Stable Plastid Transformation of Petunia for Studies in Basic Research.

Petunia hybrida is a commercial ornamental plant and is also an important model species for genetic analysis and transgenic research. Here we describe the steps required to isolate stable plastid transformants in P. hybrida using the commercial Pink Wave cultivar. Wave cultivars are popular spreading Petunias sold as ground cover and potted plants. Transgenes introduced into P. hybrida plastids exhibit stable expression over many generations. The development of plastid transformation in P. hybrida provides an enabling technology to bring the benefits of plastid engineering, including maternal inheritance and stable expression of performance-enhancing trait genes, to the important floriculture and horticulture industries.

Marker-Free Transplastomic Plants by Excision of Plastid Marker Genes Using Directly Repeated DNA Sequences.

Excision of marker genes using DNA direct repeats makes use of the efficient native homologous recombination pathway present in the plastids of algae and plants. The method is simple, efficient, and widely applicable to plants and green algae. Marker excision frequency is dependent on the length and number of directly repeated sequences. When two repeats are used a repeat size of greater than 600 bp promotes efficient excision of the marker gene. A wide variety of sequences can be used to make the direct repeats. Only a single round of transformation is required and there is no requirement to introduce site-specific recombinases by retransformation or sexual crosses. Selection is used to maintain the marker and ensure homoplasmy of transgenic plastid genomes (plastomes). Release of selection allows the accumulation of marker-free plastomes generated by marker excision, which is a spontaneous and unidirectional process. Cytoplasmic sorting allows the segregation of cells with marker-free transgenic plastids. The marker-free shoots resulting from direct repeat mediated excision of marker genes have been isolated by vegetative propagation of shoots in the T0 generation. Alternatively, accumulation of marker-free plastomes during growth, development and flowering of T0 plants allows for the collection of seeds that give rise to a high proportion of marker-free T1 seedlings. The procedure enables precise plastome engineering involving insertion of transgenes, point mutations and deletion of genes without the inclusion of any extraneous DNA. The simplicity and convenience of direct repeat excision facilitates its widespread use to isolate marker-free crops.

Rescue of Deletion Mutants to Isolate Plastid Transformants in Higher Plants.

Plastid transformation is an attractive alternative to nuclear transformation enabling manipulation of native plastid genes and the insertion of foreign genes into plastids for applications in agriculture and industrial biotechnology. Transformation is achieved using dominant positive selection markers that confer resistance to antibiotics. The very high copy number of plastid DNA means that a prolonged selection step is required to obtain a uniform population of transgenic plastid genomes. Repair of mutant plastid genes with the corresponding functional allele allows selection based on restoration of the wild type phenotype. The use of deletion rather than point mutants avoids spontaneous reversion back to wild type. Combining antibiotic resistance markers with native plastid genes speeds up the attainment of homoplasmy and allows early transfer of transplastomic lines to soil where antibiotic selection is replaced by selection for photoautotrophic growth. Here we describe our method using the wild type rbcL gene as a plastid transformation marker to restore pigmentation and photosynthesis to a pale green heterotrophic rbcL mutant.

Stable plastid transformation of petunia.

Petunia hybrida is a commercial ornamental plant and is also an important model species for genetic analysis and transgenic research. Here we describe the steps required to isolate stable plastid transformants in P. hybrida using the commercial Pink Wave cultivar. Wave cultivars are popular spreading Petunias sold as ground cover and potted plants. Transgenes introduced into P. hybrida plastids exhibit stable expression over many generations. The development of plastid transformation in P. hybrida provides an enabling technology to bring the benefits of plastid engineering, including maternal inheritance and stable expression of performance-enhancing trait genes, to the important floriculture and horticulture industries.

Excision of plastid marker genes using directly repeated DNA sequences.

Excision of marker genes using DNA direct repeats makes use of the predominant homologous recombination pathways present in the plastids of algae and plants. The method is simple, efficient, and widely applicable to plants and microalgae. Marker excision frequency is dependent on the length and number of directly repeated sequences. When two repeats are used a repeat size of greater than 600 bp promotes efficient excision of the marker gene. A wide variety of sequences can be used to make the direct repeats. Only a single round of transformation is required, and there is no requirement to introduce site-specific recombinases by retransformation or sexual crosses. Selection is used to maintain the marker and ensure homoplasmy of transgenic plastid genomes. Release of selection allows the accumulation of marker-free plastid genomes generated by marker excision, which is spontaneous, random, and a unidirectional process. Positive selection is provided by linking marker excision to restoration of the coding region of an herbicide resistance gene from two overlapping but incomplete coding regions. Cytoplasmic sorting allows the segregation of cells with marker-free transgenic plastids. The marker-free shoots resulting from direct repeat-mediated excision of marker genes have been isolated by vegetative propagation of shoots in the T0 generation. Alternatively, accumulation of marker-free plastid genomes during growth, development and flowering of T0 plants allows the collection of seeds that give rise to a high proportion of marker-free T1 seedlings. The simplicity and convenience of direct repeat excision facilitates its widespread use to isolate marker-free crops.