两种油葵自交系再生体系的建立
|
摘要
【目的】取两种自交系油葵(1538-3和1573-2)的不同外植体,建立组织再生培养体系,选出最适品系,为遗传转化奠定基础。【方法】分别取两种自交系油葵的不同组织,采用不同激素配比,从器官间接和直接发生两个方面分别诱导油葵不定芽再生。【结果】以油葵自交系1538-3的不同外植体为材料,在含有1-萘乙酸(NAA)、6-苄氨基嘌呤(6-BA)、激动素(KT)的培养基中都易形成愈伤组织,但均表现为白色、水渍化程度高,无分化能力;6-BA浓度在0~1.0 mg/L时,子叶节可诱导出不定芽,其他激素配比中未见此现象;以胚轴为外植体时,油葵1573-2自交系不定芽诱导的最佳培养基为MS+0.6 mg/L 6-BA+0.1 mg/L GA3;1538-3自交系不定芽诱导的最佳培养基为MS+0.5 mg/L 6-BA+0.05 mg/L GA3;两种自交系诱导不定芽生根时NAA的最佳浓度为0~0.2 mg/L。【结论】两种基因型油葵诱导不定芽的能力差别较大,其中自交系1538-3再生能力较强,遗传转化时选用1538-3的胚轴作为转化受体时较好。
【Objective】The explants of two inbred oil sunflower( 1538-3 and 1573-2) were used to establish the tissue regeneration culture system,and the optimum strain was selected to lay the foundation for genetic transformation.【Method】Two different inbred lines of oil sunflower were taken respectively,and the regeneration of adventitious bud of oil sunflower was induced from two aspects of indirect and direct organs by different hormone ratio.【Result】The results showed that inbred lines 1538-3 was easy to form callus in the mediums containing NAA,6-Benzylaminopurine( 6-BA) and kinetin( KT),but all the callus were white with high water damage level.The sunflower cotyledonary node could induce adventitious buds when the concentration of 6-BA was 0 to 1.0 mg/L,which couldn't be observed in other hormone combinations.The combination of MS + 0.6 mg/L 6-BA + 0.1 mg/L GA3 was most suitable for the induction of adventitious bud from inbred lines 1573-2 hypocotyls explants.While for inbred line 1538-3,the most suitable medium was MS + 0.5 mg/L 6-BA + 0.05 mg/L GA3.The best concentration of NAA for inducing the root of adventitious buds ranged from 0 to 0.2 mg/L in both inbred lines.【Conclusion】The ability of the two genotypes of oil sunflower to induce adventitious bud is different.Among them,the inbred line 1538-3 has stronger regeneration ability,and it is better to choose the 1538-3 axis as the transformation receptor in the genetic transformation.
【Objective】The explants of two inbred oil sunflower( 1538-3 and 1573-2) were used to establish the tissue regeneration culture system,and the optimum strain was selected to lay the foundation for genetic transformation.【Method】Two different inbred lines of oil sunflower were taken respectively,and the regeneration of adventitious bud of oil sunflower was induced from two aspects of indirect and direct organs by different hormone ratio.【Result】The results showed that inbred lines 1538-3 was easy to form callus in the mediums containing NAA,6-Benzylaminopurine( 6-BA) and kinetin( KT),but all the callus were white with high water damage level.The sunflower cotyledonary node could induce adventitious buds when the concentration of 6-BA was 0 to 1.0 mg/L,which couldn't be observed in other hormone combinations.The combination of MS + 0.6 mg/L 6-BA + 0.1 mg/L GA3 was most suitable for the induction of adventitious bud from inbred lines 1573-2 hypocotyls explants.While for inbred line 1538-3,the most suitable medium was MS + 0.5 mg/L 6-BA + 0.05 mg/L GA3.The best concentration of NAA for inducing the root of adventitious buds ranged from 0 to 0.2 mg/L in both inbred lines.【Conclusion】The ability of the two genotypes of oil sunflower to induce adventitious bud is different.Among them,the inbred line 1538-3 has stronger regeneration ability,and it is better to choose the 1538-3 axis as the transformation receptor in the genetic transformation.
引文
[1]Pugliesi,C.,Cecconi,F.,Mandolfo,A.,&Baroncelli,S.(1991).Plant regeneration and genetic variability from tissue cultures of sunflower(helianthus annuusl.).Plant Breeding,106(2):114-121.
