African Journal of
Biotechnology

  • Abbreviation: Afr. J. Biotechnol.
  • Language: English
  • ISSN: 1684-5315
  • DOI: 10.5897/AJB
  • Start Year: 2002
  • Published Articles: 12258

Full Length Research Paper

A protocol for Agrobacterium-mediated transformation of Kalanchoë blossfeldiana with a flavonoid 3',5' hydroxylase (F3'5'H) gene

Phopgao Buddharak
  • Phopgao Buddharak
  • Department of Biology, Faculty of Science, University of Phayao, Phayao 56000, Thailand.
  • Google Scholar
Ruttaporn Chundet
  • Ruttaporn Chundet
  • Program in Biotechnology, Faculty of Science, Maejo University, Chiang Mai 50290, Thailand.
  • Google Scholar
Warut U-kong*
  • Warut U-kong*
  • Program in Biotechnology, Faculty of Science, Maejo University, Chiang Mai 50290, Thailand.
  • Google Scholar


  •  Received: 14 July 2015
  •  Accepted: 23 September 2015
  •  Published: 30 September 2015

Abstract

In the present investigation, explants from Kalanchoë blossfeldiana were used for gene transformation. The young leaves were inoculated with Agrobacterium tumefaciens LBA4404 strain with a binary vector plasmid pArtblue containing F3'5'H gene under control of CaMV35S promoter and nptII selectable marker gene. After inoculation, the explants were transferred to the co-cultivation medium. They were then transferred to the selection medium containing kanamycin and were sub-cultured every two weeks. Leaves of the putative transgenic shoots that survived in the selection medium were used in reverse transcription polymerase chain reaction (RT-PCR) analysis to detect gene expression. The RT-PCR analysis showed the presence of 550 bp F3'5'H amplification products and had an expression of F3'5'H gene. Plants with the introduced F3'5'H gene produced totally pale red flowers.

Key words: Kalanchoë blossfeldiana, Agrobacterium-mediated transformation, young leaf, F3'5'H gene, reverse transcription-polymerase chain reaction (RT-PCR).