再生体系建立,果树学报,26(2):176-179)
Jayasankar S.,and Litz R.E.,1998,Characterization of embryogenic mango cultur es selected for resistance to Colletotrichum gloeosporioides culture filtrate and phytotoxin,Theor.Appl.Genet.,96(6-7):823-831
Jiménez V.M.,2005,Involvement of plant hormones and plant growth regulators on in vitro somatic embryogenesis,Plant Growth Regul.,47(2-3):91-110
Kermode A.R.,1990,Regulatory mechanisms involved in the transition from seed development to germination,Crit.Rev.Plant Sci.,9(2):155-195
Khair T.A.B.,Manish S.,Abdullah H.A.B.,Ghaya S.A.,Mohammed H.A.,Basem S.A.,and Hamoud D.A.,2016,In vitro regeneration of mango(Mangifera indica L.)cv.Baramasi through nucellar embryogenesis,J.Hortic.For.,8(5):37-43
Krishna H.,and Singh S.K.,2007,Biotechnological advances in mango(Mangifera indica L.)and their future implication in crop improvement:a review,Biotechnol.Adv.,25(3):223-243
Litz R.E.,Knight R.J.,and Gazit S.,1982,Somatic embryos from cultured ovules of polyembryonic Mangifera indica L.,Plant Cell Rep.,1(6):264-266
Litz R.E.,1984,In vitro somatic embryogenesis from nucellar callus of monoembryonic mango,HortScience,19:715-717
Litz R.E.,Mathews V.H.,Hendrix R.C.,and Yurgalevitch C.,1991,Mango somatic cell genetics,Acta Hortic.,291:133-140
Lu J.J.,Yan R.,He X.S.,Jin H.M.,Wang J.X.,Wang C.X.,and Sun H.M.,2016,The embryonic callus induction and somatic embryogenesis of Hippeastrum vittatum'Red Lion',Yuanyi Xuebao(Acta Horticulturae Sinica),43(12):2451-2460(鲁娇娇,严瑞,何香杉,靳宏梅,王锦霞,王春夏,孙红梅,2016,朱顶红‘Red Lion’胚性愈伤组织诱导及体细胞胚发生,园艺学报,43(12):2451-2460)
Pliego-Alfaro F.,Litz R.E.,Moon P.A.,and Gray D.J.,1996,Effect of abscisic acid,osmolarity and temperature on in vitro development of recalcitrant mango nucellar embryos,Plant Cell Tiss.Org.,44(1):53-61
Song T.,Zhou Z.P.,Liu J.,Liu C.,and Zhang L.,2018,Optimization of embryonic callus suspension culture conditions for Peperomia obtusifolia and somatic embryogenesis,Fenzi ZhiwuYuzhong(Molecular Plant Breeding),16(5):1626-1631(宋婷,周佐葡,刘洁,刘春,张黎,2018,豆瓣绿胚性愈伤悬浮培养条件优化及体细胞胚发生,分子植物育种,16(5):1626-1631)
Taniguchi T.,Kurita M.,Itahana N.,and Kondo T.,2004,Somatic embryogenesis and plant regeneration from immature zygotic embryos of Hinoki cypress(Chamaecyparis obtusa sieb.et zucc.),Plant Cell Rep.,23:26-31
Thomas P.,1999,Somatic embryogenesis and plantlet regeneration from nucellar tissue of monoembryonic mango,J.Hortic.Sci.Biotech.,74(1):135-139
Wang J.,Liu G.H.,Bao M.Z.,and Huang L.,2008,Adjustment of the status of embryogenic callus and plant regeneration of Lilium longiflorum,Yuanyi Xuebao(Acta Horticulturae Sinica),35(12):1795-1802(王杰,刘国锋,包满珠,黄莉,2008,麝香百合胚性愈伤组织状态的调整与植株再生,园艺学报,35(12):1795-1802)
Wei H.Y.,Liang H.W.,Zhang B.,and Li Z.L.,2016,Establishment of embryogenic cell suspension culture and plant regeneration of rare endangered plant Tapiscia sinensis Oliv.,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(3):756-759(韦虹宇,梁宏伟,张博,李在留,2016,珍稀濒危植物银鹊树胚性细胞悬浮系的建立和植株再生,分子植物育种,14(3):756-759)
Wu Y.J.,Huang X.L.,Chen Q.Z.,Li X.J.,and Engelmann F.,2007,Induction and cryopreservation of embryogenic cultures from nucelli and immature cotyledon cuts of mango(Mangifera indica L.var Zihua),Plant Cell Rep.,26(2):161-168
Xiao J.N.,Huang X.L.,Wu Y.J.,Li X.J.,Zhou M.D.,and Engelmann F.,2004,Direct somatic embryogenesis induced from cotyledons of mango immature zygotic embryos,In Vitro Cell.Dev-Pl.,40(2):196-199
