李玉生;吴永杰;程和禾;陈龙;赵艳华;吴雅琴;

• 1:河北省农林科学院昌黎果树研究所

摘要(Abstract)：

【目的】为解决基因型依赖性比较强的苹果品种遗传转化困难等问题,研究不同伤害处理方式下苹果茎尖碎片的再生能力及其对苹果转化率的影响。【方法】以苹果‘姬神’试管苗为试材,运用农杆菌介导法对苹果茎尖和离体叶片进行遗传转化。【结果】将茎尖纵切为4部分时,可以高比率的形成愈伤组织(88.4%)和再生不定芽(59.8%)。在20 mg·L-1的卡那霉素筛选压下,300个茎尖外植体中有50.4%的茎尖碎片形成愈伤组织并获得了19株表达GFP的转基因植株。而在同样的筛选压下有22.6%农杆菌介导的离体叶片形成愈伤组织,但不能获得转基因植株。降低筛选压可以提高植株再生率,但其中包含大量嵌合体,转化效率并没有明显提高。【结论】苹果茎尖做为转化受体基因型依赖性较低,要优于苹果离体叶片。

关键词(KeyWords)： 苹果;基因型;转基因;GFP基因;茎尖碎片

Abstract:

Keywords:

基金项目(Foundation): 河北省自然科学基金(C2012301001)

作者(Author): 李玉生;吴永杰;程和禾;陈龙;赵艳华;吴雅琴;

Email:

