油菜农杆菌介导转化体系的优化
|
摘要
以甘蓝型油菜湘油15号为试验材料,对农杆菌转化体系中外植体预培养时间、培养基中AgNO_3浓度、农杆菌侵染时的菌液浓度、共培养基pH值等进行了优化研究,确立了优化的遗传转化条件。结果表明,外植体在农杆菌侵染和共培养前预培养3 d,可明显提高子叶外植体和下胚轴外植体出愈率和抗性芽苗分化率,有利于获得较高的转化效率;在基因转化中,AgNO_3的最佳使用浓度为30μmol/L,最佳的农杆菌侵染菌液OD_(600)为0.4,共培养基pH值5.4是油菜农杆菌介导转化中优化的转化条件。
Using Xiangyou 15 as experimental materials, the pre-culture days, added AgNO_3 concentration in culture medium and the concentration of bacterial solution during coculture stage and pH value of co-culture medium in Agrobacterium-mediated transgenic system in Brassica napus L. were studied. The results showed that explants were pre-cultured for 3 days before Agrobacterium tumefaciens infection and co-culture, which could significantly improve the regeneration rate of resistant bud seedlings from cotyledon and hypocotyl explants which was beneficial to obtain higher transformation efficiency. In genetic transformation, the optimal concentration of AgNO_3 added was 30 μmol/L AgNO_3. Also, the optimal Agrobacterium tumefaciens infection OD_(600) was 0.4, and the optimal coculture medium pH was 5.4 for Agrobacterium-mediated transformation in rapeseed.
Using Xiangyou 15 as experimental materials, the pre-culture days, added AgNO_3 concentration in culture medium and the concentration of bacterial solution during coculture stage and pH value of co-culture medium in Agrobacterium-mediated transgenic system in Brassica napus L. were studied. The results showed that explants were pre-cultured for 3 days before Agrobacterium tumefaciens infection and co-culture, which could significantly improve the regeneration rate of resistant bud seedlings from cotyledon and hypocotyl explants which was beneficial to obtain higher transformation efficiency. In genetic transformation, the optimal concentration of AgNO_3 added was 30 μmol/L AgNO_3. Also, the optimal Agrobacterium tumefaciens infection OD_(600) was 0.4, and the optimal coculture medium pH was 5.4 for Agrobacterium-mediated transformation in rapeseed.
引文
[1]MOLONEY M M,WALKER J M,SHARMA K K.High efficiency transformation of Brassica napus using Agrobacterium vectors[J].Plant Cell Rep.,1989,8:238-242.
[2]PRASAD K V S K,SHARMILA P,KUMAR P A,et al.Transformation of Brassica juncea(L.)Czern with bacterial cod A gene enhances its tolerance to salt stress[J].Molecular Breeding,2000,6:489-499.
[3]刘晓庆,沈源,蔡小宁,等.甘蓝型油菜下胚轴离体培养再生植株研究[J].安徽农业科学,2008,36(31):13547-13548.
[4]黄先群.油菜子叶和下胚轴再生体系及其遗传转化的研究进展[J].西南农业学报,2006,19(1):152-158.
[5]BHUIYAN M S U,MIN S R,JEONG W J,et al.An improved method for Agrobacterium-mediated genetic transformation from cotyledon explants of Brassica juncea[J].Plant Biotechnology,2011,28(1):17-23.
[6]PENTAL D,PRADHAN A K,SODHI Y S,et al.Variation amongst Brassica juncea cultivars for regeneration from hypocotyls explants and optimization of conditions for Agrobacterium-mediated genetic transformation[J].Plant Cell Reports,1993,12:462-467.
[7]CHAREST P J,HOLBROOK L A,GABARD J,et al.Agrobacterium-mediated transformation of thin cell layer explants from Brassica napus L[J].Theor Appl.Genet.,1988,75:438-445.
[8]EAPEN S,GEORGE L.Plant regeneration from peduncle segments of oil seed Brassica species:influence of silver nitrate and silver thiosulfate[J].Plant Cell,Tissue and Organ Culture,1997,51:229-232.
