Production of hybrid calluses via donor-recipient fusion between Microcitrus papuana and Citrus sinensis

X-ray irradiated embryogenic protoplasts of Microcitrus papuana Swing. were electrically fused with iodoacetic acid-treated embryogenic protoplasts of Newhall navel orange [Citrus sinensis (L.) Osb.]. Seven cell lines were established by low-melting agarose embedding culture of fusion-treated protoplasts. Cytological examination of 4 cell lines showed that each cell line consisted of many aneuploid (45.10%, 38.98%, 32.69% and 34.85%, respectively) and diploid cells (52.94%, 59.33%, 63.46% and 62.12%. respectively), whereas only a few tetraploid cells (1.96%, 1.69%, 3.85% and 3.03%, respectively) were detected. Analyses of random amplified polymorphic DNA with four 10-mer primers confirmed the hybrid characteristics of the cell lines, which in combination with chromosome counting proved that the cell lines were asymmetric hybrids. This revised version was published online in June 2006 with corrections to the Cover Date.     

Citrus cybrids: production by donor-recipient protoplast-fusion and verification by mitochondrial-DNA restriction profiles

Summary Embryogenic nucellar-callus from three Citrus types was used for protoplast isolation. The protoplasts were fused by the donor-recipient procedure by which the nuclear-division of the donor-protoplasts was arrested by gamma-irradiation and the metabolism of unfused recipient-protoplasts was transiently inhibited by iodoacetate. The following fusion combinations were performed: (1) Poorman x Poncirus trifoliata with Villafranca lemon; (2) Poorman x P. trifoliata with Sour orange; (3) Sour orange with Villafranca. Combinations 1, 2 and 3 resulted in 120 (125), 70 (5) and 19 (89) calli (regenerated plants), respectively. The mitochondrial-DNA (mtDNA) restriction profiles of Poorman x P. trifoliata obtained by fragmentation with Bcl I, Bam H1 or Sal I differed from the respective profiles of Villafranca and Sour orange but no differences in mtDNA restriction profiles were detected between Villafranca and Sour orange. When Southern blots of Villafranca and Sour orange mtDNAs were hybridized with radiolabelled heterologous mtDNA probes, the mtDNAs of these two Citrus types could be differentiated. The fusion-derived plants from all three donor-recipient combinations had the recipients morphological (nuclear-coded) features. MtDNA restriction profiles, with and without hybridization to heterologous probes, indicated that the analysed plants were cybrids.Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel, No. 1917 E, 1986 series     

The plastome of Citrus. Physical map,variation among Citrus cultivars and species and comparison with related genera

Summary A physical plastome map was constructed for Citrus aurantium, and the plastomes of species and cultivars of Citrus and of two Citrus relatives were analysed by Southern blot-hybridisation of labelled total tobacco cpDNA to digests of total Citrus DNA. A resemblance was found between the plastomes of cultivars of C. limon (lemon), C. sinensis (orange), C. aurantium (sour orange), C. paradisii (grapefruit) and C. grandis (pomello). The plastomes of other Citrus types such as mandarin (C. reticulata) and citron (C. medico) differed from each other as well as from the plastomes of the aforementioned group. The plastomes of Poncirus trifoliata and Microcitrus sp. are distinct from each other as well as from the Citrus types.     

Inheritance of chloroplast and mitochondrial DNA in alloplasmic forms of the genus Daucus

The inheritance of mitochondrial (mt) and chloroplast (ct) DNA in the progeny from interspecific crosses between the cultivated carrot (Daucus carota sativus) and wild forms of the genus Daucus was investigated by analysis of mt and ct RFLPs in single plants of the parental and filial generations. We observed a strict maternal inheritance of the organellar DNAs in all interspecific crosses examined. Previous studies on putative F₂ plants from a cross between Daucus muricatus x D. carota sativus suggested paternal inheritance of ctDNA. Our reinvestigation of this material revealed that the mtDNA of the putative F₂ plants differed from the mtDNA of both putative parents. Therefore, our data suggest that the investigated material originated from other, not yet identified, parents. Consequently, the analysis of this material cannot provide evidence for a paternal inheritance of ctDNA.     

