桂花遗传转化条件初探
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- 英文论文题名：Preliminary Study on Genetic Transformation of Osmanthus fragrans
- 论文作者：邹晶晶 ; 曾祥玲 ; 蔡璇 ; 陈洪国 ; 王彩云
- 英文论文作者：ZOU Jing-jing ; ZENG Xiang-ling ; CAI Xuan ; CHEN Hong-guo ; WANG Cai-yun ; School of Nuclear Technology and Chemistry & Biology ; Hubei University of Science and Technology ; Key Laboratory for Biology of Horticultural Plants ; Ministry of Education ; College of Horticulture & Forestry Sciences ; Huazhong Agricultural University
- 年：2016
- 作者机构：湖北科技学院核技术与化学生物学院;华中农业大学园艺植物生物学教育部重点实验室;
- 论文关键词：桂花 ; 合子胚 ; 胚性愈伤 ; 遗传转化
- 英文论文关键词：Sweet osmanthus ; Zygotic embryos ; Embryogenic callus ; Genetic transformation
- 会议召开时间：2016-07-19
- 会议录名称：中国观赏园艺研究进展2016
- 语种：中文
- 分类号：S685.13
- 学会代码：EGYP
- 会议名称：2016年中国观赏园艺学术研讨会
- 会议地点：中国湖南长沙
- 主办单位：中国园艺学会观赏园艺专业委员会、国家花卉工程技术研究中心
- 学会名称：中国园艺学会
- 页数：8
- 文件大小：990k
- 原文格式：O
- 会议级别：全国
摘要
本研究以结实桂花(Osmanthus fragrans Lour.)早期子叶期的未成熟合子胚为外植体材料,在MS+0.5mg·L~(-1)2,4-D+1.0mg·L~(-1)6-BA初始培养基上暗培养30天后诱导得到的胚性愈伤组织作为遗传转化的受体,利用根癌农杆菌介导的方法,通过对不同的侵染及培养条件的摸索,初步探索了桂花的遗传转化体系的条件。结果表明,桂花胚性愈伤组织的卡那霉素的选择敏感性浓度为200mg·L~(-1),头孢霉素浓度为300mg·L~(-1)。GUS瞬时表达表明最优的转化条件为:农杆菌菌株为EHA105时,侵染菌液浓度为0.6,侵染时间为20min,共培养天数为2d,共培养时培养基中添加100μM AS时效率最高。选择培养基为MS+0.5mg·L~(-1)NAA+1.0mg·L~(-1)BA+200mg·L~(-1)Km+300mg·L~(-1)Cef。下一步将继续优化选择培养基抗生素的种类和浓度,解决反菌问题,以获得完整转化植株。
The embryogenic callus induced by the cotyledon segments from the ZEs explants collected at the early cotyledonary stage,with MS medium suppled with 1.0mg·L~(-1)BA and 0.5mg·L~(-1)2,4-D as the initial medium,were used for the Agrobacterium-mediated transformation in O.fragrans.The selective concentration of kanamycin(Km)was 200mg·L~(-1),and cefotaxime(Cef)was 300mg·L~(-1).According to the GUS transient assay,the opmizated transformation conditions were preliminary abtained:the strain of Agrobacterium tumefaciens was EHA105;the infection time was 20 min;the co-culture day was 2d;the co-culture medium was MS+100μM acetosyringone(AS).The selective medium was MS+0.5 mg·L~(-1)NAA+1.0mg·L~(-1)BA+200mg·L~(-1)Km+300mg·L~(-1)Cef.However,embryonic callus was infected by the agrobacterium during the selective culture process 20 days later.Type and concentration of antibiotics will be optimized in the later study to obtain the complete transformated plant
The embryogenic callus induced by the cotyledon segments from the ZEs explants collected at the early cotyledonary stage,with MS medium suppled with 1.0mg·L~(-1)BA and 0.5mg·L~(-1)2,4-D as the initial medium,were used for the Agrobacterium-mediated transformation in O.fragrans.The selective concentration of kanamycin(Km)was 200mg·L~(-1),and cefotaxime(Cef)was 300mg·L~(-1).According to the GUS transient assay,the opmizated transformation conditions were preliminary abtained:the strain of Agrobacterium tumefaciens was EHA105;the infection time was 20 min;the co-culture day was 2d;the co-culture medium was MS+100μM acetosyringone(AS).The selective medium was MS+0.5 mg·L~(-1)NAA+1.0mg·L~(-1)BA+200mg·L~(-1)Km+300mg·L~(-1)Cef.However,embryonic callus was infected by the agrobacterium during the selective culture process 20 days later.Type and concentration of antibiotics will be optimized in the later study to obtain the complete transformated plant
引文
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3.杜丽,庞振凌,周索,等.香樟胚性愈伤组织遗传转化体系建立[J].林业科学,2008,4J4:54-59.
