Plastome-genome Incompatibility Research Articles

Highly accelerated rates of genomic rearrangements and nucleotide substitutions in plastid genomes of Passiflora subgenus Decaloba

Plastid genomes (plastomes) of photosynthetic angiosperms are for the most part highly conserved in their organization, mode of inheritance and rates of nucleotide substitution. A small number of distantly related lineages share a syndrome of features that deviate from this general pattern, including extensive genomic rearrangements, accelerated rates of nucleotide substitution, biparental inheritance and plastome-genome incompatibility. Previous studies of plastomes in Passiflora with limited taxon sampling suggested that the genus exhibits this syndrome. To examine this phenomenon further, 15 new plastomes from Passiflora were sequenced and combined with previously published data to examine the phylogenetic relationships, genome organization and evolutionary rates across all five subgenera and the sister genus Adenia. Phylogenomic analyses using 68 protein-coding genes shared by Passiflora generated a fully resolved and strongly supported tree that is congruent with previous phylogenies based on a few plastid and nuclear loci. This phylogeny was used to examine the distribution of plastome rearrangements across Passiflora. Multiple gene and intron losses and inversions were identified in Passiflora with some occurring in parallel and others that extended across the Passifloraceae. Furthermore, extensive expansions and contractions of the inverted repeat (IR) were uncovered and in some cases this resulted in exclusion of all ribosomal RNA genes from the IR. The most highly rearranged lineage was subgenus Decaloba, which experienced extensive IR expansion that incorporated up to 25 protein-coding genes usually located in large single copy region. Nucleotide substitution rate analyses of 68 protein-coding genes across the genus showed lineage- and locus-specific acceleration. Significant increase in dS, dN and dN/dS was detected for clpP across the genus and for ycf4 in certain lineages. Significant increases in dN and dN/dS for ribosomal subunits and plastid-encoded RNA polymerase genes were detected in the branch leading to the expanded IR-clade in subgenus Decaloba. This subgenus displays the syndrome of unusual features, making it an ideal system to investigate the dynamic evolution of angiosperm plastomes.

Assessing Genetic Diversity and Population Differentiation of Colored Calla Lily (Zantedeschia Hybrid) for an Efficient Breeding Program.

Plastome-genome incompatibility (PGI) is prevalent in several plants including the Zantedeschia species, a worldwide commercial flower crop native to South Africa. Generally, hybrids suffering from PGI appear less vigorous and more susceptible than normal plants. Previous reports revealed that the PGI level in interspecific hybrids is correlated with the relatedness of the parental species in the genus Zantedeschia. To provide a basis for utilizing and improving resources in breeding programs, a total of 117 accessions of colored calla lily (Zantedeschia hybrid), collected from New Zealand, the Netherlands and the United States, were genotyped using 31 transferable expressed sequence tags-simple sequence repeats (EST-SSR) markers from the white calla lily (Zantedeschia aethiopica). A moderately high level of genetic diversity was observed, with 111 alleles in total, an observed/expected heterozygosity (Ho/He) of 0.453/0.478, and polymorphism information content (PIC) of 0.26. Genetic distance and STRUCTURE-based analysis further clustered all accessions into four subgroups (G-Ia, G-Ib, G-IIa and G-IIb), which mostly consisted of Zantedeschia pentlandii, Zantedeschia elliotiana, Zantedeschia albomaculata and Zantedeschia rehmannii, respectively. Significant genetic differentiation was observed between all inferred subgroup pairs, with the Fst ranging from 0.142 to 0.281. Finally, the accessions assigned into G-IIb (Z. rehmannii) were recommended as top priority parents in efficient Zantedeschia breeding program designs.

Coordinated rates of evolution between interacting plastid and nuclear genes in Geraniaceae.

