Plant Cell Reports

, Volume 25, Issue 5, pp 432–441

MicroTom—a high-throughput model transformation system for functional genomics

  • Yinghui Dan
  • Hua Yan
  • Tichafa Munyikwa
  • Jimmy Dong
  • Yanling Zhang
  • Charles L. Armstrong
Genetic Transformation and Hybridization

Abstract

We have developed a high-throughput Agrobacterium-mediated transformation model system using both nptII and the 5-enolpyruvylshikimate-3-phosphate synthase gene from Agrobacterium tumefaciens strain CP4 (cp4) based selections in MicroTom, a miniature rapid-cycling cherry tomato variety. With the NPTII selection system, transformation frequency calculated as independent transgenic events per inoculated explant ranged from 24 to 80% with an average of 56%, in industrial production scale transformation experiments. For CP4, with glyphosate selection, the average transformation frequency was 57%. Stable transformation frequency was positively correlated with transient expression (R=0.85), and variable with the genes of interest. DNA integration and germline transformation were confirmed by biological assay, Southern Blot analysis, and R1 phenotype segregation. Transgene expression was observed in leaf, root, stem, flower, and fruit tissues of the transgenic plants. Ninety-five percent of transgenic events coexpressed two introduced genes based on β-glucuronidase (GUS) and neonmycin phosphotransferase II (NPTII) expression. Seventy-five percent of transgenic events contained one to two copies of the introduced uidA (GUS) gene based on Southern analysis. Transgenic plants from the cotyledon explants to the transgenic plants transferred to soil were produced within about 2–3 months depending on the genes of interest. The utility of this MicroTom model transformation system for functional genomic studies, such as identification of genes related to important agricultural traits and gene function, is discussed.

Keywords

Agrobacterium High-throughput Kanamycin MicroTom Transformation 

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Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Yinghui Dan
    • 1
    • 2
  • Hua Yan
    • 1
  • Tichafa Munyikwa
    • 1
  • Jimmy Dong
    • 1
  • Yanling Zhang
    • 1
  • Charles L. Armstrong
    • 1
  1. 1.Monsanto CompanyChesterfieldUSA
  2. 2.Institute for Advanced Learning and ResearchVirginia Polytechnic Institute and State UniversityDanvilleUSA

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