Correction of chlorophyll-defective male-sterile winter oilseed rape (Brassica napus) through organelle exchange: molecular analysis of the cytoplasm of parental lines and corrected progeny

Summary Cytoplasmic differences between male-fertile and male-sterile Brassica napus as well as Raphanus sativus were investigated. Plastids of the male-fertile B. napus were found to differ from those of male-sterile B. napus and R. sativus with respect to DNA restriction enzyme patterns. Differences between male-fertile and male-sterile B. napus mitochondria were detected not only in the restriction fragment patterns of their DNA, but also at the level of expression by in organello translation of mitochondrial polypeptides.The chlorophyll deficiency obtained upon transferral of the male-sterility-conferring radish cytoplasm to a winter variety of B. napus had been corrected earlier through protoplast fusion. The cytoplasmic composition of the corrected lines was analysed using DNA restriction analysis and in organello translation. The stability of the recombined cytoplasm in the corrected lines was confirmed by analysis of the subsequent seed-derived generation.     

Mitochondrial DNA polymorphism induced by protoplast fusion in Cruciferae

Summary The mitochondrial genomes of five rapeseed somatic hybrid plants, which combine in a first experimentBrassica napus chloroplasts and a cytoplasmic male sterility trait coming fromRaphanus sativus, and in a second experiment chloroplasts of a triazine resistantB. compestris and a cytoplasmic male sterility trait fromR. sativus, were analyzed by restriction endonucleases. Restriction fragment patterns indicate that these genomes differ from each other and from both parents. The presence of new bands in the somatic hybrid mitochondrial DNA restriction patterns is evidence of mitochondrial recombination in somatic hybrid cells. In both parental and somatic hybrid plants large quantitative variations in a mitochondrial plasmid-like DNA have been observed. Our results suggest that the cytoplasmic support for male sterility is located in the chromosomal mitochondrial DNA instead of the plasmid-like DNA.     

Selection of Daucus cybrids based on metabolic complementation between X-irradiated D. capillifolius and iodoacetamide-treated D. carota by somatic cell fusion

Summary Protoplasts of Daucus capillifolius isolated from a suspension culture (chromosome number above 60) were X-irradiated over lethal dose (60 krad) just prior to fusion. Protoplasts from D. carota cell line (chromosome number 17) were treated with 15 mM iodoacetamide and fused with the X-irradiated protoplasts. Putative cybrid plants were regenerated on Murashige and Skoog medium (MS) lacking 2,4-D. The regenerated plants possessed chromosome numbers of 17 (2n–1) or 34 (4n–2) and an identical leaf morphology to D. carota. Their mitochondrial DNAs (mtDNAs) were analysed with restriction endonucleases. Novel restriction fragments, not present in mtDNA digests from both parents, were observed in mtDNAs of regenerated plants. These results indicate successful formation of cybrids between D. capillifolius and D. carota by protoplast fusion.     

Diploid Brassica napus somatic hybrids: characterization of nuclear and organellar DNA

Summary Five somatic hybrids between Brassica campestris and B. oleracea were obtained. Molecular, morphological and cytological information all suggest that the resynthesized B. napus plants were hybrids. All five plants were diploid (2n=38) and had mainly bivalents at meiosis. Seedset was low after selfing but normal after crossing with B. napus. Molecular proof of the hybrid nature of these plants was obtained by hybridization of a rDNA repeat to total DNA. Analysis of chloroplast DNA restriction patterns revealed that all hybrids had chloroplasts identical to the B. oleracea parent. The analysis of mitochondrial DNA indicated that three hybrids had restriction patterns identical to those of B. campestris, and the other two had restriction patterns similar to those of B. oleracea. The 11.3 kb plasmid present in mitochondria of the B. campestris parent was also found in mitochondria of all five hybrids. This suggests that the plasmid from a B. campestris type of mitochondria was transferred into mitochondria of a B. oleracea type.     

Seasonal variation in the competence of the buds of three cultivars from different Citrus species to flower

The role of bud competence in the determination of flowering seasonality was studied in three Citrus cultivars, Bearss lime (Citrus latifolia Tan.), Fino lemon (C. limon [L.] Burm. f.) and Owari satsuma (C. unshiu (Mak.) Marc.), which differ in their adaptation to hot climates and their propensity to produce off-season blooms. Potted plants were kept in a greenhouse under non-inductive conditions (minimum temperature higher than 20°C), and periodically the flowering response was determined of a group of trees exposed for 30 days to an inductive temperature regime (15/8°C). A seasonal change in bud competence was demonstrated, and both bud sprouting and flower formation were highest when the low temperature regime was imposed during February and March. During the summer months, the low temperature regime resulted in a small increase in bud sprouting as compared to non-chilled trees, but only vegetative buds developed and no flowers were formed. The influence of environmental factors on the determination of bud competence was further studied. No effect of photoperiod was found, but raising the minimum air temperature above 25°C during 60 days, eliminated bud competence in Owari satsuma. In Bearss lime trees, the buds reacquired the competence after 4 months at 25/20°C, a temperature regime that does not induce flower formation. The reacquisition of competence was much faster at a lower temperature (15/8°C). A consistent relationship between the flowering response and DNA methylation in buds could not be demonstrated in all cultivars.     