Although gene coevolution has been widely observed within individuals and between different organisms, rarely has this phenomenon been investigated within a phylogenetic framework. The Geraniaceae is an attractive system in which to study plastid-nuclear genome coevolution due to the highly elevated evolutionary rates in plastid genomes. In plants, the plastid-encoded RNA polymerase (PEP) is a protein complex composed of subunits encoded by both plastid (rpoA, rpoB, rpoC1, and rpoC2) and nuclear genes (sig1-6). We used transcriptome and genomic data for 27 species of Geraniales in a systematic evaluation of coevolution between genes encoding subunits of the PEP holoenzyme. We detected strong correlations of dN (nonsynonymous substitutions) but not dS (synonymous substitutions) within rpoB/sig1 and rpoC2/sig2, but not for other plastid/nuclear gene pairs, and identified the correlation of dN/dS ratio between rpoB/C1/C2 and sig1/5/6, rpoC1/C2 and sig2, and rpoB/C2 and sig3 genes. Correlated rates between interacting plastid and nuclear sequences across the Geraniales could result from plastid-nuclear genome coevolution. Analyses of coevolved amino acid positions suggest that structurally mediated coevolution is not the major driver of plastid-nuclear coevolution. The detection of strong correlation of evolutionary rates between SIG and RNAP genes suggests a plausible explanation for plastome-genome incompatibility in Geraniaceae.

The role of plastids in plant speciation

Understanding the molecular basis of how new species arise is a central question and prime challenge in evolutionary biology and includes understanding how genomes diversify. Eukaryotic cells possess an integrated compartmentalized genetic system of endosymbiotic ancestry. The cellular subgenomes in nucleus, mitochondria and plastids communicate in a complex way and co-evolve. The application of hybrid and cybrid technologies, most notably those involving interspecific exchanges of plastid and nuclear genomes, has uncovered a multitude of species-specific nucleo-organelle interactions. Such interactions can result in plastome-genome incompatibilities, which can phenotypically often be recognized as hybrid bleaching, hybrid variegation or disturbance of the sexual phase. The plastid genome, because of its relatively low number of genes, can serve as a valuable tool to investigate the origin of these incompatibilities. In this article, we review progress on understanding how plastome-genome co-evolution contributes to speciation. We genetically classify incompatible phenotypes into four categories. We also summarize genetic, physiological and environmental influence and other possible selection forces acting on plastid-nuclear co-evolution and compare taxa providing molecular access to the underlying loci. It appears that plastome-genome incompatibility can establish hybridization barriers, comparable to the Dobzhansky-Muller model of speciation processes. Evidence suggests that the plastid-mediated hybridization barriers associated with hybrid bleaching primarily arise through modification of components in regulatory networks, rather than of complex, multisubunit structures themselves that are frequent targets.

Relationship between Nuclear Genome Construction and the Plastome-genome Incompatibility of Progenies from Intra- and Inter-ploid Cross of Evergreen Azaleas × Rhododendron japonicum f. flavum

In intersubgeneric crosses of diploid evergreen azaleas (EV) × diploid Rhododendron japonicum f. flavum (JP), many progenies become albino as a result of plastome-genome incompatibility between a plastid genome from EV and a nuclear genome from JP. To clarify the mechanism of the incompatibility, inter- and intra-ploid crosses of diploid (2x) EV × diploid (2x) JP, tetraploid (4x) EV × tetraploid (4x) JP, 2x EV × 4x JP, and 4x EV × 2x JP were conducted. Seeds were obtained in all crosses, although the number of progenies per capsule was relatively low in crosses with 4x JP as a pollen parent. Plastome-genome incompatibility between a plastid genome from EV and a nuclear genome from JP was observed in progenies from 2x EV × 2x JP, 4x EV × 4x JP, and 2x EV × 4x JP, but not in 4x EV × 2x JP. This result indicates that plastome-genome incompatibility was broken by relative increase of the genome from EV in the nuclear genome of progenies.

The Complete Nucleotide Sequences of the 5 Genetically Distinct Plastid Genomes of Oenothera, Subsection Oenothera: II. A Microevolutionary View Using Bioinformatics and Formal Genetic Data

A unique combination of genetic features and a rich stock of information make the flowering plant genus Oenothera an appealing model to explore the molecular basis of speciation processes including nucleus–organelle coevolution. From representative species, we have recently reported complete nucleotide sequences of the 5 basic and genetically distinguishable plastid chromosomes of subsection Oenothera (I–V). In nature, Oenothera plastid genomes are associated with 6 distinct, either homozygous or heterozygous, diploid nuclear genotypes of the 3 basic genomes A, B, or C. Artificially produced plastome–genome combinations that do not occur naturally often display interspecific plastome–genome incompatibility (PGI). In this study, we compare formal genetic data available from all 30 plastome–genome combinations with sequence differences between the plastomes to uncover potential determinants for interspecific PGI. Consistent with an active role in speciation, a remarkable number of genes have high Ka/Ks ratios. Different from the Solanacean cybrid model Atropa/tobacco, RNA editing seems not to be relevant for PGIs in Oenothera. However, predominantly sequence polymorphisms in intergenic segments are proposed as possible sources for PGI. A single locus, the bidirectional promoter region between psbB and clpP, is suggested to contribute to compartmental PGI in the interspecific AB hybrid containing plastome I (AB-I), consistent with its perturbed photosystem II activity.

