谭彬,李鼎立,徐世晓,郭文武

• 1:园艺植物生物学教育部重点实验室·华中农业大学
• 2:河南农业大学园艺学院

摘要(Abstract)：

【目的】为了快速有效地利用基因工程的方法获得柑橘无核新种质,【方法】以我国特色多核柑橘优良品种锦橙实生苗上胚轴切段为外植体,采用根癌农杆菌介导法进行能导致种子败育基因CG1-400-RNase的转化;为快速有效地筛选出转化子,在实生苗上胚轴切段转化再生过程中,根据不同发育阶段的组织或器官对抗生素的敏感程度不同采用不同的选择压。【结果】结果表明,在抗性芽再生过程中卡那霉素质量浓度设定为50 mg.L-1,获得的362个抗性芽转入卡那霉素质量浓度为100 mg.L-1的伸长培养基中进行伸长培养后,进行早期PCR检测,获得28个阳性芽;经过不定芽诱导生根或试管嫁接,获得22株完整植株。【结论】再生植株经PCR和Southern杂交检测,获得2株目的基因以单拷贝的形式插入锦橙基因组的转基因植株,为最终获得具有无核性状且可稳定遗传的柑橘新种质奠定了基础。

关键词(KeyWords)： 柑橘;根癌农杆菌;CG1-400-RNase基因

Abstract:

Keywords:

基金项目(Foundation): 国家863计划(2011AA100205);; 国家自然科学基金项目(31125024)

作者(Author): 谭彬,李鼎立,徐世晓,郭文武

DOI: 10.13925/j.cnki.gsxb.2012.04.012

参考文献(References)：

• [1]DENG Xiu-xin.Advances in worldwide citrus breeding[J].Acta Hor-ticulturae Sinica,2005,32(6):1140-1146.邓秀新.世界柑橘品种改良的进展[J].园艺学报,2005,32(6):1140-1146.
• [2]MARIANI C,GOSSELE V,DE BEUCKELEER M,DE BLOCK M,GOLDBERG R B,DE GREEF W,LEEMANS J.A chimaeric ri-bonuclease-inhibitor gene restores fertility to male sterile plants[J].Nature,1992,357:384-387.
• [3]DENIS M R,DELOURME R,GOURRET J-P,MARIANI C,RE-NARD M.Expression of engineered male sterility in Brassica napus[J].Plant Physiology,1993,101:1295-1304.
• [4]REYNAERTS A,VAN DE WIELE H,DE SUTTER G,JANSSENSJ.Engineered genes for fertility control and their application in hy-brid seed production[J].Scientia Horticulturae,1993,55:125-139.
• [5]GOLDMAN M H S,GOLDBERG R B,MARIANI C.Female steriletobacco plants are produced by stigma-specific cell ablation[J].EM-BO J,1994,13:2976-2984.
• [6]KOLTUNOW A M,BRENNAN P,BARKER S J.Evaluation ofgenes to reduce seed size in Arabidopsis and tobacco and their ap-plication to Citrus[J].Molecular Breeding,1998,4:235-251.
• [7]KOLTUNOW A M,BRENNAN P.Paternal transmission of a seedsize reduction gene varies with age of a primary transformant andseed set is also influenced by gene expression in maternal tissues[J].Molecular Breeding,1998,4:253-261.
• [8]KOLTUNOW A M,BRENNAN P,PROTOPSALTIS S,NITO N.Regeneration of west indian limes(Citrus aurantifolia)containinggenes for decreased seed set[J].Acta Horticulturae(ISHS),2000,535:81-91.
• [9]GUO W W,CHENG Y J,DENG X X.Regeneration and molecularcharacterization of intergenetic somatic hybrids between Citrus retic-ulate and Poncirus trifoliata[J].Plant Cell Reports,2002,20:829-834.
• [10]DENG X X,XIAO S Y,ZHANG W C.Interspecific somatic hybridof Ichang Papeda with Valencia orange[J].Chinese Journal of Biotech-nology,1993,9:128-131.
• [11]TAN B,LI D L,XU S X,FAN G E,FAN J,GUO W W.Highly ef-ficient transformation of the GFP and MAC12.2 genes into preco-cious trifoliate orange[Poncirus trifoliata(L.)Raf],a potential modelgenotype for functional genomics studies in Citrus[J].Tree Genet-ics&Genomes,2009,5(3):529-537.
• [12]DUAN Yan-xin,FAN Jing,LI Ding-li,GUO Wen-wu.Screeningand regeneration of the transgenic embryogenic calli in Citrus[J].Journal of Huazhong Agricultural University,2008,27(6):768-771.段艳欣,范净,李鼎立,郭文武.柑橘转基因胚性愈伤组织的筛选与再生[J].华中农业大学学报,2008,27(6):768-771.
• [13]CHABAUD M,LARSONNEAU C,MARMOUGET C,HUGUET T.Transformation of barrel medic(Medicago truncatula Gaertn.)by A-grobacterium tumefaciens and regeneration via somatic embryogene-sis of transgenic plants with the MtENOD12 nodulin promoter fusedto the gus reporter gene[J].Plant Cell Reports,1996,15:305-310.
• [14]DOMINGUEZ A,FAGOAGA C,NAVARRO L,MORENO P,PE譙AL.Regeneration of transgenic citrus plants under non selective con-ditions results in high-frequency recovery of plants with silencedtransgenes[J].Molecular Genetics and Genomics,2002,267:544-556.
• [15]OLIVEIRA M L P,FEBRES V J,COSTA M G C,MOORE G A,O-TONI W C.High-efficiency Agrobacterium-mediated transformation of citrus via sonication and vacuum infiltration[J].Plant Cell Re-ports,2009,28:387-395.
• [16]CHARITY J A,HOLLAND L,DONALDSON S S,GRACE L,WAL-TER C.Agrobacterium-mediated transformation of Pinus radiata organogenic tissue using vacuum-infiltration[J].Plant Cell Tissue and Organ Culture,2002,70:51-60.