4.李君,李岩,刘德虎.植物遗传转化的替代方法及研究进展[J].生物技术通报,2011,7:31-36.
5.高洁.萱草再生体系的优化及农杆菌介导的遗传转化体系的建立[D].成都:四川农业大学图书馆,2012.
6.梁茂厂,刘友全.桂花幼胚培养及愈伤组织增殖诱导[J].经济林研究,2007,25:43-46.
7.刘萍,袁王俊.桂花愈伤组织的诱导与增殖[J].安徽农业科学,2008,36:14889-14890.
8.罗敬萍,张树珍,杨本鹏.农杆菌介导甘蔗基因转化技术的优化[J].热带作物学报,2003,24:23-28.
9.沈俊岭,赵芳,李云,等.速生型刺槐遗传转化体系的建立[J].核农学报,2006,20:477-481.
10.宋会访,葛红,周媛,等.花离体培养与快速繁殖技术的初步研究[J].园艺学报,2005,32:738-740.
11.王彩云,白吉刚,杨玉萍.桂花的组织培养[J].北京林业大学学报,2001,23:24-25.
12.王鹏良,姜福星,蔡铃,等.广林巨尾桉9号遗传转化体系的建立[J].林业科技开发,2013,27:76-80.
13.王荃,潘琪芳,袁芳,等.超声波辅助农杆菌介导的长春花(Catharanthus roseus)遗传转化[J].上海交通大学学报,2009,28:615-618,634.
14.张东.贯叶连翘茎段和叶片的离体培养及植株再生研究[J].中国农学通报,2005,21:51-52.
15.张先云,袁秀云,崔波,等.紫丁香离体再生体系研究[J].北方园艺,2010,8:155-157.
16.赵丹,赵德刚,李岩.EuFPS基因表达载体构建及对杜仲遗传转化的研究[J].基因组学与应用生物学,2009,28:27-33.
17.邹翠霞,肖荣俊.雪柳的组织培养与植株再生[J].植物生理学通讯,2000,36(2):136-137.
18.邹晶晶,袁斌,高微,等.桂花未成熟合子胚诱导体细胞胚再生[J].中国观赏园艺研究进展,2015:238-242.
19.Al Bachchu MA,Jin SB,Park JW,Sun HJ,Yun SH,Lee H Y,Lee DS,Hong QC,Kim YW,Riu KZ and Kim JH.Agrobacterium-mediated transformation using embryogenic calli in Satsuma mandarin(Citrus unshiu Marc.)cv.Miyagawa wase[J].Hortic Environ.Biote,2011,52(2):170-175.
20.Du NX and Pijut PM.Agrobacterium-mediated transformation of Fraxinus pennsylvanica hypocotyls and plant regeneration[J].Plant Cell Rep,2009,28(6):915-923.
21.Hassanein A,Hamama L,Loridon K and Dorion N.Direct gene transfer study and transgenic plant regeneration after electroporation into mesophyll protoplasts of Pelargonium x hortorum,'Panach,Sud'[J].Plant Cell Rep,2009,28(10):1521-1530.
22.Kong DM,Preece JE and Shen HL.Somatic embryogenesis in immature cotyledons of Manchurian ash(Fraxinus mandshurica Rupr.)[J].Plant Cell Tiss Org,2012,108(3):485-492.
23.Leclercq J,Lardet L,Martin F,Chapuset T,Oliver G and Montoro P.The green fluorescent protein as an efficient selection marker for Agrobacterium tumefaciens-mediated transformation in Hevea brasiliensis(Mull.Arg)[J].Plant Cell Rep,2010,29(5):513-522.
24.Liu X.M.and Pijut P.M..Agrobacterium-mediated transformation of mature Prunus serotina(black cherry)and regeneration of transgenic shoots[J].Plant Cell Tiss Org,2010,101(1):49-57.
25.Matsunaga E,Nanto K,Oishi M,Ebinuma H,Morishita Y,Sakurai N,Suzuki H,Shibata D and Shimada T.Agrobacterium-mediated transformation of Eucalyptus globulus using explants with shoot apex with introduction of bacterial choline oxidase gene to enhance salt tolerance[J].Plant Cell Rep,2012,31(1):225-235.
26.Olivicoltura Cdspl.Somatic embryogenesis and plant regeneration in olive(Olea europaea L.)[J].Plant Cell Tiss Org,1988,14:207-214.
27.Parimalan R,Venugopalan A,Giridhar P and Ravishankar GA.Somatic embryogenesis and Agrobacterium-mediated transformation in Bixa orellana L.[J].Plant Cell Tiss Org,2011,105(3):317-328.
