Transfer of defined numbers of chloroplasts into albino protoplasts by subprotoplast/protoplast microfusion: chloroplasts can be “cloned”, by using suitable plastome combinations or selective pressure

Summary Defined numbers (1–5) of (donor) chloroplasts were transferred into (acceptor) protoplasts of plastid albino mutants by subprotoplast/protoplast microfusion. Single transferred plastids gave rise to new organelle populations in the progeny of the fusion products when suitable combinations of plastomes were used or when selective pressure for the plastome transferred was applied. This process is termed chloroplast cloning and is the first reported case of cloning a cell organelle. The plastome combination and the presence or absence of selective pressure were found to influence the frequencies with which cell lines, containing both plastomes or acceptor or donor only, were obtained, and the number of cell generations needed for complete segregation — as measured by the duration of culture before the green donor plastome could be detected. The high frequency of cell lines and regenerated shoots recovered with donor plastome only, even when only a single chloroplast was transferred, leads to the conclusion that all organelles present in the fusion product contribute to the organelle population of the progeny, i.e. organelle death or loss are not regularly occurring events during plant regeneration from protoplasts in Nicotiana tabacum.Some of the results reported here were presented at the 8th International Protoplast Symposium, Uppsala 1991     

Nuclear-organelle interaction in Solanum: interspecific cybridizations and their correlation with a plastome dendrogram.

Summary Alloplasmic compatibility, namely the functional interaction between the nuclear genome of a given species with plastomes and chondriomes of alien species, is of considerable relevance in plant biology. The genus Solanum encompasses a wide spectrum of species and is therefore suitable for a study of this compatibility. We thus chose the nuclear genome of Solanum tuberosum (potato) and organelles (chloroplast and mitochondria) from 14 other Solanum species to initiate an investigation of intrageneric nucleus/organelle interactions. An assessment of the diversity of the chloroplast DNAs from these 15 species resulted in the construction of a plastome dendrogram (phylogenetic tree). In parallel we extended a previous study and performed ten additional fusion combinations by the donor-recipient protoplast fusion procedure, using potato protoplasts as recipients and protoplasts from any of ten other Solanum species as donors. We found that two fusion combinations did not yield cybrids and that the chloroplasts of S. polyadenium and the mitochondria (or mitochondrial components) from S. tarijense could not be transferred to cybrids bearing potato nuclei. In general, there is a correlation, albeit not perfect, between the cybridization data and the plastome dendrogram. These results furnish valuable information toward future transfer of plasmoneencoded breeding traits from wild Solanum species into potato. This information should also be useful for the planning of asymmetric protoplast fusion between potato and wild accessions for the improvement of pathogen and stress resistance of potato cultivars.     

Generation of heteroplastidic Nicotiana cybrids by protoplast fusion: analysis for plastid recombinant types

Summary Protoplasts of a mutant line of Nicotiana tabacum having a maternally-transmitted chlorophyll deficiency were fused with protoplasts of two alloplasmic-male-sterile Nicotiana lines by the donor-recipient technique. In both fusion experiments variegated plantlets were regenerated which were shown to contain cytoplasms of mixed chloroplast nature. This confirms that with the donor-recipient method one can obtain mixed cytoplasms of genetically different chloroplasts. We present a convenient system to assay for genetic recombination between chloroplasts by combining use of several cytoplasmic markers: vis. chlorophyll pigmentation, chloroplast DNA restriction patterns, tentoxin resistance and male sterility. Within the limits of the experiment no recombinant types were recovered.     

Phylogeography and conservation genetics of Hygrophila pogonocalyx (Acanthaceae) based on atpB–rbcL noncoding spacer cpDNA

