Restriction endonuclease analysis of plastid DNA from tomato, potato and some of their somatic hybrids

Summary The buoyant density and endonuclease restriction patterns of potato (Solanum tuberosum L.) and tomato (Lycopersicon esculentum) ptDNA were examined and compared with those of their somatic hybrids. The plastids from these plants, both of which belong to the family of Solanaceae, contain a single DNA species whose density of 1.697 gcm^-3 and size of approximately 156 kbp are similar to those of ptDNA from other higher plants. The Sal I restriction patterns were indistinguishable; however, those obtained with Kpn I, Pst I, and Eco RI disclosed that each species possesses a unique ptDNA. These observations suggest a relatively recent divergence of both species. Of the twelve hybrid lines screened, eight contained exclusively potato ptDNa and four contained only tomato ptDNA at a 0.1–3% level of detection. Rearrangements of modifications of DNA were not detected. The plastid identities of three hybrid lines that had previously been analyzed by isoelectric focusing of RuBPcase subunits (Melchers et al. 1978) agreed with those determined by restriction endonuclease analysis.Abbreviations used in the text ptDNA plastid DNA, chloroplast DNA - cDNA copy DNA - RuBPcase ribulose bisphosphate carboxylase/oxygenase - LSU large subunit of RuBPcase - kbp kilobase pairs - SDS sodium dodecyl sulfate - SSC standard saline citrate - IEF isoelectric focusing     

Plastid DNA inheritance and plastome-genome incompatibility in interspecific hybrids of Zantedeschia (Araceae)

Plastid DNA (ptDNA) probes were used in RFLP analysis to determine ptDNA inheritance in interspecific hybrids in Zantedeschia. Biparental and maternal ptDNA inheritance was found in albino hybrids between the evergreen species Z. aethiopica and several winter-dormant species. From two albino hybrids, different types of ptDNA were detected in shoots derived from different parts of an embryo. This result indicates that plastids were sorted out during embryo development. Only maternal ptDNA was detected in the hybrids of Z. aethiopica × Z. odorata (a summer-dormant species) but paternal, biparental, and maternal ptDNA were found in the hybrids of the reciprocal cross. Z. odorata × Z. aethiopica. By correlating these ptDNA inheritance patterns with the leaf colour (albino, pale-green, and green) of the hybrids, it is suggested that the Z. odorata plastome is incompatible with the Z. aethiopica genome. The Z. aethiopica plastome is partially compatible with the Z. odorata genome but the development of Z. aethiopica plastids appears to be blocked by the presence of the Z. odorata plastids.     

The model plant Medicago truncatula exhibits biparental plastid inheritance

The plastid, which originated from the endosymbiosis of a cyanobacterium, contains its own plastid DNA (ptDNA) that exhibits a unique mode of inheritance. Approximately 80% of angiosperms show maternal inheritance, whereas the remainder exhibit biparental inheritance of ptDNA. Here we studied ptDNA inheritance in the model legume, Medicago truncatula. Cytological analysis of mature pollen with DNA-specific fluorescent dyes suggested that M. truncatula is one of the few model plants potentially showing biparental inheritance of ptDNA. We further examined pollen by electron microscopy and revealed that the generative cell (a mother of sperm cells) indeed has many DNA-containing plastids. To confirm biparental inheritance genetically, we crossed two ecotypes (Jemalong A17 and A20), and the transmission mode of ptDNA was investigated by a PCR-assisted polymorphism. Consistent with the cytological observations, the majority of F(1) plants possessed ptDNAs from both parents. Interestingly, cotyledons of F(1) plants tended to retain a biparental ptDNA population, while later emergent leaves tended to be uniparental with either one of the parental plastid genotypes. Biparental transmission was obvious in the F(2) population, in which all plants showed homoplasmy with either a paternal or a maternal plastid genotype. Collectively, these data demonstrated that M. truncatula is biparental for ptDNA transmission and thus can be an excellent model to study plastid genetics in angiosperms.     

