水稻成熟胚愈伤组织染色体加倍的探究
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摘要
本研究以水稻成熟胚诱导的胚性愈伤为材料,通过直接在培养基中添加秋水仙素,或用秋水仙素溶液浸泡愈伤组织,研究了培养基中秋水仙素浓度、浸泡处理方式、胚性愈伤大小、恢复培养时间等因素对染色体加倍效率的影响。结果表明,在分化培养基中添加4～10 mg/L的秋水仙素能直接得到四倍体水稻植株,加倍率为3.33%～18.86%。浸泡法中高浓度短时间(500 mg/L+48 h)处理比低浓度长时间(300 mg/L+65 h)处理更利于染色体加倍。较小的胚性愈伤组织(2～3 mm)加倍效率更高。延长恢复培养时间能极显著提高处理后的绿苗分化率,从而提高加倍效率。
In this study, the embryonic callus induced from rice mature embryo was used as the material. By adding colchicine directly to the medium or soaking the callus with colchicine solution, the effects of colchicine concentration in the medium, soaking treatment method, embryonic callus size, and recovery culture time on chromosome doubling efficiency were studied. The results showed that the addition of 4～10 mg/L colchicine in the differentiation medium could directly obtain tetraploid rice plantlets with a doubling rate of 3.33%～18.86%. The soaking treatment of high concentration and short time(500 mg/L+48 h) was more beneficial to chromosome doubling than the treatment of low concentration and long time(300 mg/L+65 h). Smaller embryogenic callus(2～3 mm) had higher doubling efficiency. Prolonged recovery culture time could significantly improve the green seedling differentiation rate after treatment, thus increasing the doubling efficiency.
In this study, the embryonic callus induced from rice mature embryo was used as the material. By adding colchicine directly to the medium or soaking the callus with colchicine solution, the effects of colchicine concentration in the medium, soaking treatment method, embryonic callus size, and recovery culture time on chromosome doubling efficiency were studied. The results showed that the addition of 4～10 mg/L colchicine in the differentiation medium could directly obtain tetraploid rice plantlets with a doubling rate of 3.33%～18.86%. The soaking treatment of high concentration and short time(500 mg/L+48 h) was more beneficial to chromosome doubling than the treatment of low concentration and long time(300 mg/L+65 h). Smaller embryogenic callus(2～3 mm) had higher doubling efficiency. Prolonged recovery culture time could significantly improve the green seedling differentiation rate after treatment, thus increasing the doubling efficiency.
引文
Alix K.,Ge'rard P.R.,Schwarzacher T.,and Heslop-Harrison J.S.2017,Polyploidy and interspecific hybridization:partners for adaptation,speciation and evolution in plants,Annals of Botany,120(2):183-194
Allario T.,Brumos J.,Colmenero-Flores J.M.,Iglesias D.J.,Pina J.A.,Navarro L.,Talon M.,Ollitrault P.,and Morillon R.2013,Tetraploid Rangpur lime rootstock increases drough tolerance via enhanced constitutive root abscisicacid production,Plant Cell Envi.,36(4):856-868
An T.G.,Zhong G.C.,Li J.M.,Wang Z.G.,Wang Y.M.,and Ji J.2003,Chromosome doubling methods of immature embryo plants of wheat distant hybridization,Zuowu Xuebao(Acta Agronomica Sinica),29(6):955-957(安调过,钟冠昌,李俊明,王志国,王彦梅,纪军,2003,半浸根法加倍小麦远缘杂交幼胚再生植株染色体,作物学报,29(6):955-957)
Cai D.T.,Yuan L.P.,and Lu X.G.,2001,A new strategy of rice breeding in the 21stcenturyⅡ.Searching a new pathway of rice breeding by utilization of double heterosis of wide cross and polyploidization,Zuowu Xuebao(Acta Agronomica Sinica),27(1):110-116(蔡得田,袁隆平,卢兴桂,2001,21世纪水稻育种的新战略(Ⅱ):利用远缘杂交和多倍体双重优势进行超级稻育种,作物学报,27(1):110-116)
