Micro-Tom番茄转基因植株再生体系的优化
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摘要
转基因植株的再生频率较低一直是植物基因工程研究的瓶颈之一.以番茄的MicroTom突变体为材料,探索了外植体植物激素配比、预培养时间、共培养时间、筛选剂对愈伤组织发生和不定芽生长的影响.结果表明:子叶比下胚轴更适合作为愈伤组织和不定芽诱导的外植体;当玉米素(zeatin,ZT)质量浓度为0.5 mg/L,吲哚乙酸(indole acetic acid,IAA)质量浓度为1.0 mg/L时最有利于不定芽诱导;外植体预培养的适宜时间为3 d,农杆菌和外植体共培养的适宜时间为2 d;外植体对卡那霉素的耐受上限为40 mg/L,替门汀的适宜质量浓度为150 mg/L.实验所建立的转基因再生体系为Micro-Tom番茄的遗传工程应用奠定了基础.
Low redifferentiation frequency of transgenic plant is a major barrier in plant gene engineering. Tomato species Micro-Tom was used as the experimental material.Effects of plant hormone combination, preculture time, and co-culture time of kanamycin and timentin on callusogenesis and adventitious bud growth were explored. The results show that cotyledon is more suitable than hypocotyledon as explants for callusogenesis and adventitous bud induction. Combination of 0.5 mg/L zeatin(ZT) with 1.0 mg/L indole acetic acid(IAA) brings about the highest adventitous bud induction. Suitable preculture time for explants is 3 d, and that for co-culture with Agrobacterium tumefaciens is 2 d.The upper tolerance limit of explant to kanamycin for selection is 40 mg/L and suitable timentin concentration for Agrobacterium tumefaciens inhibition is 150 mg/L. The established system may provide a good base for genetic engineering for Micro-Tom tomato.
Low redifferentiation frequency of transgenic plant is a major barrier in plant gene engineering. Tomato species Micro-Tom was used as the experimental material.Effects of plant hormone combination, preculture time, and co-culture time of kanamycin and timentin on callusogenesis and adventitious bud growth were explored. The results show that cotyledon is more suitable than hypocotyledon as explants for callusogenesis and adventitous bud induction. Combination of 0.5 mg/L zeatin(ZT) with 1.0 mg/L indole acetic acid(IAA) brings about the highest adventitous bud induction. Suitable preculture time for explants is 3 d, and that for co-culture with Agrobacterium tumefaciens is 2 d.The upper tolerance limit of explant to kanamycin for selection is 40 mg/L and suitable timentin concentration for Agrobacterium tumefaciens inhibition is 150 mg/L. The established system may provide a good base for genetic engineering for Micro-Tom tomato.
引文
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[3]张丽华,许小勇,魏榕.加工番茄品种卡那霉素抗性的筛选[J].西北农业学报,2006,15(3):124-127.
[4]罗素兰,陈若,长孙东亭.番茄组培苗的不同阶段对抗生素和PPT的抗性筛选试验[J].海南大学学报(自然科学版),2003,21(1):58-64.
[5]杨亚萍,李永兰,梁月荣,等.发根农杆菌抑菌剂的抑菌效果及对茶组培苗丛生芽的影响[J].茶叶科学,2015,35(5):437-442.
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[11]王傲雪,赵越,陈秀玲,等.不同激素组合对番茄芽分化率的影响[J].东北农业大学学报,2013,44(7):85-90.
[12]陈丽萍,张丽华,程智慧.加工番茄离体再生体系的建立[J].西北农业学报,2007,16(1):162-167.
[13]欧阳波,李汉霞,叶志彪.玉米素和IAA对番茄子叶再生的影响[J].植物生理学通讯,2003,39(3):217-218.
[14]王关林,方宏筠.植物基因工程[M].2版.北京:科学出版社,2009:85.
[15]张赛群,叶志彪.异戊烯基转移酶基因导入番茄及转基因植株再生[J].园艺学报,1999,26(6):376-379.
