Chloroplast transformation in plants: Polyethylene glycol (PEG) treatment of protoplasts is an alternative to biolistic delivery systems

Carmel O'Neill, G. Horváth, Éva Horváth, Philip J. Dix, Péter Medgyesy

Research output: Contribution to journalArticle

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Abstract

Nicotiana plumbaginifolia protoplasts were directly transformed by PEG treatment with a cloned 16S rRNA gene isolated from a double antibiotic-resistant Nicotiana tabacum plastid mutant. Putative plastid transformants were selected in cell culture by their spectinomycin resistance and identified by their unselected streptomycin resistance. Alternatively, cell lines were selected in the presence of both antibiotics. The cell line (and its regenerated plants) selected solely for spectinomycin resistance demonstrated an extensive segregation of streptomycin resistance in subsequent tests, while the double-selected line showed stable resistance for both antibiotics. The resistance markers were inherited maternally. In the putative plastid transformants the origin of the resistance mutations was identified by the absence of an Aatll site, missing in the donor N. tabacum plastid gene (spectinomycin resistance site) but present in that of wild-type N. plumbaginifolia, and a sequence analysis of the particular nucleotide changes in both resistance sites. Restriction enzyme analysis of total plastid DNA (ptDNA), and the recloning and full sequencing of the fragment introduced, investigated in one of the plastid transformants, showed no DNA rearrangements accompanied with the integration process. Sequence analysis indicated a targeted, homologous integration of the DNA fragment introduced but an unexpectedly complete homology of the parental ptDNA sequences in this region prevented the location of borders. Although the frequency of plastid transformant colonies (2 × 10-5) should still be improved, this method for stable chloroplast DNA transformation is comparable with or more efficient than the particle bombardment techniques.

Original languageEnglish
Pages (from-to)729-738
Number of pages10
JournalPlant Journal
Volume3
Issue number5
Publication statusPublished - 1993

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Biolistics
biolistics
Plastids
Protoplasts
Chloroplasts
polyethylene glycol
protoplasts
plastids
chloroplasts
spectinomycin
Spectinomycin
Nicotiana plumbaginifolia
plastid DNA
streptomycin
Tobacco
Nicotiana tabacum
Streptomycin
Therapeutics
sequence analysis
antibiotics

ASJC Scopus subject areas

  • Plant Science

Cite this

Chloroplast transformation in plants : Polyethylene glycol (PEG) treatment of protoplasts is an alternative to biolistic delivery systems. / O'Neill, Carmel; Horváth, G.; Horváth, Éva; Dix, Philip J.; Medgyesy, Péter.

In: Plant Journal, Vol. 3, No. 5, 1993, p. 729-738.

Research output: Contribution to journalArticle

O'Neill, Carmel ; Horváth, G. ; Horváth, Éva ; Dix, Philip J. ; Medgyesy, Péter. / Chloroplast transformation in plants : Polyethylene glycol (PEG) treatment of protoplasts is an alternative to biolistic delivery systems. In: Plant Journal. 1993 ; Vol. 3, No. 5. pp. 729-738.
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