Agrobacterium tumefaciens-mediated transformation of Narcissus tazzeta var. chinensis.
Phytoene synthase (PSY), as a key regulatory enzyme for carotene biosynthesis, plays an important role in regulating color formation in many species. In the present study, a protocol was developed for the transformation of Narcissus tazzeta var chinensis using Agrobacterium tumefaciens strain LBA4404 harboring a binary vector pCAMBIA1301 plasmid which contained an antisense phytoene synthase gene, a reporter beta-glucuronidase gene and a selectable marker hygromycin phosphotransferase gene. Effects of some factors on efficiency of transformation and regeneration were examined. Preculture of the explants for 6 days before inoculation enhanced the transient GUS expression. The addition of acetosyringone (AS) at 100 micromol l(-1) for inoculation and a period of 3 days co-cultivation yielded efficient transient GUS expression. Transformants were obtained through selection on MS medium containing 5 mg l(-1) 6-benzylaminopurine (BA), 0.1 mg l(-1)alpha-naphthalene acetic acid (NAA) and 40 mg l(-1) hygromycin. The transformation frequency was 1.24% based on PCR analysis of gus gene. One or two copies of transgene were demonstrated in different transformations by Southern blotting analyses. Northern blotting results confirmed that the transcription of the endogenous psy gene in transgenic plants was inhibited or silenced. The method reported here provides new opportunities for improvement of quality traits of Narcissus tazzeta via genetic transformation.
Lu
Gang
G
Agr Minist China, Key Lab Hort Plant Growth Dev and Biotechnol, Department of Horticulture, Zhejiang University, Hangzhou 310029, People's Republic of China. glu@zju.edu.cn
Zou
Qingcheng
Q
Guo
Deping
D
Zhuang
Xiaoying
X
Yu
Xiaolin
X
Xiang
Xun
X
Cao
Jiashu
J
D016960:Agrobacterium tumefaciens / Q000187:drug effects / Q000502:physiology*; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002934:Cinnamates / Q000494:pharmacology; D004269:DNA, Bacterial / Q000378:metabolism; D017343:Genes, Plant; D005822:Genetic Vectors; D005966:Glucuronidase / Q000378:metabolism; D006921:Hygromycin B / Q000031:analogs & derivatives / Q000494:pharmacology; D031427:Narcissus / Q000166:cytology / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D030821:Plants, Genetically Modified; D012038:Regeneration / Q000187:drug effects; D014170:Transformation, Genetic / Q000187:drug effects
D002934:Cinnamates; D004269:DNA, Bacterial; C036483:T-DNA; D006921:Hygromycin B; C026273:hygromycin A; D005966:Glucuronidase