Genetic variation in the atpB–rbcL intergenic spacer region of chloroplast DNA (cpDNA) was investigated in Hygrophila pogonocalyx Hayata (Acanthaceae), an endangered and endemic species in Taiwan. In this aquatic species, seed dispersal from capsules via elasticity is constrained by gravity and is thereby confined within populations, resulting in limited gene flow between populations. In this study, a total of 849 bp of the cpDNA atpB–rbcL spacer were sequenced from eight populations of H. pogonocalyx. Nucleotide diversity in the cpDNA is low (=0.00343±0.00041). The distribution of genetic variation among populations agrees with an isolation-by-distance model. Two geographically correlated groups, the western and eastern regions, were identified in a neighbor-joining tree and a minimum-spanning network. Phylogeographical analyses based on the cpDNA network suggest that the present-day differentiation between western and eastern groups of H. pogonocalyx resulted from past fragmentation. The differentiation between eastern and western populations may be ascribed to isolation since the formation of the Central Mountain Range about 5 million years ago, which is consistent with the rate estimates based on a molecular clock of cpDNA.Huang JC and Wang WK equally contributed to this work.     

Nicotiana cybrids with Petunia chloroplasts

Summary Protoplasts of a chloroplast-defective cultivar of Nicotiana tabacum were fused with gamma-irradiated protoplasts of Petunia hybrida. Over 100 photoautotrophic plants were regenerated; of these 94 were tested for Petunia chloroplast traits and all but one had Petunia chloroplasts based on their sensitivity to the fungal toxin, tentoxin. Chloroplast DNA was analysed for 3 of the sensitive plants and was shown to be identical to Petunia chloroplast DNA. Most of the plants (about 70%) appeared to be normal N. tabacum plants, based on morphology and chromosome number. They were fully fertile with normal pollen viability, seed set, and seed viability. The remaining 30% of the plants showed varying degrees of vegetative and reproductive abnormalities.The techniques of somatic cell genetics have led to many possible nuclear-organellar combinations that may be considered as cybrids. In this paper, we use the term to include the combination of nucleus from one species and chloroplast from another species     

Comparative studies on the structural gene for the ribosomal protein S1 in ten bacterial species

Summary Callus protoplasts of a plastome chlorophyll-deficient mutant of tobacco, Nicotiana tabacum, were fused with mesophyll protoplasts from one of the following five sources: cms-analogs of tobacco bearing the cytoplasms of N. suaveolens, N. undulata, N. repanda, and N. plumbaginifolia, respectively, and the wild species N. glauca. In the sixth experiment, callus cells of the tobacco chlorophyll-deficient genome mutant, homozygous for the Su gene, were hybridized with mesophyll protoplasts of the plastome chlorophyll-deficient mutant of tobacco. Individual dividing heteroplasmic fusion products were isolated mechanically and cloned in microdroplets of nutrient medium. Among the regenerants in all parental combinations, though not in all clones, besides pure green and pure chlorophyll-deficient plants, numerous variegated plant forms were obtained. The variegation of cybrid and hybrid plants was connected with their heterozygocity for chloroplast DNA composition, as demonstrated by restriction analysis. In analytical crosses, the variegation was inherited maternally by part of the sexual progeny, and variegated F₁ progeny were also heterozygous for chloroplast DNA composition. As demonstrated by electron microscopic studies, the variegation is connected with the presence of mixed, i.e., heteroplastidic cells in leaves. The results obtained demonstrate that (1) upon somatic cell fusion, plastome genes are inherited biparentally in most fusion products, and (2) despite an evident mitotic segregation process, heterozygosity for plastome genes is a relatively durable state and can be revealed in cell hybrids of different specific combinations of plasmons after a great number of cell generations. In this regard the plastome cytogetes obtained by somatic cell fusion do not differ qualitatively from the cytoplasmic heterozygotes that arise as a result of the mutation process.     

Reconstitution of cytoplasmic streaming inCharaceae

Summary The active sites of actin of oneCharaceae species were found to interact with the endoplasmic factor from a different species. Protoplasm was suqueezed out of cells ofChara australis with vacuoles that had been perfused beforehand with a medium containing EGTA and Mg · ATP. Centrifugation of this protoplasmic mixture divided it into the supernatant composed of endoplasmic granules and the precipitate composed of chloroplasts and nuclei. When the endoplasmic granular aggregates were introduced into a tonoplast-freeNitella axilliformis cell treated with NEM to inactivate the endoplasmic factor, they became attached to theNitella gel and streamed longitudinally with the polarity. Treatment of the endoplasmic granules with the strong Mg²⁺chelator CyDTA (1,2-cyclohexane diamineN, N-tetraacetic acid) irreversibly inhibited reconstitution of the cytoplasmic streaming.Abbreviations APW artificial pond water - ATP adenosine-5-triphosphoric acid - CyDTA cyclohexanediamine-N,N-tetraacetic acid - DTT dithiothreitol - EGTA ethyleneglycol-bis-(-aminoethylether)-N,N-tetraacetic acid - HMM heavy meromyosin - NEM N-ethylmaleimide - PEP phosphoenolypyruvate - PIPES piperazine-N,N-bis-(2-ethane-sulfonic acid) - PK pyruvate kinase - PMSF phenylmethylsulfonylfluoride     