Plastid genomes in a regenerating tobacco shoot derive from a small number of copies selected through a stochastic process

The plastid genome (ptDNA) of higher plants is highly polyploid, and the 1000-10 000 copies are compartmentalized with up to approximately 100 plastids per cell. The problem we address here is whether or not a newly arising genome can be established in a developing tobacco shoot, and be transmitted to the seed progeny. We tested this by generating two unequal ptDNA populations in a cultured tobacco cell. The parental tobacco plants in this study have an aurea (yellowish-golden) leaf color caused by the presence of a bar(au) gene in the ptDNA. In addition, the ptDNA carries an aadA gene flanked with the phiC31 phage site-specific recombinase (Int) attP/attB target sites. The genetically distinct ptDNA copies were obtained by Int, which either excised only the aadA marker gene (i.e. did not affect the aurea phenotype) or triggered the deletion of both the aadA and bar(au) transgenes, and thereby restored the green color. The ptDNA determining green plastids represented only a small fraction of the population and was not seen in a transient excision assay, and yet three out of the 53 regenerated shoots carried green plastids in all developmental layers. The remaining 49 Int-expressing plants had either exclusively aurea (24) or variegated (25) leaves with aurea and green sectors. The formation of homoplastomic green shoots with the minor green ptDNA in all developmental layers suggests that the ptDNA population in a regenerating shoot apical meristem derives from a small number of copies selected through a stochastic process.     

Homologies between nuclear and plastid DNA in spinach

Summary Homologies between spinach nuclear (n) DNA and Chloroplast (pt) DNA, have been detected with a clone bank of spinach ptDNA as hybridization probes to restriction fragments of nDNA prepared from purified root nuclei. Every cloned fragment of ptDNA showed homologies to discrete restriction fragments of nDNA, different from those of ptDNA, indicating integration of these homologies into nDNA. While most ptDNA clones were relatively large and probably contained several genes, sequence homologies were also found to the cloned plastid gene for RuBP carboxylase and the subunit of ptATPase. Many of the homologies in nDNA occur in regions of the genome that are highly methylated and are not digested by the methylation sensitive restriction endonucleases HpaII and MspI. In contrast these enzymes cleave ptDNA into small fragments which allows the nDNA homologies to be distinguished in total root DNA. The sequence homologies observed were not due to contaminating non nuclear sequences as shown by hybridization to mitochondrial (mt) and bacterial DNAs. The total amount of homology to ptDNA in nDNA is equivalent to about five copies of the plastome per haploid nuclear genome. The homologies generally appear to be in individual segments of less than 2 kbp in length, integrated into several different places in the genome.On sabbatical leave from Department of Botany, University College, Dublin, Ireland     

三种愈伤组织中质体的DNA含量变化及其与水稻花粉白苗形成的关系

以质体为单位的愈伤组织ptDNA的荧光强度变化显示出了特有的规律性,结合光培养花粉白苗叶质体结构的电镜观察及叶绿素等色素的测定等,这种规律性似提示花粉白苗的ptDNA由于发生了相继缺失而显示出高度异质性,花粉白苗的形成则在于其占优势ptDNA的严重缺失,花粉白苗ptDNA的这种相继缺失不是随机的,似反映了ptDNA顺序组织上的特点,故ptDNA发生缺失的潜在可能性具有普遍意义,它与父系细胞质体类核的消失存在着并行关系,花粉白苗的占优势ptDNA指导的植株的性状,而次要ptDNA亦在较小程度上显示出影响。     

Structural changes in the plastid DNA of rice (Oryza sativa L.) during tissue culture

To investigate the rearrangement of the plastid genome during tissue culture, DNA from rice callus lines, which had been derived individually from single protoplasts isolated from seed or pollen callus (protoclones), was analyzed by Southern hybridization with rice chloroplast DNA (ctDNA) clones as probes. Among 44 long-term cultured protoclones, maintained for 4, 8 or 11 years, 28 contained plastid DNA (ptDNA) from which portions had been deleted. The ptDNA of all protoclones that had been maintained for 11 years had a deletion that covered a large region of the plastid genome. The deletions could be classified into 15 types from their respective sizes and positions. By contrast, no deletions were found in the ptDNA of 38 protoclones that had been maintained for only 1 month. These results indicate that long-term culture causes deletions in the plastid genome. Detailed hybridization experiments revealed that plastid genomes with deletions in several protoclones were organized as head-to-head or tail-to-tail structures. Furthermore, ptDNAs retained during long-term culture all had a common terminus at one end, where extensive rearrangement is known to have occurred during the speciation of rice and tobacco. Morphological analysis revealed the accumulation of starch granules in plastids and amyloplasts in protoclones in which the plastid genome had undergone deletion. Our observations indicated that novel structural changes in the plastid genome and morphological changes in the plastid had occurred in rice cells during long-term tissue culture. Moreover, the morphological changes in plastids were associated with deletions in the plastid genome.     

