Stable Plastid Transformation in PEG-treated Protoplasts of Nicotiana tabacum

Golds, T. J.; Maliga, Pál; Koop, Hans‐Ulrich

doi:10.1038/nbt0193-95

Cited by 118 publications

(51 citation statements)

References 16 publications

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“…The frequent homologous recombinations during plastid DNA transformation (Kavanagh et al 1999) may also decrease the co-integration frequency of the resistance mutation and the transgene. Nevertheless, it has been used in several instances with different Nicotiana genotypes (Golds et al 1993;O'Neill et al 1993;Kavanagh et al 1999;Horváth et al 2000;Rumeau et al 2004;Buhot et al 2006) and also with tomato (Nugent et al 2005), exploiting either homologous or homeologous recombination for transgene integration.…”

Section: Discussionmentioning

confidence: 99%

Transplastomic tobacco plants expressing a fatty acid desaturase gene exhibit altered fatty acid profiles and improved cold tolerance

Craig¹,

Lenzi²,

Scotti³

et al. 2008

Transgenic Res

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The possibility of altering the unsaturation level of fatty acids in plant lipids by genetic transformation has implications for the stress tolerance of higher plants as well as for their nutritional value and industrial utilisation. While the integration and expression of transgenes in the plastome has several potential advantages over nuclear transformation, very few attempts have been made to manipulate fatty acid biosynthesis using plastid transformation. We produced transplastomic tobacco plants that express a D 9 desaturase gene from either the wild potato species Solanum commersonii or the cyanobacterium Anacystis nidulans, using PEG-mediated DNA uptake by protoplasts. Incorporation of chloroplast antibioticinsensitive point mutations in the transforming DNA was used to select transformants. The presence of the transcript and the D 9 desaturase protein in transplastomic plants was confirmed by northern and western blot analyses. In comparison with control plants, transplastomic plants showed altered fatty acid profiles and an increase in their unsaturation level both in leaves and seeds. The two transgenes produced comparable results. The results obtained demonstrate the feasibility of using plastid transformation to engineer lipid metabolic pathways in both vegetative and reproductive tissues and suggest an increase of cold tolerance in transplastomic plants showing altered leaf fatty acid profiles. This is the first example of transplastomic plants expressing an agronomically relevant gene produced with the ''binding-type'' vectors, which do not contain a heterologous marker gene. In fact, the transplastomic plants expressing the S. commersonii gene contain only plant-derived sequences, a clear attraction from a public acceptability perspective.

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Section: Discussionmentioning

confidence: 99%

Transplastomic tobacco plants expressing a fatty acid desaturase gene exhibit altered fatty acid profiles and improved cold tolerance

Craig¹,

Lenzi²,

Scotti³

et al. 2008

Transgenic Res

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“…All of these investigators used biolistics to deliver DNA into chloroplasts. Direct DNA uptake into protoplasts is an alternative means of plastid transformation for Nicotiana species (Golds et al 1993;O'Neill et al 1993;Kavanagh et al 1999) but has not been reported for any other genera.…”

Section: Introductionmentioning

confidence: 99%

“…Binding-type markers, where cloned plastid genes contain point mutations conferring antibiotic insensitivity, offers an alternative means of selection, at least in Nicotiana, without the need for foreign antibiotic resistance genes (e.g. Svab et al 1990;Golds et al 1993;O'Neill et al 1993;Kavanagh et al 1999). However, when bindingtype markers are used, the plastid transformation frequency of tobacco is much lower in both biolistic transformation and direct DNA uptake experiments than when dominant selectable markers such as aadA (Koop et al 1996) are used.…”

Section: Introductionmentioning

confidence: 99%

Plastid transformants of tomato selected using mutations affecting ribosome structure

Nugent

Have²,

Gulik³

et al. 2005

Plant Cell Rep

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Tomato plastid transformants were obtained using two vectors containing cloned plastid DNA of either Nicotiana tabacum or Solanum nigrum and including point mutations conferring resistance to spectinomycin and streptomycin. Transformants were recovered after PEGmediated direct DNA uptake into protoplasts, followed by selection on spectinomycin-containing medium. Sixteen lines contained the point mutation, as confirmed by mapping restriction enzyme sites. One line obtained with each vector was analysed in more detail, in comparison with a spontaneous spectinomycin-resistant mutant. Integration of the cloned Solanum or Nicotiana plastid DNA, by multiple recombination events, into the tomato plastome was confirmed by sequence analysis of the targeted region of plastid DNA in the inverted repeat region. Maternal inheritance of spectinomycin and streptomycin resistances or sensitivity in seedlings also confirmed the transplastomic status of the two transformants. The results demonstrate the efficacy in tomato of a selection strategy which avoids the integration of a dominant bacterial antibiotic resistance gene.Communicated by H. Lörz

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“…The process of transgene integration into the plastome following delivery into the chloroplast by means of particle bombardment (Svab et al 1990) or PEG-mediated transformation (Golds et al 1993) is mediated by homologous recombination. This mechanism has enabled the introduction of point mutations in a site-specific manner (Przibilla et al 1991).…”

Section: Cr Boehm Et Almentioning

confidence: 99%

Synthetic Botany

Boehm

Pollak

Purswani³

et al. 2017

Cold Spring Harb Perspect Biol

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Plants are attractive platforms for synthetic biology and metabolic engineering. Plants' modular and plastic body plans, capacity for photosynthesis, extensive secondary metabolism, and agronomic systems for large-scale production make them ideal targets for genetic reprogramming. However, efforts in this area have been constrained by slow growth, long life cycles, the requirement for specialized facilities, a paucity of efficient tools for genetic manipulation, and the complexity of multicellularity. There is a need for better experimental and theoretical frameworks to understand the way genetic networks, cellular populations, and tissue-wide physical processes interact at different scales. We highlight new approaches to the DNA-based manipulation of plants and the use of advanced quantitative imaging techniques in simple plant models such as Marchantia polymorpha. These offer the prospects of improved understanding of plant dynamics and new approaches to rational engineering of plant traits.

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Stable Plastid Transformation in PEG-treated Protoplasts of Nicotiana tabacum

Cited by 118 publications

References 16 publications

Transplastomic tobacco plants expressing a fatty acid desaturase gene exhibit altered fatty acid profiles and improved cold tolerance

Transplastomic tobacco plants expressing a fatty acid desaturase gene exhibit altered fatty acid profiles and improved cold tolerance

Plastid transformants of tomato selected using mutations affecting ribosome structure

Synthetic Botany

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