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Maliga, P, Svab Z.  2011.  Engineering the plastid genome of Nicotiana sylvestris, a diploid model species for plastid genetics. Methods in Molecular Biology. 701:37-50. AbstractWebsite
The plastids of higher plants have their own approximately 120-160-kb genome that is present in 1,000-10,000 copies per cell. Engineering of the plastid genome (ptDNA) is based on homologous recombination between the plastid genome and cloned ptDNA sequences in the vector. A uniform population of engineered ptDNA is obtained by selection for marker genes encoded in the vectors. Manipulations of ptDNA include (1) insertion of transgenes in intergenic regions; (2) posttransformation excision of marker genes to obtain marker-free plants; (3) gene knockouts and gene knockdowns, and (4) cotransformation with multiple plasmids to introduce nonselected genes without physical linkage to marker genes. Most experiments on plastome engineering have been carried out in the allotetraploid Nicotiana tabacum. We report here for the first time plastid transformation in Nicotiana sylvestris, a diploid ornamental species. We demonstrate that the protocols and vectors developed for plastid transformation in N. tabacum are directly applicable to N. sylvestris with the advantage that the N. sylvestris transplastomic lines are suitable for mutant screens.
Thyssen, G, Svab Z, Maliga P.  2012.  Exceptional inheritance of plastids via pollen in Nicotiana sylvestris with no detectable paternal mitochondrial DNA in the progeny. Plant J.. 72:84-8. AbstractWebsite
Plastids and mitochondria, the DNA-containing cytoplasmic organelles, are maternally inherited in the majority of angiosperm species. Even in plants with strict maternal inheritance, exceptional paternal transmission of plastids has been observed. Our objective was to detect rare leakage of plastids via pollen in Nicotiana sylvestris and to determine if pollen transmission of plastids results in co-transmission of paternal mitochondria. As father plants, we used N. sylvestris plants with transgenic, selectable plastids and wild-type mitochondria. As mother plants, we used N. sylvestris plants with Nicotiana undulata cytoplasm, including the CMS-92 mitochondria that cause cytoplasmic male sterility (CMS) by homeotic transformation of the stamens. We report here exceptional paternal plastid DNA in approximately 0.002% of N. sylvestris seedlings. However, we did not detect paternal mitochondrial DNA in any of the six plastid-transmission lines, suggesting independent transmission of the cytoplasmic organelles via pollen. When we used fertile N. sylvestris as mothers, we obtained eight fertile plastid transmission lines, which did not transmit their plastids via pollen at higher frequencies than their fathers. We discuss the implications for transgene containment and plant evolutionary histories inferred from cytoplasmic phylogenies.
Azhagiri, AK, Maliga P.  2007.  Exceptional paternal inheritance of plastids in Arabidopsis suggests that low-frequency leakage of plastids via pollen may be universal in plants. Plant J.. 52:817-23. AbstractWebsite
Plastid DNA is absent in pollen or sperm cells of Arabidopsis thaliana. Accordingly, plastids and mitochondria, in a standard genetic cross, are transmitted to the seed progeny by the maternal parent only. Our objective was to test whether paternal plastids are transmitted by pollen as an exception. The maternal parent in our cross was a nuclear male sterile (ms1-1/ms1-1), spectinomycin-sensitive Ler plant. It was fertilized with pollen of a male fertile RLD-Spc1 plant carrying a plastid-encoded spectinomycin resistance mutation. Seedlings with paternal plastids were selected by spectinomycin resistance encoded in the paternal plastid DNA. Our data, in general, support maternal inheritance of plastids in A. thaliana. However, we report that paternal plastids are transmitted to the seed progeny in Arabidopsis at a low (3.9 x 10(-5)) frequency. This observation extends previous reports in Antirrhinum majus, Epilobium hirsutum, Nicotiana tabacum, Petunia hybrida, and the cereal crop Setaria italica to a cruciferous species suggesting that low-frequency paternal leakage of plastids via pollen may be universal in plants previously thought to exhibit strict maternal plastid inheritance. The genetic tools employed here will facilitate testing the effect of Arabidopsis nuclear mutations on plastid inheritance and allow for the design of mutant screens to identify nuclear genes controlling plastid inheritance.
Svab, Z, Maliga P.  2007.  Exceptional transmission of plastids and mitochondria from the transplastomic pollen parent and its impact on transgene containment. Proc. Natl. Acad. Sci. U.S.A.. 104:7003-8. AbstractWebsite
Plastids in Nicotiana tabacum are normally transmitted to the progeny by the maternal parent only. However, low-frequency paternal plastid transmission has been reported in crosses involving parents with an alien cytoplasm. Our objective was to determine whether paternal plastids are transmitted in crosses between parents with the normal cytoplasm. The transplastomic father lines carried a spectinomycin resistance (aadA) transgene incorporated in the plastid genome. The mother lines in the crosses were either (i) alloplasmic, with the Nicotiana undulata cytoplasm that confers cytoplasmic male sterility (CMS92) or (ii) normal, with the fertile N. tabacum cytoplasm. Here we report that plastids from the transplastomic father were transmitted in both cases at low (10(-4)-10(-5)) frequencies; therefore, rare paternal pollen transmission is not simply due to breakdown of normal controls caused by the alien cytoplasm. Furthermore, we have found that the entire plastid genome was transmitted by pollen rather than small plastid genome (ptDNA) fragments. Interestingly, the plants, which inherited paternal plastids, also carried paternal mitochondrial DNA, indicating cotransmission of plastids and mitochondria in the same pollen. The detection of rare paternal plastid transmission described here was facilitated by direct selection for the transplastomic spectinomycin resistance marker in tissue culture; therefore, recovery of rare paternal plastids in the germline is less likely to occur under field conditions.