玉米单倍体胚性愈伤的诱导与鉴定
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摘要
为建立一套适合玉米单倍体胚性愈伤培养和快速筛选体系,以单倍体诱导系MT-1为父本,18-599红为母本进行单倍体诱导,设置暗培养、全光照培养和光暗周期培养3种光照培养方式,均分别培养0、1、5、10、20、40 h后,观察幼胚形态和颜色。结果表明,自交系18-599红的单倍体和二倍体幼胚均能正常诱导形成胚性愈伤组织;不同光照培养方式对紫色(二倍体)愈伤率的检出效果依次为光培养>光暗周期培养>暗培养。通过光照筛选的方法可在早期鉴定愈伤组织,其二倍体愈伤的筛选率在培养20 h时可达68%,40 h时为71%,剔除了大部分非单倍体愈伤,综合分析确定光照强度为2 000 lx、20℃处理20 h为最适光照筛选处理。染色体压片技术获得的拟单倍体愈伤中有二倍体愈伤的检出,但经流式细胞仪检出获得的拟单倍体愈伤再经染色体压片检测,无二倍体愈伤,表明流式细胞仪检测单倍体愈伤的准确性高于染色体压片技术。通过光照初步筛选结合流式细胞仪的精确鉴定,迅速从3 000个单倍体愈伤中获得110个单倍体愈伤,单倍体愈伤率3. 67%。本研究结果为以玉米单倍体愈伤为转基因受体,快速获得转基因植株提供了一定的技术支撑和理论参考。
In order to establish maize haploid embryogenic callus culture and rapid screening system,MT-1 was used as inducer to induce haploid on maternal material 18-599( red). And 0,1,5,10,20 and 40 h were cultured under dark light and light/dark photoperiod for haploid callus induction. The results showed that calluses could be induced from all young embryos of inbred 18-599( red). The effects of light culture on the callus rate of purple( diploid) were: light culture > light/dark alternate culture > dark culture. Haploid callus could be identified early by light screening,and the detectable rates were 68% and 71% for exposed 20 h and 40 h,respectively,which could delete eliminate most of none haploid callus. It was concluded that illumination intensity 2 000 Lx,and treatment at 20℃ for 20 h was the best screening process. Furthermore,the remaining putative haploid calluses were observed through flow cytometer and chromosome tableting technology,and some diploid calluses were detected from candidate haploid calluses after tableting technology with flow cytometer,but no diploid callus were detected after flow cytometer,which suggest that flow cytometer is more accurate than tableting technology in detecting haploid calluses. By illumination screening and flow cytometer detection,100 haploid callus were obtained rapidly from 3 000 haploid callus,and the haploid callus rate was3. 67%. The result of this study provided a reference for utilizing maize haploid embryogenic callus as a transgenic receptor and also lays the foundation for obtaining transgenic plant rapidly.
In order to establish maize haploid embryogenic callus culture and rapid screening system,MT-1 was used as inducer to induce haploid on maternal material 18-599( red). And 0,1,5,10,20 and 40 h were cultured under dark light and light/dark photoperiod for haploid callus induction. The results showed that calluses could be induced from all young embryos of inbred 18-599( red). The effects of light culture on the callus rate of purple( diploid) were: light culture > light/dark alternate culture > dark culture. Haploid callus could be identified early by light screening,and the detectable rates were 68% and 71% for exposed 20 h and 40 h,respectively,which could delete eliminate most of none haploid callus. It was concluded that illumination intensity 2 000 Lx,and treatment at 20℃ for 20 h was the best screening process. Furthermore,the remaining putative haploid calluses were observed through flow cytometer and chromosome tableting technology,and some diploid calluses were detected from candidate haploid calluses after tableting technology with flow cytometer,but no diploid callus were detected after flow cytometer,which suggest that flow cytometer is more accurate than tableting technology in detecting haploid calluses. By illumination screening and flow cytometer detection,100 haploid callus were obtained rapidly from 3 000 haploid callus,and the haploid callus rate was3. 67%. The result of this study provided a reference for utilizing maize haploid embryogenic callus as a transgenic receptor and also lays the foundation for obtaining transgenic plant rapidly.
