“Gros Michel”香蕉胚性细胞悬浮系及遗传转化体系的建立
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摘要
本研究以"Gros Michel"香蕉未成熟雄花序为外植体,建立了其胚性细胞悬浮系及遗传转化体系。结果表明,90 d后可诱导产生浅黄色松散的胚性愈伤组织,经悬浮培养120 d后获得含有均质细胞团的胚性细胞悬浮系(ECS);将3个月龄的ECS置于胚诱导培养基30 d后有大量白色半透明状球形的体细胞胚发生,继代培养60 d后发育为成熟不透明的体细胞胚;将成熟的体胚在萌发培养基上培养10 d后有幼芽产生,转移至生根培养基30 d后得到幼苗。同时开展了含绿色荧光蛋白(GFP)的pCAMBIA0380载体的遗传转化研究,经过3次液体培养基筛选转移至半固体筛选培养基90 d后,在胚萌发培养基和生根培养基中不再添加抗生素。经对转化植株的根尖镜检及PCR鉴定,均为阳性植株。研究结果将为进一步开展"Gros Michel"优质抗病基因功能研究提供材料和技术支撑。
In the research, embryogenic cell suspension and genetic transformation of "Gros Michel" banana were established with the immature male inflorescence as explants. The results showed that yellowish and loose embryogenic callus could be developed after 90 days. Embryogenic cell suspensions(ECS) with homogeneous cell mass were obtained after 120 days of suspension culture. 3-month-old ECS were cultured in embryo-inducing medium for 30 days, followed by a large number of white translucent spherical somatic embryos, which would be developed into mature opaque somatic embryos after subculture for 60 days. Mature somatic embryos werecultured on the germination medium for 10 days, and then buds were produced and transferred to the rooting medium for 30 days to obtain seedlings. At the same time, the genetic transformation of pCAMBIA0380 vector containing green fluorescent protein(GFP) was carried out. The embryogenic cells were selected after three times of liquid screening medium and transferred to semi-solid screening culture medium for 90 days, followed by culturing in embryo germination medium and rooting medium without any antibiotic. After root tip microscopic examination and PCR identification of the transformed plants, they were found to be positive. Those results would provide materials and technical support for the further research on the function of high-quality disease resistance gene in "Gros Michel".
In the research, embryogenic cell suspension and genetic transformation of "Gros Michel" banana were established with the immature male inflorescence as explants. The results showed that yellowish and loose embryogenic callus could be developed after 90 days. Embryogenic cell suspensions(ECS) with homogeneous cell mass were obtained after 120 days of suspension culture. 3-month-old ECS were cultured in embryo-inducing medium for 30 days, followed by a large number of white translucent spherical somatic embryos, which would be developed into mature opaque somatic embryos after subculture for 60 days. Mature somatic embryos werecultured on the germination medium for 10 days, and then buds were produced and transferred to the rooting medium for 30 days to obtain seedlings. At the same time, the genetic transformation of pCAMBIA0380 vector containing green fluorescent protein(GFP) was carried out. The embryogenic cells were selected after three times of liquid screening medium and transferred to semi-solid screening culture medium for 90 days, followed by culturing in embryo germination medium and rooting medium without any antibiotic. After root tip microscopic examination and PCR identification of the transformed plants, they were found to be positive. Those results would provide materials and technical support for the further research on the function of high-quality disease resistance gene in "Gros Michel".
