金钗石斛转基因体系的建立
|
摘要
以金钗石斛种子为外植体建立组织培养体系,在含1.5 mg/L 6-BA MS培养基中添加0.5 mg/L NAA和2%蔗糖能有效地诱导原球茎,诱导率达91.67%;添加0.25 mg/L NAA和3%蔗糖则更适于原球茎分化形成不定芽;添加0.10 mg/L NAA和2%蔗糖适宜于不定根的诱导.同时建立了根癌农杆菌(Agrobacterium tumefaciens)转化金钗石斛的方法和体系:使用根癌农杆菌EHA105侵染30 min,共培养3 d时,金钗石斛的根段、根尖和组培幼苗的转化率最高,而含腋芽的茎节则需要共培养5 d.文章所建立的组培体系与转基因体系将为探讨金钗石斛的基因功能和调控机制等生物学问题及建立稳定转化的再生植株提供技术手段.
This study attempts the establishment of the tissue culturing system using Dendrobium nobile seeds as the starting materials. It is found that the MS medium supplemented with 1.5 mg/L 6-BA+0.5 mg/L NAA+2% sucrose is the most suitable for the induction of protocorm,showing an induction rate of 91.67% after being cultured for 15 days. Adding 0.25 mg/L NAA and 3% sucrose to the MS medium with 1.5 mg/L 6-BA is more suitable to form adventitious seedlings with dark green sturdy leaves,and the addition of 0.10 mg/L NAA and 2% sucrose to the MS medium with 1.5 mg/L 6-BA is suitable for root emergency and growth. The transgenic system for Dendrobium nobile tissues is also studied. It is found that compared with GV1301 strain,the Agrobacterium tumefaciens EHA105 strain can be used to obtain higher transformation rates. The rate of transformation using root segments,root tips and tissuecultured seedlings as the starting materials reaches the highest when they are soaked with EHA105 for 30 minutes and then co-cultured for 3 days,but the rate of transformation using nodes with axillary buds reaches the highest if co-cultured for 5 days. The results of the present study can provide meaningful techniques for further research on Dendrobium nobile and the breeding of Dendrobium varieties.
This study attempts the establishment of the tissue culturing system using Dendrobium nobile seeds as the starting materials. It is found that the MS medium supplemented with 1.5 mg/L 6-BA+0.5 mg/L NAA+2% sucrose is the most suitable for the induction of protocorm,showing an induction rate of 91.67% after being cultured for 15 days. Adding 0.25 mg/L NAA and 3% sucrose to the MS medium with 1.5 mg/L 6-BA is more suitable to form adventitious seedlings with dark green sturdy leaves,and the addition of 0.10 mg/L NAA and 2% sucrose to the MS medium with 1.5 mg/L 6-BA is suitable for root emergency and growth. The transgenic system for Dendrobium nobile tissues is also studied. It is found that compared with GV1301 strain,the Agrobacterium tumefaciens EHA105 strain can be used to obtain higher transformation rates. The rate of transformation using root segments,root tips and tissuecultured seedlings as the starting materials reaches the highest when they are soaked with EHA105 for 30 minutes and then co-cultured for 3 days,but the rate of transformation using nodes with axillary buds reaches the highest if co-cultured for 5 days. The results of the present study can provide meaningful techniques for further research on Dendrobium nobile and the breeding of Dendrobium varieties.
引文
[1]FAY M F,PAILLER T,DIXON K W.Orchid conservation:making the links[J].Annals of Botany,2015,116(3):377-379.
[2]YU H,GOH C J.Molecular genetics of reproductive biology in orchids[J].Plant Physiology,2001,127(4):1390-1393.
[3]中国科学院中国植物志编辑委员会.中国植物志:第十九卷[M].北京:科学出版社,1999:291-294.
[4]周威,夏杰,孙文博,等.金钗石斛的化学成分和药理作用研究现状[J].中国新药杂志,2017(22):2693-2700.ZHOU W,XIA J,SUN W B,et al.Current research status of chemical constituents and pharmacological effects of Dendrobium nobile[J].Chinese Journal of New Drugs,2017(22):2693-2700.
[5]VELLUPILLAI M,SWARUP S,GOH C J.Histological and protein changes during early stages of seed germination in the orchid,Dendrobium crumenatum[J].The Journal of Horticultural Science&Biotechnology,1997,72(6):941-948.
[6]张明,夏鸿西,朱利泉,等.石斛组织培养研究进展[J].中国中药杂志,2000,25(6):323-326.ZHANG M,XIA H X,ZHU L Q,et al.Research progress on tissue culture of Dendrobiumc[J].China Journal of Chinese Materia Medica,2000,25(6):323-326.
[7]张建勇,刘涛,袁佐清.石斛属植物组织培养及遗传转化研究进展[J].安徽农业科学,2007,35(3):656-657.ZHANG J Y,LIU T,YUAN Z Q.Research progress in dendrobium tissue culture and genetic transformation[J].Journal of Anhui Agricultural Sciences,2007,35(3),656-657.