Construction of marker-free transplastomic plants

Because of its prokaryotic-type gene expression machinery, maternal inheritance and the opportunity to express proteins at a high level, the plastid genome (plastome or ptDNA) is an increasingly popular target for engineering. The ptDNA is present as up to 10,000 copies per cell, making selection for marker genes essential to obtain plants with uniformly transformed ptDNA. However, the marker gene is no longer desirable when homoplastomic plants are obtained. Marker-free transplastomic plants can now be obtained with four recently developed protocols: homology-based excision via directly repeated sequences, excision by phage site-specific recombinanses, transient cointegration of the marker gene, and the cotransformation-segregation approach. Marker excision technology will benefit applications in agriculture and in molecular farming.     

The pattern of amyloplast DNA accumulation during wheat endosperm development

The accumulation of amyloplast DNA during endosperm development was studied in two cultivars of spring wheat, Triticum aestivum L. Chinese Spring (CS) and Spica, small and relatively larger-grained cultivars, respectively. Endosperms were isolated between 9 and 45 days post anthesis (dpa) and the amyloplast DNA content of endosperm nucleic-acid extracts was measured by quantitative hybridisation with a homologous chloroplast-DNA probe. The endosperm cells of CS and Spica accumulated amyloplast DNA during development in a similar way. In both cultivars there was a large increase in the amount of plastid DNA (ptDNA) per endosperm between 9 and about 15 dpa, after which there was no further increase. Because nuclear DNA continued to accumulate until 24 dpa, the percentage contribution of amyloplast DNA to total DNA fluctuated in both cultivars during development, reaching maxima at 12 dpa of about 1.00% and 0.85%, and dropping to apparently constant levels of 0.60% and 0.52% in CS and Spica, respectively, by 24 dpa. In both cultivars, the average number of ptDNA copies per amyloplast was calculated to increase from about 10 copies at 9 dpa to about 50 copies in the mature amyloplasts at 31 dpa. However, the heavier endosperms of Spica contain more cells than those of CS and the varieties therefore differed in the amount of ptDNA that accumulated per endosperm: Spica endosperms accumulated 110 ng of ptDNA by 15 dpa, compared with only 85 ng in CS. The apparent accumulation of ptDNA copies in wheat amyloplasts during endosperm development contrasts with the decline in chloroplast-DNA copies in wheat chloroplasts during leaf development.Abbreviations CS Chinese Spring - ctDNA chloroplast DNA - dpa days post anthesis - kbp 10³ base pairs - nDNA nuclear DNA - ptDNA plastid DNA - mtDNA mitochondrial DNA     

Unparalleled GC content in the plastid DNA of Selaginella

One of the more conspicuous features of plastid DNA (ptDNA) is its low guanine and cytosine (GC) content. As of February 2009, all completely-sequenced plastid genomes have a GC content below 43% except for the ptDNA of the lycophyte Selaginella uncinata, which is 55% GC. The forces driving the S. uncinata ptDNA towards G and C are undetermined, and it is unknown if other Selaginella species have GC-biased plastid genomes. This study presents the complete ptDNA sequence of Selaginella moellendorffii and compares it with the previously reported S. uncinata plastid genome. Partial ptDNA sequences from 103 different Selaginella species are also described as well as a significant proportion of the S. moellendorffii mitochondrial genome. Moreover, S. moellendorffii express sequence tags are data-mined to estimate levels of plastid and mitochondrial RNA editing. Overall, these data are used to show that: (1) there is a genus-wide GC bias in Selaginella ptDNA, which is most pronounced in South American articulate species; (2) within the Lycopsida class (and among plants in general), GC-biased ptDNA is restricted to the Selaginella genus; (3) the cause of this GC bias is arguably a combination of reduced AT-mutation pressure relative to other plastid genomes and a large number of C-to-U RNA editing sites; and (4) the mitochondrial DNA (mtDNA) of S. moellendorffii is also GC biased (even more so than the ptDNA) and is arguably the most GC-rich organelle genome observed to date—the high GC content of the mtDNA also appears to be influenced by RNA editing. Ultimately, these findings provide convincing support for the earlier proposed theory that the GC content of land-plant organelle DNA is positively correlated and directly connected to levels of organelle RNA editing.     