Zavattieri M.A.,Frederico A.M.,Lima M.,Sabino R.,and Arnholdt-Schmitt B.,2010,Induction of somatic embryogenesis as an example of stress-related plant reactions,Electr.J.Biotechnol.,13(1):1-9
Zeng W.D.,Lu L.Y.,Xie X.Y.,and Yan H.B.,2015,Research on somatic embryogenesis and plant regeneration of cassava,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(7):1522-1526(曾文丹,陆柳英,谢向誉,严华兵,2015,木薯体细胞胚的发生及植株再生的研究,基因组学与应用生物学,34(7):1522-1526)
Zhang Y.Y.,2010,Primary study in vitro culture of mango(Mangifera indica L.),Thesis for M.S.,Hainan university,Supervisors:Li K.M.,and Wei Y.J.,pp.28-43(张越阳,2010,芒果离体培养的初步研究,硕士学位论文,海南大学,导师:李开绵,魏亚军,pp.28-43)
Zhou X.H.,Zheng R.H.,Liu G.G.,Xu Y.,Zhou Y.W.,Laux T.,Zhen Y.,Harding S.A.,Shi J.,and Chen J.H.,2017,Desiccation treatment and endogenous IAA levels are key factors influencing high frequency somaticembryogenesis in Cunninghamia lanceolata(Lamb.)hook,Front.Plant Sci.,8:2054
Aronen T.,Pehkonen T.,and Ryynanen L.,2009,Enhancement of somatic embryogenesis from immature zygotic embryos of Pinus sylvestris,Scand.J.For.Res.,24(5):372-383
Cheng Y.,Wan D.Y.,Zhang B.L.,Wang Y.J.,and Wen Y.Q.,2017,Induction,preservation,propagation and cytological observation of proembryonic masses of grapevine,Guoshu Xuebao(Journal of Fruit Science),34(8):968-987(程远,万东艳,张宝林,王跃进,文颖强,2017,葡萄原胚团的诱导、保存、扩繁及其细胞学观察,果树学报,34(8):968-987)
Dewald S.G.,Litz R.E.,and Moore G.A.,1989,Optimizing somatic embryo production in mango,J.Am.Soc.Hortic.Sci.,114(4):712-716
He Y.H.,Fang S.Q.,Ma J.,Hu Z.Y.,Lu M.,and Peng B.,2010,Histocytology observation on the somatic embryogenesis in Ananas comosus callus,Yuanyi Xuebao(Acta Horticulturae Sinica),37(5):689-696(何业华,方少秋,马均,胡中沂,卢敏,彭兵,2010,菠萝愈伤组织中体细胞胚起源过程的组织细胞学观察,园艺学报,37(5):689-696)
Hu R.Y.,Sun Y.H.,Wu B.,Duan H.,Zheng H.Q.,Hu D.H.,Lin H.Z.,Tong Z.K.,Xu J.L.,and Li Y.,2017,Somatic embryogenesis of immature Cunninghamia lanceolata(Lamb.)hook zygotic embryos,Sci.Rep.,7(1):56
Huang J.H.,Xie J.H.,Wang S.B.,Liang G.L.,and Ma W.H.,2009,Studies on somatic embryogenesis and plant regeneration of Mangifera persiciformis,Guoshu Xuebao(Journal of Fruit Science),26(2):176-179(黄镜浩,谢江辉,王松标,梁国鲁,马蔚红,2009,扁桃杧(Mangifera persiciformis)体细胞胚发生及再生体系建立,果树学报,26(2):176-179)
Jayasankar S.,and Litz R.E.,1998,Characterization of embryogenic mango cultur es selected for resistance to Colletotrichum gloeosporioides culture filtrate and phytotoxin,Theor.Appl.Genet.,96(6-7):823-831
Jiménez V.M.,2005,Involvement of plant hormones and plant growth regulators on in vitro somatic embryogenesis,Plant Growth Regul.,47(2-3):91-110
Kermode A.R.,1990,Regulatory mechanisms involved in the transition from seed development to germination,Crit.Rev.Plant Sci.,9(2):155-195
Khair T.A.B.,Manish S.,Abdullah H.A.B.,Ghaya S.A.,Mohammed H.A.,Basem S.A.,and Hamoud D.A.,2016,In vitro regeneration of mango(Mangifera indica L.)cv.Baramasi through nucellar embryogenesis,J.Hortic.For.,8(5):37-43
Krishna H.,and Singh S.K.,2007,Biotechnological advances in mango(Mangifera indica L.)and their future implication in crop improvement:a review,Biotechnol.Adv.,25(3):223-243
Litz R.E.,Knight R.J.,and Gazit S.,1982,Somatic embryos from cultured ovules of polyembryonic Mangifera indica L.,Plant Cell Rep.,1(6):264-266