引文
Becker D.K.,Dugdale B.,Smith M.K.,Harding R.M.,and Dale J.,2000,Genetic transformation of Cavendish banana(Musa spp.AAA group)cv.'Grand Nain'via microprojectile bombardment,Plant Cell Rep.,19(3):229-234
Bretagne-Sagnard B.,and Chupeau Y.,1996,Selection of transgenic flax plants facilitated by spectinomycin,Transgene R-es.,5(2):131-137
Butler D.,2013,Fungus threatens top banana,Nature,504(7479)195-196
Cote F.X.,Domergue R.,Monmarson S.,Schwendiman J.,Teisson C.,and Escalant J.V.,1996,Embryogenic cell suspensions from the male flower of Musa AAA cv.Grand Nain,Physiol.Plant,97(2):285-290
Dash P.K.,and Rai R.,2016,Translating the"Banana Genome"to delineate stress resistance,dwarfing,parthenocarpy and mechanisms of fruit ripening,Front.Plant Sci.,7:1543
Dheda D.,Dumortier F.,Panis B.,Vuylsteke D.,and Langhe E.,1991,Plant regeneration in cell suspension cultures of the cooking banana cv.Bluggoe(Musa spp.ABB group),Fruits,46(2):125-135
Escalant J.V.,Teisson C.,and Cote F.X.,1994,Amplified somatic embryogenesis from male flowers of triploid banana and plantain varieties(Musa sp.),In Vitro Cell.Dev.Biol.Plant,30(4):181-186
Grapin A.,Ortíz J.L.,Lescot T.,Ferrière N.,and Cote F.X.,2000,Recovery and regeneration of embryogenic cultures from female flowers of false horn plantain,Plant Cell Tiss.Org.Cult.,61(3):237-244
Hu C.H.,Wei Y.R.,Yi G.J.,Huang B.Z.,and Huang Y.H.,2010,Establishment of a high efficient Agrobacterium tumefaciensmediated transformation system for banana,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),8(1):172-178(胡春华,魏岳荣,易干军,黄秉智,黄永红,2010,根癌农杆菌介导的香蕉高效遗传转化系统的建立,分子植物育种,8(1):172-178)
Huo M.J.,Wei Y.R.,Hu J.J.,Huang B.Z.,and Yi G.J.,2008,Factors affecting Agrobacterium-mediated genetic transformation of embryogenic cell suspension of banana,Guoshu Xuebao(Journal of Fruit Science),25(4):597-600(霍妙娟,魏岳荣,胡家金,黄秉智,易干军,2008,影响根癌农杆菌介导香蕉胚性悬浮细胞遗传转化的因素,果树学报,25(4):597-600)
Kuang R.B.,Li C.Y.,Yang J.,Wei Y.R.,Yang Q.S.,Hu C.H.,Sheng O.,and Yi G.J.,2013,Cell structure alteration of banana cultivars with different resistance to Fusarium Oxysporum f.sp.Cubense,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),11(2):193-198(邝瑞彬,李春雨,杨静,魏岳荣,杨乔松,胡春华,盛鸥,易干军,2013,抗感枯萎病香蕉的细胞结构抗性研究,分子植物育种,11(2):193-198)
Lorenzen J.,Tenkouano A.,Bandyopadhyay R.,Vroh B.,Coyne D.,and Tripathi L.,2010,Overview of banana and plantain(Musa spp.)improvement in Africa:past and future,Acta Hortic.,879(4):595-603
Lu Y.,Li H.P.,and Zheng F.C.,2006,Factors affecting interaction of Agrobacterium and materials in Agrobacterium-mediated genetic transformation on banana,Redai Zuowu Xuebao(Chinese Journal of Tropical Crops),27(1):84-87(陆英,李华平,郑服从,2006,电镜观察农杆菌介导香蕉基因转化的影响因子,热带作物学报,27(1):84-87)
Marroquin C.G.,Paduscheck C.,Escalant J.V.,and Teisson C.1993,Somatic embryogenesis and plant regeneration through cell suspension in Musa acuminate,In Vitro Cell.Dev Biol.Plant,29(1):43-46
