László Menczel

Streptomycin resistant and sensitive somatic hybrids of Nicotiana tabacum + Nicotiana knightiana: correlation of resistance to N. tabacum plastids.

SummaryProtoplasts of Nicotiana tabacum SRI (streptomycin resistant) and of Nicotiana knightiana (streptomycin sensitive) were fused using polyethylene glycol treatment. From three heterokaryons 500 clones were obtained. From the 43 which were further investigated, 6 resistant, 3 sensitive, and 34 chimeric (consisting of resistant and sensitive sectors) calli were found. From eight clones, a total of 39 plants were regenerated and identified as somatic hybrids. Chloroplast type (N. tabacum = NT or N. knightiana = NK) in the plants was determined on the basis of the species specific EcoRI restriction pattern of the chloroplast DNA. Regenerates contained NT (13 plants) or NK (15 plants) plastids but only the plants with the NT chloroplasts were resistant to streptomycin. This finding and our earlier data on uniparental inheritance points to the chloroplasts as the carriers of the streptomycin resistance factor.

Chloroplast transfer in Nicotiana based on metabolic complementation between irradiated and iodoacetate treated protoplasts

Protoplasts of a light sensitive plastome mutant of Nicotiana tabacum (2 n=48) were irradiated and fused with iodoacetate-treated Nicotiana plumbaginifolia (2 n=20) protoplasts. Treated parental protoplasts were unable to divide. Metabolic complementation, however, helped the recovery of interspecific fusion products which survived and formed calli. Altogether 40 clones were investigated. N. plumbaginifolia plants were obtained in 15 clones (38%), somatic hybrids in 23 clones, and both types of regenerates were found in 2 clones. Irradiation therefore significantly increased the frequency of segregant formation with the non-irradiated N. plumbaginifolia nuclei (the frequency was 1.4% in the absence of irradiation). Regenerated plants in most cases (31 out of 34) contained chloroplasts from the irradiated parent. In 6 clones plants were obtained with both types of chloroplast. Thus, irradiated N. tabacum chloroplasts had an improved chance of dominating the heterokaryonderived cells, many of which contained N. plumbaginifolia nucleus. The system described should be generally applicable for the transfer of chloroplasts without the use of selectable genetic markers.

The use of cytoplasmic streptomycin resistance: Chloroplast transfer from Nicotiana tabacum into Nicotiana sylvestris, and Isolation of their somatic hybrids

SummaryLeaf protoplasts of Nicotiana tabacum SR1 (2n=4x=48) treated with iodoacetate (10 mM; 25 C; 30 min) and consequently unable to divide, and untreated leaf protoplasts of Nicotiana sylvestris (2n=2x=24) were fused using polyethylene glycol (PEG). The SR1 line is resistant to streptomycin because of a maternally inherited mutation, and has streptomycin-insensitive chloroplast ribosomes.After 1 month of growth in the absence of streptomycin protoplast-derived calli were plated into selective medium (1,000 μg ml-1 streptomycin) and the resistant clones were isolated. Out of 106 PEG-treated protoplasts (1:1 mixture of parental types) 137 resistant (green) clones were obtained, whereas in the same number of parental cells, not subjected to fusion induction, no resistant callus was found.At least four plants were regenerated from each of the clones. The regenerates were identified as somatic hybrids (H), N. sylvestris (Ns) or N. tabacum (Nt) by looking at esterase and peroxidase isoenzymes and morphology. The three types of regenerates were distributed amongst the clones as follows: H only (105 clones); Ns (16 clones); Ns+H (6 clones); Nt only (3 clones); Nt+H (6 clones); Nt+Ns (1 clone). The high proportion of hybrid regenerates indicates that nuclear fusion has occured in the overwhelming majority of the heterokaryocytes. Cytoplasmic mutations in combination with inactivation by iodoacetate, therefore, are suitable markers to produce somatic hybrids. Segregation of nuclei after fusion resulted in new combinations of organelles and nuclei, the final outcome being the transfer of resistant chloroplasts into N. sylvestris, some of which have the original diploid (2n=24) chromosome number. Data suggest that segregants were in most cases obtained from multiple fusions. Streptomycin resistance was inherited maternally in the N. sylvestris (six clones) tested and the hybrid (three clones) regenerates.

High frequency of fusion induced in freely suspended protoplast mixtures by polyethylene glycol and dimethylsulfoxide at high pH.

Fusion of freely suspended protoplast mixtures (hypocotyl protoplasts of Brassica napus mixed with mesophyll protoplasts of either B. campestris or Nicotiana plumbaginifolia) was induced by a solution containing 10% polyethylene glycol, 10% dimethyl-sulfoxide and 0.1M glycine-NaOH buffer (pH 10.0). The fusion products represented 15 to 17 percent of the surviving cells. More than 50% of the fusion products divided within two days after fusion, indicating that the fusion procedure did not significantly affect the viability of fused cells. The fusion products were not bound to the surface of the fusion vessel, so they could be isolated with a micropipette immediately after fusion.