[2]Jeannin,G.,Bronner,R.,&Hahne,G.(1995).Somatic embryogenesis and organogenesis induced on the immature zygotic embryo of sunflower(helianthus annuml.)cultivated in vitro:role of the sugar.Plant Cell Reports,15(3-4):200-204.
[3]Sarrafi,A.,Roustan,J.P.,Fallot,J.,&Alibert,G.(1996).Genetic analysis of organogenesis in the cotyledons of zygotic embryos of sunflower(helianthus annuusl.).Theoretical and Applied Genetics,92(2):225-229.
[4]Fiore,M.C.,Trabace,T.,&Sunseri,F.(1997).High frequency of plant regeneration in sunflower from cotyledons via somatic embryogenesis.Plant Cell Reports,16(5):295-298.
[5]Zezul',T.G.,Gorbatenko,E.V.,&Raldugina,G.N.(1995).Regeneration of sunflower(helianthus annuusl.)plants via somatic embryogenesis in vitro.Genetika.(3):197-202.
[6]Fambrini,M.,Cionini,G.,&Pugliesi,C.(1997).Acquisition of high embryogenic potential in regenerated plants of helianthus annuus x h.tuberosus.Plant Cell,Tissue and Organ Culture(PCTOC),51(2):103-110.
[7]Laparra,H.,Bronner,R.,&Hahne,G.(1997).Histological analysis of somatic embryogenesis induced in leaf explants ofhelianthus smithii heiser.Protoplasma,196(1):1-11.
[8]贺宾,高燕,向理军,等.油葵茎尖周缘分生区培养及高频率芽再生[J].新疆农业科学,2004,41(2):86-88.HE Bin,GAO Yan,XIANG Li-jun,et al.(2004)The culture of apical shoot of oil sunflower and its high frequent regeneration[J].Xinjiang Agricultural Sciences,41(2):86-88.(in Chinese)
[9]徐培洲,吴先军,胡保民,等.油葵子叶外植体不定芽再生体系的建立[J].中国油料作物学报,2004,26(4):16-19.XU Pei-zhou,WU Xian-jun,HU Bao-min,et al.(2004)A shoot regeneration system from cotyledon of Helianthus annuus L.[J].Chinese Journal of Oil Crop Sciences,26(4):16-19.(in Chinese)
[10]胡文冉,黄乐平,孟庆玉,等.油葵叶片离体再生体系的建立[J].新疆农业科学,2006,43(1):82-85.HU Wen-ran,HUANG Le-ping,MENG Qing-yu,et al.(2006)A regeneration system from cotyledon of sunflower(Helianthus annuus L.)[J].Xinjiang Agricultural Sciences,43(1):82-85.(in Chinese)
[11]刘海臣.向日葵下胚轴组织培养及其遗传转化的研究[D].长春:长春理工大学硕士学位论文,2007.LIU Hai-chen.(2007).Study on the tissue culture and genetic transformation with hypocotyl of sunflower[D].Master Dissertation.Changchun University of Science and Technology,Changchun.(in Chinese)
[12]刘海学,王罡,季静,等.向日葵(Helianthus annuus L.)不同外植体组织培养及其再生的研究[J].中国农业科学,2006,39(11):2 208-2 213.LIU Hai-xue,WANG Gang,JI Jing,et al.(2006).The research of tissue culture and regenerated of the different explants of sunflower(Helianthus annuus L.)[J].Scientia Agricultura Sinica,39(11):2,208-2,213.(in Chinese)
[13]刘宝,顾德锋,邬信康,等.向日葵(Helianthus annus)自交系的组织培养和芽的分化[J].吉林农业大学学报,1991,13(1):6-10.LIU Bao,GU De-feng,WU Xin-kang,et al.(1991)Tissue culture and plantlet regeneration of sunflower(Helianthus annuus L.)inbred lines[J].Journal of Jilin Agriculture University,13(1):6-10.(in Chinese)
[14]王园园,李彩凤,张翼飞,等.向日葵离体再生体系的建立[J].生物工程学报,2011,27(9):379-389.WANG Yuan-yuan,LI Cai-feng,ZHANG Yi-fei,et al.(2011).Establishing the regeneration system of sunflower[J].Chinese Journal of Biotechnology,27(9):379-389.(in Chinese)
[15]Paterson,K.E.,&Everett,N.P.(1985).Regeneration of helianthus annuus,inbred plants from callus.Plant Science,42(2):125-132.
