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Polymerase Chain Reaction Used for Monitoring multiple Gene Integration in Agrobacterium ‐Mediated Transformation
Author(s) -
Blake Nancy K.,
Ditterline Raymond L.,
Stout Richard G.
Publication year - 1991
Publication title -
crop science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.76
H-Index - 147
eISSN - 1435-0653
pISSN - 0011-183X
DOI - 10.2135/cropsci1991.0011183x003100060061x
Subject(s) - biology , agrobacterium tumefaciens , agrobacterium , transformation (genetics) , kanamycin , polymerase chain reaction , southern blot , gene , genetically modified crops , rhizobiaceae , microbiology and biotechnology , genetics , transgene , bacteria , symbiosis
Characterizing plants following Agrobacterium ‐mediated transformation has become an important consideration in the creation of transgenic plants. As an alternative to Southern blot analysis, polymerase chain reaction (PCR) was used to screen for the simultaneous integration of two foreign genes for neomycin phosphotransferase II (NFT II) and β‐glucuronidase (GUS) in alfalfa ( Medicago saliva L.) transformed with Agrobacterium tumefaciens . We were able to distinguish between plants that contained only the NFT II gene and those that had both the NFT II and GUS genes. Of our putative transformants selected on kanamycin, 28% contained only the NFT II gene and apparently had lost the GUS gene during the transformation process. Requiring only nanogram amounts of DNA, PCR provided a rapid method to determine which genes had been integrated to our putative transformants much earlier in the plant regenerative process than was feasible by Southern analysis.