Navarro C.,Escobedo R.M.,and Mayo A.,1997,In vitro plan regeneration from embryogenic cultures of a diploid and a triploid,Cavendish banana,Plant Cell Tiss.Org.Cult.,51(1):17-25
Novak F.J.,Afza R.,Duren M.V.,Perea-Dallos M.,Conger B.V.and Tang X.L.,1989,Somatic embryogenesis and plant regeneration in suspension cultures of dessert(AA and AAA)and cooking(ABB)bananas(Musa spp.),Nat.Biotechnol.7(2):147-158
Sreeramanan S.,Mazial M.,Abadullah M.P.,Rosli N.M.,and Xavier R.,2006,Potential selectable marker for genetic transformation in banana,Biotechnology,5(2):189-197
Strosse H.,Schoofs H.,Panis B.,Andre E.,Reyniers K.,and Swennen R.,2006,Development of embryogenic cell suspensions from shoot meristematic tissue bananas and plantains(Musa spp.),Plant Sci.,170(1):104-112
Tripathi J.,Muwonge A.,and Tripathi L.,2012,Highly efficient regeneration and transformation protocol for plantain cv.Gonja manjaya(Musa spp.AAB)using embryogenic cell suspension,In Vitro Cell.Dev.Biol.Plant,48(2):216-224
Tripathi J.N.,Oduor R.O.,and Tripathi L.,2015,A high-throughput regeneration and transformation platform for production of genetically modified banana,Front.Plant Sci.,6(2):538-543
Wei Y.R.,Huang X.L.,Li J.,Huang X.,Li Z.,and Li X.J.,2005,Establishment of embryogenic cell suspension culture and plant regeneration of edible banana Musa acuminate cv.M-as(AA),Shengwu Gongcheng Xuebao(Chinese Journal of Biotechnology),21(1):58-65(魏岳荣,黄学林,李佳,黄霞,李哲,李筱菊,2005,贡蕉胚性细胞悬浮系的建立和植株再生,生物工程学报,21(1):58-65)
Yao J.L.,Cohen D.,Atkinson R.,Richardson K.,and Morris B.,1995,Regeneration of transgenic plants from the commercial apple cultivar Royal Gala,Plant Cell Rep.,14(7):407-412
Bretagne-Sagnard B.,and Chupeau Y.,1996,Selection of transgenic flax plants facilitated by spectinomycin,Transgene R-es.,5(2):131-137
Butler D.,2013,Fungus threatens top banana,Nature,504(7479)195-196
Cote F.X.,Domergue R.,Monmarson S.,Schwendiman J.,Teisson C.,and Escalant J.V.,1996,Embryogenic cell suspensions from the male flower of Musa AAA cv.Grand Nain,Physiol.Plant,97(2):285-290
Dash P.K.,and Rai R.,2016,Translating the"Banana Genome"to delineate stress resistance,dwarfing,parthenocarpy and mechanisms of fruit ripening,Front.Plant Sci.,7:1543
Dheda D.,Dumortier F.,Panis B.,Vuylsteke D.,and Langhe E.,1991,Plant regeneration in cell suspension cultures of the cooking banana cv.Bluggoe(Musa spp.ABB group),Fruits,46(2):125-135
Escalant J.V.,Teisson C.,and Cote F.X.,1994,Amplified somatic embryogenesis from male flowers of triploid banana and plantain varieties(Musa sp.),In Vitro Cell.Dev.Biol.Plant,30(4):181-186
Grapin A.,Ortíz J.L.,Lescot T.,Ferrière N.,and Cote F.X.,2000,Recovery and regeneration of embryogenic cultures from female flowers of false horn plantain,Plant Cell Tiss.Org.Cult.,61(3):237-244
Hu C.H.,Wei Y.R.,Yi G.J.,Huang B.Z.,and Huang Y.H.,2010,Establishment of a high efficient Agrobacterium tumefaciensmediated transformation system for banana,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),8(1):172-178(胡春华,魏岳荣,易干军,黄秉智,黄永红,2010,根癌农杆菌介导的香蕉高效遗传转化系统的建立,分子植物育种,8(1):172-178)