[16]刘宇新.抗氧化蛋白Ss Prx Q在向日葵中的表达研究[D].天津:天津大学硕士学位论文,2009.LIU Yu-xin.(2009).Studies of expression of antioxidant protein Ssprx Q in sunflower[D].Master Dissertation.Tianjin University,Tianjin.(in Chinese)
[17]杨长友.向日葵离体再生及农杆菌介导的遗传转化研究[D].重庆:重庆师范大学硕士学位论文,2014.YANG Chang-you.(2014).Regeneration in vitro and agrobacterium-mediated transformation of sunflower(Helianthus annuus L.)[D].Master Dissertation.Chongqing Normal University,Chongqing.(in Chinese)
[18]卫志明,许智宏.向日葵的组织培养和植株再生[J].植物生理学通讯,1983,(5):42.WEI Zhi-ming,XU Zhi-hong.(1983).Tissue culture and plantlet regeneration of sunflower[J].Plant Physiology Journal,(5):42.(in Chinese)
[19]梁国婷.生长素和细胞分裂素调节拟南芥离体器官的再生[D].泰安:山东农业大学硕士学位论文,2009.LIANG Guo-ting.(2009)Auxin and cytokinin regulate organ regeneration of Arabidopsis in vitro[D].Master Dissertation.Shandong Agricultural University,Taian.(in Chinese)
[20]勾畅,武慧,将达,等.激素配比对不同基因型菊花再生体系的调控[J].北方园艺,2013,(13):135-137.GOU Chang,WU Hui,JIANG Da,et al.(2011)Effects of different hormone combinations on the regeneration of four different chrysanthemum genotypes[J].Northern Hourticulture,(13):135-137.(in Chinese)
[21]Vanegas,P.E.,Cruz-Hernández,A.,Valverde,M.E.,&Paredes-López,O.(2002).Plant regeneration via organogenesis in marigold.Plant Cell,Tissue and Organ Culture(PCTOC),69(3):279-283.
[22]齐迎春,叶要妹,刘国锋,等.不同基因型孔雀草高效植株再生体系的建立[J].中国农业科学,2005,38(7):1 414-1 417.QI Ying-chun,YE Yao-mei,LIU Guo-feng,et al.(2005)The establishment of efficient regeneration system for different genotypes of Tagetes patula L.[J].Scientia Agricultura Sinica,38(7):1,414-1,417.(in Chinese)
[2]Jeannin,G.,Bronner,R.,&Hahne,G.(1995).Somatic embryogenesis and organogenesis induced on the immature zygotic embryo of sunflower(helianthus annuml.)cultivated in vitro:role of the sugar.Plant Cell Reports,15(3-4):200-204.
[3]Sarrafi,A.,Roustan,J.P.,Fallot,J.,&Alibert,G.(1996).Genetic analysis of organogenesis in the cotyledons of zygotic embryos of sunflower(helianthus annuusl.).Theoretical and Applied Genetics,92(2):225-229.
[4]Fiore,M.C.,Trabace,T.,&Sunseri,F.(1997).High frequency of plant regeneration in sunflower from cotyledons via somatic embryogenesis.Plant Cell Reports,16(5):295-298.
[5]Zezul',T.G.,Gorbatenko,E.V.,&Raldugina,G.N.(1995).Regeneration of sunflower(helianthus annuusl.)plants via somatic embryogenesis in vitro.Genetika.(3):197-202.
[6]Fambrini,M.,Cionini,G.,&Pugliesi,C.(1997).Acquisition of high embryogenic potential in regenerated plants of helianthus annuus x h.tuberosus.Plant Cell,Tissue and Organ Culture(PCTOC),51(2):103-110.
[7]Laparra,H.,Bronner,R.,&Hahne,G.(1997).Histological analysis of somatic embryogenesis induced in leaf explants ofhelianthus smithii heiser.Protoplasma,196(1):1-11.