[8]徐云鹃,于力文.霍山石斛种子试管苗的培养[J].植物生理学通讯,1984(4):35-36.
[9]王立安,沈淑瑜,李煜照,等.铁皮石斛种子试管苗的大量繁殖[J].安徽大学学报(自然科学版),1989(1):83-86.WANG L A,SHEN S Y,LI Y Z,et al.A mass propagation of tube-plantlets by seeds in dendrodium officinalie[J].Journal of Anhui University,1989(1):83-86.
[10]史永锋,付开聪,张宁,等.束花石斛快繁育苗技术的研究[J].中草药,2005,36(3):438-441.SHI Y F,FU K C,ZHANG N,et al.Rapid propagation and breeding seedling technique for dendrobium chrysanthum[J].Chinese Traditional and Herbal Drugs,2005,36(3):438-441.
[11]杜刚,来天超,杨海英.兜唇石斛的组织培养研究[J].北方园艺,2012(8):140-141.DU G,LAI T C,YANG H Y.Study on the tissue culture of Dendrobium aphyllum(Roxb.)C.E.C.Fisch[J].Northern Horticulture,2012(8):140-141.
[12]JEFFERSON R A,KAVANAGH T A,BEVAN M W.GUSfusions:beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants[J].The EMBO Journal,1987,6(13):3901-3907.
[13]MEN S,MING X,LIU R,et al.Agrobacterium-mediated genetic transformation of a Dendrobium,orchid[J].Plant Cell Tissue&Organ Culture,2003,75(1):63-71.
[14]BAI Y,QU R.Factors influencing tissue culture responses of mature seeds and immature embryos in turf-type tall fescue[J].Plant Breeding,2010,120(3):239-242.
[15]UDOMDEE W,WEN P J,LEE C Y,et al.Effect of sucrose concentration and seed maturity on in vitro germination of Dendrobium nobile hybrids[J].Plant Growth Regulation,2014,72(3):249-255.
[16]杨玉萍,李宜芸,李茜雯,等.紫锥菊叶柄高效再生体系的建立[J].华南师范大学学报(自然科学版),2015,47(4):94-97.YANG Y P,LI Y Y,LI Q W,et al.Establishment of an efficient regeneration system for echinacea purpurea petiol[J].Journal of South China Normal University(Natural Science Edition),2015,47(4):94-97.
[17]HOOD E E,HELMER G L,FRALEY R T,et al.The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of p Ti Bo542 outside of T-DNA[J].Journal of Bacteriology,1986,168(3):1291-1301.
[18]JIN S G,KOMARI T,GORDON M P,et al.Genes responsible for the supervirulence phenotype of Agrobacterium tumefaciens A281[J].Journal of Bacteriology,1987,169(10):4417-4425.
[19]贾彩红,张建斌,金志强,等.不同转基因方法转化石斛兰的研究[C]∥2006年中国园艺学会观赏园艺专业委员会年会论文集.北京:中国农业出版社,2006:651-654.
[20]柴明良,金斗焕.农杆菌介导的蝴蝶兰基因转化系统的建立[J].园艺学报,2004,31(4):537-539.CHAI M L,JIN D H.Establishment of agrobacteriummediated transformation in phalaenopsis[J].Acta Horticulturae Sinica,2004,31(4):537-539.
[21]李学农.常用中药石斛显微鉴别[J].海峡药学,2008,20(3):84-85.
[22]BELARMINO M M,MII M.Agrobacterium-mediated genetic transformation of a phalaenopsis orchid[J].Plant Cell Reports,2000,19(5):435-442.
[23]陈之林,段俊,曾宋君,等.原球茎为转化受体的农杆菌介导石斛遗传转化[J].中山大学学报(自然科学版),2007,46(1):86-90.CHEN Z L,DUAN J,ZENG S J,et al.Agrobacteriummediated transformation of dendrobium orchid by targeting protocorms[J].Acta Scientiarum Naturalium Universitatis Sunyatseni,2007,46(1):86-90.
[24]MISHIBA K I,CHIN D P,MII M.Agrobacterium-mediated transformation of Phalaenopsisby targeting protocorms at an early stage after germination[J].Plant Cell Reports,2005,24(5):297-303.
[2]YU H,GOH C J.Molecular genetics of reproductive biology in orchids[J].Plant Physiology,2001,127(4):1390-1393.
[3]中国科学院中国植物志编辑委员会.中国植物志:第十九卷[M].北京:科学出版社,1999:291-294.
[4]周威,夏杰,孙文博,等.金钗石斛的化学成分和药理作用研究现状[J].中国新药杂志,2017(22):2693-2700.ZHOU W,XIA J,SUN W B,et al.Current research status of chemical constituents and pharmacological effects of Dendrobium nobile[J].Chinese Journal of New Drugs,2017(22):2693-2700.
[5]VELLUPILLAI M,SWARUP S,GOH C J.Histological and protein changes during early stages of seed germination in the orchid,Dendrobium crumenatum[J].The Journal of Horticultural Science&Biotechnology,1997,72(6):941-948.