Plastid‐targeted forms of restriction endonucleases enhance the plastid genome rearrangement rate and trigger the reorganization of its genomic architecture

Plant cells have acquired chloroplasts (plastids) with a unique genome (ptDNA), which developed during the evolution of endosymbiosis. The gene content and genome structure of ptDNAs in land plants are considerably stable, although those of algal ptDNAs are highly varied. Plant cells seem, therefore, to be intolerant of any structural or organizational changes in the ptDNA. Genome rearrangement functions as a driver of genomic evolutionary divergence. Here, we aimed to create various types of rearrangements in the ptDNA of Arabidopsis genomes using plastid‐targeted forms of restriction endonucleases (pREs). Arabidopsis plants expressing each of the three specific pREs, i.e., pTaqI, pHinP1I, and pMseI, were generated; they showed the leaf variegation phenotypes associated with impaired chloroplast development. We confirmed that these pREs caused double‐stranded breaks (DSB) at their recognition sites in ptDNAs. Genome‐wide analysis of ptDNAs revealed that the transgenic lines exhibited a large number of rearrangements such as inversions and deletions/duplications, which were dominantly repaired by microhomology‐mediated recombination and microhomology‐mediated end‐joining, and less by non‐homologous end‐joining. Notably, pHinP1I, which recognized a small number of sites in ptDNA, induced drastic structural changes, including regional copy number variations throughout ptDNAs. In contrast, the transient expression of either pTaqI or pMseI, whose recognition site numbers were relatively larger, resulted in small‐scale changes at the whole genome level. These results indicated that DSB frequencies and their distribution are major determinants in shaping ptDNAs.     

Functional loss of all ndh genes in an otherwise relatively unaltered plastid genome of the holoparasitic flowering plant Cuscuta reflexa

We have cloned and sequenced an area of about 9.0 kb of the plastid DNA (ptDNA) from the holoparasitic flowering plant Cuscuta reflexa to investigate the evolutionary response of plastid genes to a reduced selective pressure. The region contains genes for the 16S rRNA, a subunit of a plastid NAD(P)H dehydrogenase (ndhB), three transfer RNAs (trnA, trnI, trnV) as well as the gene coding for the ribosomal protein S7 (rps7). While the other genes are strongly conserved in C. reflexa, the ndhB gene is a pseudogene due to many frameshift mutations. In addition we used heterologous gene probes to identify the other ndh genes encoded by the plastid genome in higher plants. No hybridization signals could be obtained, suggesting that these genes are either lost or strongly altered in the ptDNA of C. reflexa. Together with evidence of deleted genes in the ptDNA of C. reflexa, the plastid genome can be grouped into four classes reflecting a different evolutionary rate in each case. The phylogenetic position of Cuscuta and the significance of ndh genes in the plastid genome of higher plants are discussed.     

Purification of High-quality Plastid DNA from the Brown Alga Laminaria japonica Sporophytes

Extracting DNA from a variety of algae is rather difficult because of high levels of polysaccharides, tannins, and phenolics as these interfere with DNA isolation and downstream applications. High-quality plastid DNA (ptDNA) purification is particularly difficult because of its small proportion in total genomic DNA. This report describes an improved protocol for ptDNA purification that efficiently produces high-quality ptDNA from sporophytes of Laminaria japonica and several other algae. This improved protocol simplifies procedures for ptDNA purification and improves yield to 150–200 μg of ptDNA per 100 g of frozen algal tissue. Polymerase chain reaction (PCR) amplification of conserved sequences has been used to verify purity of the ptDNA product.     

Streptomycin and lincomycin resistances are selective plastid markers in cultured Nicotiana cells

Summary Resistance to streptomycin and lincomycin in plant cell culture is used as a color marker: resistant cells are green whereas sensitive cells are white on the selective medium. Streptomycin and lincomycin at appropriate concentrations do not kill sensitive Nicotiana cells. The selective value of plastid ribosomal DNA mutations, conferring resistance to streptomycin and lincomycin, was investigated by growing heteroplastidic cells on a selective medium. The heteroplastidic cells were obtained by protoplast fusion, and contained a mixed population of streptomycin resistant plastids from the N. tabacum line Nt-SR1-Kan2, and lincomycin resistant plastids from the N. plumbaginifolia line Np-LR400-Hyg1. Clones derived from protoplast fusion were selected by kanamycin and hygromycin resistance, transgenic nuclear markers. Somatic hybrids were then grown on a selective streptomycin or lincomycin medium, or in the absence of either drug to a 50 to 100 mg size callus. Southern analysis of a polymorphic region of plastid DNA (ptDNA) revealed that somatic hybrids grown on streptomycin contained almost exclusively ptDNA from the streptomycin resistant parent, somatic hybrids grown on lincomycin contained almost exclusively ptDNA from the lincomycin resistant parent whereas somatic hybrids grown in the absence of either drug contained mixed parental plastids. Sensitive ptDNA was below detection level in most clones on selective medium, but could be recovered upon subsequent culture in the presence of the appropriate drug. The drugs streptomycin and lincomycin provide a powerful selection pressure that should facilitate recovery of plastid transformants.     