[8]贺宾,高燕,向理军,等.油葵茎尖周缘分生区培养及高频率芽再生[J].新疆农业科学,2004,41(2):86-88.HE Bin,GAO Yan,XIANG Li-jun,et al.(2004)The culture of apical shoot of oil sunflower and its high frequent regeneration[J].Xinjiang Agricultural Sciences,41(2):86-88.(in Chinese)
[9]徐培洲,吴先军,胡保民,等.油葵子叶外植体不定芽再生体系的建立[J].中国油料作物学报,2004,26(4):16-19.XU Pei-zhou,WU Xian-jun,HU Bao-min,et al.(2004)A shoot regeneration system from cotyledon of Helianthus annuus L.[J].Chinese Journal of Oil Crop Sciences,26(4):16-19.(in Chinese)
[10]胡文冉,黄乐平,孟庆玉,等.油葵叶片离体再生体系的建立[J].新疆农业科学,2006,43(1):82-85.HU Wen-ran,HUANG Le-ping,MENG Qing-yu,et al.(2006)A regeneration system from cotyledon of sunflower(Helianthus annuus L.)[J].Xinjiang Agricultural Sciences,43(1):82-85.(in Chinese)
[11]刘海臣.向日葵下胚轴组织培养及其遗传转化的研究[D].长春:长春理工大学硕士学位论文,2007.LIU Hai-chen.(2007).Study on the tissue culture and genetic transformation with hypocotyl of sunflower[D].Master Dissertation.Changchun University of Science and Technology,Changchun.(in Chinese)
[12]刘海学,王罡,季静,等.向日葵(Helianthus annuus L.)不同外植体组织培养及其再生的研究[J].中国农业科学,2006,39(11):2 208-2 213.LIU Hai-xue,WANG Gang,JI Jing,et al.(2006).The research of tissue culture and regenerated of the different explants of sunflower(Helianthus annuus L.)[J].Scientia Agricultura Sinica,39(11):2,208-2,213.(in Chinese)
[13]刘宝,顾德锋,邬信康,等.向日葵(Helianthus annus)自交系的组织培养和芽的分化[J].吉林农业大学学报,1991,13(1):6-10.LIU Bao,GU De-feng,WU Xin-kang,et al.(1991)Tissue culture and plantlet regeneration of sunflower(Helianthus annuus L.)inbred lines[J].Journal of Jilin Agriculture University,13(1):6-10.(in Chinese)
[14]王园园,李彩凤,张翼飞,等.向日葵离体再生体系的建立[J].生物工程学报,2011,27(9):379-389.WANG Yuan-yuan,LI Cai-feng,ZHANG Yi-fei,et al.(2011).Establishing the regeneration system of sunflower[J].Chinese Journal of Biotechnology,27(9):379-389.(in Chinese)
[15]Paterson,K.E.,&Everett,N.P.(1985).Regeneration of helianthus annuus,inbred plants from callus.Plant Science,42(2):125-132.
[16]刘宇新.抗氧化蛋白Ss Prx Q在向日葵中的表达研究[D].天津:天津大学硕士学位论文,2009.LIU Yu-xin.(2009).Studies of expression of antioxidant protein Ssprx Q in sunflower[D].Master Dissertation.Tianjin University,Tianjin.(in Chinese)
[17]杨长友.向日葵离体再生及农杆菌介导的遗传转化研究[D].重庆:重庆师范大学硕士学位论文,2014.YANG Chang-you.(2014).Regeneration in vitro and agrobacterium-mediated transformation of sunflower(Helianthus annuus L.)[D].Master Dissertation.Chongqing Normal University,Chongqing.(in Chinese)
[18]卫志明,许智宏.向日葵的组织培养和植株再生[J].植物生理学通讯,1983,(5):42.WEI Zhi-ming,XU Zhi-hong.(1983).Tissue culture and plantlet regeneration of sunflower[J].Plant Physiology Journal,(5):42.(in Chinese)
[19]梁国婷.生长素和细胞分裂素调节拟南芥离体器官的再生[D].泰安:山东农业大学硕士学位论文,2009.LIANG Guo-ting.(2009)Auxin and cytokinin regulate organ regeneration of Arabidopsis in vitro[D].Master Dissertation.Shandong Agricultural University,Taian.(in Chinese)
[20]勾畅,武慧,将达,等.激素配比对不同基因型菊花再生体系的调控[J].北方园艺,2013,(13):135-137.GOU Chang,WU Hui,JIANG Da,et al.(2011)Effects of different hormone combinations on the regeneration of four different chrysanthemum genotypes[J].Northern Hourticulture,(13):135-137.(in Chinese)
[21]Vanegas,P.E.,Cruz-Hernández,A.,Valverde,M.E.,&Paredes-López,O.(2002).Plant regeneration via organogenesis in marigold.Plant Cell,Tissue and Organ Culture(PCTOC),69(3):279-283.
[22]齐迎春,叶要妹,刘国锋,等.不同基因型孔雀草高效植株再生体系的建立[J].中国农业科学,2005,38(7):1 414-1 417.QI Ying-chun,YE Yao-mei,LIU Guo-feng,et al.(2005)The establishment of efficient regeneration system for different genotypes of Tagetes patula L.[J].Scientia Agricultura Sinica,38(7):1,414-1,417.(in Chinese)