[6]张明,夏鸿西,朱利泉,等.石斛组织培养研究进展[J].中国中药杂志,2000,25(6):323-326.ZHANG M,XIA H X,ZHU L Q,et al.Research progress on tissue culture of Dendrobiumc[J].China Journal of Chinese Materia Medica,2000,25(6):323-326.
[7]张建勇,刘涛,袁佐清.石斛属植物组织培养及遗传转化研究进展[J].安徽农业科学,2007,35(3):656-657.ZHANG J Y,LIU T,YUAN Z Q.Research progress in dendrobium tissue culture and genetic transformation[J].Journal of Anhui Agricultural Sciences,2007,35(3),656-657.
[8]徐云鹃,于力文.霍山石斛种子试管苗的培养[J].植物生理学通讯,1984(4):35-36.
[9]王立安,沈淑瑜,李煜照,等.铁皮石斛种子试管苗的大量繁殖[J].安徽大学学报(自然科学版),1989(1):83-86.WANG L A,SHEN S Y,LI Y Z,et al.A mass propagation of tube-plantlets by seeds in dendrodium officinalie[J].Journal of Anhui University,1989(1):83-86.
[10]史永锋,付开聪,张宁,等.束花石斛快繁育苗技术的研究[J].中草药,2005,36(3):438-441.SHI Y F,FU K C,ZHANG N,et al.Rapid propagation and breeding seedling technique for dendrobium chrysanthum[J].Chinese Traditional and Herbal Drugs,2005,36(3):438-441.
[11]杜刚,来天超,杨海英.兜唇石斛的组织培养研究[J].北方园艺,2012(8):140-141.DU G,LAI T C,YANG H Y.Study on the tissue culture of Dendrobium aphyllum(Roxb.)C.E.C.Fisch[J].Northern Horticulture,2012(8):140-141.
[12]JEFFERSON R A,KAVANAGH T A,BEVAN M W.GUSfusions:beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants[J].The EMBO Journal,1987,6(13):3901-3907.
[13]MEN S,MING X,LIU R,et al.Agrobacterium-mediated genetic transformation of a Dendrobium,orchid[J].Plant Cell Tissue&Organ Culture,2003,75(1):63-71.
[14]BAI Y,QU R.Factors influencing tissue culture responses of mature seeds and immature embryos in turf-type tall fescue[J].Plant Breeding,2010,120(3):239-242.
[15]UDOMDEE W,WEN P J,LEE C Y,et al.Effect of sucrose concentration and seed maturity on in vitro germination of Dendrobium nobile hybrids[J].Plant Growth Regulation,2014,72(3):249-255.
[16]杨玉萍,李宜芸,李茜雯,等.紫锥菊叶柄高效再生体系的建立[J].华南师范大学学报(自然科学版),2015,47(4):94-97.YANG Y P,LI Y Y,LI Q W,et al.Establishment of an efficient regeneration system for echinacea purpurea petiol[J].Journal of South China Normal University(Natural Science Edition),2015,47(4):94-97.
[17]HOOD E E,HELMER G L,FRALEY R T,et al.The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of p Ti Bo542 outside of T-DNA[J].Journal of Bacteriology,1986,168(3):1291-1301.
[18]JIN S G,KOMARI T,GORDON M P,et al.Genes responsible for the supervirulence phenotype of Agrobacterium tumefaciens A281[J].Journal of Bacteriology,1987,169(10):4417-4425.
[19]贾彩红,张建斌,金志强,等.不同转基因方法转化石斛兰的研究[C]∥2006年中国园艺学会观赏园艺专业委员会年会论文集.北京:中国农业出版社,2006:651-654.
[20]柴明良,金斗焕.农杆菌介导的蝴蝶兰基因转化系统的建立[J].园艺学报,2004,31(4):537-539.CHAI M L,JIN D H.Establishment of agrobacteriummediated transformation in phalaenopsis[J].Acta Horticulturae Sinica,2004,31(4):537-539.
[21]李学农.常用中药石斛显微鉴别[J].海峡药学,2008,20(3):84-85.
[22]BELARMINO M M,MII M.Agrobacterium-mediated genetic transformation of a phalaenopsis orchid[J].Plant Cell Reports,2000,19(5):435-442.
[23]陈之林,段俊,曾宋君,等.原球茎为转化受体的农杆菌介导石斛遗传转化[J].中山大学学报(自然科学版),2007,46(1):86-90.CHEN Z L,DUAN J,ZENG S J,et al.Agrobacteriummediated transformation of dendrobium orchid by targeting protocorms[J].Acta Scientiarum Naturalium Universitatis Sunyatseni,2007,46(1):86-90.
[24]MISHIBA K I,CHIN D P,MII M.Agrobacterium-mediated transformation of Phalaenopsisby targeting protocorms at an early stage after germination[J].Plant Cell Reports,2005,24(5):297-303.