Variation of plastid and mitochondrial DNAs in the genus Hedysarum

Summary Plastid and mitochondrial DNAs from Hedysarum species of the western Mediterranean basin, H. spinosissimum ssp eu-spinosissimum, H. spinosissimum ssp capitatum, H. carnosum, H. coronarium and H. flexuosum, were compared by restriction endonuclease fragment analysis. ctDNA fragment patterns for ssp eu-spinosissimum and ssp capitatum were indistinguishable in different enzyme digests. An identical ctDNA variation was found in Hpa II digests with two Sardinian populations of ssp capitatum. Each of the two subspecies was characterized by specific mt DNA patterns with Pst I, Bam HI, Sma I and EcoRI. No variation was detected in populations of different geographical origins for a given subspecies. H. carnosum, H. coronarium and H. flexuosum generated specific ct and mt DNA patterns. Comparison of mitochondrial fragments indicated: — a strong homology between the two subspecies, — a closer homology among the three other diploids, each being closer to the other two than to H. spinosissimum subspecies — as was also the case for the plastid genomes.     

Improvement of<Emphasis Type="Italic"> Citrus clementina</Emphasis> Hort. ex Tan. microspore-derived embryoid induction and regeneration

An improvement of the protocol for haploid induction through anther culture of Citrus clementina Hort. ex Tan. cv. Nules was achieved following the evaluation of a number of the factors affecting androgenesis. The influence of thidiazuron (TDZ) and three temperature pre-treatments (4°C, 25°C, 32°C) on the floral buds with respect to anther culture of C. clementina Hort. ex Tan., cv. Nules was investigated. An increased embryoid production was induced in the medium supplemented with TDZ. Pre-treatment temperatures of 4°C and 25°C were more favorable for embryo production than 32°C. Regeneration of androgenic haploid plantlets from cv. SRA 63 of C. clementina is reported here for the first time.Abbreviations 6-BA 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA₃ Gibberellic acid - KI Kinetin - NAA -Naphthaleneacetic acid - TDZ Thidiazuron (N-phenyl-1,2,3,-thi-diazol-5-ylurea) - ZEA Zeatin Communicated by L. PeñaBoth authors have contributed equally to this article.     

Induction of triploid Citrus plants from endosperm calli in vitro

Summary Triploid hybrid Citrus plants were regenerated by somatic embryogenesis in vitro from endosperm derived calli. A sequence of media formulations was used to induce and support proliferation of primary callus from endosperm, to induce embryogenesis from primary callus, and to allow embryo development leading to viable plantlets. Calli were induced from cellular endosperm of Citrus sinensis (sweet orange), C. Xparadisi (grapefruit), and C. grandis (pummelo) excised 12–14 weeks post-anthesis. Induction of embryogenesis from sweet orange and pummelo primary calli required gibberellic acid and double mineral nutrient concentrations. Embryogenesis was not induced from grapefruit calli in these experiments. Only sweet orange embryos developed sufficiently to allow plant regeneration. Triploid axillary buds were minigrafted onto etiolated diploid rootstock seedlings in vitro in order to transfer triploid regenerants to soil and the external environment. Triploidy (2n = 3x = 27) was observed consistently in all phases of regeneration and in recovered plants. These results demonstrate that triploid hybrid plant recovery from Citrus endosperm can overcome barriers to sexual hybridization resulting from apomixis.Florida Agricultural Experiment Station Journal Series No. R-00627     

Absence of mitochondrial and chloroplast DNA recombinations in Brassica napus plants regenerated from protoplasts,protoplast fusions and anther culture

Summary Over 400 Brassica napus plants regenerated from individual protoplasts, from protoplast fusions and from anther culture were analysed for chloroplast and mitochondrial genome rearrangements by restriction fragment length polymorphisms. None were detected, attesting to the fidelity of the tissue culture procedures employed. In the majority of protoplast fusion products, the cytoplasmic organelles had completely sorted out at the callus stage but three regenerated plants possessed mixed parental populations of mitochondrial genomes and one regenerant contained mixed chloroplast genomes. In all four examples, the cytoplasmic genome sorted out in planta in favor of one parental type which was faithfully maternally transmitted to progeny.     