DOI: 10.13925/j.cnki.gsxb.2013.06.009

参考文献(References)：

• [1]SHAO Jian-zhu,MA Bao-kun.Advances in research of transgenic apples[J].Journal of Fruit Science,2003,20(1):49-53.邵建柱,马宝馄.转基因苹果研究进展[J].果树学报,2003,20(1):49-53.
• [2]YANG Yue,ZHANG Zhi-hong,LIU Yue-xue,HE Ping,ZHOU Yu,DAI Hong-yan,ZHAO Gui-ling.Estanblishment of Agrobacterium tumefaciens-mediated transformation system for Hanfu apple[J].Journal of Fruit Science,2010,27(2):174-178.杨越,张志宏,刘月学,何平,周宇,代红艳,赵桂玲.根癌农杆菌介导寒富苹果转化体系的建立[J].果树学报,2010,27(2):174-178.
• [3]LIU Q,SALIH S,HAMMERSCHLAG F.Etiolation of Royal Gala apple(Malus domestica Borkh)shoots promotes high frequenty shoot organogenesis and enhance B-glucuronidase expression from stem internodes[J].Plant Cell Rep,1998,18:32-36.
• [4]SALAMI A,EBADI A,ZAMANI Z,GHASEMI M.Improvement in apex culture in an Iranian grapevine(Vitis vinifera L.‘Bidaneh Sefid’)through fragmented shoot apices[J].Int J Agric Biol,2005,7:333-336.
• [5]MBANASO E A,CROUCH J,ONOFEGHARA F.Respons of cooking banana genotypes to fragmentation and incision during shoot tips culture[J].InfoMusa,2006,15:30-32.
• [6]DUTT M,LI Z T,DHEKNEY S A,GRAY D J.Transgenic plants from shoot apical meristems of Vitis vinifera L.‘Thompson Seedless’via Agrobacterium-mediated transformation[J].Plant Cell Rep,2007,26:2101-2110.
• [7]JAMES D J,PASSEY A J,WEBSTER A D,BARBARA D J,DANDEKAR A M,URATSU S L,VISS P.Transgenic apples and strawberries:advances in transformation,introduction of genes for insect resistance and field studies of tissue cultured plants[J].Acta Horticulture,1993,336:179-184.
• [8]NORELLI J L,MILLS J,ALDWINCKLE H S.Leaf wounding increases efficiency of Agrobacterium-mediated transformation of apple[J].Hort Sci,1996,31:1-2.
• [9]LI Hou-hua,QUE Yi,FEI Zhao-xue,WANG Ya-jie.Estabilishment and optimization of tissue culture and genetic transformation system of Malus sieversii var.niedzwetzkyana[J].Northern Horticulture,2011,35(15):175-179.李厚华,阙怡,费昭雪,王亚杰.红肉苹果组织培养及转基因体系的建立与优化[J].北方园艺,2011,35(15):175-179.
• [10]MAHESWARAN G,WELANDER M,HUTCHINSON J F,GRAHAM M W,RICHARDS D.Transforamtion of apple rootstock M26with Agrobacterium tumefaciens[J].J Plant Physiol,1992,39:560-568.
• [11]NORELLI J L,ALDWINCKLE H S.The role of aminoglycoside antibitics in the regeneration and selection of neomycin phosphotransferase transgenic apple tissue[J].J Am Soc Hort Sci,1993,118:311-316.
• [12]LIU Qing-zhong,ZHAO Hong-jun,LIU Peng,Mong Rengong,Freddi A H.Regerneration of tetraploid plants with cecropin MB39gene from‘Royal Gala’apple[J].Acta Horticulturace Sinica,2001,28(5):392-398.刘庆忠,赵红军,刘鹏,Mong Rengong,Freddi A Hammerschlag.抗菌肽MB39基因导入‘皇家嘎拉’苹果及其四倍体植株的培育[J].园艺学报,2001,28(5):392-398.
• [13]FASOLO R H,ZIMMERMAN I H,FORDHAM I.Adventitious shoot formation on excised leaves of in vitro grown shoots of apple cultivars[J].Plant Cell Tissue Organ Cult,1989,16:75-87.
• [14]DE BONDT A,EGGERMONT K,DRUART P,VIL M,DE GODERIS L,VANDERLEYDEN J,BROEKAERT W F.Agrobacterium-mediated transformation of apple(Malus domestica Borkh.):an assessment of factors affecting gene transfer efficiency during early transformation steps[J].Plant Cell Rep,1994,13:587-593.
• [15]PUITE K J,SCHAART J G.Genetic modification of the commercial apple cultivars Gala,Golden Delicious and Elstar via an Agrobacterium tumefaciens-mediated transformation method[J].Plant Sci,1996,119:125-133.
• [16]DE BONDT A,EGGERMONT K,PENNINCKXT I,GODERIS I,BRAEKAERT W E.Agrobacterium mediated transformation of apple(Malus domestica Borkh.):an assessment of factors affecting regeneration of transgenic plant[J].Plant Cell Rep,1996,15:549-554.
• [17]DOMINGUEZ A,CERVERA M,PEREZ R M,ROMERO J,CARMEN C,CUVERO J,LOPEZ M M,JUAREZ J A,NAVARRO L,PENA L.Characterisation of regenerants obtained under selective conditions after Agrobacterium-mediated transformation of citrus explants reveals production of silenced and chimeric plants at unexpected high frequencies[J].Mol Breed,2004,14:171-183.
• [18]FLACHOWSKY H,RIEDEL M,REIM S,HANKE M.Evaluation of the uniformity and stability of T-DNA integration and gene expression in transgenic apple plants[J].Electron J Biotechnol,2008,11:1-13.
• [19]MATTHEWS H,DEWEY V,WAGONER W,BESTWICK R K.Molecular and cellular evidence of chimaeric tissues in primary transgenics and elimination of chimaerism through improved selection protocols[J].Trans Res,1998,7:123-129.
• [20]MAXIMOVA S N,DANDEKAR A M,GUILTINAN M J.Investigation of Agrobacterium-mediated transformation of apple using green fluorescent protein:high transient expression and low stable transformation suggest that factors other than T-DNA transfer are ratelimiting[J].Plant Mol Biol,1998,37:549-559.
• [21]HILY J M,LIU Z.A simple and sensitive high-throughput GFP screening in woody and herbaceous plants[J].Plant Cell Rep,2009,28:493-501.