Huo M.J.,Wei Y.R.,Hu J.J.,Huang B.Z.,and Yi G.J.,2008,Factors affecting Agrobacterium-mediated genetic transformation of embryogenic cell suspension of banana,Guoshu Xuebao(Journal of Fruit Science),25(4):597-600(霍妙娟,魏岳荣,胡家金,黄秉智,易干军,2008,影响根癌农杆菌介导香蕉胚性悬浮细胞遗传转化的因素,果树学报,25(4):597-600)
Kuang R.B.,Li C.Y.,Yang J.,Wei Y.R.,Yang Q.S.,Hu C.H.,Sheng O.,and Yi G.J.,2013,Cell structure alteration of banana cultivars with different resistance to Fusarium Oxysporum f.sp.Cubense,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),11(2):193-198(邝瑞彬,李春雨,杨静,魏岳荣,杨乔松,胡春华,盛鸥,易干军,2013,抗感枯萎病香蕉的细胞结构抗性研究,分子植物育种,11(2):193-198)
Lorenzen J.,Tenkouano A.,Bandyopadhyay R.,Vroh B.,Coyne D.,and Tripathi L.,2010,Overview of banana and plantain(Musa spp.)improvement in Africa:past and future,Acta Hortic.,879(4):595-603
Lu Y.,Li H.P.,and Zheng F.C.,2006,Factors affecting interaction of Agrobacterium and materials in Agrobacterium-mediated genetic transformation on banana,Redai Zuowu Xuebao(Chinese Journal of Tropical Crops),27(1):84-87(陆英,李华平,郑服从,2006,电镜观察农杆菌介导香蕉基因转化的影响因子,热带作物学报,27(1):84-87)
Marroquin C.G.,Paduscheck C.,Escalant J.V.,and Teisson C.1993,Somatic embryogenesis and plant regeneration through cell suspension in Musa acuminate,In Vitro Cell.Dev Biol.Plant,29(1):43-46
Navarro C.,Escobedo R.M.,and Mayo A.,1997,In vitro plan regeneration from embryogenic cultures of a diploid and a triploid,Cavendish banana,Plant Cell Tiss.Org.Cult.,51(1):17-25
Novak F.J.,Afza R.,Duren M.V.,Perea-Dallos M.,Conger B.V.and Tang X.L.,1989,Somatic embryogenesis and plant regeneration in suspension cultures of dessert(AA and AAA)and cooking(ABB)bananas(Musa spp.),Nat.Biotechnol.7(2):147-158
Sreeramanan S.,Mazial M.,Abadullah M.P.,Rosli N.M.,and Xavier R.,2006,Potential selectable marker for genetic transformation in banana,Biotechnology,5(2):189-197
Strosse H.,Schoofs H.,Panis B.,Andre E.,Reyniers K.,and Swennen R.,2006,Development of embryogenic cell suspensions from shoot meristematic tissue bananas and plantains(Musa spp.),Plant Sci.,170(1):104-112
Tripathi J.,Muwonge A.,and Tripathi L.,2012,Highly efficient regeneration and transformation protocol for plantain cv.Gonja manjaya(Musa spp.AAB)using embryogenic cell suspension,In Vitro Cell.Dev.Biol.Plant,48(2):216-224
Tripathi J.N.,Oduor R.O.,and Tripathi L.,2015,A high-throughput regeneration and transformation platform for production of genetically modified banana,Front.Plant Sci.,6(2):538-543
Wei Y.R.,Huang X.L.,Li J.,Huang X.,Li Z.,and Li X.J.,2005,Establishment of embryogenic cell suspension culture and plant regeneration of edible banana Musa acuminate cv.M-as(AA),Shengwu Gongcheng Xuebao(Chinese Journal of Biotechnology),21(1):58-65(魏岳荣,黄学林,李佳,黄霞,李哲,李筱菊,2005,贡蕉胚性细胞悬浮系的建立和植株再生,生物工程学报,21(1):58-65)
Yao J.L.,Cohen D.,Atkinson R.,Richardson K.,and Morris B.,1995,Regeneration of transgenic plants from the commercial apple cultivar Royal Gala,Plant Cell Rep.,14(7):407-412