Chao D.Y.,Dilkes B.,Luo H.B.,Douglas A.,Yakubova E.,Lahner B.,and Salt D.E.,2013,Polyploids exhibit higher potassium uptake and salinity tolerance in Arabidopsis,Science,341(6146):658-659
Godfree R.C.,Marshall D.J.,Young A.G.,Miller C.H.,and Mathews S.,2017,Empirical evidence of fixed and homeostatic patterns of polyploidy advantage in a keystone grass exposed to drought and heat stress,Royal Society Open Science,4(11):170934
Huang Q.C.,2000,Effect of Oryza sativa×Pennisetum alopecuroides,Zhongguo Shuidao Kexue(Chinese Journal of Rice Sciene),14(1):48-50(黄群策,2000,同源四倍体水稻与狼尾草杂交效果,中国水稻科学,14(1):48-50)
Huang Q.C.,Sun M.Y.,and Deng Q.Y.,2001,Ployploid rice and its potential value,Zajiao Shuidao(Hybrid Rice),16(1):1-3(黄群策,孙梅元,邓启云,2001,多倍体水稻及其潜在价值,杂交水稻,16(1):1-3)
Li Y.,Mao S.L.,Wang Y.C.,and Li Q.,2016,Effects of temperature and seed size on seed germination in Potentilla aurea,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(5):1248-1251(李阳,毛少利,王宇超,李倩,2016,温度和种子大小对黄花委陵菜种子萌发特征的影响,基因组学与应用生物学,35(5):1248-1251)
Luan L.,Long W.B.,Wang X.,Chen Y.,Qiu L.,Tu S.B.,Kong F.L.,and Xiao X.Y.,2009,Research on creating autotetrapioid rice using colchicum to induce the panicle callus of diploid rice,Sichuang Daxue Xuebao(Journal of Sichuan U-niversity),46(3):829-837(栾丽,龙文波,王兴,陈英,邱琳,涂升斌,孔繁伦,肖小余,2009,秋水仙素诱导水稻幼穗愈伤组织创制同源四倍体种质的研究,四川大学学报,46(3):829-837)
Ramsey J.,and Schemske D.W.,1998,Pathways,mechanisms,and rates of polyploid formation in flowering plants,Annu.Rev.Ecol.Syst.,29(1):467-501
Ruiza M.,Qui觡ones A.,Martínez-Alcántara B.,Aleza P.,Morillon R.,Navarro L.,Primo-Millo E.,and Martínez-Cuenca M.,2016,Effects of salinity on diploid(2x)and doubled diploid(4x)Citrus macrophylla genotypes,Scientia Horticulturae,207:33-40
Van de Peer Y.,Mizrachi E.,and Marchal K.,2017,The evolutionary significance of polyploidy,Nat.Rev.Genet.,15(10)1-14
Xu C.,Wang Y.H.,Yu Y.C.,Duan J.B.,Liao Z.G.,Xiong G.S.Meng X.B.,Liu G.F.,Qian Q.,and Li J.Y.,2012,Degradation of monoCuLm 1 by APC/CTAD1regulates rice tillering Nat.Commun.,20(10):1-9
Yang P.M.,Huang Q.C.,Qin G.Y.,Zhao S.P.,and Zhou J.G.2014,Different drought-stress responses in photosynthesis and reactive oxygen metabolism between autotetraploid and diploid rice,Photosynthetica,52(2):193-202
Zhang X.H.,Wang A.Y.,Du C.Q.,Song Z.J.,Wang W.,He Y.C.,and Cai D.T.,2014,An efficient method of developing synthetic allopolyploid rice(Oryza spp),Genet.Resour.Crop E-vol.,61(4):809-816
Zhou X.J.,and Cheng Z.H.,2008,Establishment of induction system of garlic clove-tip by injection with colchicine and effect of mutation,Xibei Nongye Xuebao(Acta Agriculturae Boreali-occidentalis Sinica),17(5):267-271(周香君,程智慧,2008,蒜瓣茎尖注射秋水仙素的诱变效果和诱变方法的建立,西北农业学报,17(5):267-271)
Allario T.,Brumos J.,Colmenero-Flores J.M.,Iglesias D.J.,Pina J.A.,Navarro L.,Talon M.,Ollitrault P.,and Morillon R.2013,Tetraploid Rangpur lime rootstock increases drough tolerance via enhanced constitutive root abscisicacid production,Plant Cell Envi.,36(4):856-868