[9]CHI G L,BARFIELD D G,SIM G E,et al.Effect of Ag NO3and aminoethoxyvinglglycine on in vitro shoot and root organogenesis from seedling explants of recalcitrant Brassica genotypes[J].Plant Cell Rep.,1990,9:195-198.
[10]WILLIAMS J,PINK D A C,BIDDINGTON N L.Effect of silver nitrate of long-term culture and regeneration of callus from Brassica oleracea var.gemmifera[J].Plant Cell,Tissue and Organ Culture,1990,21:61-66.
[11]SETHI U,BASU A,MUKHERJEE S G.Role of inhibitors in the induction of differentiation in callus cultures of Brassica,Datura and Nicotiana[J].Plant Cell Reports,1990,8:598-600.
[12]PALMER C E.Enhanced shoot regeneration from Brassica campestris by silver nitrate[J].Plant Cell Reports,1992,11:541-545.
[13]PUA E C,CHI G L.De novo shoot morphogenesis and plant growth of mustard(Brassica juncea)in vitro in relation to ethylene[J].Physiol.Plantarum,1993,88:467-474.
[14]DE BLOCK M,DE BROUWER D,TENNING P.Transformation of Brassica napus and Brassica olercea using Agrobacterium tumefaciens and the expression of bar and neo genes in the transgenic plants[J].Plant Physiol.,1989,91:694-701.
[15]KUNTZON D S,THOMPSON G A,RADKE S E,et al.Modification of Brassica seed oil by antisense expression of a stearoyl-acyl carrier protein desaturase gene[J].Proc.Natl.Acad.Sci.USA,1992,89:2624-2628.
[16]MUKHOPADHYAY A,ARUMUGAMN,NANDAKUMAR P B A,et al.Agrobacterium-mediated genetic transformation of oilseed Brassica campestris:transformation frequency is strong influenced by the mode of shoot regeneration[J].Plant Cell Reports,1992,11:506-513.
[17]RADKE S E,ANDREWS B M,MOLONEY M M,et al.Transfornmation of Brassica napus L.using Agrobacterium tumefaciens:developmentally regulated expression of a reintroduced napin gene[J].Theor.Appl.Genet.,1988,75:685-694.
[18]MISRA S.Transformation of Brassica napus L.with a disarmed octopine plasmid of Agrobacterium tumefaciens:Molecular analysis and inheritance of the transformed phenotype[J].Experimental Botany,1990,41:269-275.
[19]OVESN魣J,PTACEK L,OPATRNY Z.Factors influencing the regeneration capacity of oilseed rape and cauliflower in transformation experiments[J].Biologia Plantarum,1993,35:107-112.
[20]SCHR魻DER M,DIXELIUS C,R魡HL魬N L,et al.Transformation Brassica napus by using the aad A gene as selectable marker and inheritance studies of the marker genes[J].Physiol.Plantarum,1994,92:37-46.
[21]GONG H,ENG-CHONG PUA.Identification and expression of genes associated with shoot regeneration from leaf disc explants of mustard(Brassica juncea)in vitro[J].Plant Science,2004,167(6):1191-1201.
[22]刘品,赵万千,史团省,等.油菜农杆菌介导转化体系的建立[J].郑州大学学报,2012,44(3):97-100.
[23]李韩帅,周秒依,杨凤玲,等.农杆菌介导的玉米自交系Z21遗传转化体系的研究[J].山西农业科学,2018,46(4):517-521,547.
[24]BABIC V,DATLA SCOLES G J,KELLER W A.Dvelopment of an efficient Agrobacterium-mediated transformation system for Brassica carinata[J].Plant Cell Reports,1998,17:183-188.
[25]CARDOZA V,STEWART C N.Increased Agrobacterium-mediated transformation and rooting efficiencies in canola(Brassica napus L.)from hypocotyls segment explants[J].Plant Cell Rep.,2003,21:599-604.
