A novel two T-DNA binary vector allows efficient generation of marker-free transgenic plants in three elite cultivars of rice (Oryza sativa L.)

被引:51
作者
Breitler, JC
Meynard, D
Van Boxtel, J
Royer, M
Bonnot, F
Cambillau, L
Guiderdoni, E
机构
[1] Cirad Amis, Biotrop Programme, UMR PIA1096, F-34398 Montpellier 05, France
[2] Cirad Amis, Crop Protect Programme, UMR PIA1096, F-34398 Montpellier, France
[3] Cirad Cp, F-34398 Montpellier 5, France
关键词
co-transformation; segregation analysis; selectable marker gene; transgenic rice;
D O I
10.1023/B:TRAG.0000034626.22918.0a
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
A pilot binary vector was constructed to assess the potential of the 2 T-DNA system for generating selectable marker-free progeny plants in three elite rice cultivars (ZhongZuo321, Ariete and Khao Dawk Mali 105) known to exhibit contrasting amenabilities to transformation. The first T-DNA of the vector, delimited by Agrobacterium tumefaciens borders, contains the hygromycin phosphotransferase (hpt) selectable gene and the green fluorescent protein (gfp) reporter gene while the second T-DNA, delimited by Agrobacterium rhizogenes borders, bears the phosphinothricin acetyl transferase ( bar) gene, featuring the gene of interest. 82 - 90% of the hygromycin-resistant primary transformants exhibited tolerance to ammonium glufosinate mediated by the bar gene suggesting very high co-transformation frequency in the three cultivars. All of the regenerated plants were analyzed by Southern blot which confirmed co-integration of the T-DNAs at frequencies consistent with those of co-expression and allowed determination of copy number for each gene as well as detection of two different vector backbone fragments extending between the two T-DNAs. Hygromycin susceptible, ammonium glufosinate tolerant phenotypes represented 14.4, 17.4 and 14.3% of the plants in T1 progenies of ZZ321, Ariete and KDML105 primary transformants, respectively. We developed a statistical model for deducing from the observed copy number of each T-DNA in T0 plants and phenotypic segregations in T1 progenies the most likely constitution and linkage of the T-DNA integration locus. Statistical analysis identified in 40 out of 42 lines a most likely linkage configuration theoretically allowing genetic separation of the two T-DNA types and out segregation of the T-DNA bearing the bar gene. Overall, though improvements of the technology would be beneficial, the 2 T-DNA system appeared to be a useful approach to generate selectable marker-free rice plants with a consistent frequency among cultivars.
引用
收藏
页码:271 / 287
页数:17
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