引文
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[24]张如养,段民孝,赵久然,刘新香,邢锦丰. 6个玉米单倍体诱导系诱导率的差异性研究[J].玉米科学,2013,21(2):6-10
[25]杜志钊,陆瑞菊,黄剑华.以单倍体材料为转化受体的植物转基因研究进展[J].核农学报,2010,24(2):302-306
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[29] Coe E H. A line of maize with high haploid frequency[J]. The American Naturalist,1959,93(873):381-382
[30]汤飞宇,丁菲,王国英.从玉米传粉子房培养出单倍体植株[J].福建农林大学学报(自然科学版),2004,4(21):489-493
[31]吴雅琴,常瑞丰,程和禾.流式细胞术进行倍性分析的原理和方法[J].云南农业大学学报,2006,21(4):407-409
[32] Tao D,Li X,Zhou J,Chen Y,Huo W. Progresses on determination of cell chromosome ploidy level of plants[J]. Life Science Research,2009,13(5):453-458
[33]田新民,周香艳,弓娜.流式细胞技术在植物学研究中的应用-检测植物核DNA含量的倍性水平[J].中国农学通报,2011,27(9):21-27
[34] Mallón R,Rodríguez-Oubi1a J,González M L. In vitro propagation of the endangered plant Centaurea ultreiae:assessment of genetic stability by cytological studies,flow cytometry and RAPD analysis[J]. Plant Cell Tissue&Organ Culture,2010,101(1):31-39
[2] Singh R K,Prasad M. Advances in Agrobacterium tumefaciensmediated genetic transformation of graminaceous crops[J].Protoplasma,2016,253(3):691-707
[3] Nuccio M L. A brief history of promoter development for use in transgenic maize applications[J]. Methods in Molecular Biology,2018,1676:61-93
[4] Miluse T,Odd Gunnar W,Niklaus Z,Alex W,Angelika H.Transgene expression and Bt protein content in transgenic Bt maize(MON810)under optimal and stressful environmental conditions[J]. PLoS One,2015,10(4):e0123011
[5] de Cerqueira D T R,Schafer A C,Fast B J,Herman R A.Agronomic performance of insect-protected and herbicide-tolerant MON 89034×TC1507×NK603×DAS-40278-9 corn is equivalent to that of conventional corn[J]. GM Crops&Food,2017,8(3):149-155
[6] Planta J,Messing J. Quality protein maize based on reducing sulfur in leaf cells[J]. Genetics,2017,207(4):1687-1697
[7] Armstrong C L. The first decade of maize transformation:a review and future perspectives[J]. Maydica,1999,44(1)101-109
[8] Chen L, Cong Y, He H, Yu Y. Maize(Zea mays L.)transformation by Agrobacterium tumefaciens infection of pollinated ovules[J]. Journal of Biotechnology,2014,171(19):8-16
[9] Raji J A,Frame B,Little D,Santoso T J,Wang K. Agrobacteriumand biolistic-mediated transformation of maize B104 inbred[J].Methods in Molecular Biology,2018,1676:15-40.