The complete nucleotide sequences of the five genetically distinct plastid genomes of Oenothera, subsection Oenothera: I. sequence evaluation and plastome evolution.

The flowering plant genus Oenothera is uniquely suited for studying molecular mechanisms of speciation. It assembles an intriguing combination of genetic features, including permanent translocation heterozygosity, biparental transmission of plastids, and a general interfertility of well-defined species. This allows an exchange of plastids and nuclei between species often resulting in plastome–genome incompatibility. For evaluation of its molecular determinants we present the complete nucleotide sequences of the five basic, genetically distinguishable plastid chromosomes of subsection Oenothera (=Euoenothera) of the genus, which are associated in distinct combinations with six basic genomes. Sizes of the chromosomes range from 163 365 bp (plastome IV) to 165 728 bp (plastome I), display between 96.3% and 98.6% sequence similarity and encode a total of 113 unique genes. Plastome diversification is caused by an abundance of nucleotide substitutions, small insertions, deletions and repetitions. The five plastomes deviate from the general ancestral design of plastid chromosomes of vascular plants by a subsection-specific 56 kb inversion within the large single-copy segment. This inversion disrupted operon structures and predates the divergence of the subsection presumably 1 My ago. Phylogenetic relationships suggest plastomes I–III in one clade, while plastome IV appears to be closest to the common ancestor.

Cross Compatibility of Intersubgeneric Hybrids of Azaleas on Backcross with Several Evergreen Species

For the reduction of several undesirable traits, such as deciduousness and weak heat tolerance, of intersubgeneric hybrids between interspecific F1 hybrids of evergreen azaleas and a deciduous species (Rhododendron japonicum f. flavum), back cross with evergreen azaleas was conducted. Though pollen of the intersubgeneric hybrids was highly sterile, fertility was restored in a tetraploid plant (9027-5). Unilateral cross incompatibility was observed between the intersubgeneric tetraploid hybrid 9027-5 and evergreen azaleas, i.e., progenies obtained only in the cross with the tetraploid plant 9027-5 used as a pollen parent. Leaf color of most of the progenies was green, and hybridity was confirmed by isozyme analysis. PtDNA of the green progenies inherited from the seed parent (evergreen azaleas), and plastome-genome incompatibility between the plastid genome from evergreen azaleas and nuclear genome from R. japonicum f. flavum was not observed in the back cross.

BIPARENTAL PLASTID INHERITANCE IN ZANTEDESHIA ALBOMACULATA (ARACEAE)

Most flowering plants exhibit maternal plastid inheritance. In Zantedeschia, however, biparental is known to occur in interspecific hybrids. Moreover, plastome-genome incompatibility exists between the different species. The evolutionary significance of biparental plastid inheritance and plastome-genome incompatibility is unclear. It is possible that the unusual genomic background of interspecific crosses affects plastid inheritance and that plastids inherit maternally in intraspecific hybrids as in most other flowering plants. The mode of plastid inheritance between Z. albomaculata subsp. albomaculata and Z. albomaculata subsp. macrocarpa was, therefore, investigated. Subspecies specific CAPS markers were developed and used to demonstrate that biparental plasmid inheritance occurs between these subspecies. This finding suggests that biparental plastid inheritance exists within Zantedeschia spp. and is not a result from the unusual genomic background of interspecific crosses.

Plastid Inheritance and Plastome-genome Incompatibility of Intergeneric Hybrids between Menziesia and Rhododendron

Intergeneric hybridization between Menziesia and Rhododendron generates many albino or pale-green seedlings. To clarify the factors that cause albino phenotype, we analyzed the inheritance of plastid (pt) DNA by PCR-RFLP analysis on the intergeneric hybrids between two genera and an intraspecific hybrid within Menziesia by reciprocal cross-pollination, respectively. All albino and pale-green intergeneric progenies contained the ptDNA of Rhododendron, whereas green progenies contained that of Menziesia, indicating that Rhododendron plastome is incompatible with Menziesia nuclear genome. Moreover, when M. multiflora was used as a female parent, most progenies transmitted their plastids paternally. Plastome-genome incompatibility seems to be independent from ptDNA inheritance in Menziesia-Rhododendron hybrids.