Fusion-mediated combination of Ogura-type cytoplasmic male sterility with Brassica napus plastids using X-irradiated CMS protoplasts

X-irradiated protoplasts of a Brassica napus line carrying the Ogura Raphanus sativus male sterile cytoplasm were fused to protoplasts of male fertile B. napus cv. Olga. Plants were regenerated from six out of 34 randomly selected clones. In one clone, Bn(RS)26, a plant with male sterile flowers was obtained. Mitochondria of this plant are non-parental as revealed by DNA-DNA hybridization using a species specific probe. Its chloroplasts, however, derive from the fertile parent which results in loss of the sensitivity to low temperatures associated with R. sativus plastids in the male sterile parent. The novel cytoplasm of the Bn(RS)26 cybrid was transmitted through seed.

A heteroplasmic state induced by protoplast fusion is a necessary condition for detecting rearrangements in Nicotiana mitochondrial DNA.

SummaryMitochondrial DNA (mtDNA) restriction patterns were studied in mutant, cybrid and somatic hybrid plants regenerated from Nicotiana protoplasts.It has been shown that neither components of the culture media used for protoplast culture and plant regeneration, nor the antibiotics streptomycin and lincomycin used for the mutant selection induced alterations in the mtDNA. No rearrangements were detected in the mtDNA of plants derived from homoplasmic fusions where the mtDNA of the parents was identical as judged by mtDNA restriction patterns.There were rearrangements, however, in the mtDNA of each of the cybrid plants derived from heteroplasmic fusions. Restriction patterns generated by BamHI and SalI restriction endonucleases were different from those of both parents, and were composed of parental and non-parental fragments.

Transfer of cytoplasmic male sterility by selection for streptomycin resistance after protoplast fusion in Nicotiana

SummaryCytoplasmic male sterility (CMS) in Nicotiana is located on mitochondrial DNA (mtDNA) on which no selectable mutation has been isolated. The possibility of co-transfer of CMS and a selectable chloroplast trait, streptomycin resistance, was investigated by marker rescue from irradiated protoplasts, a method described from this laboratory (Menczel et al. 1982). As the source of the CMS factor, the Nicotiana tabacum St-R701 line was chosen. This line is male sterile due to replacement of its original cytoplasm by that of N. megalosiphon, and carries a cytoplasmic streptomycin resistance mutation (Umiel 1979).Following fusion of irradiated (lethal dose) St-R701 protoplasts with untreated N. plumbaginifolia protoplasts, streptomycin-resistant clones were selected. Regenerated plants representing 27 clones were studied in detail. These plants have a N. plumbaginifolia nuclear genome and the resistance factor from the St-R701 parent.In each clone transfer of streptomycin resistance resulted in the transfer of St-R701 chloroplasts suggesting the involvement of chloroplast DNA in determining streptomycin resistance. All regenerated streptomycin resistant N. plumbaginifolia plants were male sterile. In plants from three clones, in addition to male sterile flowers, a few male fertile flowers were obtained.

Efficient transient expression system based on square pulse electroporation and in vivo luciferase assay of fertilized fish eggs

Electroporation mediated DNA transfer into fish eggs has been improved by using a train of square pulses. Fertilized eggs of African catfish (Clarias gariepinus), zebrafish (Brachydanio rerio) and rosy barb (Barbus conchonius) were dechorionated enzymatically followed by application of pulses. Efficiency of plasmid DNA delivery was significantly increased by applying multiple pulses on dechorionated eggs. Optimization of physical parameters such as field strength, pulse width and pulse numbers resulted in reproducible transient expression in 25–50% of embryos and larvae by using the firefly luciferase and the E. coli β‐galactosidase (lacZ) genes both driven by CMV IE1 promoter. Temporal luceferase expression was assayed using both qualitative (sheet film) and quantitative (scintillation counting) methods in developing embryos and fry in vivo. Spatial expression of lacZ was assayed by histochemical staining. A number of embryos revealed foreign gene product also localised in the vegetal pole of the embryo.

Isolation of somatic hybrids by cloning Nicotiana heterokaryons in nurse cultures.

Fusion of Nicotiana knightiana Goodsp. and kanamycin resistant Nicotiana sylvestris Speg. et Com. protoplasts was induced by polyethylene glycol treatment. Heterokaryons were isolated by micropipette and transferred to nurse cultures of albino cells. Colonies originating from the heterokaryons could subsequently be distinguished by their green colour. The somatic hybrid nature of four such colonies was confirmed by isoenzyme pattern, kanamycin resistance and restored morphogenic potential. An additional kanamycin resistant line with characteristic Nicotiana knightiana isoenzymes was also found indicating that the drug resistance in the kanamycin resistant parent is under cytoplasmic control.

Fusion-mediated transfer of triazine-resistant chloroplasts: Characterization of Nicotiana tabacum cybrid plants

SummaryTerbutryn-resistant plastids of the Nicotiana plumbaginifolia TBR2 mutant were introduced into N. tabacum plants by protoplast fusion following X-irradiation of TBR2 protoplasts. The N. tabacum chloroplast recipient line, SR1-A15, carried mutant (albino) plastids. Following protoplast fusion, potential cybrid cell lines with an N. tabacum (SR1-A15) nucleus and N. plumbaginifolia (TBR2) chloroplasts were identified by their green color. The presence of TBR2 plastids in regenerated green N. tabacum plants was confirmed by hybridization with a chloroplast DNA probe and by the modified chloroplast fluorescence transients characteristic of the TBR2 mutant. Cybrid plants were resistant to high levels of atrazine (10 kg/ha). The protruding stigma and shorter than normal filaments of the cybrids resulted in male sterility. In the cybrids atrazine resistance was associated with reduced vigour, suggesting a causal relationship.