[10] Brettschneider R,Becker D,Lorz H. Efficient transformation of scutellar tissue of immature maize embryos[J]. Theoretical and Applied Gentics,1997,94(6):737-748
[11]杜文平,徐利远,余桂容,王一,钟昌松.玉米幼胚离体培养体系的建立[J].玉米科学,2007,15(2):73-75,78
[12] Pathi K M,Tula S,Huda K M,Srivastava V K,Tuteja N. An efficient and rapid regeneration via multiple shoot induction from mature seed derived embryogenic and organogenic callus of Indian maize(Zea mays L.)[J]. Plant Signal Behaviar,2013,8(10):e25891
[13]陈靖,董浩,马海珍,孙全喜,刘江,亓宝秀,李新征,董树亭.玉米幼胚和源于幼苗的幼嫩叶段愈伤诱导及其植株再生的比较[J].中国农业科学,2011,44(17):3676-3682
[14]杨爱国,刘爽,赵琦,赵玉锦,张世煌,潘光堂.农杆菌介导玉米胚性愈伤的遗传转化研究[J].生物技术通报,2008,4(1):104-108,121
[15]金万梅,潘青华,尹淑萍,董静,姜立杰,杨丽,陈青华,赵剑波.外源基因在转基因植物中的遗传稳定性及其转育研究进展[J].分子植物育种,2005,6(3):864-868
[16] Prigge V,Xu X,Li L,Babu R,Chen S,Atlin G N,Melchinger A E. New insights into the genetics of in vivo induction of maternal haploids,the backbone of doubled haploid technology in maize[J].Genetics,2012,190(2):781-793
[17] Rober F K,Gordillo G A,Geiger H H. In vivo haploid induction in maize-performance of new inducers and significance of doubled haploid lines in hybrid breeding[J]. Maydica,2005,50(3):275-284
[18]郑企成,陈文华.单倍体在突变育种中的利用[J].核农学报,1980(1):60-63
[19] Shen Y,Pan G,Lübberstedt T. Haploid strategies for functional validation of plant genes[J]. Trends in Biotechnology,2015,10(33):611-620
[20]才卓,徐国良,CHANG Ming-tang,路明(译),张洪艳(校).玉米单倍体育种研究进展[J].玉米科学,2008,16(1):1-5
[21] Prigge V,Schipprack W,Mahuku G,Atlin G N,Melchinger A E.Development of in vivo haploid inducers for tropical maize breeding programs[J]. Euphytica,2012,185(3):481-490
[22]刘志增,宋同明.玉米高频率孤雌生殖单倍体诱导系的选育与鉴定[J].作物学报,2000,26(5):587-590
[23]才卓,徐国良,刘向辉,董亚琳,代玉仙.玉米高频率单倍生殖诱导系吉高诱系3号的选育[J].玉米科学,2007,15(1):1-4
[24]张如养,段民孝,赵久然,刘新香,邢锦丰. 6个玉米单倍体诱导系诱导率的差异性研究[J].玉米科学,2013,21(2):6-10
[25]杜志钊,陆瑞菊,黄剑华.以单倍体材料为转化受体的植物转基因研究进展[J].核农学报,2010,24(2):302-306
[26]陈琦.玉米孤雌生殖单倍体胚遗传转化体系建立的初步研究[D].成都:四川农业大学,2015
[27]蔡华,马传喜,乔玉强.小麦×玉米诱导小麦单倍体的形态学及细胞学鉴定[J].核农学报,2008,22(2):127-130
[28]张虹,龙宏周,路国栋,陈任.黑果枸杞多倍体诱导及鉴定[J].核农学报,2017,31(1):58-65
[29] Coe E H. A line of maize with high haploid frequency[J]. The American Naturalist,1959,93(873):381-382
[30]汤飞宇,丁菲,王国英.从玉米传粉子房培养出单倍体植株[J].福建农林大学学报(自然科学版),2004,4(21):489-493
[31]吴雅琴,常瑞丰,程和禾.流式细胞术进行倍性分析的原理和方法[J].云南农业大学学报,2006,21(4):407-409
[32] Tao D,Li X,Zhou J,Chen Y,Huo W. Progresses on determination of cell chromosome ploidy level of plants[J]. Life Science Research,2009,13(5):453-458
[33]田新民,周香艳,弓娜.流式细胞技术在植物学研究中的应用-检测植物核DNA含量的倍性水平[J].中国农学通报,2011,27(9):21-27
[34] Mallón R,Rodríguez-Oubi1a J,González M L. In vitro propagation of the endangered plant Centaurea ultreiae:assessment of genetic stability by cytological studies,flow cytometry and RAPD analysis[J]. Plant Cell Tissue&Organ Culture,2010,101(1):31-39