An T.G.,Zhong G.C.,Li J.M.,Wang Z.G.,Wang Y.M.,and Ji J.2003,Chromosome doubling methods of immature embryo plants of wheat distant hybridization,Zuowu Xuebao(Acta Agronomica Sinica),29(6):955-957(安调过,钟冠昌,李俊明,王志国,王彦梅,纪军,2003,半浸根法加倍小麦远缘杂交幼胚再生植株染色体,作物学报,29(6):955-957)
Cai D.T.,Yuan L.P.,and Lu X.G.,2001,A new strategy of rice breeding in the 21stcenturyⅡ.Searching a new pathway of rice breeding by utilization of double heterosis of wide cross and polyploidization,Zuowu Xuebao(Acta Agronomica Sinica),27(1):110-116(蔡得田,袁隆平,卢兴桂,2001,21世纪水稻育种的新战略(Ⅱ):利用远缘杂交和多倍体双重优势进行超级稻育种,作物学报,27(1):110-116)
Chao D.Y.,Dilkes B.,Luo H.B.,Douglas A.,Yakubova E.,Lahner B.,and Salt D.E.,2013,Polyploids exhibit higher potassium uptake and salinity tolerance in Arabidopsis,Science,341(6146):658-659
Godfree R.C.,Marshall D.J.,Young A.G.,Miller C.H.,and Mathews S.,2017,Empirical evidence of fixed and homeostatic patterns of polyploidy advantage in a keystone grass exposed to drought and heat stress,Royal Society Open Science,4(11):170934
Huang Q.C.,2000,Effect of Oryza sativa×Pennisetum alopecuroides,Zhongguo Shuidao Kexue(Chinese Journal of Rice Sciene),14(1):48-50(黄群策,2000,同源四倍体水稻与狼尾草杂交效果,中国水稻科学,14(1):48-50)
Huang Q.C.,Sun M.Y.,and Deng Q.Y.,2001,Ployploid rice and its potential value,Zajiao Shuidao(Hybrid Rice),16(1):1-3(黄群策,孙梅元,邓启云,2001,多倍体水稻及其潜在价值,杂交水稻,16(1):1-3)
Li Y.,Mao S.L.,Wang Y.C.,and Li Q.,2016,Effects of temperature and seed size on seed germination in Potentilla aurea,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(5):1248-1251(李阳,毛少利,王宇超,李倩,2016,温度和种子大小对黄花委陵菜种子萌发特征的影响,基因组学与应用生物学,35(5):1248-1251)
Luan L.,Long W.B.,Wang X.,Chen Y.,Qiu L.,Tu S.B.,Kong F.L.,and Xiao X.Y.,2009,Research on creating autotetrapioid rice using colchicum to induce the panicle callus of diploid rice,Sichuang Daxue Xuebao(Journal of Sichuan U-niversity),46(3):829-837(栾丽,龙文波,王兴,陈英,邱琳,涂升斌,孔繁伦,肖小余,2009,秋水仙素诱导水稻幼穗愈伤组织创制同源四倍体种质的研究,四川大学学报,46(3):829-837)
Ramsey J.,and Schemske D.W.,1998,Pathways,mechanisms,and rates of polyploid formation in flowering plants,Annu.Rev.Ecol.Syst.,29(1):467-501
Ruiza M.,Qui觡ones A.,Martínez-Alcántara B.,Aleza P.,Morillon R.,Navarro L.,Primo-Millo E.,and Martínez-Cuenca M.,2016,Effects of salinity on diploid(2x)and doubled diploid(4x)Citrus macrophylla genotypes,Scientia Horticulturae,207:33-40
Van de Peer Y.,Mizrachi E.,and Marchal K.,2017,The evolutionary significance of polyploidy,Nat.Rev.Genet.,15(10)1-14
Xu C.,Wang Y.H.,Yu Y.C.,Duan J.B.,Liao Z.G.,Xiong G.S.Meng X.B.,Liu G.F.,Qian Q.,and Li J.Y.,2012,Degradation of monoCuLm 1 by APC/CTAD1regulates rice tillering Nat.Commun.,20(10):1-9
Yang P.M.,Huang Q.C.,Qin G.Y.,Zhao S.P.,and Zhou J.G.2014,Different drought-stress responses in photosynthesis and reactive oxygen metabolism between autotetraploid and diploid rice,Photosynthetica,52(2):193-202
Zhang X.H.,Wang A.Y.,Du C.Q.,Song Z.J.,Wang W.,He Y.C.,and Cai D.T.,2014,An efficient method of developing synthetic allopolyploid rice(Oryza spp),Genet.Resour.Crop E-vol.,61(4):809-816
Zhou X.J.,and Cheng Z.H.,2008,Establishment of induction system of garlic clove-tip by injection with colchicine and effect of mutation,Xibei Nongye Xuebao(Acta Agriculturae Boreali-occidentalis Sinica),17(5):267-271(周香君,程智慧,2008,蒜瓣茎尖注射秋水仙素的诱变效果和诱变方法的建立,西北农业学报,17(5):267-271)