[16]Nap J P,Bijv J,司家钢.转基因植物中的卡那霉素抗性[J].生物技术通报,1998(1):29-31.
[17]梁美霞,许向阳,李景富.抗生素对番茄愈伤组织诱导和分化的影响[J].东北农业大学学报,2005,36(1):19-22.
[18]Labia R,Morand A,Peduzzi J.Timentin andβ-lactamases[J].Journal of Antimicrobial Chemotherapy,1986,17(S1):17-26.
[19]陆荣生,韩美丽,吴耀军,等.农杆菌介导AFT基因转入四季桔的研究[J].广东农业科学,2013,40(11):145-148.
[20]Cheng Z M,Schnurr J,Kapaun J.Timentin as an alternative antibiotic for suppression of Agrobacterium tumefaciens in genetic transformation[J].Plant Cell Reports,1998,17(8):646-649.
[2]Scott J W.Micro-Tom—a miniature dwarf tomato[J].Florida Agric Exp Stn Circ,1989,370:1-6.
[3]张丽华,许小勇,魏榕.加工番茄品种卡那霉素抗性的筛选[J].西北农业学报,2006,15(3):124-127.
[4]罗素兰,陈若,长孙东亭.番茄组培苗的不同阶段对抗生素和PPT的抗性筛选试验[J].海南大学学报(自然科学版),2003,21(1):58-64.
[5]杨亚萍,李永兰,梁月荣,等.发根农杆菌抑菌剂的抑菌效果及对茶组培苗丛生芽的影响[J].茶叶科学,2015,35(5):437-442.
[6]Bhatia P,Ashwath N,Senaratna T,et al.Tissue culture studies of tomato(Lycopersicon esculentum)[J].Plant Cell,Tissue and Organ Culture,2004,78(1):1-21.
[7]卫志明,许智宏.番茄叶组织培养中植株再生的初步研究[J].植物生理学通讯,1979(1):10-11.
[8]佟新萍.番茄幼叶愈伤组织诱导与再生植株[J].植物生理学通讯,1993,29(3):190-192.
[9]李浚明,朱登云.植物组织培养教程[M].3版.北京:中国农业大学出版社,2005:128.
[10]叶志彪,李汉霞.番茄子叶离体培养与再生成株[J].华中农业大学学报,1994,13(3):291-295.
[11]王傲雪,赵越,陈秀玲,等.不同激素组合对番茄芽分化率的影响[J].东北农业大学学报,2013,44(7):85-90.
[12]陈丽萍,张丽华,程智慧.加工番茄离体再生体系的建立[J].西北农业学报,2007,16(1):162-167.
[13]欧阳波,李汉霞,叶志彪.玉米素和IAA对番茄子叶再生的影响[J].植物生理学通讯,2003,39(3):217-218.
[14]王关林,方宏筠.植物基因工程[M].2版.北京:科学出版社,2009:85.
[15]张赛群,叶志彪.异戊烯基转移酶基因导入番茄及转基因植株再生[J].园艺学报,1999,26(6):376-379.
[16]Nap J P,Bijv J,司家钢.转基因植物中的卡那霉素抗性[J].生物技术通报,1998(1):29-31.
[17]梁美霞,许向阳,李景富.抗生素对番茄愈伤组织诱导和分化的影响[J].东北农业大学学报,2005,36(1):19-22.
[18]Labia R,Morand A,Peduzzi J.Timentin andβ-lactamases[J].Journal of Antimicrobial Chemotherapy,1986,17(S1):17-26.
[19]陆荣生,韩美丽,吴耀军,等.农杆菌介导AFT基因转入四季桔的研究[J].广东农业科学,2013,40(11):145-148.
[20]Cheng Z M,Schnurr J,Kapaun J.Timentin as an alternative antibiotic for suppression of Agrobacterium tumefaciens in genetic transformation[J].Plant Cell Reports,1998,17(8):646-649.