EFFECTIVENESS OF INTER-PLOID CROSSES FOR OVERCOMING PLASTOME-GENOME INCOMPATIBILITY IN INTERSECTIONAL CROSSES OF AZALEAS

We clarified the appearance of albino seedlings caused by plastome-genome incompatibility in intersectional hybridisation between evergreen azaleas and deciduous Rhododendron japonicum f. flavum. Intra- and inter-ploid crosses of 2x and 4x evergreen azaleas x 2x R. japonicum f. flavum, respectively, showed high capsule set, but their reversed crosses resulted no capsule set. Green and albino hybrids were obtained from 2x evergreen azaleas x 2x R. japonicum f. flavum where ptDNAs of the green and albino plants were derived from R. japonicum f. flavum (paternal parent) and evergreen azaleas (maternal parents), respectively. All the progenies in the crosses of 4x evergreen azaleas x 2x R. japonicum f. flavum were green triploids with evergreen azalea-derived ptDNA. The efficiency of obtaining green hybrids in inter-ploid crosses was higher than that in intra-ploid crosses. These results suggest that inter-ploid crosses of 4x evergreen azaleas x 2x R. japonicum f. flavum can overcome plastome-genome incompatibility between evergreen azalea-derived plastome and hybrid nuclear genome.

Genetic control of resistance to soft rot caused by Erwinia carotovora subsp. carotovora in Zantedeschia spp. (Araceae), section Aestivae

The pattern of heredity of resistance to Erwinia carotovora subsp. carotovora in Zantedeschia spp. is investigated. Four species with different resistance levels (Z. albomaculata, Z. elliotiana, Z. pentlandii, Z. rehmannii) were compared to their reciprocal offspring. The occurrence of plastome-genome incompatibility (PGI) affected plant resistance in all families. Therefore, plants that suffered from PGI were omitted from genetic analyses. Resistance was quantitative and the correlation between resistance levels of parents and offspring (h2= 0.33;r2= 0.66) indicated a genetic basis of resistance. Z. rehmannii and Z. albomaculata contributed more resistance genes than Z. elliotiana or Z. pentlandii. Transgression among some of the offspring of Z. rehmannii and Z. albomaculata indicated the presence of complementary resistance genes in these two species and good potential for resistance breeding.

Plastome-genome Incompatibility of Rhododendron serpyllifolium (A. Gray) Miq. to Evergreen Azalea Species Belonging to Series Kaempferia.

To confirm that unilateral cross-incompatibility exists between Rhododendron serpyllifolium, series Serpyllifolia, and six evergreen azaleas belonging to series Kaempferia, interspecific crosses were conducted. In crosses with R. serpyllifolium as seed parents, few seeds of the progenies germinated ; the survivors developed into plants with green leaves which PCR-RFLP analysis showed as having R. serpyllifolium type ptDNA. In the reciprocal crosses with R. serpyllifolium as pollen parents, many seeds germinated but many were non-viable albinos which had Kaempferia type ptDNA. In crosses between R. transiens (Accession 1)×R. serpyllifolium (Accession 1), however, relatively high frequency of green seedlings having R. serpyllifolium type ptDNA appeared. From these results, we conclude that it is better to use R. serpyllifolium as pollen parent for obtaining many seedlings, and that, in this case, R. transiens (Accession 1) is useful as bridge plants for overcoming plastome-genome incompatibility between R. serpyllifolium and series Kaempferia species.

Extensive developmental and metabolic alterations in cybrids Nicotiana tabacum (+ Hyoscyamus niger) are caused by complex nucleo-cytoplasmic incompatibility.