28.Qiao GR,Zhou J,Jiang J,Sun YH,Pan LY,Song HG,Jiang JM,Zhuo RY,Wang XJ and Sun ZX.Transformation of Liquidambar formosana L.via Agrobacterium tumefaciens using a mannose selection system and recovery of salt tolerant lines[J].Plant Cell Tiss Org,2010,102(2):163-170.
29.Ribas AF,Dechamp E,Champion A,Bertxand B,Combes MC,Verdeil JL,Lapeyre F,Lashermes P and Etienne H.Agrobacterium-mediated genetic transformation of Coffea arabica(L.)is greatly enhanced by using established embryogenie callus cultures[J].Bmc Plant Biol,2011,11:1-15.
30.Santos CV,Brito G,Pinto G and Fonseca HMAC.In vitro plantlet regeneration of Olea europaea ssp.maderensis[J].Sci Hon,2003,97(1):83-87.
31.Silva TER,Cidade LC,Alvim FC,Cascardo JCM and Costa MGC.Studies on genetic transformation of Theobroma cacao L.;evaluation of different polyamines and antibiotics on.somatic embryogenesis and the efficiency of uidA gene transfer by Agrobacterium tumefaciens[J].Plant Cell Tiss Org,2009,99(3):287-298.
32.Stevens M.E.and Pijut PM.Hypocotyl derived in vitro regeneration of pumpkin ash(Fraxinus profunda)[J].Plant Cell Tiss Org,2012,108(1):129-135.
33.Yang JL,Zhao B,Kim YB,Zhou CG,Li CY,Chen YL,Zhang HZ and Li CH.Agrobacterium tumefaciens-mediated transformation of Phellodendron amurense Rupr.using mature-seed explants[J].Mol Biol Rep,2013,40(1):281-288.
34.Zaragozaa C,Munoz-Bertomeu J and Arrillaga I.Regeneration of herbicide-tolerant black locust transgenic plants by SAAT[J].Plant Cell Rep,2004,22(11):832-838.
35.Zou JJ,Gao W,Cai X,Zeng XL and Wang CY.Somatic embryogenesis and plant regeneration in Osmanthus fragrans Lour[J].Propag Ornam Plants,2014 b,14(1):32-39.
2.陈玉玉,苏乔,祖勇,等.拟青山海关杨高效遗传转化系统的建立[J].中国农学通报,2009,25:89-92.
3.杜丽,庞振凌,周索,等.香樟胚性愈伤组织遗传转化体系建立[J].林业科学,2008,4J4:54-59.
4.李君,李岩,刘德虎.植物遗传转化的替代方法及研究进展[J].生物技术通报,2011,7:31-36.
5.高洁.萱草再生体系的优化及农杆菌介导的遗传转化体系的建立[D].成都:四川农业大学图书馆,2012.
6.梁茂厂,刘友全.桂花幼胚培养及愈伤组织增殖诱导[J].经济林研究,2007,25:43-46.
7.刘萍,袁王俊.桂花愈伤组织的诱导与增殖[J].安徽农业科学,2008,36:14889-14890.
8.罗敬萍,张树珍,杨本鹏.农杆菌介导甘蔗基因转化技术的优化[J].热带作物学报,2003,24:23-28.
9.沈俊岭,赵芳,李云,等.速生型刺槐遗传转化体系的建立[J].核农学报,2006,20:477-481.
10.宋会访,葛红,周媛,等.花离体培养与快速繁殖技术的初步研究[J].园艺学报,2005,32:738-740.
11.王彩云,白吉刚,杨玉萍.桂花的组织培养[J].北京林业大学学报,2001,23:24-25.
12.王鹏良,姜福星,蔡铃,等.广林巨尾桉9号遗传转化体系的建立[J].林业科技开发,2013,27:76-80.
13.王荃,潘琪芳,袁芳,等.超声波辅助农杆菌介导的长春花(Catharanthus roseus)遗传转化[J].上海交通大学学报,2009,28:615-618,634.
14.张东.贯叶连翘茎段和叶片的离体培养及植株再生研究[J].中国农学通报,2005,21:51-52.
15.张先云,袁秀云,崔波,等.紫丁香离体再生体系研究[J].北方园艺,2010,8:155-157.
16.赵丹,赵德刚,李岩.EuFPS基因表达载体构建及对杜仲遗传转化的研究[J].基因组学与应用生物学,2009,28:27-33.
17.邹翠霞,肖荣俊.雪柳的组织培养与植株再生[J].植物生理学通讯,2000,36(2):136-137.
18.邹晶晶,袁斌,高微,等.桂花未成熟合子胚诱导体细胞胚再生[J].中国观赏园艺研究进展,2015:238-242.
