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Somaclonal variation does not preclude the use of rice transformants for genetic screening
Authors:Fu‐Jin Wei  Lin‐Yun Kuang  Hui‐Min Oung  Sin‐Yuan Cheng  Hshin‐Ping Wu  Lin‐Tzu Huang  Yi‐Tzu Tseng  Wan‐Yi Chiou  Vicki Hsieh‐Feng  Cheng‐Han Chung  Su‐May Yu  Lan‐Ying Lee  Stanton B Gelvin  Yue‐Ie C Hsing
Institution:1. Institute of Plant and Microbial Biology, Academia Sinica, No. 128, Section 2. 2, Taipei, Taiwan;3. Department of Agronomy, National Taiwan University, No. 1, Section 4. 4, Taipei, Taiwan;5. Transgenic Plant Core Facility, Academia Sinica, No. 128, Section 6. Institute of Plant Biology, National Taiwan University, No. 1, Section 7. Institute of Molecular Biology, Academia Sinica, No. 128, Section 8. Department of Biological Sciences, Purdue University, West Lafayette, IN, USA
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.
Keywords:Agrobacterium‐mediated transformation  indels  regeneration  rice  single‐nucleotide polymorphisms  somaclonal variation
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