一种花色突变雄性不育油菜的发现

在甘蓝型油菜杂交种C022(其母本为由3对基因控制育性的隐性细胞核雄性不育系9012A)不育株开放受粉的后代中,发现一种稀有的黄白双色嵌合花瓣的甘蓝型油菜突变体991S。其具有3个形态特征:(1)4片花瓣每片中央均为条带状黄色色斑,而两侧为白色,为嵌合双色花瓣;(2)4片花萼也可发生中间条带状白化;(3)目前只在不同群体的雄性不育株中出现,与同源的黄色花不育株形态相似,植株纤细矮小,花器也较小,花瓣较为平整,雌蕊弯曲,雄蕊退化,花药干缩。通过对其材料来源及后代花色表型分析,初步认为黄白双色性状由可局部表达的隐性白化基因控制。     

Plastid-DNA levels in the different tissues of potato

The plastid-(pt) DNA levels in the different tissues of potato (Solanum tuberosum L.), including tubers of differing ages, have been studied. The DNA could be detected as a single nucleoid in amyloplasts of cells from young potato tubers by fluorescence microscopy, following staining of glutaraldehyde-fixed tissue with 4,6-diamidino-2-phenyl indole (DAPI). The renaturation kinetics of spinach ptDNA in the presence of total DNA from potato tissues and the fragments generated by restriction-enzyme digestion of potato-tuber DNA and chloroplast DNA indicated that the ptDNA of potato-tuber amyloplasts and of potato-leaf chloroplasts is essentially the same. Expressed as a percentage of the total DNA the level of ptDNA (5.2%) found in tubers, while less than that found in leaves (7.6%) was more than that found in petioles (3.4%), stems (3.0%) and roots (1.0%). There was a high level of both nuclear and plastid ploidy in mature potato-tuber cells and, on average, nuclei contained 32 pg of DNA (equivalent to 14C) and the 40 amyloplasts per cell contained DNA equivalent to 7800 copies of ptDNA, or 195 copies per amyloplast.Abbreviations DAPI 4,6-diamidino-2-phenyl-indole - LSU large sub-unit of ribulose-1,5-bisphosphate carboxylase - mtDNA mitochondrial DNA - ptDNA chloroplast or plastid DNA     

Species delimitation and biogeography of two fir species (Abies) in central China: cytoplasmic DNA variation

It remains unclear how speciation history might contribute to species-specific variation and affect species delimitation. We examined concordance between cytoplasmic genetic variation and morphological taxonomy in two fir species, Abies chensiensis and A. fargesii, with overlapping distributions in central China. Range-wide genetic variation was investigated using mitochondrial (mt) and plastid (pt) DNA sequences, which contrast in their rates of gene flow. Four mtDNA haplotypes were recovered and showed no obvious species' bias in terms of relative frequency. In contrast, a high level of ptDNA variation was recorded in both species with 3 common ptDNA haplotypes shared between them and 21 rare ptDNA haplotypes specific to one or other species. We argue that the lack of concordance between morphological and molecular variation between the two fir species most likely reflects extensive ancestral polymorphism sharing for both forms of cytoplasmic DNA variation. It is feasible that a relatively fast mutation rate for ptDNA contributed to the production of many species-specific ptDNA haplotypes, which remained rare due to insufficient time passing for their spread and fixation in either species, despite high levels of intraspecific ptDNA gene flow. Our phylogeographic analyses further suggest that polymorphisms in both organelle genomes most likely originated during and following glacial intervals preceding the last glacial maximum, when species distributions became fragmented into several refugia and then expanded in range across central China.     