Selection of anthocyanin-accumulating potato (Solanum tuberosum L.) cell lines from calli derived from seedlings produced by gamma-irradiated seeds

Callus cell lines of potato (Solanum tuberosum L. cv. Zarevo) were obtained from seedlings germinated from gamma-irradiated seeds (200 Gy). Some of these cell lines produce red-violet pigments which were identified as acylated anthocyanins. The major anthocyanin was determined to be peonidin 3-O-[6-O-(4-O-E-p-coumaroyl-rhamnosyl)-glucoside]-5-O-glucoside (peonanin). Single cell-derived protoclones from non-pigmented protoplasts sometimes also gave rise to pigmented cell clusters thus indicating that the changes in the expression of the anthocyanin pathway can also occur after the stage of initial callus induction.     

Surface charge of protoplasts and their significance in cell-cell interaction

-potential of mesophyll protoplasts of tobacco (Nicotiana tabacum L.), petunia (Petunia hybrida Hort.), turnip (Brassica rapa L.) and cowpea (Vigna unguiculata L. Walpers) was determined by use of a cell electrophoresis apparatus. All protoplasts examined showed a constant negative value of-10 to-35 mV. The addition of CaCl₂ nullified the -potential of tobacco protoplasts. This phenomenon is explained by DLVO theory of colloid science, which has been successfully applied to animal cells. Furthermore, positively charged polymers reversed the -potential to positive values. Treatment of the protoplast surface with several enzymes was carried out to characterize the chemical nature of suface charges. The removal of surface charges was most conspicuous by the treatment of acid phosphatase (EC 3.1.3.2), but did not occur upon treatment with -neuraminidase (EC 3.2.1.18) or Streptomyces griseus pronase. Thus a major part of the surface charge originates from the phosphate groups at the cell membrane. The significance of these studies for the properties of the protoplast surface in cell adhesion is discussed.     

Mutation proximal to the tRNA binding region of the Nicotiana plastid 16S rRNA confers resistance to spectinomycin.

Summary Nicotiana tabacum lines carrying maternally inherited resistance to spectinomycin were obtained by selection for green callus in cultures bleached by spectinomycin. Two levels of resistance was found. SPC1 and SPC2 seedlings are resistant to high levels (500 g/ml), SPC23 seedlings are resistant to low levels (50 g/ml) of spectinomycin. Lines SPC2 and SPC23 are derivatives of the SR1 streptomycin-resistant plastome mutant. Spectinomycin resistance is due to mutations in the plastid 16S ribosomal RNA: SPC1, an A to C change at position 1138; SPC2, a C to U change at position 1139; SPC23, a G to A change at position 1333. Mutations similar to those in the SPC1 and SPC2 lines have been previously described, and disrupt a conserved 16S ribosomal RNA stem structure. The mutation in the SPC23 line is the first reported case of a mutation close to the region of the 16S rRNA involved in the formation of the initiation complex. The new mutants provide markers for selecting plastid transformants.     

