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.
Key wordsChloroplast transformation Plastid transformation Nicotiana tabacum Tobacco Transplastomic plants Aminoglycoside 3″-adenylyltransferase aadA Homologous recombination Large subunit of ribulose bisphosphate carboxylase/oxygenase LS RuBisCO rbcL Genome editing
Supported in part by the Biotechnology and Biological Sciences Research Council (BB/I011552/1) and a University of Manchester (investment in success award). MEH was supported by a President’s Doctoral Scholar Award (University of Manchester), MFBH by the University Malaysia Terengganu and EMA by a Biotechnology and Biological Sciences Research Council PhD studentship.
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