title: Somaclonal variation does not preclude the use of rice transformants for genetic screening.
abstract: Rice (Oryza sativa) is one of the world's most important crops. Rice researchers make extensive use of insertional mutants for the study of gene function. Approximately half a million flanking sequence tags from rice insertional mutant libraries are publicly available. However, the relationship between genotype and phenotype is very weak. Transgenic plant assays have been used frequently for complementation, overexpression or antisense analysis, but sequence changes caused by callus growth, Agrobacterium incubation medium, virulence genes, transformation and selection conditions are unknown. We used high-throughput sequencing of DNA from rice lines derived from Tainung 67 to analyze non-transformed and transgenic rice plants for mutations caused by these parameters. For comparison, we also analyzed sequence changes for two additional rice varieties and four T-DNA tagged transformants from the Taiwan Rice Insertional Mutant resource. We identified single-nucleotide polymorphisms, small indels, large deletions, chromosome doubling and chromosome translocations in these lines. Using standard rice regeneration/transformation procedures, the mutation rates of regenerants and transformants were relatively low, with no significant differences among eight tested treatments in the Tainung 67 background and in the cultivars Taikeng 9 and IR64. Thus, we could not conclusively detect sequence changes resulting from Agrobacterium-mediated transformation in addition to those caused by tissue culture-induced somaclonal variation. However, the mutation frequencies within the two publically available tagged mutant populations, including TRIM transformants or Tos17 lines, were about 10-fold higher than the frequency of standard transformants, probably because mass production of embryogenic calli and longer callus growth periods were required to generate these large libraries.
authors.affiliation: Institute of Plant and Microbial Biology, Academia Sinica, No. 128, Section 2, Yien-Chu-Yuan Road, Taipei, 11529, Taiwan.|Department of Agronomy, National Taiwan University, No. 1, Section 4, Roosevelt Rd, Taipei, 10617, Taiwan.
authors.forename: Fu-Jin
authors.initials: FJ
authors.lastname: Wei
authors.affiliation: Transgenic Plant Core Facility, Academia Sinica, No. 128, Section 2, Yien-Chu-Yuan Road, Taipei, 11529, Taiwan.
authors.forename: Lin-Yun
authors.initials: LY
authors.lastname: Kuang
authors.affiliation: Institute of Plant and Microbial Biology, Academia Sinica, No. 128, Section 2, Yien-Chu-Yuan Road, Taipei, 11529, Taiwan.
authors.forename: Hui-Min
authors.initials: HM
authors.lastname: Oung
authors.affiliation: Institute of Plant and Microbial Biology, Academia Sinica, No. 128, Section 2, Yien-Chu-Yuan Road, Taipei, 11529, Taiwan.
authors.forename: Sin-Yuan
authors.initials: SY
authors.lastname: Cheng
authors.affiliation: Institute of Plant and Microbial Biology, Academia Sinica, No. 128, Section 2, Yien-Chu-Yuan Road, Taipei, 11529, Taiwan.
authors.forename: Hshin-Ping
authors.initials: HP
authors.lastname: Wu
authors.affiliation: Institute of Plant and Microbial Biology, Academia Sinica, No. 128, Section 2, Yien-Chu-Yuan Road, Taipei, 11529, Taiwan.
authors.forename: Lin-Tzu
authors.initials: LT
authors.lastname: Huang
authors.affiliation: Institute of Plant and Microbial Biology, Academia Sinica, No. 128, Section 2, Yien-Chu-Yuan Road, Taipei, 11529, Taiwan.|Institute of Plant Biology, National Taiwan University, No. 1, Section 4, Roosevelt Rd, Taipei, 10617, Taiwan.
authors.forename: Yi-Tzu
authors.initials: YT
authors.lastname: Tseng
authors.affiliation: Institute of Plant and Microbial Biology, Academia Sinica, No. 128, Section 2, Yien-Chu-Yuan Road, Taipei, 11529, Taiwan.
authors.forename: Wan-Yi
authors.initials: WY
authors.lastname: Chiou
authors.affiliation: Department of Agronomy, National Taiwan University, No. 1, Section 4, Roosevelt Rd, Taipei, 10617, Taiwan.
authors.forename: Vicki
authors.initials: V
authors.lastname: Hsieh-Feng
authors.affiliation: Institute of Plant and Microbial Biology, Academia Sinica, No. 128, Section 2, Yien-Chu-Yuan Road, Taipei, 11529, Taiwan.
authors.forename: Cheng-Han
authors.initials: CH
authors.lastname: Chung
authors.affiliation: Institute of Molecular Biology, Academia Sinica, No. 128, Section 2, Yien-Chu-Yuan Road, Taipei, 11529, Taiwan.
authors.forename: Su-May
authors.initials: SM
authors.lastname: Yu
authors.affiliation: Department of Biological Sciences, Purdue University, 201 South University St., West Lafayette, IN, 47907-1392, USA.
authors.forename: Lan-Ying
authors.initials: LY
authors.lastname: Lee
authors.affiliation: Department of Biological Sciences, Purdue University, 201 South University St., West Lafayette, IN, 47907-1392, USA.
authors.forename: Stanton B
authors.initials: SB
authors.lastname: Gelvin
authors.affiliation: Institute of Plant and Microbial Biology, Academia Sinica, No. 128, Section 2, Yien-Chu-Yuan Road, Taipei, 11529, Taiwan.
authors.forename: Yue-Ie C
authors.initials: YI
authors.lastname: Hsing
mesh_terms: D060054:Agrobacterium / Q000235:genetics; D002999:Clone Cells / Q000378:metabolism; D018556:Crops, Agricultural / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics; D056726:Genetic Association Studies / Q000379:methods*; D014644:Genetic Variation; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D054643:INDEL Mutation; D016254:Mutagenesis, Insertional; D012275:Oryza / Q000145:classification / Q000235:genetics*; D010641:Phenotype; D030821:Plants, Genetically Modified; D011003:Ploidies; D020641:Polymorphism, Single Nucleotide; D013045:Species Specificity; D013624:Taiwan; D046509:Tissue Culture Techniques / Q000379:methods; D014170:Transformation, Genetic / Q000235:genetics*
chemical_list: D004269:DNA, Bacterial; D018744:DNA, Plant; C036483:T-DNA
keywords: Agrobacterium-mediated transformation; indels; regeneration; rice; single-nucleotide polymorphisms; somaclonal variation