Protoplast-fusion-mediated transfer of organelles from Microcitrus into Citrus and regeneration of novel alloplasmic trees

Summary Iodoacetate-treated Citrus protoplasts from embryogenic nucellar calli of Sour orange (C. aurantium) or from Rough lemon (C. jambhiri) were fused with -irradiated protoplasts from a related genus, Microcitrus. The fused protoplasts were cultured to obtain colonies and micro-calli. Micro-calli derived from these two fusion combinations were isolated, propagated and differentiated into embryos, which subsequently regenerated trees having the morphology of Sour orange or Rough lemon. These intergeneric fusions resulted in mitochondria with novel DNA, indicating recombination between the chondriomes of Citrus and Microcitrus. Chloroplast DNA analyses of fusion-derived embryos indicated that they contained the chloroplasts of either fusion-partner or a mix of these chloroplasts. Later plastome analyses of leaves from fully differentiated plants showed that cybrids having Rough lemon morphology had either Rough lemon or Microcitrus chloroplast DNA, indicating complete sorting out of chloroplasts. Likewise, sorting out of Microcitrus chloroplasts was detected in a cybrid plant having Sour orange morphology.Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, 50250 Israel. No. 2663-E, 1989 series     

The Cucumis plastome: physical map,intrageneric variation and phylogenetic relationships

Summary A restriction map of the Cucumis melo L. (melon) plastome was constructed by using several mapping approaches: single and double digestions of the chloroplast DNA (chlDNA) with endonucleases (XhoI, SmaI, SacI and PvuII) and hybridization to heterologous chlDNA probes and to isolated melon chlDNA fragments. Four plastome-coded genes were located using heterologous probes. The overall organization and gene position of the melon plastome was found to be similar to that of tobacco and other angiosperm species. Restriction patterns based on digestion of the chlDNA with nine endonucleases were obtained in over 20 wild species and cultivated varieties of Cucumis. These led to mutational analysis of the restiction sites yielding the most parsimonious phylogenetic tree of the Cucumis plastome. Most African species from a compact group (Anguria group) which is distant from the melon, the cucumber and a few other species (C. sagittatus, C. metuliferus and C. humifructus). All of these are also far apart from each other. The distribution of polymorphic restriction sites along the Cucumis plastome is described and conservative regions as well as hot spots are suggested.     

Structural requirements for the binding of oligosaccharides to immobilized lectin ofErythrina variegata (Linn) var.Orientalis

The structural requirements for the interaction of asparagine-linked oligosaccharide moieties of glycoproteins withErythrina variegata agglutinin (EVA) were investigated by means of affinity chromatography on an EVA-Sepharose column. Some of the branched poly-N-acetyllactosamine-type oligosaccharides obtained from human erythrocyte band 3 glycoprotein were found to show high affinity to EVA-Sepharose, whereas complex-type oligosaccharides were shown to have low affinity. Hybrid type, oligomannose-type and unbranched poly-N-acetyllactosamine-type oligosaccharides bound very little or not at all to EVA-Sepharose. To further study the carbohydrate-binding specificity of this lectin, we investigated the interaction of immobilized EVA and oligosaccharide fragments obtained through partial hydrolysis from branched poly-N-acetyllactosamine-type oligosaccharides. Branched poly-N-acetyllactosamine-type oligosaccharides were subjected to limited hydrolysis with 0.1% trifluoroacetic acid at 100°C for 40 min and then separated on an amino-bonded silica column. One of pentasaccharides thus prepared strongly bound to the EVA-Sepharose column. Structural analysis of this pentasaccharide showed that the Gal1-4GlcNAc1-3(Gal1-4GlcNAc1-6)Gal sugar sequence, which is an l-antigen determinant, was essential for the high affinity binding of the oligosaccharides to the EVA-Sepharose column.Abbreviations EVA Erythrina variegata agglutinin - WGA wheat germ agglutinin - STA potato lectin - LEA tomato lectin - DSA Datura stramonium agglutinin - PBS 0.01 M sodium phosphate buffer, pH 7.3, containing 0.15 M NaCl - Galol galactitol     

Geographic structuring of chloroplast DNA genotypes inTiarella trifoliata (Saxifragaceae)

