Investigation of Agrobacterium-mediated transformation of apple using green fluorescent protein: High transient expression and low stable transformation suggest that factors other than T-DNA transfer are rate-limiting

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Abstract

To investigate early events of Agrobacterium-mediated transformation of apple cultivars, a synthetic green fluorescent protein gene (SGFP) was used as a highly sensitive, vital reporter gene. Leaf explants from four apple cultivars ('Delicious', 'Golden Delicious', 'Royal Gala' and 'Greensleeves') were infected with Agrobacterium EHA101 harboring plasmid pDM96.0501. Fluorescence microscopy indicated that SGFP expression was first detected 48 h after infection and quantitative analysis revealed a high T-DNA transfer rate. Plant cells with stably incorporated T-DNA exhibited cell division and developed transgenic calli, followed by formation of transgenic shoots at low frequencies. The detection of SGFP expression with an epifluorescence stereomicroscope confirmed the effectiveness of SGFP as a reporter gene for detection of very early transformation events and for screening of putative transformants. The efficiency of the transformation and regeneration process decreased ca. 10 000-fold from Agrobacterium infection to transgenic shoot regeneration, suggesting that factors other than Agrobacterium interaction and T-DNA transfer are rate-limiting steps in Agrobacterium-mediated transformation of apple.

Original languageEnglish (US)
Pages (from-to)549-559
Number of pages11
JournalPlant molecular biology
Volume37
Issue number3
DOIs
StatePublished - Jun 1 1998

All Science Journal Classification (ASJC) codes

  • Agronomy and Crop Science
  • Genetics
  • Plant Science

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