ATP binding and hydrolysis-driven rate-determining events in the RFC-catalyzed PCNA clamp loading reaction

The multi-subunit replication factor C (RFC) complex loads circular proliferating cell nuclear antigen (PCNA) clamps onto DNA where they serve as mobile tethers for polymerases and coordinate the functions of many other DNA metabolic proteins. The clamp loading reaction is complex, involving multiple components (RFC, PCNA, DNA, and ATP) and events (minimally: PCNA opening/closing, DNA binding/release, and ATP binding/hydrolysis) that yield a topologically linked clamp·DNA product in less than a second. Here, we report pre-steady-state measurements of several steps in the reaction catalyzed by Saccharomyces cerevisiae RFC and present a comprehensive kinetic model based on global analysis of the data. Highlights of the reaction mechanism are that ATP binding to RFC initiates slow activation of the clamp loader, enabling it to open PCNA (at ~2 s(-1)) and bind primer-template DNA (ptDNA). Rapid binding of ptDNA leads to formation of the RFC·ATP·PCNA(open)·ptDNA complex, which catalyzes a burst of ATP hydrolysis. Another slow step in the reaction follows ATP hydrolysis and is associated with PCNA closure around ptDNA (8 s(-1)). Dissociation of PCNA·ptDNA from RFC leads to catalytic turnover. We propose that these early and late rate-determining events are intramolecular conformational changes in RFC and PCNA that control clamp opening and closure, and that ATP binding and hydrolysis switch RFC between conformations with high and low affinities, respectively, for open PCNA and ptDNA, and thus bookend the clamp loading reaction.     

Changes in Chloroplast DNA Levels during Growth of Spinach Leaves

In young spinach leaves, 1–4 mm long, 7–10% of thetotal DNA of the leaf was chloroplast (pt) DNA. Growth in theseleaves was mainly by cell division with plastid division keepingpace with cell division and maintaining about 10 plastids percell. About 1% of the leaf cells were formed in 4.0 mm leaves.Both cell division and cell expansion contribute to the nextstage of leaf growth, which was quantitatively the major periodof new cell formation, nuclear DNA synthesis and ptDNA synthesis.Relative to the nuclear DNA level ptDNA levels rose to 21% ofthe total DNA and chloroplast.plastome copy numbers from 1500to 5000 per cell while chloroplast numbers rose from 10 to 30per cell. In the final period of leaf growth, cell expansionwas the main determinant of growth and chloroplast number percell rose to 180. In contrast to young leaves, newly emergedcotyledons contained 20% of their DNA as ptDNA and, during cellexpansion, cell number per cotyledon doubled. On average, thecells became octoploid, and chloroplast numbers and plastomecopy numbers rose to 500 and 22 000 per cell respectively. Similarlevels of nuclear ploidy, chloroplast number and plastome copynumber were induced in the first leaf pair of spinach followingdecapitation. When senescence was induced in mature leaves byshading, no loss of nuclear or ptDNA occurred. Following theonset of leaf yellowing and a form of senescence induced bynitrogen deficiency in leaves which had not fully expanded,there was preferential loss of ptDNA which fell from 8200 to3700 plastome copies per cell over an 11 d period. Key words: Spinach, Chloroplast, DNA, Ploidy     

Chloroplast transformation in plants: polyethylene glycol (PEG) treatment of protoplasts is an alternative to biolistic delivery systems

Nicotiana plumbaginifolia protoplasts were directly transformed by PEG treatment with a cloned 16S rRNA gene isolated from a double antibiotic-resistant Nicotiana tabacum plastid mutant. Putative plastid transformants were selected in cell culture by their spectinomycin resistance and identified by their unselected streptomycin resistance. Alternatively, cell lines were selected in the presence of both antibiotics. The cell line (and its regenerated plants) selected solely for spectinomycin resistance demonstrated an extensive segregation of streptomycin resistance in subsequent tests, while the double-selected line showed stable resistance for both antibiotics. The resistance markers were inherited maternally. In the putative plastid transformants the origin of the resistance mutations was identified by the absence of an Aat ll site, missing in the donor N. tabacum plastid gene (spectinomycin resistance site) but present in that of wild-type N. plumbaginifolia , and a sequence analysis of the particular nucleotide changes in both resistance sites. Restriction enzyme analysis of total plastid DNA (ptDNA), and the recloning and full sequencing of the fragment introduced, investigated in one of the plastid transformants, showed no DNA rearrangements accompanied with the integration process. Sequence analysis indicated a targeted, homologous integration of the DNA fragment introduced but an unexpectedly complete homology of the parental ptDNA sequences in this region prevented the location of borders. Although the frequency of plastid transformant colonies (2 × 10⁻⁵) should still be improved, this method for stable chloroplast DNA transformation is comparable with or more efficient than the particle bombardment techniques.