Tiarella trifoliata comprises varietieslaciniata, trifoliata, andunifoliata, and is distributed from southeastern Alaska to northern California. We analyzed restriction site variation of chloroplast DNA (cpDNA) using 23 endonucleases in 76 populations representing the entire geographic range of the species and the three recognized varieties. We also employed comparative restriction site mapping of PCR-amplified chloroplast DNA fragments using 16 restriction endonucleases. This species exhibits low cpDNA restriction site variation. No differentiation is evident among varieties of this species based on cpDNA data; some plants of each variety were characterized by each of the two major cpDNA types detected. The two major cpDNA clades, which differ by only a single restriction site mutation, are geographically structured. A northern clade comprises populations from Alaska to central Oregon; most populations analyzed from southern Oregon and California form a southern clade. Populations that possess the typical northern cpDNA type also occur disjunctly to the south at high elevations in the Siskiyou—Klamath Mountain area of southern Oregon and northern California. Conversely, the southern cpDNA type is found disjunctly to the north in the Olympic Peninsula of Washington. Both geographic areas characterized by disjunct cytoplasms are considered glacial refugia.Tiarella trifoliata joins two other species,Tolmiea menziesii andTellima grandiflora, in having well-demarcated northern and southern cpDNA lineages. All three species have similar life-history traits and geographic distributions. We suggest that glaciation may have played a major role in the formation of the cpDNA discontinuities present in these three taxa. The pronounced relationship between cpDNA variation and geographic distribution suggests the potential applicability of intraspecific phylogeography to plants via the analysis of intraspecific cpDNA variation. These three examples also join a rapidly growing data base which indicates that cytoplasms are often geographically structured within species and species complexes.     

Introgression of the Haynaldia villosa genome into γ-ray-induced asymmetric somatic hybrids of wheat

To study the effect of -ray treatment on donor and derived somatic hybrids, we carried out -ray donor treatment experiments with a wide range of -ray dosages and asymmetric somatic hybridization between protoplasts of wheat (Triticum aestivum L. Jinan 177) and protoplasts of Haynaldia villosa Schur. treated with different dosages of -rays (40, 60 and 80 Gy, respectively). We first screened the putative hybrids by isozyme analysis, followed by characterization of nuclear and organellar genome composition of the hybrids. Genomic in situ hybridization on mitotic metaphases demonstrated that the donor chromosome elimination in the hybrids increased with increased -ray dosage. Intergenomic chromosome recombination/translocations were observed in the hybrids from different dosages of -rays. PCR amplification of 5S rDNA spacer sequences showed that only some of the regenerated hybrid clones inherited donor 5S rDNA sequences, suggesting that the donor DNA was also eliminated randomly. Restriction fragment length polymorphism analysis using mitochondrion (mt) and chloroplast (cp) gene-specific probes showed that the hybrid calli contained mt genomes of both parents and the cp genome of only one of the parents. Recombinations between parental mt as well as cp genes were found in the hybrid clones. Furthermore, development of the hybrid clones was dependent on the -ray dosage used for the donor treatment. Regenerated plants were only obtained from fusion combinations of low (40 Gy) and intermediate (60 Gy) dose irradiation. The possible role and significance of -rays on the introgression of small segments of donor chromosomes to the receptor is discussed.     

Stability of cloned Brassica napus chloroplast DNA fragments in the cyanobacterium Anacystis nidulans R2

Summary Brassica napus (cv. Triton) chloroplast (cp) DNA BamHI gragments were inserted into a bacteria-cyanobacteria shuttle vector pCB4. The chloroplast genomic library was screened in Escherichia coli and 28 individual clones, which represent 94% of the total chloroplast genome, were isolated. Cyanobacterium Anacystis nidulans R2 was transformed with each member of the clone bank by selection for ampicillin resistance. A study of transformation efficiency showed dramatic variation (up to 200-fold) among recombinant clones. Furthermore, plasmid DNA reisolated from some cyanobacterial transformants exhibited instability. Variations in transformation efficiency and plasmid instability were shown to be DNA sequence specific. B. napus cpDNA clones were thus classified into three types according to their stability in the cyanobacterial host.     

Limited DNA elimination from the irradiated potato parent in fusion products of albino Lycopersicon esculentum and Solanum tuberosum

Summary This paper describes the analysis of the elimination of potato DNA from potato-tomato somatic cell hybrids. The hybrids were obtained by fusion of protoplasts of a cytoplasmic albino tomato genotype with leaf mesophyll protoplasts of a potato genotype carrying the -glucuronidase (GUS) gene of Escherichia coli. The potato protoplasts were either isolated from unirradiated plants or from plants irradiated with 50 or 500 Gy of -rays. Green calli were selected as putative fusion products. The hybridity of these calli was confirmed by isoenzyme analysis. All of the green calli tested contained a potato-specific chloroplast DNA restriction fragment, and most of the calli analysed were positive for -glucuronidase activity. In 72 of the hybrid calli we determined the percentage of potato nuclear DNA using species-specific probes. All of the tested green calli contained a considerable amount of potato genomic DNA, irrespective of the dose of irradiation of the potato parent. The limited degree of potato DNA elimination and the absence of true cybrids are discussed.     