Composition of soluble cuticular lipids and water permeability of cuticular membranes from Citrus leaves

Water permeability and composition of soluble cuticular lipids of isolated cuticular membranes from leaves of Citrus aurantium L. were investigated for 3 successive years. The average water permeability coefficient determined using 169 cuticular membranes was 1.09·10^–7 cm s^–1 with a standard deviation of 0.78·10^–7 cm s^–1. There were no significant differences in water permeability between years. Cuticular membranes are characterized by a great variability in water permeability both within and between years. Both water permeability of individual membranes and variability between membranes are shown to be determined by soluble cuticular lipids contained within the cuticular membranes. The soluble cuticular lipids of Citrus leaves are composed of fatty acids, primary alcohols, esters, and hydrocarbons. They occur in amounts of 9.84 g cm^–2, which represents approx. 3% of the total mass of isolated cuticular membranes. The specific weight of cuticular membranes (365.4 g cm^–1) and total amount of soluble cuticular lipids did not vary significantly between years. Significant differences were observed for the amounts and composition of the constituent classes of lipids. Six homologues comprise 86% of the fatty acids (C₁₆; C₁₈; C₁₉; C₂₁; C₂₄; C₂₆), 83% of the primary alcohols (C₂₄; C₂₆; C₂₈; C₃₀; C₃₂; C₃₄) and 88% of the esters (C₃₆; C₃₈; C₄₀; C₄₁; C₄₂; C₄₄). Eleven major homologues amount only to 62% of the total hydrocarbons (C₁₆; C₁₇; C₁₈; C₂₀; C₂₆; C₂₇; C₂₉; C₃₀; C₃₁; C₃₂; C₃₃). Variability in the composition of soluble cuticular lipids between years was much smaller than variability of water permeability and, therefore, no relation between composition of soluble cuticular lipids and water permeability could be found. It is suggested that this may be due to the fact that the lipid composition observed represents the averages of 20 to 30 membranes analyzed so that differences between individual membranes may have been leveled out.Abbreviations CM cuticular membranes - MX polymer matrix - P_d permeability coefficient for diffusion of water - SCL soluble cuticular lipids - MES morpholinoethane sulphonic acid     

Mitochondrial DNA rearrangements in somatic hybrids of Solanum tuberosum and Solanum brevidens

Summary Thirty somatic hybrids between Solanum tuberosum and Solanum brevidens were analysed for mitochondrial and chloroplast genome rearrangements. In all cases, the chloroplast genomes were inherited from one of the parental protoplast populations. No chloroplast DNA alterations were evident but a range of mitochondrial DNA alterations, from zero to extensive intra- and inter-molecular recombinations, were found. Such recombinations involved specific recombination hot spots in the mitochondrial genome. Not all hybrids regenerated from a common callus possessed identical mitochondrial genomes, suggesting that sorting out of mitochondrial populations in the callus may have been incomplete at the plant regeneration stage. Sorting out of organelles in planta was not observed.     

Ethylene-binding characteristics in Phaseolus,Citrus, and Ligustrum plants

A number of plants were tested for their ability to bind ethylene and the number of binding sites present in each was calculated. Primary leaves of laboratory-grown beans (Phaseolus vulgaris) bound 140 dpm/g fwt (1794 dpm/g dry wt) when exposed to 1.0 Ci/1 of [¹⁴C]ethylene (110 ci/mol). Phytotron-grown leaves were less succulent but only bound 90 dpm/g fwt (1046 dpm/g dry wt). Bean roots bound 30 dpm/g fwt. Citrus and Ligustrum bound 207 and 240 dpm/g fwt, respectively. The time required to achieve equilibrium of leaves with the gas phase was 15 min for bean, 30 min for Citrus, and 30–60 min for Ligustrum. The time for 1/2 of the bound ethylene to diffuse out of the leaves was 20 min for bean, 10 min for Citrus, and 30 min for Ligustrum. The amount of ethylene needed to occupy 1/2 of the binding sites was obtained from Scatchard plots. This value (K_d) was 0.2 l/1 for bean, 0.15 for Citrus, and 0.31 for Ligustrum. The quantity of binding sites in the tissues was 2.0×10^-9 mol of binding sites/kg tissue for bean leaves, 5.7×10^-9 for Citrus leaves, and 6.8×10^-9 for Ligustrum. Pretreatment with indoleacetic acid (IAA), ehtylene, and cycloheximide (1 mg/1) had little effect on the level of ethylene-binding sites in Citrus.Contribution from the Department of Biochemistry, School of Agriculture and Life Sciences and School of Physical and Mathematical Sciences, North Carolina State University. Paper No. 8445 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina 27695-7601.North Carolina-Israel exchange Scholar for 1981 at the Department of Biochemistry, North Carolina State University Raleigh, North Carolina, USA     

