Visual marker and Agrobacterium-delivered recombinase enable the manipulation of the plastid genome in greenhouse-grown tobacco plants

Tarinee Tungsuchat-Huang, Pal Maliga

Research output: Contribution to journalArticlepeer-review

11 Scopus citations

Abstract

Summary Successful manipulation of the plastid genome (ptDNA) has been carried out so far only in tissue-culture cells, a limitation that prevents plastid transformation being applied in major agronomic crops. Our objective is to develop a tissue-culture independent protocol that enables manipulation of plastid genomes directly in plants to yield genetically stable seed progeny. We report that in planta excision of a plastid aurea bar gene (bar αau) is detectable in greenhouse-grown plants by restoration of the green pigmentation in tobacco leaves. The P1 phage Cre or PhiC31 phage Int site-specific recombinase was delivered on the Agrobacterium T-DNA injected at the axillary bud site, resulting in the excision of the target-site flanked marker gene. Differentiation of new apical meristems was forced by decapitating the plants above the injection site. The new shoot apex that differentiated at the injection site contained bar αau-free plastids in 30-40% of the injected plants, of which 7% transmitted the bar αau-free plastids to the seed progeny. The success of obtaining seed with bar αau-free plastids depended on repeatedly forcing shoot development from axillary buds, a process that was guided by the size and position of green sectors in the leaves. The success of in planta plastid marker excision proved that manipulation of the plastid genomes is feasible within an intact plant. Extension of the protocol to in planta plastid transformation depends on the development of new protocols for the delivery of transforming DNA encoding visual markers.

Original languageEnglish (US)
Pages (from-to)717-725
Number of pages9
JournalPlant Journal
Volume70
Issue number4
DOIs
StatePublished - May 2012

ASJC Scopus subject areas

  • Genetics
  • Plant Science
  • Cell Biology

Keywords

  • Agrobacterium injection
  • Nicotiana tabacum
  • marker-free plastids
  • phage recombinases
  • technical advance
  • tobacco
  • visual plastid markers

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