KARAKTERISASI T-DNA pGAS102 PEMBAWA GEN PaFT1 UNTUK TRANSFER GEN PEMBUNGAAN PADA ANGGREK Dendrobium aphyllum Roxb. DENGAN AGROBAKTERIUM

• Main Authors: , Muhamad Wisnu, , Dr. Endang Semiarti, M.S., M.Sc.
• Format: Theses and Dissertations NonPeerReviewed
• Published: [Yogyakarta] : Universitas Gadjah Mada 2012
• Subjects: ETD
• Online Access: https://repository.ugm.ac.id/97681/; http://etd.ugm.ac.id/index.php?mod=penelitian_detail&sub=PenelitianDetail&act=view&typ=html&buku_id=54183
• Institution: Universitas Gadjah Mada

Agrobacterium-mediated genetic transformation has been widely used for gene transfer into plants. Stability of T-DNA structure plays important roles for the successful of genetic transformation. The aim of this research is to confirm the stability of T-DNA structure that harbor interest genes prior to gene transfer into plant. The method was carried out using pPZP2Ha3 and pGAS102 containing A.tumefaciens LBA4404. The Agrobacterium was six times subcultured in LB medium. The T-DNA structures were checked by PCR using a pair of primer for Hd3a gene and a pair of primer for pUbi-NOS. The 120 bp Hd3a gene fragment was amplified from pPZP2Ha and 1148 bp pUbi::PaFT1 fragment was amplified from pGAS102. The structure of T-DNA was also confirmed with endonuclease restriction enzymes, i.e. : EcoRI, BamHI, Bgl II, and Sal I. The most stable plasmid was transferred into lateral shoot meristem of Dendrobium aphyllum Roxb. orchid plant by in planta technique mediated by Agrobacterium. The emerged shoots from the orchid stems were then analyzed for the existence of the T-DNA fragment. The results show that the pGAS102 plasmid was more stable than pPZP2Ha3. An Hd3a gene was not exist anymore in the first subculture Agrobacterium pPZP2Ha3. On the other hand, the plasmid from all six colonies of Agrobacterium pGAS102 contained 1148 bp pUbi::PaFT1 fragment. Confirmation of the T-DNA structure by restriction enzyme showed that BamHI or Bgl II digestion resulted in one linear fragment of 15086 bp. Double digestion by BamHI and Bgl II resulted in two fragments: 10832 bp and 4254 bp. Sal I digested of plasmid DNA resulted in three fragments: 10832 bp, 3472 bp and 2680 bp. These data indicate that the T-DNA structure of pUbi::PaFT1 is stable until six subculture analyzed. There are six shoots transformant candidates obtained from in planta genetic transformation. The 1148 bp of T-DNA fragment had been amplified from all transformant candidates. Taken together, these data indicate that the structure of T-DNA in pUbi::PaFT1 is stable and it has been successfully transferred into D.aphyllum plant genom.