Differential patterns of mitochondrial,chloroplastic and nuclear DNA synthesis in the synchronous cell cycle of Chlamydomonas reinhardtii

Nuclear DNA (ncDNA) synthesis in Chlamydomonas reinhardtii was measured by both ³²P[or-thophosphoric acid] (³²P) and [¹⁴C]adenine incorporation and found to be highly synchronous. Ca. 85% of incorporation was confined to the first 6 h of the dark period of a synchronized regime consisting of an alternating light-dark period of 12 h each. In contrast, no such synchronous incorporation pattern was found for chloroplast (cp) and mitochondrial (mt) DNAs in the same cell population. These two organellar DNAs also exhibited different ³²P-incorporation patterns in the cell cycle. Considerable amounts of ³²P were incorporated into cpDNA throughout the light-dark synchronous cycle under both mixo- and phototrophic growth conditions, although the second 6-h light period under phototrophy showed an increase not apparent under mixotrophy. This change in growth conditions did not affect ³²P incorporation into mtDNA, which was found throughout the cell cycle, with a modest peak in the first 6-h of the dark period. The pattern of [³H]thymidine incorporation into cpDNA was also determined. Under synchronous phototrophic conditions, this pattern was quite different from that obtained with ³²P. Most [³H]thymidine incorporation occurred during the light period of the synchronous cycle; this period had been shown previously by density transfer experiments to be the time of cpDNA duplication. Such preferential [³H]thymidine incorporation into cpDNA in the light period was not observed under mixotrophic synchronous growth conditions; in these, [³H]thymidine incorporation was detected throughout the cell cycle. This lack of coincidence between the patterns of ³²P- and of [³H]thymidine incorporation into cpDNA during the synchronous cell cycle indicates that in addition to replication, the considerably reiterated organelle-DNA molecules may also regularly undergo an extensive repair process during each cell cycle.     

Phytogeny of Brassica and allied genera based on variation in chloroplast and mitochondrial DNA patterns: molecular and taxonomic classifications are incongruous

Summary Chloroplast DNA (cpDNA) variability of 60 taxa of the genus Brassica and allied genera comprising 50 species was studied. RFLPs for seven enzymes were generated and F values were estimated from five frequently cutting enzymes. Phenetic clusterings indicated a clear division of Brassica coenospecies into two distinct lineages referred to as the Brassica and Sinapis lineages. Two unexplored genera, Diplotaxis and Erucastrum, also exhibited two lineages in addition to the genera Brassica and Sinapis. This finding is inconsistent with the existing taxonomic classification based on morphology. Mitochondrial DNA (mtDNA) variability studied from EcoRI RFLP patterns, by hybridizing total DNA with four cosmid clones containing non-overlapping mtDNA fragments, did not show any congruence with cpDNA variation patterns. However, at the cytodeme level, the patterns of genetic divergence suggested by the cpDNA data could be correlated with mtDNA variation. In the Brassica lineage, Diplotaxis viminea was identified as the female parent of the allotetraploid D. muralis. The chloroplast DNAs of Erucastrum strigosum and Er. abyssinicum were found to be very closely related. In the Sinapis lineage, Brassica maurorum was found to be the diploid progenitor of autotetraploid B. cossoneana. B. amplexicaulis showed a very different cpDNA pattern from other members of the subtribe. Brassica adpressa was closest to Erucastrum laevigatum and could be the diploid progenitor of autotetraploid Er. laevigatum. Based on the close similarity of the cpDNA pattern of Diplotaxis siifolia with that of D. assurgens, we have proposed the retention of this species in the genus Diplotaxis. The taxonomic positions of some other species have also been discussed.