19.Al Bachchu MA,Jin SB,Park JW,Sun HJ,Yun SH,Lee H Y,Lee DS,Hong QC,Kim YW,Riu KZ and Kim JH.Agrobacterium-mediated transformation using embryogenic calli in Satsuma mandarin(Citrus unshiu Marc.)cv.Miyagawa wase[J].Hortic Environ.Biote,2011,52(2):170-175.
20.Du NX and Pijut PM.Agrobacterium-mediated transformation of Fraxinus pennsylvanica hypocotyls and plant regeneration[J].Plant Cell Rep,2009,28(6):915-923.
21.Hassanein A,Hamama L,Loridon K and Dorion N.Direct gene transfer study and transgenic plant regeneration after electroporation into mesophyll protoplasts of Pelargonium x hortorum,'Panach,Sud'[J].Plant Cell Rep,2009,28(10):1521-1530.
22.Kong DM,Preece JE and Shen HL.Somatic embryogenesis in immature cotyledons of Manchurian ash(Fraxinus mandshurica Rupr.)[J].Plant Cell Tiss Org,2012,108(3):485-492.
23.Leclercq J,Lardet L,Martin F,Chapuset T,Oliver G and Montoro P.The green fluorescent protein as an efficient selection marker for Agrobacterium tumefaciens-mediated transformation in Hevea brasiliensis(Mull.Arg)[J].Plant Cell Rep,2010,29(5):513-522.
24.Liu X.M.and Pijut P.M..Agrobacterium-mediated transformation of mature Prunus serotina(black cherry)and regeneration of transgenic shoots[J].Plant Cell Tiss Org,2010,101(1):49-57.
25.Matsunaga E,Nanto K,Oishi M,Ebinuma H,Morishita Y,Sakurai N,Suzuki H,Shibata D and Shimada T.Agrobacterium-mediated transformation of Eucalyptus globulus using explants with shoot apex with introduction of bacterial choline oxidase gene to enhance salt tolerance[J].Plant Cell Rep,2012,31(1):225-235.
26.Olivicoltura Cdspl.Somatic embryogenesis and plant regeneration in olive(Olea europaea L.)[J].Plant Cell Tiss Org,1988,14:207-214.
27.Parimalan R,Venugopalan A,Giridhar P and Ravishankar GA.Somatic embryogenesis and Agrobacterium-mediated transformation in Bixa orellana L.[J].Plant Cell Tiss Org,2011,105(3):317-328.
28.Qiao GR,Zhou J,Jiang J,Sun YH,Pan LY,Song HG,Jiang JM,Zhuo RY,Wang XJ and Sun ZX.Transformation of Liquidambar formosana L.via Agrobacterium tumefaciens using a mannose selection system and recovery of salt tolerant lines[J].Plant Cell Tiss Org,2010,102(2):163-170.
29.Ribas AF,Dechamp E,Champion A,Bertxand B,Combes MC,Verdeil JL,Lapeyre F,Lashermes P and Etienne H.Agrobacterium-mediated genetic transformation of Coffea arabica(L.)is greatly enhanced by using established embryogenie callus cultures[J].Bmc Plant Biol,2011,11:1-15.
30.Santos CV,Brito G,Pinto G and Fonseca HMAC.In vitro plantlet regeneration of Olea europaea ssp.maderensis[J].Sci Hon,2003,97(1):83-87.
31.Silva TER,Cidade LC,Alvim FC,Cascardo JCM and Costa MGC.Studies on genetic transformation of Theobroma cacao L.;evaluation of different polyamines and antibiotics on.somatic embryogenesis and the efficiency of uidA gene transfer by Agrobacterium tumefaciens[J].Plant Cell Tiss Org,2009,99(3):287-298.
32.Stevens M.E.and Pijut PM.Hypocotyl derived in vitro regeneration of pumpkin ash(Fraxinus profunda)[J].Plant Cell Tiss Org,2012,108(1):129-135.
33.Yang JL,Zhao B,Kim YB,Zhou CG,Li CY,Chen YL,Zhang HZ and Li CH.Agrobacterium tumefaciens-mediated transformation of Phellodendron amurense Rupr.using mature-seed explants[J].Mol Biol Rep,2013,40(1):281-288.
34.Zaragozaa C,Munoz-Bertomeu J and Arrillaga I.Regeneration of herbicide-tolerant black locust transgenic plants by SAAT[J].Plant Cell Rep,2004,22(11):832-838.
35.Zou JJ,Gao W,Cai X,Zeng XL and Wang CY.Somatic embryogenesis and plant regeneration in Osmanthus fragrans Lour[J].Propag Ornam Plants,2014 b,14(1):32-39.
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