[26]KHAN M R,RASHID H,ANSAR M,et al.High frequency shoot regeneration and Agrobacterium-mediated DNA transfer in Canola(Brassica napus)[J].Plant Cell,Tissue and Organ Culture,2003,75:223-231.
[27]高武军,王景雪,卢龙斗,等.硝酸银在油菜基因转化中的影响作用[J].中国农学通报,2002,18(4):43-45.
[28]何业华,熊兴华,官春云,等.根癌农杆菌介导TA29-Barnase基因转化甘蓝型油菜的研究[J].作物学报,2003,29(4):615-620.
[29]杨广东,朱桢,李燕娥,等.大白菜高效遗传转化体系的建立[J].农业生物技术学报,2002,10(2):127-132.
[30]STACHEL S E,MESSENS E,VAN MONTUGU M,et al.Identification of the signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens[J].Nature,1986,318:624-629.
[31]TAKASAKI T,HATEKEYAMA K,OJIMA K,et al.Factors influencing Agrobacterium-mediated transformation of Brassica rapa L.[J].Breed Sci.,1997,47:127-134.
[32]任伟,王嘉莹,任华华,等.PuCAT,Bar抗逆抗除草剂基因共转化紫花苜蓿的研究[J].华北农学报,2017,32(Z):1-6.
[2]PRASAD K V S K,SHARMILA P,KUMAR P A,et al.Transformation of Brassica juncea(L.)Czern with bacterial cod A gene enhances its tolerance to salt stress[J].Molecular Breeding,2000,6:489-499.
[3]刘晓庆,沈源,蔡小宁,等.甘蓝型油菜下胚轴离体培养再生植株研究[J].安徽农业科学,2008,36(31):13547-13548.
[4]黄先群.油菜子叶和下胚轴再生体系及其遗传转化的研究进展[J].西南农业学报,2006,19(1):152-158.
[5]BHUIYAN M S U,MIN S R,JEONG W J,et al.An improved method for Agrobacterium-mediated genetic transformation from cotyledon explants of Brassica juncea[J].Plant Biotechnology,2011,28(1):17-23.
[6]PENTAL D,PRADHAN A K,SODHI Y S,et al.Variation amongst Brassica juncea cultivars for regeneration from hypocotyls explants and optimization of conditions for Agrobacterium-mediated genetic transformation[J].Plant Cell Reports,1993,12:462-467.
[7]CHAREST P J,HOLBROOK L A,GABARD J,et al.Agrobacterium-mediated transformation of thin cell layer explants from Brassica napus L[J].Theor Appl.Genet.,1988,75:438-445.
[8]EAPEN S,GEORGE L.Plant regeneration from peduncle segments of oil seed Brassica species:influence of silver nitrate and silver thiosulfate[J].Plant Cell,Tissue and Organ Culture,1997,51:229-232.
[9]CHI G L,BARFIELD D G,SIM G E,et al.Effect of Ag NO3and aminoethoxyvinglglycine on in vitro shoot and root organogenesis from seedling explants of recalcitrant Brassica genotypes[J].Plant Cell Rep.,1990,9:195-198.
[10]WILLIAMS J,PINK D A C,BIDDINGTON N L.Effect of silver nitrate of long-term culture and regeneration of callus from Brassica oleracea var.gemmifera[J].Plant Cell,Tissue and Organ Culture,1990,21:61-66.
[11]SETHI U,BASU A,MUKHERJEE S G.Role of inhibitors in the induction of differentiation in callus cultures of Brassica,Datura and Nicotiana[J].Plant Cell Reports,1990,8:598-600.
[12]PALMER C E.Enhanced shoot regeneration from Brassica campestris by silver nitrate[J].Plant Cell Reports,1992,11:541-545.
[13]PUA E C,CHI G L.De novo shoot morphogenesis and plant growth of mustard(Brassica juncea)in vitro in relation to ethylene[J].Physiol.Plantarum,1993,88:467-474.