The genetic basis of multiple phenotypic alterations was studied in cell-engineered cybrids Nicotiana tabacum (+ Hyoscyamus niger) combining the nuclear genome of N. tabacum, plastome of H. niger and recombinant mitochondria. The plants possess a complex, maternally inheritable syndrome of nucleo-cytoplasmic incompatibility, severely affecting growth, metabolism and development. In vivo, the syndrome was manifested as: late germination of seeds; dramatic decrease of chlorophyll and carotenoids in cotyledons and leaves; altered morphology of cotyledons, leaves and flowers; and dwarfism. The leaf phenotype depended on light intensity. In 'green flowers' (an extreme phenotype), homeotic function B was downregulated. In vitro, the incompatibility syndrome was restricted to the pigment deficiency of cotyledons. Electron microscopy revealed perturbations in the differentiation of chloroplasts and palisade parenchyma cells in bleached leaves. The pigment deficiency accompanied by retarded growth is discussed as a result of plastome-genome incompatibility, whereas other features are likely to be due to nucleo-mitochondrial incompatibilities.

Multiple gene control of plastome-genome incompatibility and plastid DNA inheritance in interspecific hybrids of Zantedeschia

From interspecific hybridisation in the genus Zantedeschia, we have previously produced albino, variegated and virescent hybrids. The inhibition of chloroplast development in these hybrids is due to plastome-genome incompatibility. The albino hybrids did not develop prolamellar bodies in their etioplasts in darkness and formed only distended membranes, but no grana in light. This indicates that the block to chloroplast development occurs before or during the development of etioplasts. In albino leaf sectors of variegated hybrids, chlorophyll and carotenoid contents were only 2–4% of those in green plants. The mRNA levels for five plastid-development-related genes were also severely reduced, but they were still readily detectable using Northern hybridisation techniques. In the green sectors of variegated hybrids, chlorophyll content and mRNA levels of the fives genes were slightly reduced. We have earlier shown that F1 hybrids between Z. aethiopica and Z. odorata were albino when Z. odorata plastids were present but virescent when Z. aethiopica plastids were present. When the F1 hybrids with Z. aethiopica plastids were backcrossed to Z.odorata, the progeny were albino, virescent or green. Z. odorata plastids were inherited only from maternal parents in the F1 progenies but inherited from either maternal or paternal parents in the backcross. The increased compatibility with, and inheritance of Z. odorata plastids in the backcross suggests that multiple genes play a major role in the plastome-genome incompatibility. An increase in the number of genes from the parent contributing the plastids improves chloroplast development in the hybrids of the genus Zantedeschia.

Functional cybrid plants of Lycopersicon peruvianum var 'dentatum ' with chloroplasts of Lycopersicon esculentum.

Fertile cybrid plants of three subclones, B1A, B3A, B4A were regenerated from the single colony obtained after the fusion of mesophyll protoplasts of plastome chlorophyll-deficient mutant Lycopersicon peruvianum var 'dentatum' (line 3767) and γ-irradiated mesophyll protoplasts of L. esculentum (cv 'Quedlinburger Frühe Liebe'). Cytogenetic, isozyme, RAPD, morphological and restriction analyses all showed that the subclones had the nuclear genome of L. peruvianum var 'dentatum' and plastome genome of L. esculentum, while the mitochondrial genome was altered. No phenotypical traits that could be taken as evidence of plastome-genome incompatibility in the cybrid subclones were observed. Genetic functionality of all subclones was proven by the backcrossing analysis. To study the functionality of the cybrid plants we also carried out an analysis of their photosynthetic system. Data on chlorophyll-a and -b content, analyses of the fluorescence induction curves, intensity of CO2 assimilation, pigment-protein complexes and polypeptides of thylakoid membranes showed the absence of structural and functional abnormalities in the photosynthetic apparatus of the cybrid plants. We concluded that the plastome of L. esculentum is able to effectively interract with the nuclear genome of L. peruvianum var 'dentatum' and together with the recombined chondriome can support genetic functionality of cybrid plants of the peruvianum tomato.

Interspecific albino and variegated hybrids in the genus Zantedeschia

Following interspecific hybridization between the two sections of the genus Zantedeschia, embryo development was restricted to the globular stage and the endosperm became degenerate and watery. Embryo culture was used to rescue hybrid embryos and the 178 hybrid plants produced were all albino because of plastome-genome incompatibility. A newly described species Z. odorata, which falls in between the two sections of the genus, was used in an attempt to transfer genes between the two sections. Hybrids from section I × Z. odorata were virescent and fertile. Hybrids from the reciprocal cross, Z. odorata × section I, were either virescent, albino or variegated. When Z. odorata was crossed with section II, embryo and endosperm development improved considerably compared with crosses between sections I and II. However, all hybrids were albino. Hybrids were confirmed using RFLP analysis with a ribosomal DNA probe.