Identifying populations useful for improving parents of a single cross based on net transfer of alleles

Summary Theory and methods for identifying populations (P _y ) with the highest frequency of favorable dominant alleles not present in an elite single cross (I ₁× I ₂) have been developed recently. During selection, new favorable alleles can be transferred from P _y to either I ₁ or I ₂ only at the risk of losing favorable alleles already present in the single cross. A net improvement (NI) statistic, which estimates the relative number of favorable alleles that can be gained from P _y minus the relative number of favorable alleles that can be lost from I ₁ or I ₂, is presented. NI is calculated as maximum [(I ₁×P _y –I ₁×I ₂)/2,(I ₂×P _y –I ₁×I ₂)/2]. Because I ₁ × I ₂ is constant in an experiment, the method reduces to choosing P _y populations with the best mean performance in combination with either I ₁ or I ₂. For a set of maize (Zea mays L.) grain yield data, NI was highly correlated to three other statistics proposed for choosing populations, namely: (1) minimally biased estimate (l ) of the relative number of favorable dominant alleles present in P _y but not in I ₁ and I ₂; (2) minimum upper bound on l ; and (3) predicted performance of the three-way cross [P _y (I ₁× I ₂)]. While l estimates potential improvement likely to be achieved only through long-term recurrent selection, NI is probably a better predictor of short-term improvement in single-cross performance.A contribution from Lifaco Genetics, a subsidiary of Groupe Limagrain     

Somatic hybridization in potato: use of γ-irradiated protoplasts of Solanum pinnatisectum in genetic reconstruction

Summary Leaf mesophyll protoplasts of Solanum pinnatisectum (2n=24) -irradiated at doses of 200 Gy and consequently unable to divide were fused with untreated protoplasts of genomic chlorophyll deficient mutant IvP 841-1 (2n=24) containing the germplasms of S. tuberosum and S. phureja. Two types of plants differing in their pigmentation characteristics were selected. The regenerants of one group were identified as true somatic hybrids by using isozyme analyses of esterase and aspartate aminotransferase. The anthocyanin marker of S. pinnatisectum was phenotypically expressed in these regenerants and could be used as an additional selection trait for hybrid screening in this species combination. The regenerants of the second group were corrected for the gene controlling chlorophyll deficiency but contained species-specific isozymes of the potato cultivar only. Restriction analysis of chloroplast DNA revealed chloroplasts of the S. pinnatisectum type in all but one of the plants tested. The fusion experiments involving -irradiated protoplasts show that this approach in potato reconstruction has the advantage of producing a wide range of genetically novel plants.Dedicated to Prof. H. F. Linskens on his 65th birthday     

Unusual characteristics of Codium fragile chloroplast DNA revealed by physical and gene mapping

Summary A complete physical map of the Codium fragile chloroplast genome was constructed and the locations of a number of chloroplast genes were determined. Several features of this circular genome are unusual. At 89 kb in size, it is the smallest chloroplast genome known. Unlike most chloroplast genomes it lacks any large repeat elements. The 8 kb spacer region between the 16 S and 23 S rRNA genes is the largest such spacer characterized to date in chloroplast DNA. This spacer region is also unusual in that it contains the rps12 gene or at least a portion thereof. Three regions polymorphic for size are present in the Codium chloroplast genome. The psbA and psbC genes map closely to one of these regions, another region is in the spacer between the 16 S and 23 S rRNA genes and the third is very close to or possibly within the 16 S rRNA gene. The gene order in the Codium genome bears no marked resemblance to either the consensus vascular plant order or to that of any green algal or bryophyte genome. Present address: Department of Biology, Texas A&M University, College Station, TX 77843; USA