[14]DE BLOCK M,DE BROUWER D,TENNING P.Transformation of Brassica napus and Brassica olercea using Agrobacterium tumefaciens and the expression of bar and neo genes in the transgenic plants[J].Plant Physiol.,1989,91:694-701.
[15]KUNTZON D S,THOMPSON G A,RADKE S E,et al.Modification of Brassica seed oil by antisense expression of a stearoyl-acyl carrier protein desaturase gene[J].Proc.Natl.Acad.Sci.USA,1992,89:2624-2628.
[16]MUKHOPADHYAY A,ARUMUGAMN,NANDAKUMAR P B A,et al.Agrobacterium-mediated genetic transformation of oilseed Brassica campestris:transformation frequency is strong influenced by the mode of shoot regeneration[J].Plant Cell Reports,1992,11:506-513.
[17]RADKE S E,ANDREWS B M,MOLONEY M M,et al.Transfornmation of Brassica napus L.using Agrobacterium tumefaciens:developmentally regulated expression of a reintroduced napin gene[J].Theor.Appl.Genet.,1988,75:685-694.
[18]MISRA S.Transformation of Brassica napus L.with a disarmed octopine plasmid of Agrobacterium tumefaciens:Molecular analysis and inheritance of the transformed phenotype[J].Experimental Botany,1990,41:269-275.
[19]OVESN魣J,PTACEK L,OPATRNY Z.Factors influencing the regeneration capacity of oilseed rape and cauliflower in transformation experiments[J].Biologia Plantarum,1993,35:107-112.
[20]SCHR魻DER M,DIXELIUS C,R魡HL魬N L,et al.Transformation Brassica napus by using the aad A gene as selectable marker and inheritance studies of the marker genes[J].Physiol.Plantarum,1994,92:37-46.
[21]GONG H,ENG-CHONG PUA.Identification and expression of genes associated with shoot regeneration from leaf disc explants of mustard(Brassica juncea)in vitro[J].Plant Science,2004,167(6):1191-1201.
[22]刘品,赵万千,史团省,等.油菜农杆菌介导转化体系的建立[J].郑州大学学报,2012,44(3):97-100.
[23]李韩帅,周秒依,杨凤玲,等.农杆菌介导的玉米自交系Z21遗传转化体系的研究[J].山西农业科学,2018,46(4):517-521,547.
[24]BABIC V,DATLA SCOLES G J,KELLER W A.Dvelopment of an efficient Agrobacterium-mediated transformation system for Brassica carinata[J].Plant Cell Reports,1998,17:183-188.
[25]CARDOZA V,STEWART C N.Increased Agrobacterium-mediated transformation and rooting efficiencies in canola(Brassica napus L.)from hypocotyls segment explants[J].Plant Cell Rep.,2003,21:599-604.
[26]KHAN M R,RASHID H,ANSAR M,et al.High frequency shoot regeneration and Agrobacterium-mediated DNA transfer in Canola(Brassica napus)[J].Plant Cell,Tissue and Organ Culture,2003,75:223-231.
[27]高武军,王景雪,卢龙斗,等.硝酸银在油菜基因转化中的影响作用[J].中国农学通报,2002,18(4):43-45.
[28]何业华,熊兴华,官春云,等.根癌农杆菌介导TA29-Barnase基因转化甘蓝型油菜的研究[J].作物学报,2003,29(4):615-620.
[29]杨广东,朱桢,李燕娥,等.大白菜高效遗传转化体系的建立[J].农业生物技术学报,2002,10(2):127-132.
[30]STACHEL S E,MESSENS E,VAN MONTUGU M,et al.Identification of the signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens[J].Nature,1986,318:624-629.
[31]TAKASAKI T,HATEKEYAMA K,OJIMA K,et al.Factors influencing Agrobacterium-mediated transformation of Brassica rapa L.[J].Breed Sci.,1997,47:127-134.
[32]任伟,王嘉莹,任华华,等.PuCAT,Bar抗逆抗除草剂基因共转化紫花苜蓿的研究[J].华北农学报,2017,32(Z):1-6.