Genetically Programmed Chloroplast Dedifferentiation as a Consequence of Plastome-Genome Incompatibility in Oenothera.

Comparision of chloroplast from plants with one of four plastome types (I, II, III, IV) in the nuclear background of Oenothera elata strain Johansen addressed the effects of plastome-genome incompatibility with respect to leaf pigmentation, plastid ultrastructure, chlorophyll a/chlorophyll b ratio, and photosynthetic electron transport. Previous observations of plastomes I, II, and IV in this nuclear background have revealed no indications of incompatibility, but the studies reported here demonstrate that chloroplasts of plastome IV have subtle alterations in their photosynthetic abilities, in particular, deficiencies in photosystem II. The well-characterized "hybrid bleaching" of plants with the AA genotype and plastome III involves leaves that become bleached in the center while remaining green at the tips, edges, and veins. Electron transport assays performed on fractionated bleached and green tissue from the same plants show photosynthetic defects in both the green and bleached regions, although defects in the latter are more severe. Ultrastructural studies show that chloroplasts in the bleached areas enlarge, thylakoid membranes become swollen and vesiculated, and production of new thylakoids is blocked, with chloroplasts appearing to undergo a programmed senescence. A time course revealed that the senescence is actually a reversible dedifferentiation. Alterations in the composition of medium to which AA/III seedlings were transferred showed that the presence of auxin can prevent the development of the typical incompatibility response, with leaf tissue remaining green rather than bleaching. It is proposed that differences in concentrations of plant growth regulators may be responsible for the persistence of normal chloroplasts near the vascular tissue and leaf blade edges and that seasonal fluctuations in auxin levels could explain the periodic bleaching that occurs in older plants.

Limited chloroplast gene transfer via recombination overcomes plastomegenome incompatibility between Nicotiana tabacum and Solanum tuberosum

Green cybrids with a new nucleus-chloroplast combination cannot be selected after protoplast fusion in the intersubfamilial Nicotiana-Solanum combination. As an approach to overcome the supposed plastomegenome incompatibility, a partial plastome transfer by genetic recombination has been considered. After fusions of protoplasts of a light-sensitive Nicotiana tabacum (tobacco) plastome mutant and lethally irradiated protoplasts of wild-type Solanum tuberosum (potato), a single green colony was recovered among 2.5×10(4) colonies. The regenerated plants had tobacco-like (although abnormal) morphology, but were normally green, and sensitive to tentoxin, demonstrating chloroplast markers of the potato parent. Restriction enzyme analysis of the chloroplast DNA (cpDNA) revealed recombinant, nonparental patterns. A comparison with physical maps of the parental cpDNA demonstrated the presence of a considerable part of the potato plastome flanked by tobacco-specific regions. This "potacco" plastome proved to be stable in backcross and backfusion experiments, and normally functional in the presence solely of N. tabacum nucleus.

Intertrubal chloroplast transfer by protoplast fusion between Nicotiana tabacum and Salpiglossis sinuata

Chloroplast tranfer was achieved by protoplast fusion between Nicotiana tobacum (Cestreae, Cestroideae) and Salpiglossis sinuata (Salpiglossideae, Cestroideae) in the family Solanaceae. Isolation of cybrid clones was facilitated by irradiation of the cytoplasm donor protoplasts, and the use of appropriate plastid mutants, streptomycin-resistant as donor, or light-sensitive as recipient. Cybrid colonies were selected by their green colour against the background of bleached (light-sensitive or streptomycin-sensitive) colonies. In the Nicotiana (Salpiglossis) cybrid plants possessing normal tobacco morphology and chromsome number, the presence of Salpiglossis, plastids was verified by restriction analysis of the chloroplast DNA. A similar analysis of the mitochondrial DNA of these lines revealed unique, recombinant patterns in the case of both fertile and sterile plants. Progeny showed no appearance of chlorophyll-deficiency in F1 and an additional back-cross generation. Attempts at transfer of entire chloroplasts between Nicotiana tabacum and Solanum nigrum (Solaneae, Solanoideae) did not result in any cybrid cell lines in a medium suitable for green colony formation of both species. These results suggest that fusion-mediated chloroplast transfer can surmount a considerable taxonomical distance, but might be hampered by a plastome-genome incompatibility in more remote combinations.