Litz R.E.,1984,In vitro somatic embryogenesis from nucellar callus of monoembryonic mango,HortScience,19:715-717
Litz R.E.,Mathews V.H.,Hendrix R.C.,and Yurgalevitch C.,1991,Mango somatic cell genetics,Acta Hortic.,291:133-140
Lu J.J.,Yan R.,He X.S.,Jin H.M.,Wang J.X.,Wang C.X.,and Sun H.M.,2016,The embryonic callus induction and somatic embryogenesis of Hippeastrum vittatum'Red Lion',Yuanyi Xuebao(Acta Horticulturae Sinica),43(12):2451-2460(鲁娇娇,严瑞,何香杉,靳宏梅,王锦霞,王春夏,孙红梅,2016,朱顶红‘Red Lion’胚性愈伤组织诱导及体细胞胚发生,园艺学报,43(12):2451-2460)
Pliego-Alfaro F.,Litz R.E.,Moon P.A.,and Gray D.J.,1996,Effect of abscisic acid,osmolarity and temperature on in vitro development of recalcitrant mango nucellar embryos,Plant Cell Tiss.Org.,44(1):53-61
Song T.,Zhou Z.P.,Liu J.,Liu C.,and Zhang L.,2018,Optimization of embryonic callus suspension culture conditions for Peperomia obtusifolia and somatic embryogenesis,Fenzi ZhiwuYuzhong(Molecular Plant Breeding),16(5):1626-1631(宋婷,周佐葡,刘洁,刘春,张黎,2018,豆瓣绿胚性愈伤悬浮培养条件优化及体细胞胚发生,分子植物育种,16(5):1626-1631)
Taniguchi T.,Kurita M.,Itahana N.,and Kondo T.,2004,Somatic embryogenesis and plant regeneration from immature zygotic embryos of Hinoki cypress(Chamaecyparis obtusa sieb.et zucc.),Plant Cell Rep.,23:26-31
Thomas P.,1999,Somatic embryogenesis and plantlet regeneration from nucellar tissue of monoembryonic mango,J.Hortic.Sci.Biotech.,74(1):135-139
Wang J.,Liu G.H.,Bao M.Z.,and Huang L.,2008,Adjustment of the status of embryogenic callus and plant regeneration of Lilium longiflorum,Yuanyi Xuebao(Acta Horticulturae Sinica),35(12):1795-1802(王杰,刘国锋,包满珠,黄莉,2008,麝香百合胚性愈伤组织状态的调整与植株再生,园艺学报,35(12):1795-1802)
Wei H.Y.,Liang H.W.,Zhang B.,and Li Z.L.,2016,Establishment of embryogenic cell suspension culture and plant regeneration of rare endangered plant Tapiscia sinensis Oliv.,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(3):756-759(韦虹宇,梁宏伟,张博,李在留,2016,珍稀濒危植物银鹊树胚性细胞悬浮系的建立和植株再生,分子植物育种,14(3):756-759)
Wu Y.J.,Huang X.L.,Chen Q.Z.,Li X.J.,and Engelmann F.,2007,Induction and cryopreservation of embryogenic cultures from nucelli and immature cotyledon cuts of mango(Mangifera indica L.var Zihua),Plant Cell Rep.,26(2):161-168
Xiao J.N.,Huang X.L.,Wu Y.J.,Li X.J.,Zhou M.D.,and Engelmann F.,2004,Direct somatic embryogenesis induced from cotyledons of mango immature zygotic embryos,In Vitro Cell.Dev-Pl.,40(2):196-199
Zavattieri M.A.,Frederico A.M.,Lima M.,Sabino R.,and Arnholdt-Schmitt B.,2010,Induction of somatic embryogenesis as an example of stress-related plant reactions,Electr.J.Biotechnol.,13(1):1-9
Zeng W.D.,Lu L.Y.,Xie X.Y.,and Yan H.B.,2015,Research on somatic embryogenesis and plant regeneration of cassava,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(7):1522-1526(曾文丹,陆柳英,谢向誉,严华兵,2015,木薯体细胞胚的发生及植株再生的研究,基因组学与应用生物学,34(7):1522-1526)
Zhang Y.Y.,2010,Primary study in vitro culture of mango(Mangifera indica L.),Thesis for M.S.,Hainan university,Supervisors:Li K.M.,and Wei Y.J.,pp.28-43(张越阳,2010,芒果离体培养的初步研究,硕士学位论文,海南大学,导师:李开绵,魏亚军,pp.28-43)
Zhou X.H.,Zheng R.H.,Liu G.G.,Xu Y.,Zhou Y.W.,Laux T.,Zhen Y.,Harding S.A.,Shi J.,and Chen J.H.,2017,Desiccation treatment and endogenous IAA levels are key factors influencing high frequency somaticembryogenesis in Cunninghamia lanceolata(Lamb.)hook,Front.Plant Sci.,8:2054